The Role of the Transcription Factor Nfat in the Regulation of Smooth Muscle Contractility Jeff Layne University of Vermont

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Graduate College Dissertations and Theses Dissertations and Theses

2008 The Role of the Transcription Factor nFAT in the Regulation of Smooth Muscle Contractility Jeff Layne University of Vermont

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THE ROLE OF THE TRANSCRIPTION FACTOR NFAT IN THE REGULATION OF SMOOTH MUSCLE CONTRACTILITY

A Dissertation Presented

Jeff Layne

The Faculty of the Graduate College

The University of Vermont

In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Pharmacology

October, 2007

ABSTRACT

The goals of this dissertation were to explore the role of the Ca2+-sensitive transcription factor NFAT in the regulation of smooth muscle contractility. We have identified a conserved NFAT binding site that overlaps an intronic SRF- binding CArG element that has previously been demonstrated to be essential for expression of smooth muscle α-actin. Transfection of a reporter construct containing the composite CArG/NFAT element, designated SNAP, into SMCs resulted in robust basal reporter activity that was sensitive to the calcineurin/NFAT pathways inhibitors FK506 and CsA. Mutations to either the NFAT or adjacent SRF binding site essentially abolished reporter activity, indicating that both were required. Co-immunoprecipitation assays revealed that NFATc3 and SRF formed a complex in solution, and that the formation of this complex was facilitated by the presence of the SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased α-actin expression in cultured SMCs, suggesting that NFAT plays a role in the expression of smooth muscle contractile proteins such as α-actin. To determine if NFAT transcriptional activity is involved in modulating urinary bladder smooth muscle contractility, we compared the contractile and electrophysiological properties of NFATc3-null mice to wild-type mice. UBSM strips taken from NFATc3-null mice displayed an elevated contractile response to EFS compared to strips from wild-type mice. This increased contractility was due to a decrease in IBTX-sensitive BK current and was supported at the molecular level by reduced expression of mRNA for the pore-forming α-subunit of the BK channel. Single-channel recordings revealed that the β-subunit of the BK channel, which modulates the sensitivity of the BK 2+ channel to voltage and Ca , was not altered. Interestingly, TEA-sensitive KV currents, and expression of the pore-forming KV2.1 subunit, were increased in the NFATc3-null myocytes. However, the increased contractile response of UBSM strips from NFATc3-null mice indicates that, at least in response to electrical field stimulation, the downregulation of BK current plays a more significant role than does the increase in KV current. Presumably, this is due to the prominent role that BK channels play in shaping the UBSM action potential. Thus, this dissertation provides evidence that NFAT plays a role in modulating smooth muscle contractility via its role in regulating the expression of contractile proteins and ion channels and, furthermore, lays the foundation for future investigations into the specific role of NFAT in the pathological response of the urinary bladder to outlet obstruction. CITATIONS

Material from this dissertation has been published in the following form:

Gonzalez Bosc LV, Layne JJ, Nelson M, Hill-Eubanks DC. Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an alpha-actin intronic enhancer. J Biol Chem. 2005; 280:26113-20.

Material from this dissertation has been submitted for publication to the Journal of Physiology on January 24, 2007 in the following form:

Layne JJ, Werner ME, Hill-Eubanks DC, and Nelson MT. NFATc3 regulates BK channel function in murine urinary bladder smooth muscle.

ACKNOWLEDGEMENTS

I would like to thank all of the people who have contributed to my growth

as a not-so-young scientist at UVM. I will mention those who, as I write this

acknowledgement, I am able to recall, but I am sure that there are also many

others that I have forgotten to mention. I would like to thank the members of my

Thesis committee including Drs. Mark Nelson, Mercedes Rincon, David Hill-

Eubanks, and David Warshaw for their guidance and patience with a graduate student who does not always do a good job of informing them of his progress on his dissertation. In particular, I would like to thank Drs. Mark Nelson and David

Hill-Eubanks, the two scientists with whom I have had the closest working relationship during the term of my PhD studies at UVM. Mark was kind enough to allow me to do my Dissertation research in his laboratory, and his guidance has proven invaluable to my work. There were numerous occasions during my tenure here at UVM where my research seemed to be at a standstill and I had no idea how to proceed. However, Mark was able to pick out the important points from my data and point me in the correct direction to proceed. I will not say that we spent a lot of time together, but those rare moments when I was able to drag

Mark away from his busy schedule for a few minutes made all the difference in my research. If Mark Nelson was my Yoda, then Dr. David Hill-Eubanks has been by Obi-Wan Kenobi, my most intimate guidance counselor who has been able to support me both with his lore and counsel, and also with his hands-on

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guidance in the laboratory. From him I learned not only the philosophy of

science, but also many of the nuts-and-bolts of molecular biology techniques. My

growth and development as a scientist are due, in large part, to the efforts of

these two scientists.

In addition to Mark and David, I have had the pleasure to work with many

other people who have helped me to achieve my goals. Drs. Adrian Bonev,

Thomas Heppner, Kevin Thorneloe, Laura Gonzalez Bosc, and Matthias Werner

all took time out of their busy schedules to instruct me on various scientific

techniques in the Nelson Lab. I consider my time here at UVM to be quite rich, and I have had the opportunity to learn a many techniques, ranging from molecular assays such as Western Blotting, to more physiological practices such as myography and patch-clamp analysis. These scientists were kind and patient enough to share their knowledge with me, and for this I am very grateful.

Lastly, I would like to thank my family for their love and support and, most importantly, for their patience over the last five years. I am a very private man when it comes to personal and family matters, but I would like to thank my wife,

Julie, my daughter Anna, and my parents and my parent-in-laws. None of this would have been possible without their unwavering support.

TABLE OF CONTENTS

Citations ...... ii

Acknowledgements ...... iii

List of figures ...... viii

Chapter 1. Literature Review ...... 1 Overview of smooth muscle physiology...... 2 Ion channels in smooth muscle physiology ...... 6 Regulation of gene expression in UBSM ...... 16 Phenotypic modulation of vascular SM ...... 16 Phenotypic modulation of UBSM ...... 17 SRF-dependent gene regulation in SM ...... 19 Ca2+-dependent gene regulation in SM ...... 23 NFAT...... 29 The structure of NFAT proteins ...... 29 General features of NFAT activation...... 34 SM-specific features of NFAT activation...... 50 The role of NFAT in smooth muscle ...... 56 Hypotheses ...... 62

Chapter 2. Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an α-actin intronic enhancer ...... 67 Abstract ...... 69 Introduction ...... 71 Experimental Procedures...... 76 Cell culture ...... 76 Construction of plasmids ...... 76 Transient transfection and luciferase assay ...... 77 Whole-cell extracts ...... 78 Avidin biotin conjugated DNA binding assay ...... 79 Co-immunoprecipitation ...... 80 Western blot analysis ...... 80 Immunofluorescence ...... 81 In-cell western analysis ...... 82

Results ...... 84 A CArG box-containing region of the mouse α-actin first intron acts as an NFAT- and SRF-dependent enhancer element ...... 84 NFAT/AP-1 pathway stimulation increases SNAP enhancer activity in HEK293 cells ...... 86 NFATc3 and SRF simultaneously bind to the SNAP enhancer element ...... 87 NFATc3 exhibits constitutive nuclear localization in A7r5 cells but not HEK293 cells ...... 89 Calcineurin/NFAT pathway inhibition reduces α-actin expression in smooth muscle cells ...... 89 Discussion ...... 91 Acknowledgements ...... 98 References ...... 113

Chapter 3. NFATc3 Regulates BK Channel Function in Murine Urinary Bladder Smooth Muscle ...... 118 Abstract ...... 120 Introduction ...... 121 Methods ...... 127 Isolation of myocytes ...... 127 Electrophysiology ...... 127 UBSM contractility studies ...... 129 Immunohistochemistry and nuclear localization assay. 129 Semi-quantitative RT-PCR ...... 130 Statistical analysis ...... 131 Results ...... 132 Expression and stimulation of NFAT nuclear localization in urinary bladder smooth muscle ...... 132 UBSM strips from NFATc3-null mice exhibit enhanced contractility ...... 133 UBSM strips from NFATc3-null mice exhibit reduced sensitivity to the BK channel blocker, IBTX...... 134 BK currents are reduced in UBSM myocytes from NFATc3-null mice ...... 136 BK channel expression is downregulated in NFATc3-null mice ...... 137 UBSM myocytes isolated from NFATc3-null mice have increased KV currents ...... 138 Biophysical and molecular evidence for increased KV2.1 expression in NFATc3-null mice ...... 140

Discussion ...... 142 Acknowledgements ...... 147 References ...... 166

Chapter 4. Synopsis and future directions ...... 172

Comperehensive bibliography ...... 181

vii

LIST OF FIGURES

Chapter 1: Literature Review

Figure 1. Comparison of skeletal and smooth muscle contractile organization...... 4 Figure 2. Ca2+-dependent regulation of smooth muscle contraction ...... 7 Figure 3. Calcium sparks in smooth muscle tissues ...... 19 Figure 4. Possible pathways of SMC gene expression involve RhoA/ROK and CaMK ...... 27 Figure 5. Schematic diagrams of the NFAT isoforms ...... 30 Figure 6. Conserved NFAT domains and sequence motifs ...... 32 Figure 7. NFAT activation pathway ...... 35 Figure 8. Intracellular masking of the NFAT nuclear localization signal ...... 40 Figure 9. Integration of E-C and E-T coupling pathways ...... 63

Chapter 2: Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an α-actin intronic enhancer

Figure 1. Region of the mouse α-actin gene containing 5' CArG boxes A and B, exon 1, and intron 1 ...... 100 Figure 2. A CArG box-containing region of the mouse α-acti first intron acts as an NFAT- and SRF-dependent enhancer under basal conditions ...... 102 Figure 3. NFAT/AP-1 pathway stimulation increases SNAP reporter activity in HEK293 cells ...... 104 Figure 4. ABCD assay showing NFAT and SRF binding to SNAP ...... 106 Figure 5. SNAP facilitates co-immunoprecipitation of SRF and NFAT ...... 108 Figure 6. Differential subcellular localization of NFATc3 between HEK293 and A7r5 cells ...... 110 Figure 7. Calcineurin/NFAT pathway inhibition reduces α-actin expression in aorta-derived smooth muscle cells (A7r5) ...... 112

viii

Chapter 3. NFATc3 Regulates BK Channel Function in Murine Urinary Bladder Smooth Muscle

Figure 1. NFAT expression in murine UBSM cultured cells and tissue ...... 149 Figure 2. UBSM strips from NFATc3-null mice are more contractile than strips from wild-type mice ...... 151 Figure 3. UBSM strips from NFATc3-null mice display a reduced responsiveness to IBTX ...... 153 Figure 4. UBSM myocytes from wild-type and NFATc3-null + mice have similar total K currents (IK) ...... 155 Figure 5. UBSM myocytes from NFATc3-null mice have reduced BK currents (IBK) ...... 157 Figure 6. Reduced expression of the BK α-subunit in UBSM from NFATc3-null mice ...... 159 Figure 7. Single channel recordings and BK channel density from wild-type and NFATc3-null myocytes ...... 161 Figure 8. UBSM myocytes from NFATc3-null mice have elevated KV currents (IKV) ...... 163 Figure 9. Molecular and electrophysiological evidence for elevated KV2.1 expression in NFATc3-null mice ...... 165

CHAPTER 1: LITERATURE REVIEW

OVERVIEW OF SMOOTH MUSCLE PHYSIOLOGY

Smooth Muscle Contractility

The walls of the vasculature and hollow organs of the body, such as the

urinary bladder, are comprised of smooth muscle. In order to generate force, muscle fibers must shorten, or contract. In skeletal muscle, the fundamental

contractile unit is the sarcomere, which consists of interdigitating arrays of thick

and thin filaments, comprised of myosin and actin, respectively (Figure 1A).

Structures known as Z-discs (Figure 1A) serve as attachment points for the thin

filaments, and the region encompassed by two Z-discs comprises a single

sarcomere. Sarcomeres stack in series to form myofibrils, and bundles of these myofibrils, in turn, form muscle fibers (Meiss, 1995).

The contractions of smooth muscle tissues are also mediated by the interaction of thick and thin filaments. However, the ultrastructure of the smooth muscle contractile machinery is much less clearly defined (Small & North, 1995).

Smooth muscle lacks sarcomeres and, therefore, does not have the highly ordered, striated pattern that accounts for the appearance of skeletal muscle

(Figure 1B). The thin filaments of the smooth muscle contractile units are associated with α-actinin rich structures known as dense bodies that serve as attachment points in much the same way as the Z-discs of skeletal muscle

(Figure 1B; Hathaway et al., 1991; Andersson & Arner, 2004; Meiss, 1995; Small

& North, 1995). Intermediate filament proteins, including desmin and vimentin, are thought to play a role in maintaining the three-dimensional network of dense bodies (Bond & Somlyo, 1982).

Smooth muscle myosin is a hexamer consisting of two heavy chains

(MHC) associated with two pairs of light chains - regulatory light chains of ~20 kDa each and essential light chains of ~17 kDa (Andersson & Arner, 2004). Two myosin heavy chain isoforms, SM1 (~200 kDa) and SM2 (~204 kDa), are formed by alternative splicing of a transcript arising from the single MHC gene. The SM1 isoform contains a 43 amino acid loop in the carboxyl-terminus of the molecule, while the same region of the SM2 isoform contains a smaller 9 amino acid sequence (Nagai et al., 1989). In addition to the SM1 and SM2 isoforms, additional variants can be made via alternative splicing at the amino terminal, giving rise to the SM-A and SM-B isoforms. Thus, there are four possible myosin heavy chain isoforms expressed in smooth muscle: SM1-A, SM1-B, SM2-A, and

SM2-B. Although the functional consequences of the splice variants has not been fully explored, there have been reports that the SM1/SM2 ratio affects the maximal shortening velocities (Hewett et al., 1993), while the SM-A and SM-B variants may influence actin-myosin interaction kinetics (Sweeney et al., 1998).

According to the sliding filament model of muscle contraction (Huxley &

Niedergerke, 1954; Huxley & Hanson, 1954), force is generated as the thick and thin filaments of each individual contractile unit “slide” past one another, resulting

Figure 1. Comparison of skeletal and smooth muscle contractile organization. (A) Electron micrograph of a skeletal muscle sarcomere. (Modified from Alberts et al., 1989). (B) Schematic illustration of smooth muscle cytoskeletal and contractile unit organization (Meiss, 1995).

in a shortening of the contractile unit, although the lengths of the individual thick and thin filaments themselves do no change. This process is dependent upon

Ca2+, as will be discussed in the next section.

The Central Role of Calcium in Contractility

An elevation of intracellular Ca2+ levels is the trigger that initiates muscle contraction (Murphy, 1993). In skeletal muscle tissue, a protein complex consisting of tropomyosin and troponin is intimately associated with the actin filament. In the resting, non-contractile state, the tropomyosin molecules sterically protect myosin binding sites located on the actin filament. Ca2+ can bind

to troponin, and this binding, in turn, induces an allosteric change in tropomyosin that results in the exposure of the myosin binding sites in the actin molecule. The globular myosin heads of the thick filament can then interact with these exposed actin binding sites. The hydrolysis of ATP provides the energy required for the myosin heads to ratchet along the actin filament in a process known as cross- bridge cycling. This results in a shortening of the individual sarcomeric units and

the generation of force.

Troponin is not found in smooth muscle tissues such as UBSM (Hathaway

et al., 1991; Small & North, 1995). Thus, the Ca2+-sensitivity of smooth muscle

contractility is not conferred to it by this thin filament-associated process. Rather,

the Ca2+-dependent regulation of contractility in smooth muscle depends instead

upon the phosphorylation state of the regulatory myosin light chain molecules

located in the thick filament (Bremel, 1974; Hathaway et al., 1991; Pfitzer, 2001).

In brief, under low cytosolic Ca2+ conditions, the myosin regulatory light chains

are not phosphorylated and there is no interaction between the myosin heads and actin filaments. When intracellular Ca2+ is elevated, myosin light chain kinase

(MLCK) can be activated by Ca2+-calmodulin (CaM). MLCK phosphorylates the

regulatory myosin light chains, shifting the conformation of the myosin molecules

into one in which cross-bridge formation, and force generation, can occur. A

second enzyme, myosin phosphatase, dephosphorylates the regulatory light

chains to reverse this process and return the muscle to the relaxed state (Pfitzer,

2001) (Figure 2).

Ion Channels in Smooth Muscle Physiology

The contractions of both skeletal and smooth muscle tissues are

dependent upon intracellular Ca2+ levels as Ca2+ is required to expose the actin

binding sites in skeletal muscle and to activate MLCK in smooth muscle tissues.

Given the dependence of the contractile process on Ca2+, intracellular Ca2+ levels must be precisely regulated to meet the contractile needs of the cell. To accomplish this regulatory feat, smooth muscle cells rely upon a repertoire of ion channels and pumps that serve to modulate, directly or indirectly, intracellular

Ca2+ levels.

Figure 2. Ca2+-dependent regulation of smooth muscle contraction.

Extracellular Ca2+ influx triggered by receptor activation or membrane

depolarization initiates the formation of the Ca2+-CaM complex. Ca2+-CaM

activates the enzyme MLCK, which phosphorylates the regulatory light chains of

myosin. This shifts the conformation of myosin into one in which interaction

between the myosin globular heads and actin is favorable. (From Pfitzer, (2001),

Regulation of myosin phosphorylation in smooth muscle, J. Appl. Physiol.

91:497-503).

Channels mediating extracellular Ca2+ entry

In smooth muscle tissues, depolarization of the plasma membrane leads

to Ca2+ entry through voltage-dependent Ca2+ channels (VDCC) (Thorneloe &

Nelson, 2005). Under basal conditions, the concentration of cytosolic Ca2+ is low

(~120 nmol/L; (Williams & Fay, 1986; Knot & Nelson, 1998), while Ca2+ levels in the sarcoplasmic reticulum (SR) and extracellular space are in the millimolar range. This steep chemical gradient, combined with the negative resting membrane potential of SM cells provides a powerful electrochemical driving force favoring the flow of Ca2+ into the cytosol from the extracellular space. Stimuli that

depolarize the cell membrane will open VDCC, trigger a rapid increase in

intracellular Ca2+ levels, and promote contraction. Conversely, those factors that

tend to hyperpolarize the membrane, namely outward current through potassium

(K+) channels, will oppose the influx of extracellular Ca2+ through VDCC and tend

to relax the cell. The net contractile state of a given cell will be determined,

therefore, by the balance of forces tending to depolarize or to hyperpolarize the

cell membrane.

The VDCC superfamily of ion channels includes both high voltage- and

low voltage-sensitive members (Gollasch & Nelson, 1997; Thorneloe & Nelson,

2005). The high voltage-sensitive members (L-type), consisting of channel

proteins Cav1.1-1.4 and Cav2.1-2.3, require a larger degree of membrane

depolarization to be activated than the low voltage-sensitive members (T-type),

which include Cav3.1-3.3 (Gollasch & Nelson, 1997; Thorneloe & Nelson, 2005).

Structurally, VDCCs consist of a single pore-forming subunit (α1) which contains four transmembrane-spanning domains (I-IV). Accessory subunits (β, α2/δ, and

γ) modulate the activity of the α1 subunit. Members of the Cav1 family are

blocked by dihydropyridines such as nisoldipine and nifedipine, while Cav2 and

Cav3 are insensitive to these drugs. Although low-voltage sensitive T-type VDCC

have been identified in urinary bladder preparations from mouse and guinea pig

(Sui et al., 2001; Wegener et al., 2004) it is currently thought that, at the physiologically relevant resting membrane potential of UBSM (-30 to -50 mV), T-

type channels are largely inactivated (half-inactivated at -70 mV) and contribute

little to the physiology of UBSM tissues (Isenberg et al., 1992). Thus, Ca2+ conductance through L-type channels (L-VDCC) is believed to account for the bulk of extracellular Ca2+ influx into UBSM.

In addition to Ca2+ influx via VDCC, purinergic P2X receptors can also serve as a conduit for the influx of extracellular Ca2+. In the urinary bladder,

parasympathetic stimulation triggers the release of both acetylcholine and ATP

from nerve varicosities. The released ATP activates P2X1 receptor channels in

the post-synaptic smooth muscle and leads to spatially restricted Ca2+ and Na+ entry which can be detected optically (Heppner et al., 2005; Thorneloe & Nelson,

2005; Burnstock, 2006). In rodents, P2X receptor signaling plays a significant role in the nerve-mediated contractile response (Brading & Williams, 1990), but in humans it appears that the cholinergic response dominates (Sibley, 1984), although the importance of P2X-mediated signaling may increase during disease

states, such as occurs during bladder hypertrophy (Palea et al., 1993; O'Reilly et

al., 2002).

Intracellular Ca2+channels

Ca2+ is released from the SR internal Ca2+ stores by activation of either

ryanodine receptors (RyR) or IP3 receptors (IP3R). RyR are located on the SR

membrane. RyR channels can be activated by cytosolic Ca+2, with channel

opening favored under low cytosolic Ca2+ (1-10 µM) , and inhibition occurring at

high Ca2+ (1-10 mM) levels (Fill & Copello, 2002). RyRs are also sensitive to

luminal Ca2+ levels (Ching et al., 2000), an effect that may be mediated by the

Ca2+-binding proteins, such as calsequestrin, that are located in the SR lumen

(Beard et al., 2002). Thus, while the activation of RyR in SM is dependent upon

an elevation in intracellular Ca2+ through VDCC (Collier et al., 2000), the SR Ca2+ load also influence Ca2+ release through RyR, and factors that deplete the SR

(e.g. thapsigargin or cyclopiazonic acid) have been demonstrated to modulate

RyR Ca2+ release (Nelson et al., 1995; Jaggar et al., 2000; Herrera et al., 2001).

Whereas in cardiac and skeletal muscle tissues, activation of RyR releases the SR Ca2+ that contributes the bulk of the Ca2+ needed for contraction,

in smooth muscle tissues such as UBSM, activation of RyR is believed to

contribute to relaxation rather than contraction (Heppner et al., 1997; Jaggar et

al., 2000; Herrera et al., 2001; Wellman & Nelson, 2003). This is due to the fact

that, in SM, Ca2+ release from RyRs can lead to a transient, localized elevation of

Ca2+ (10-100 µM) known as a Ca2+ spark (Figure 3) (Nelson et al., 1995; Jaggar

et al., 1998). These Ca2+ sparks can activate juxtaposed BK (large conductance

Ca2+ -sensitive K+) channels, leading to a transient outward K+ current known as

a STOC (spontaneous transient outward current) (Benham & Bolton, 1986). This

outward current hyperpolarizes the cell membrane and, thus, reduces the open

probability of VDCC and promotes SM relaxation (Nelson et al., 1995; Jaggar et

al., 2000).

Inositol-1,4,5-triphosphate receptors (IP3R) are also located on the surface of the SR and are opened by the binding of IP3 to the cytosolic side of the

2+ receptor. Intracellular Ca levels can modulate the sensitivity of the IP3R to IP3 in

a bell-shaped manner, i.e. Ca2+ promotes channel opening at low concentrations and inhibits channel opening at higher concentrations (Thrower et al., 2001). The constrictive effect of many vasoactive substances such as endothelin-1 (ET-1) or angiotensin II (ANG II) is due to the activation of Gq-protein coupled receptors

and the subsequent generation of IP3 (Andersson & Arner, 2004; Thorneloe &

Nelson, 2005). Agonist binding to Gq-coupled receptors activates phospholipase

C (PLC), which hydrolyzes phosphatidylinositol (PI)-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). IP3 binds to the IP3 receptor on the sarcoplasmic reticulum and triggers the release of internal Ca2+.

In the urinary bladder, one of the most important types of Gq-coupled receptors is the M3 muscarinic receptor

Figure 3. Ca2+ sparks in smooth muscle tissues. Ca2+release from RyRs in

smooth muscle can activate juxtaposed BK cahnnels, triggering an outward K+ current which tends to hyperpolarize, and therefore relax, the cell. (From

Wellman and Nelson, (2004), Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca2+-sensitive ion channels, Cell

Calcium, 34:211-229).

(Thorneloe & Nelson, 2005). Parasympathetic stimulation of UBSM leads to the

release of acetylcholine (Ach) from nerve varicosities. This Ach activates post-

synaptic M3 muscarinic receptors and leads to muscle contraction. The

importance of this signaling pathway in UBSM physiology and function is

underscored by the fact that blocking muscarinic receptors in UBSM from wild-

type mice using atropine has been reported to reduced the amplitude of

neuronally-induced contractions by up to 60% (Werner et al., 2007).

Potassium (K+) channels

The majority of hyperpolarizing current in SM is carried by potassium (K+)

channels (Thorneloe & Nelson, 2005). Five classes of K+ channels have been

+ identified in various SM preparations: 1) voltage-dependent K channels (KV), 2) large-conductance Ca2+-sensitive K+ channels (BK), 3) small-conductance Ca2+-

+ + activated K channels (SK), 4)ATP-dependent K channels (KATP), and 5)

+ inward-rectifying K channels (Kir). Although both KATP and Kir channels have been demonstrated to play significant roles in vascular smooth muscle (Quayle et al., 1997), there is no evidence for a role for these ion channels in UBSM and

2+ neither the KATP blocker glibenclamide nor the Kir blocker Ba has any effect on

spontaneous contractile activity in UBSM (Imai et al., 2001). The two voltage-

+ dependent K channels, BK and KV channels, as well as SK channels, have been

shown to play vital roles the physiology of UBSM.

As mentioned previously, in smooth muscle, K+ efflux through BK

channels serves as a negative feedback mechanism to limit cellular contractility

by hyperpolarizing the cell membrane (Nelson et al., 1995; Heppner et al., 1997;

Jaggar et al., 2000; Herrera et al., 2001). These channels are activated by membrane depolarization and by local elevation in intracellular Ca2+ levels. BK channels are composed of four pore-forming α-subunits (encoded by the Slo1

gene) that may be associated with modulatory β-subunits. In smooth muscle, the

β1-subunit increases the apparent voltage- and Ca2+-sensitivity of the BK

channel by shifting the current-voltage (I-V) relationship to more negative

membrane potentials (Brenner et al., 2000; Cox & Aldrich, 2000). BK channels are sensitive to block by iberiotoxin (IBTX) and charybdotoxin (CTX), and these scorpion venoms have been used extensively to analyze the functional

significance of BK channels. In isolated myocytes, IBTX treatment increases the

size and duration of the action potential (Heppner et al., 1997). At the tissue level, blocking BK channels increases the duration and peak amplitude of action potentials (Kaczorowski et al., 1996; Heppner et al., 1997) and increases the force of muscle contraction (Herrera et al., 2000; Imai et al., 2001; Hashitani &

Brading, 2003). BK block also leads to an increase in the amplitude of spontaneous phasic contractions (Herrera et al., 2000; Petkov et al., 2001;

Hashitani & Brading, 2003).

+ Voltage-gated K channels (KV), like BK channels, are composed of 4 pore-forming α-subunits (Jan & Jan, 1997; Robertson, 1997; Yellen, 2002). The

α-subunits from the broad family of KV channel subunits (KV1-11) can form homo-

or heteromultimeric channel complexes. However, only KV1-4 proteins can form

functional channels. Proteins from the KV5-11 families do not form functional

channels alone, but combine KV1-4 subunits to form channels with unique

conductances (Post et al., 1996; Salinas et al., 1997a; Salinas et al., 1997b;

Kramer et al., 1998; Thorneloe & Nelson, 2003).

In summary, the excitability of smooth muscle is dictated by the level of

intracellular Ca2+ which, in turn, is dependent primarily upon Ca2+ influx through

L-VDCC. Outward current through K+ channels will hyperpolarize the cell

membrane and tend to relax the cells by reducing the open probability of L-

VDCC. Given the importance of this feedback loop in determining the contractile state of smooth muscle, the modulation of K+ channel activity is a fundamental

aspect in the maintenance of contractility, and transcription factors that contribute

to the regulation of K+ channel expression should figure prominently in dictating

the overall contractile status of smooth muscle tissues.

REGULATION OF GENE EXPRESSION IN UBSM

Phenotypic modulation of vascular SM

Smooth muscle has evolved a tissue-specific repertoire of contractile

proteins, ion channel proteins, and signal transducer molecules, that collectively

defines the mature contractile phenotype (Owens, 1995). Even in the mature,

fully differentiated state, vascular SMC are typified by a degree of phenotypic

plasticity and, upon the proper stimulus, can dedifferentiate from a quiescent,

contractile phenotype to a proliferative, non-contractile phenotype (Owens, 1995;

Sobue et al., 1999; Kumar & Owens, 2003; Owens et al., 2004). Dedifferentiation

is typified by a reduction in the expression of classical SM contractile genes such

as SM α-actin, SM myosin heavy chain, calponin, SM22α, and smoothelin

(Wamhoff et al., 2006). This phenotypic modulation can be adaptive in nature, such as when vascular smooth muscle cells muscle dedifferentiate and proliferate to repair damaged vascular tissues. However, paradoxically, this very plasticity can also contribute to pathologies associated with neointimal thickening and the narrowing of vascular lumens such as occurs during atherosclerosis and restostenosis (Owens et al., 2004).

Phenotypic modulation of UBSM

This process of phenotypic modulation of smooth muscle is not limited to

vascular smooth muscle tissues. In response to a chronic resistance to urinary

flow as the result of partial blockage of the urethra, i.e. partial bladder outlet

obstruction (PBOO), the urinary bladder smooth muscle wall undergoes a

dramatic remodeling process. The most immediate and obvious response to

PBOO is a significant increase in the mass of the urinary bladder loosely referred

to as bladder hypertophy, although the increase in bladder mass is due to both

hypertrophy and hyerplasia of the UBSM cells (Malkowicz et al., 1986; Levin et

al., 1990; Levin et al., 1994; Andersson & Arner, 2004), as well as an increase in

the deposition of connective tissue (Uvelius & Mattiasson, 1984; Tekgul et al.,

1996; Kim et al., 2000). In addition to the increase in bladder mass, chronic outlet

obstruction also leads to changes in the contractile and urodynamic properties of

the urinary bladder (Ghoniem et al., 1986; Malkowicz et al., 1986; Levin et al.,

1990; Gosling et al., 2000; Andersson & Arner, 2004).

Significant alterations in the expression of contractile proteins are associated with outlet obstruction. The hypertrophic growth of bladder muscle cells is associated with an increase in the synthesis of contractile proteins (Levin et al., 1990; Malmqvist et al., 1991a, b; Levin et al., 1994; Andersson & Arner,

2004). However, it has been reported that the synthesis of myosin does not keep up with the overall increase in tissue mass, and the net result is a reduction in the

amount of myosin per unit volume, which contributes to the overall changes in

the contractile properties of the hypertrophied cells (Malmqvist et al., 1991a).

Hypertrophied bladders also display a decrease in the ratio of the SM2:SM1

myosin heavy chain isoforms in rat and rabbit models (Malmqvist et al., 1991a;

Chiavegato et al., 1993; Burkhard et al., 2001; Austin et al., 2004), although the physiological implication of this shift is unclear. In addition to changes in the relative expression of SM1:SM2 MHC isoforms, Sjuve et al. (1996) found that the hypertrophied bladders of rats expressed more of the myosin heavy chain SM-B isoform, which should translate into slower contractile phenotype as compared to the faster SM-A MHC isoform. In support of this, a reduction in maximal shortening velocity has been reported to occur in hypertrophied bladders (Sjuve

et al., 1996).

Actin levels are also reported to vary as a function of outlet obstruction.

The bladders of hypertrophied rabbits have been reported to contained less smooth muscle α-actin and more γ-actin (Kim et al., 1994). In humans, though, hypertrophic growth of the bladder secondary to outlet obstruction is typified by an increase in α-actin expression and drop in β-actin (Malmqvist et al., 1991a).

The implications of these changes in myosin and actin expression are unclear,

although the alterations in myosin isoform expression seem to indicate that the

hypertrophied bladder is shifting to a slower muscle phenotype. In support of this

hypothesis, bladder strips taken from obstructed rabbits were slower to reach

maximal force development in response to KCl (110 mM), but also maintained

peak force for a longer period of time and decayed at a much slower rate than

tissues from control animals (Su et al., 2003).

In summary, smooth muscle tissues are characterized by an ability to modulate their functional phenotype in response to environmental cues. This phenotypic modulation is associated with changes in the expression of the repertoire of proteins that are associated with the contractile phenotype, and represents a distinct modulation of genetic expression which, at the most fundamental level, derives from alterations in the regulation of transcription of contractile-associated genes. In the sections to follow, I will discuss two major paradigms of smooth muscle transcriptional regulation: (1) transcriptional regulation mediated by serum response factor (SRF) and (2) Ca2+-dependent

regulation of smooth muscle transcription.

SRF-dependent gene regulation in SM

Sequence analysis of the regulatory regions of many smooth muscle specific genes, such as SM α-actin or myosin heavy chain (SM MHC), reveals the presence of a conserved, 10bp motif (CC- (A/T)6-GG) known as the CArG

box (Kim et al., 1997; Mack & Owens, 1999; Manabe & Owens, 2001; Wamhoff et al., 2004). The CArG box was first identified in 1985 as the cis-element required for activation of the immediate-early gene c-fos (Treisman, 1985).

Tresiman, in addition to identifying the CArG box, also identified the transcription

factor that interacted with it and termed it Serum Response Factor (SRF). As of

2003, over 60 genes have been identified that contain conserved CArG boxes

(Miano, 2003), and virtually all of the CArG-containing genes are either growth-

related or expressed specifically in muscle tissues. The puzzling observation that

a transcriptional cis-element (CArG) and its trans-acting partner (SRF) could be involved in seemingly paradoxical regulatory pathways - i.e. that SRF/CArG

could be involved in both proliferative (growth genes) and quiescent (contractile

proteins) pathways - can be explained, in part, by the dependence of SRF

transcriptional activity on multiple levels of regulatory control.

Many smooth-muscle specific genes are characterized by the presence of

multiple CArG elements (Li et al., 1996; Mack & Owens, 1999; Mericskay et al.,

1999; Manabe & Owens, 2001), while the growth-related genes, such as c-fos,

are typified by having a single CArG element in the promoter (Treisman, 1985).

For example, transcriptional regulation of SM α-actin is dependent upon the integrity of two CArG elements, designated CArG A and B, located in the promoter region of the gene (Mack & Owens, 1999). Transcriptional regulation of two other smooth muscle-specific contractile-associated proteins, SM myosin heavy chain (Manabe & Owens, 2001) and SM22α (Li et al., 1996), is also dependent upon two CArG boxes located in their respective promoter regions. It has been proposed that the presence of these two CArG elements acts to restrict the expression SMC-specific genes to the appropriate tissues, or rather, that the presence of multiple CArG boxes insures that these genes will not be expressed

in non-SMC tissues. One explanation for this is that the CArG boxes in these

SMC-related genes tend to be "imperfect" and to have a relatively lower affinity

for SRF than the CArG elements of growth related genes (Hautmann et al., 1997;

Mack et al., 2000). Thus, these imperfect CArG elements would require the relatively high level of SRF typically found in smooth muscle tissue to be activated or, alternatively, may require the presence of other factors, such as the

homeodomain protein Mhox (Hautmann et al., 1997), to increase the binding

affinity of SRF to these "imperfect" CArG elements. In non-smooth muscle tissues, which tend to express SRF at lower levels (Croissant et al., 1996;

Belaguli et al., 1997), these SM-specific genes would not be activated while

CArG-containing growth genes such as c-fos, which express the higher-affinity

SRF-binding element and require only a single SRF to be bound, could still be

transcriptionally active.

An alternate model of SRF-dependent, SMC-specific gene expression

involves the transcriptional co-regulator myocardin (Mycd). The specificity of

gene expression in this model is not determined by SRF itself, which is

ubiquitously expressed, but rather by the expression patterns of myocardin,

which is restricted in its expression to smooth muscle and cardiac tissues (Kumar

& Owens, 2003; Miano, 2003; Yoshida et al., 2003). Myocardin cannot directly

bind to DNA itself, but instead associates with SRF and greatly enhances its

transcriptional activity by up to 3 orders of magnitude (Wang et al., 2001).

Blocking myocardin expression using siRNA results in a downregulation in the

expression of several SM marker genes such as MHC, α-actin, and SM22α (Du

et al., 2003; Yoshida et al., 2003). Conversely, transfecting non-SMC cells with

myocardin can result in the expression of the same SMC-specific subset of

genes (Wang et al., 2001; Chen et al., 2002; Du et al., 2003; Yoshida et al.,

2003). Thus, the transcription of CArG-containing SMC genes may be mediated by the interaction of SRF with myocardin, resulting in the tissue-specific expression of proteins that are regulated by the ubiquitous transcription factor

SRF.

Transcriptional regulation of SM α-actin, MHC, as well as the SMC protein calponin, is dependent not only upon the CArG boxes located in the promoter region, but also upon a CArG element located in the first intronic region (Mack &

Owens, 1999; Miano et al., 2000; Manabe & Owens, 2001). Owens and colleagues (Mack and Owens, 1999) analyzed the functional significance of the three CArG elements (two promoter and one intronic) located in the SM α-actin gene using transgenic mice. A 2600bp segment of the α-actin gene encompassing the two promoter CArG boxes and the intronic CArG element was inserted into a Lac Z reporter construct. Selective mutations to each of the CArG elements, and the resultant constructs were used to make a series of transgenic mice in which the in vivo expression patterns of the reporter could be monitored as a function of the specific CArG mutations. Mutations to the distal promoter

CArG element (CArG B) abolished expression in all cell types (SM, cardiac, and skeletal muscle). However, a mutation to the intronic CArG element specifically

blocked expression in SMC tissues, indicating that the intronic CArG played a

role in dictating the smooth muscle-specific expression patterns of α-actin.

Thus, the transcription of many smooth-muscle related genes is dependent upon the interaction between SRF and the CArG box(es). SM-specific expression patterns can be modified by the relative stringency of SRF binding to a given CArG or CArG-like elements, and may also be influenced by the dependence of SRF interaction with other transcriptional cofactors such as myocardin.

Ca2+-dependent gene regulation in SM

While it has been established that the contractility of smooth muscle is

dependent upon an elevation of intracellular Ca2+ levels (excitation-contraction

coupling; E-C coupling), we have also come to realize that cytosolic Ca2+ levels can also affect gene expression patterns (excitation-transcription coupling; E-T

coupling). Ca2+-dependent transcriptional regulation in SMC in mediated by two

prominent transcription factors: CREB (Ca2+-cAMP responsive element binding

protein) and NFAT. In addition, recent data suggests that the RhoA/ROK

pathway may also play a crucial role in dictating the phenotypic expression

patterns of SMC.

Ca2+/cAMP-responsive element binding protein (CREB)

A rise in cytosolic Ca2+ can trigger the formation of a Ca2+-CaM complex.

Ca2+-CaM, in turn, modulates the activity of a wide range of target substrates.

Ca2+-CaM is involved in the activation of myosin light chain kinase (MLCK) and, therefore, plays a critical role in directly regulating SM contraction. In addition,

Ca2+-CaM plays a crucial role in the regulation of transcription in SM by activating

Ca2+-CaM-dependent protein kinase (CaMK), which is responsible for

phosphorylating and activating CREB. Activated CREB regulates the

transcription of target genes by binding to the Ca2+/cAMP responsive element

(CRE), a process which requires the transcriptional coactivator CREB-binding

protein (CBP) (Brindle & Montminy, 1992).

Although CREB is a Ca2+-dependent transcription factor and may play a

role in the regulation of SMC phenotype, one of the major targets of CREB

regulation is c-fos, a protein whose expression is normally associated with growth and proliferation. It has been previously demonstrated that, in cerebral vascular smooth muscle, phosphorylation and activation of CREB can be triggered by membrane depolarization using 60 mM K+, and that this activation is

mediated by Ca2+ influx through L-VDCC (Cartin et al., 2000; Stevenson et al.,

2001). The activation of CREB correlated with an increase in the transcription of

the immediate-early gene c-fos (Cartin et al., 2000), a gene which is known to

contain a single CRE located just downstream from its CArG box. Thus, in the context of the phenotypic plasticity exhibited by SMC, the Ca2+ dependent

activation of CREB might actually contribute to the loss of the contractile

phenotype, or rather, the shift from the contractile to the proliferative phenotype.

The RhoA/ROK pathway

This simplified view is challenged by the recent observations that membrane depolarization and the influx of intracellular Ca2+ via L-VDCC can lead

to the increased expression of not only c-fos, but also of various SM-specific marker genes that are associated with the contractile phenotype (Wamhoff et al.,

2004). How is it possible that two contrasting physiological end-points both share

a common signaling stimulus - Ca2+ influx via L-VDCC?

While the answer to this fundamental question is still not clear, it is

believed that the RhoA/ROK (Rho Kinase) pathway plays a crucial role in the

ability of SMC to selectively follow one phenotypic pathway. The RhoA/ROK

signaling pathway can be activated by Ca2+ influx via either receptor stimulated

pathways (e.g. G-protein coupled receptors), or via direct membrane

depolarization (Sakurada et al., 2003). Expression of a constitutively active form of RhoA has been shown to trigger increased expression of SM marker genes including SM α-actin, SM MHC, and SM22α (Mack et al., 2001). Owens and colleagues (Wamhoff et al., 2004) showed that depolarization of rat aortic VSMC with 60mM K+ leads to an increase in the transcription of SM α-actin, SM MHC,

SM22α, myocardin, and c-fos . However, blocking the RhoA/ROK pathway in these cells using the drug Y27632 completely abrogates the increased

expression of the contractile proteins (SM MHC, SM α-actin, and myocardin), but

not of the growth-associated gene c-fos. Conversely, treatment of these VSMC

with KN-93, a selective CaMK inhibitor, blocks the increased expression of c-fos,

but not of the SM genes. These studies provide compelling evidence that

activation of the transcription of SMC marker genes (i.e. SM MHC, SM α-actin),

many of which are CArG-containing, is mediated by the RhoA/ROK pathway in

accordance with SRF/myocardin, and the CArG-containing growth genes, such

as c-fos, are regulated via activation of the CaMK/CREB pathway and SRF

interaction (Figure 4). A question yet to be resolved by this model, however, is

how the cell commits to either activation of the CaMK or RhoA/ROK pathways

since, presumably, concomitant activation of both pathways would result in the

expression of both proliferative and contractile proteins.

NFAT

Ca2+-CaM, in addition to activating CaMK, can also activate the protein

phosphatase, calcineurin (Cn). The phosphatase activity of calcineurin, in turn,

triggers the translocation of the transcription factor NFAT into the nucleus where it can modulate the expression of target genes. NFAT was first identified and characterized in T lymphocytes (Shaw et al., 1988), but has since been shown to be active in a wide variety of cell types, including smooth muscle, cardiac and skeletal muscle tissues (Rao et al., 1997; Im & Rao, 2004). In the sections to

Figure 4. Possible pathways of SMC gene expression involve RhoA/ROK

and CaMK. Owens and colleagues have proposed that activation of SMC

differentiation genes such as α-actin or SM MHC may involve the RhoA/ROK pathways and SRF/myocardin interaction at the respective CArG elements.

Growth-responsive genes, however, may require SRF and CREB binding for

maximal transcriptional activity. (From Wamhoff et al., (2004), L-type voltage- gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism, Circ.

Res., 95:406-416).

follow, I will discuss the major aspects of NFAT activation and function in smooth muscle, with some emphasis on its potential role in UBSM.

NFAT (NUCLEAR FACTOR OF ACTIVATED T CELLS)

The Structure of NFAT Proteins

The NFAT proteins are related to the Rel/NK-κB family of transcription factors. Structurally, the similarity derives from the namesake Rel-homology region (RHR) that, in NF-κB proteins, is proposed to make contact with the underlying DNA (Ghosh et al., 1995; Jain et al., 1995; Chytil & Verdine, 1996).

Functionally, both NFAT and NF-κB proteins share the common characteristics of being localized to the cytosol under basal conditions and, upon stimulation, translocating to the nucleus where they modulate the transcription of target genes (Sen & Baltimore, 1986; Baeuerle, 1991).

There are currently 5 identified NFAT isoforms, NFATc1-c4, and NFAT5

(Figure 5). Differential splicing leads to variants of the NFATc1, NFATc2, and

NFATc3 isoforms. NFAT5 is the primordial NFAT isoform and is identical to the tonicity element binding protein (TonEBP), a protein that, as the name implies, plays a role in the cellular response to hypertonic stress (Miyakawa et al., 1999).

This isoform also shows the highest degree of homology to the NF-κB proteins

(Lopez-Rodriguez et al., 2001; Stroud et al., 2002). However, unlike other members of the NFAT family, NFAT5 appears to be constitutively nuclear and is

Figure 5. Schematic diagrams of the NFAT isoforms. Note that the nomenclature for NFAT has changed since this publication. Currently, the accepted format is NFAT1 = NFATc2, NFAT2 = NFATc1, NFAT3 = NFATc4,

NFAT4 = NFATc3. (From Rao et al., (1997), Transcription factors of the NFAT family: regulation and function, Ann. Rev. Immunol., 15:707-747).

regulated by pathways not shared with other members of the NFAT (Lopez-

Rodriguez et al., 2001). This review will focus on the Ca2+-sensitive members of the NFAT family (NFATc1-c4).

Structurally, there are two major conserved regions that characterize the

NFAT proteins (Hogan et al., 2003)(Figure 6): (1) the NFAT homology region

(NHR), which is the regulatory domain of NFAT and (2) the DNA binding domain

(DBD), also known as the Rel-homology region (RHR). The NHR lies between amino acids ~100-400 and contains a series of highly conserved sequence motifs that are essential for the regulation of NFAT activity. These include a series of serine-rich sequences, designated as SP-1, SP-2, SP-3, SRR-1 and

SRR-2, and KTS. Under non-stimulated conditions, the serine residues located in

these sequences are maintained in a phosphorylated state by various kinases

(Beals et al., 1997; Okamura et al., 2000). At the N-terminal portion of the NHR

lies the docking site for calcineurin, the protein phosphatase that is responsible

for dephosphorylating the NFAT serine residues, with the consensus sequence

PxIxIT. Another noteworthy sequence located within the NHR is the nuclear localization signal (NLS), which, as the name implies, plays a role in the nuclear translocation of NFAT.

The DBD of NFAT lies between amino acids ~400-700. It contains a highly

conserved sequence RAHYETEG, of which the R, Y, T and E residues are

believed to make contact with the DNA (Jain et al., 1995). While the NFAT proteins show a degree of evolutionary similarity to the Rel/NF-κB proteins, one

Figure 6. Conserved NFAT domains and sequence motifs. The regulatory domain, also known as the NFAT homology region (NHR) contains the serine- rich sequences that are essential to the regulation of NFAT activity. The DNA binding domain, or Rel homology region (RHR), is structurally similar to the DNA binding domain of Rel proteins. (From Hogan et al., (2003), Transcriptional regulation by calcium, calcineurin, and NFAT, Genes Dev, 17:2205-2232).

significant difference is that, while NF-κB proteins are bound to DNA as obligate

dimers, NFAT proteins rarely interact at DNA as dimers, instead interacting with

a wide range of transcriptional cofactors (to be discussed in sections to follow).

An exception to this is NFAT5, which typically binds as a dimer at NF-κb-like

sites (Jin et al., 2003). Interestingly, while residues located in the C-terminal portion of the RHR of NF-κB and NFAT5 stabilize the dimer formation, the residues that are involved in the stabilizing the interaction of NFAT and AP-1, the canonical NFAT cofactor, are located within the N-terminus portion of the RHR, the same region responsible for DNA binding (Chen et al., 1998).

The transcriptional activation domains (TADs) of NFAT have not been fully characterized. However, some preliminary studies have determined that portions of both the N- and C-terminal regions of NFAT may possess TADs. A ~100 residue region located in the N-terminal of NFAT1 (NFATc2) that resembles the acidic activation domains that have been implicated in transcriptional activation in other transcription factors, including NF-κB (Schmitz et al., 1994), has been identified (Luo et al., 1996). Although these residues are not very well conserved across the NFAT isoforms, NFATc1b, NFATc1, and NFATc4 all have acidic regions located within the N-terminal portions of the molecule (Rooney et al.,

1995; Park et al., 1996).

General Features of NFAT Activation

Overview of the NFAT activation pathway

The transcriptional activity of NFAT is dictated by its subcellular distribution. In general, in resting or non-stimulated cells, the regulatory serine

residues located within the NHR are phosphorylated and NFAT remains localized

to the cytosol. The classical paradigm of NFAT activation and nuclear translocation, first elucidated in immune cells, is shown in Figure 7. Antigen binding to the TCR results in the activation of phospholipase C (PLC). PLC

hydrolyzes phosphatidylinositol (PI)-4,5-bisphosphate (PIP2) into diacylglycerol

(DAG) and inositol-1,4,5-triphosphate (IP3). IP3 binds to the IP3 receptor on the

endoplasmic reticulum (ER) and triggers the release of internal Ca2+ . The

elevated intracellular Ca2+ pool caused by the release of store Ca2+, coupled with

the influx of extracellular Ca2+, can lead to the formation of a Ca2+/CaM

(calmodulin) complex that subsequently activates the protein phosphatase

calcineurin (Cn). Calcineurin dephosphorylates the regulatory serine residues in

the NHR, leading to a conformation shift in NFAT that exposes a nuclear

localization signal and facilitates nuclear translocation. In most instances, a

sustained elevation in intracellular Ca2+ is required to maintain NFAT activation

and nuclear localization. If intracellular Ca2+ levels drop, or calcineurin activity is

blocked, nuclear kinases rapidly rephosphorylate NFAT and promote its removal

from the nucleus (Negulescu et al., 1994; Timmerman et al., 1996).

Figure 7. NFAT activation pathway in immune cells. Activation of the cell-

surface T cell receptor, or Gq-coupled receptors, triggers the formation of IP3,

2+ which subsequently binds to the IP3Rs and releases SR Ca stores. In immune cells, NFAT activation is dependent upon Ca2+ influx via CRAC channels. In

smooth muscle cells, however, the principle source of Ca2+ for NFAT activation is

through L-VDCC. (From Hogan et al., (2003), Transcriptional regulation by Ca2+, calcineurin, and NFAT, Genes Dev, 17:2205-2232).

NFAT nuclear import: Dephosphorylation by calcineurin

In unstimulated cells, the serine residues located in the NHR of NFAT are

phosphorylated and the proteins are confined to the cytosol. The first step in the

activation of NFAT is the dephosphorylation of these regulatory serine residues

by calcineurin (Cn), also known as protein phosphatase 2B (PP2B), a Ca2+/CaM dependent serine/threonine phosphatase (Klee et al., 1998). Structurally, calcineurin is a heterodimer consisting of a Ca2+/CaM-binding catalytic subunit

(CnA) and a Ca2+-binding regulatory subunit (CnB). Under low cytosolic Ca2+

conditions (< 10-7 M), Ca2+ can be found bound to the high affinity Ca2+ binding

site located in the regulatory subunit, but the enzyme remains in an inactive state. An elevation in cytosolic Ca2+ levels, triggered by extracellular Ca2+ influx and/or the release of SR Ca2+, can lead to the formation of a Ca2+/CaM complex

which can bind to the catalytic CnA subunit, displacing an autoinhibitory domain

and exposing the calcineurin active site (Kissinger et al., 1995). This results in a

significant increase (up to 20-fold) in the phosphatase activity of Cn. The

cooperative interaction between Ca2+/CaM and free cytosolic Ca2+ allows calcineurin to functionally respond to narrow changes in intracellular Ca2+ levels

(Stemmer & Klee, 1994)

Calcineurin is resistant to okadaic acid, the potent inhibitor of protein

phosphatase 1 and 2A (Cohen et al., 1996), but is specifically inhibited by the

immunosuppresants FK506 (tacrolimus) and cyclosporine (CsA). Both FK506

and CsA bind to proteins - FK binding proteins and cyclophilins, respectively -

and the resultant drug/protein complexes bind to calcineurin and inhibit its activity

(Baumann et al., 1992; Sigal & Dumont, 1992; Wiederrecht et al., 1993). CsA has

been historically used in transplant surgery to reduce the effects of immune

rejection of transplanted tissues (Sigal & Dumont, 1992). The ability of CsA to

block the NFAT-dependent activation of T-cells provided a mechanistic rationale

for this clinically important property. Experimentally, these selective calcineurin

blockers are invaluable for defining the NFAT dependence of a given

physiological or biochemical response.

In vitro assays have demonstrated that calcineurin is able to

dephosphorylate NFAT from a variety of sources including purified NFAT

(Flanagan et al., 1991), recombinantly expressed NFAT (Luo et al., 1996), and endogenous NFAT (Park et al., 1996). In addition, substantial evidence indicates that exposure of NFAT-expressing cells, such as cultured immune cells, with

NFAT activating-stimuli results in the rapid dephosphorylation of NFAT proteins

(Ho et al., 1994; Northrop et al., 1994; Ruff & Leach, 1995). The fact that the okadaic acid, at levels sufficient to block the activity of protein phosphatase 1 and

2A, does not inhibit the dephosphorylation of NFAT in COS cell extracts indicates that calcineurin is directly responsible for the phosphatase activity, and the dephosphorylation of NFAT is not the result of calcineurin-initiated phosphatase cascade (Luo et al., 1996).

The docking site for the NFAT/calcineurin interaction is located within the

N-terminal region of the regulatory domain and bears the sequence PxIxIT. In

addition to the PxIxIT site, NFAT/calcineurin interactions for NFATc1 and

NFATc3 may be mediated by a second calcineurin docking site located on the C-

terminal side of the regulatory domain (Park et al., 2000; Liu et al, 1999). The

non-covalent interactions between NFAT and Cn are sufficiently strong that, in

cells transfected with a constitutively active form of calcineurin, Cn is found to be

localized to the nucleus with NFAT (Shibasaki et al., 1996; Timmerman et al.,

1996).

Dephosphorylation of the serine residues located within the NFAT regulatory domain induces a conformational change which exposes the nuclear localization signal (NLS) and leads to nuclear import (Shaw et al., 1995; Luo et al., 1996; Okamura et al., 2000). There is evidence that an intramolecular mechanism is involved in the masking of the NFATc3 NLS (Figure 8) (Zhu et al.,

1998; Zhu & McKeon, 2000). The NLS of NFATc3 lacks any residues that might be subject to direct phosphorylation. However, there is a domain containing several potential phosphorylation sites located upstream from the NLS that was proposed to directly interact with the NLS of NFATc3 in non-stimulated cells. In this conformation, the NLS is not accessible to the proteins responsible for mediating NFAT nuclear shuttling and, hence, the proteins remain in the cytosol.

Dephosphorylation of these elements by calcineurin disrupts this intramolecular masking, resulting in a conformational shift that exposes the NLS. This allows the importins responsible for NFAT translocation to recognize the signal and translocate NFAT into the nucleus.

Figure 8. Intracellular masking of the NFAT nuclear localization signal.

When NFAT is inactive and phosphorylated, the nuclear localization signal (NLS)

is masked by interaction with a phosphorylated region located upstream (region

Z). A rise in cytosolic Ca2+ levels can activate calcineurin which, in turn, dephosphorylates the residues located in the Z region and releases the NLS.

NFAT can then be translocated to the nucleus. The enzyme CK1α catalyzes the

reverse reaction and acts to maintain NFAT in the cytosol. (From Zhu and

McKeon., (2000), Nucleocytoplasmic shuttling and the control of NF-AT

signaling, Cell Mol. Life Sci., 57:411-420).

NFAT nuclear export: The role of nuclear and cytosolic kinases

Under resting conditions, NFAT proteins are located in the cytosol as phosphoproteins. There are several different kinases, both cytosolic and nuclear, that phosphorylate the NFAT regulatory serines and promote the cytosolic retention, or nuclear export, of NFAT. Among the best characterized are glycogen synthase kinase-3β (GSK-3β), members of the Jun N-terminal kinase

(JNK) family, casein kinase 1α (CK1α), and Crm-1.

GSK-3β is a constitutively active nuclear kinase that was found to be responsible for catalyzing the nuclear export of NFATc1 (Beals et al., 1997).

Transfection and overexpression of GSK-3β in COS cells led to the blockade of ionomycin-induced NFATc1 translocation. When NFATc1 proteins containing

Ser→Ala mutations at several critical regulatory serine residues were co- transfected with GSK-3β, there was no blockade of ionomycin-induced NFATc1 nuclear accumulation, indicating that the effect was mediated via phosphorylation of the regulatory serine residues of NFATc1. Interestingly, GSK-3β could only rephosphorylate NFATc1 after it had also been subject to a priming kinase reaction by cAMP-dependent protein kinase (PKA), thus indicating that the regulation of NFATc1 subcellular distribution was also dependent upon PKA activity.

In neuronal cells transfected with a constitutively active form of calcineurin, it was found that co-transfection and overexpression of GSK-3β led to a 3-fold increase in the rate of NFATc4 nuclear export, while co-transfection

with other kinases including JNK, MEKK, CK1α did not inhibit the nuclear

accumulation of NFATc4 (Graef et al., 1999). Given the fact that JNK, MEKK, and CK1α have all been reported to oppose NFAT nuclear accumulation in other studies, these findings suggests that there may be tissue-specific differences in the responsiveness of NFAT to kinase activity and/or that different NFAT isoforms may be regulated by different protein kinases. In support of the latter explanation, it has been speculated that there exists enough variation amongst the regulatory serine-rich motifs located in the NHR of different NFAT isoforms to allow for different kinases to recognize and act upon isoform-specific regulatory domains (Hogan et al., 2003).

In addition to GSK-3β, c-Jun N-terminal kinases (JNK1-3), which are downstream effectors of the Ras/MAPK signaling cascade, have also been demonstrated to regulate the phosphorylation status, and thus the subcellular distribution and activity, of NFAT proteins. Using nuclear extracts derived from

COS cells co-transfected with JNK, NFATc3, and NFATc4, Chow et al. (2000)

found that both JNK1 and JNK2 bound to NFATc3 in vitro, but not to NFATc4.

The NFAT/JNK interaction was dependent upon Ser163 and Ser165, which are located in the N-terminal NHR of the human NFATc3 molecule. NFATc3 molecules with Ser→Ala mutations at the Ser163 and Ser165 sites were not

phosphorylated in vitro, and cells transfected with these mutant NFATc3 proteins

displayed a nuclear distribution of the NFATc3 even in the absence of calcineurin

stimulation. The observation that mutations to these two serines leads to

constitutively nuclear NFATc3 localization indicates that their phosphorylation state is a key determinant in the subcellular distribution of NFATc3. Another interesting observation from these studies was that JNK activity does not affect the nuclear accumulation of NFATc1, NFATc2, or NFATc4, indicating that the

JNK activity is specifically involved in the regulation of NFATc3. This finding leads to the possibility that other NFAT isoforms may be regulated by isoform- specific kinases as well. Thus, the regulatory dependence of NFATc3 on JNK activity integrates the signaling pathways and downstream targets of NFATc3 with the Ras/MAPK pathway that is required for JNK activation. In addition, the specificity of JNK regulation of NFATc3 is one of the examples of the functional differences among the various NFAT isoforms.

McKeon and colleagues (Zhu et al., 1998; Zhu & McKeon, 2000) have identified 2 other kinases, MEKK1 (mitogen-activated protein kinase kinase 1) and CK1α (casein kinase 1α), that cooperatively regulate NFATc3 nuclear accumulation. However, unlike JNK and GSK3β, which exert their effect in the nucleus, MEKK1 and CK1α act in the cytosol to promote the cytosolic retention of

NFATc3. Transfection and overexpression of CK1α in BHK (baby hamster kidney) cells led to a significantly reduction in the rate of NFATc3 nuclear accumulation in response to ionomycin treatment, while transfection with a dominant-negative form of CK1α led to a net increase in NFATc3 nuclear accumulation. NFATc3 and CK1α co-immunoprecipitated in vivo, and this interaction was dependent upon the A domain (Figure 9). CK1α phosphorylated

residues located within the A domain as well as within the neighboring Z domain, which is the putative region that interacts with and masks the NLS. Although the

activation of MEKK1 also suppressed NFATc3 nuclear accumulation, this action

was dependent upon the presence of CK1α, and MEKK1 was ineffective when co-transfected with DN CK1α. It was determined that MEKK1 did not interact directly with NFAT, but instead interacted with CK1α by an undefined mechanism to increase its binding affinity to NFATc3 nearly 10-fold. Thus, the authors provide a model in which CK1α, stabilized by MEKK1, phosphorylates residues located within the A and Z domains of NFATc3. This induces a conformational shift that promotes an intramolecular interaction of the NLS of NFAT with the Z domain, resulting in masking of the NLS.

CK1α, JNK, and GSK3β all exert their effects by phosphorylating serine residues located in the NHR. Another nuclear kinase, Crm-1, regulates the distribution and activity of NFAT by recognizing and binding to a nuclear export signal (NES) and, thus, facilitating the export of NFAT from the nucleus. Zhu and

McKeon (1999) found that overexpression of Crm-1 had no effect on the distribution of NFATc3 in non-stimulated cells, but blocked the ionomycin- stimulated nuclear accumulation of NFATc3. This effect was dependent upon two motifs (NES1 and NES2) located within the NHR, upstream of the A domain from

Figure 9. Significantly, overexpression of Crm-1 was still able to exert its effect on mutant NFAT proteins lacking the NLS, indicating the effects of Crm-1 were not mediated by interaction with the nuclear localization signal, as is the case

with MEKK1/CK1α (Zhu et al., 1998). They also reported that mutations to both

the NES1 and NES2 sites resulted in an NFAT mutant that did not translocate to

the nucleus even in response to co-transfection with a constitutively active form

of Cn, indicating that the NES sites overlaps with the calcineurin-binding domain.

Co-transfection with constitutively active Cn also abolished NFATc3-Crm-1

interactions as determined by co-immunoprecipitation, indicating that, not only do

the NES sites and Cn-binding domains overlap, but that Crm-1 and calcineurin

actively compete for this composite binding site. The implication of this model is

that, if Crm-1 activity is not blocked, it can displace Cn from their overlapping

binding site(s) and prevent a net nuclear accumulation of NFAT, leading to a

futile cycle of nuclear import and export.

Thus, the activity of both nuclear and cytosolic kinases contributes to the

cytosolic distribution and, hence, activity of NFAT by phosphorylating key

residues located within the NHR. The activities of GSK3β, JNK, and Crm-1

promote the nuclear export of NFAT proteins, while the cooperative effects of

MEKK1 and CK1α contribute to the masking of the NLS and, thus, oppose the

activity of calcineurin and promote the cytosolic retention of NFAT.

NFAT transcriptional cofactors

One of the most striking characteristics of NFAT is its dependence upon a wide variety of transcriptional cofactors (Rao et al., 1997; Hill-Eubanks et al.,

2003; Hogan et al., 2003). NFAT proteins have since been found to interact with

an enormous range of proteins including, but not limited to, basic leucine-zipper

(bZIP) transcription factors (AP-1; Ho et al., 1994), the GATA zinc finger proteins

(Molkentin et al., 1998; Wada et al., 2002), helix-turn-helix proteins such as Oct

(Bert et al., 2000), IRF-4 (Furstenau et al., 1999; Hu et al., 2002; Rengarajan et

al., 2002), and HNF3 (Furstenau et al., 1999), and transcription factors from the

MADS family including MEF2 (Youn et al., 2000; Meissner et al., 2007) and SRF,

a topic which will be addressed in Chapter 2 of this dissertation.

AP-1, a dimer of the proteins Fos and Jun, whose synthesis is stimulated

by activation of the PKC/MAPK pathway, was the first identified NFAT partner

(Ho et al., 1994; Macian et al., 2001). NFAT/AP-1 composite binding sites have been identified and sequenced from a wide range of immune response genes.

They consist of a ~15 base pair segment of DNA in which the AP-1 site is

invariably located downstream of the NFAT binding site. A classic example of this

is the distal ARRE-2 from the human IL-2 gene, which bears the sequence:

AGGAAAaacTGttTCA, with the bold letters representing the NFAT recognition

nucleotides, and the underlined nucleotides representing the consensus AP-1

binding site (Jain et al., 1993). The NFAT/AP-1 complex is stabilized by amino

acids located in the N-terminal region of the NFAT RHR, which interact with basic

amino acids located in the leucine zipper region of AP-1. This complex is not

stable in solution, however, and the formation of the NFAT/AP-1/DNA complex is

~20x more stable than the DNA/NFAT complex alone (Chen et al., 1995).

In addition to AP-1, NFAT has also been demonstrated to interact with

other members of the bZIP family. The proto-oncogene c-Maf has been reported to synergistically transactivate expression of IL-4 expression in immune cells with

NFATc2 (Ho et al., 1996). However, two other bZIP proteins, ICER (inducible cAMP early repressor) (Bodor et al., 2000)and p21SNFT (21-kDa small nuclear factor isolated from T cells) (Bower et al., 2002) have been reported to negatively affect NFAT activity by blocking NFAT-mediated transactivation of cytokine expression. Transcription of the CD28 responsive element (CD28RE) gene requires a cooperative interaction between CREB and NFAT at a composite binding site located in the CD28RE promoter region. ICER blocks transactivation of this gene by competing with CREB for binding to its recognition site, and the resultant NFAT/ICER complex is not transcriptionally active. p21SNFT also inhibits NFAT-mediated transcriptional activation. At NFAT/AP-1 composite elements, p21SNFT can replace Fos in the Fos/Jun dimer that comprises AP-1.

The resultant p21SNFT/Jun/NFAT complex is not transcriptionally active. Thus, while effective NFAT transactivation in many cases is based upon a synergistic interaction with one bZIP protein (AP-1), two other bZIP proteins, p21SNFT and

ICER, have inhibitory effects on NFAT activity.

Cooperative interactions between NFAT and zinc-finger proteins from the

GATA family of transcription factors have been reported from skeletal muscle

(Musaro et al., 1999), cardiomyocytes (Molkentin et al., 1998), as well as in T cells (Avni et al., 2002). Musaro et al. (1999) found that cultured skeletal

myocytes exposed to insulin-like growth factor (IGF-1) developed a hypertrophic

phenotype, and that this effect was blocked by co-treatment with CsA or by

transfection with a dominant-negative form of Cn, indicating that NFAT was playing a central role in this response. Immunostaining revealed that the GATA-2 and NFATc1 were both co-localized to the same subset of nuclei in hypertrophic myocytes. However, despite the presence of NFATc1, NFATc2 and NFATc3 in these cells, GATA-2 only co-immunoprecipitated with NFATc1, implying that that

NFAT-mediated regulation of target genes may be modulated by isoform-specific interaction with various transcriptional cofactors. Thus, in skeletal muscle,

NFATc1 and GATA-2 appear to synergistically regulate the expression of the genes that may contribute to the hypertrophic phenotype of skeletal muscle, although the specific transcriptional targets were not identified in that study.

Similarly, Molkentin and colleagues (Molkentin et al., 1998) have demonstrated that NFAT and GATA can synergistically drive a reporter probe containing the promoter region of BNP (B-type natriuretic peptide), a marker of cardiac hypertrophy. Using a yeast two-hybrid screening system as well as co- immunoprecipitation studies, they found that NFATc4 interacted with GATA-4, and that the formation of the NFATc4/GATA-4 complex was dependent upon an interaction between the second zinc finger of GATA-4 and the c-terminal portion of the NFATc4 RHR, the same portion of the NFAT RHR that mediates the

NFAT/AP-1 interaction. Co-expression of NFATc4 and GATA-4 synergistically activated the BNP reporter probe 100-fold greater than did GATA-4 alone.

Although the results of these experiments would seem to suggests that NFATc4 plays an essential role in the development of pathological cardiac hypertrophy, more recent work by this same group (Wilkins et al., 2002) clearly indicate that

NFATc3, not NFATc4, is mandatory for the development of cardiac hypertrophy.

Using NFATc3 and NFATc4 knock-out mice, the authors found that the NFATc3- null mice displayed a significant reduction in calcineurin-induced cardiac hypertrophy, while the loss of NFATc4 expression did not attenuate the hypertrophic response. Thus, although NFATc4 can interact with GATA to induce cardiac hypertrophy, it appears that, in a more physiological context, it is the

NFATc3 isoform that is actually more critical in the development of this pathology.

Proteins from the MADS (MCM1 agamous deficies SRF) family of transcription factors have also been widely reported to interact with NFAT proteins to cooperatively modulate gene transcription. NFATc2 and MEF2 cooperatively regulate the apoptosis of thymocytes by activating the immediate- early gene Nur77 (Youn et al., 2000). The transcriptional activity was mediated by the recruitment of p300 and did not involve NFAT binding directly to the DNA.

Immunoprecipitation assays were used to determine that a full length NFATc2 protein or an NFAT fragment containing only the c-terminal region (residues 680-

927) were able to bind to MEF2 in vivo. However, an NFAT fragment containing only the N-terminal region (residues 1-407) or only the RHR of NFATc2 were unable to interact with to MEF2 in vivo. Thus, the region of NFATc2 that interacts

with MEF2 is located within the C-terminus of the protein, which is distinct from

the regions of NFAT that interact with both calcineurin and p300.

NFAT has also been demonstrated to interact with MEF2 in the

modulation of skeletal muscle phenotype (Chin et al., 1998). When cultured

skeletal myocytes were co-transfected with constitutively active calcineurin and

reporter constructs containing promoters from a variety of marker genes specific for fast- or slow-twitch skeletal muscle, calcineurin selectively stimulated the activity of myoglobin (Mb) and troponin I slow (TnIs), genes that are associated with the slow-twitch phenotype. Maximal transcriptional activation of these slow- twitch reporters was dependent upon the presence of two NFAT consensus sites in the construct. However, in addition to the conserved NFAT elements, maximal reporter expression of the Mb gene also required conserved binding sites for the factors Sp1 and MEF2, and reporter constructs containing deletions, or mutations, to any other these sites display reduced transcriptional activity in response to the constitutive calcineurin.

Thus, maximal NFAT transactivation of target genes is dependent upon interaction with a wide range of transcriptional cofactors prominently including

AP-1, GATA, and MEF. The majority of the reported NFAT/cofactor interactions results in transcriptional upregulation of gene expression. However, downregulation of transcriptional activity is also possible as is evidenced by the effects of p21SNFT and ICER on NFAT transcriptional activity. The dependence of NFAT, a Ca2+-sensitive transcription factor, on transcriptional cofactors that

may be stimulated by alternative pathways (e.g. the MAP kinase pathway for AP-

1) serves to integrate these signaling cascades with the dynamic Ca2+

environment of the cell.

SM-specific Features of NFAT activation

NFAT in smooth muscle: The role of extracellular Ca2+

The activity of NFAT is determined by its subcellular distribution which, in

turn, is dictated by intracellular Ca2+ levels. In smooth muscle, the influx of extracellular Ca2+ is conducted primarily L-VDCC (Knot & Nelson, 1998). It has

been demonstrated that this influx of Ca2+ via L-type VDCCs is indispensable to

NFAT activation in smooth muscle tissues. Stimulation of ileal smooth muscle

tissues with PDGF (platelet derived growth factor) leads to a significant increase

in NFATc3 nuclear accumulation (Stevenson et al., 2001). However, this is

completely inhibited by nisoldipine, a dihydropyridine that specifically blocks L-

VDCC and, hence, prevents the influx of extracellular Ca2+. Furthermore, co-

treatment with pinacidil, a KATP channel opener that induces membrane

hyperpolarization, also completely abrogates NFAT activation by PDGF,

supporting the crucial role of L-VDCC in the activation of NFATc3 in ileal SM.

Likewise, the stimulation of NFATc3 nuclear translocation in cerebral vascular smooth muscle by UTP is also dependent upon Ca2+ influx via L-type VDCC, and

is blocked by treatment with pinacidil (Gomez et al., 2002).

Although these previous reports have indicated that membrane

depolarization and activation of L-VDCC is required for NFAT activation in SM,

membrane depolarization alone may not be a sufficient signal to drive NFAT

nuclear translocation SM tissues. Nelson and colleagues (Stevenson et al., 2001)

found that direct membrane depolarization of ileal SM using 60 mM K+ did not

trigger an increase NFAT nuclear accumulation (a topic which will be more fully

discussed in sections to follow), and neither did treatment with the Ca2+

ionophore ionomycin (Io). This stands in contrast to the responsiveness of

neurons, where a brief membrane depolarization using high K+ is sufficient to

trigger sustained NFATc4 nuclear accumulation (Graef et al., 1999). These

differences may reflect isoform-specific (i.e. NFATc3 vs. NFATc4) and/or tissue- specific differences (neuronal vs. SM tissue) in NFAT activation patterns.

NFAT in smooth muscle: The role of the SR Ca2+ stores

In addition to being dependent upon external Ca2+ influx via L-VDCC,

NFAT activation has also been shown to be dependent upon the nature of Ca2+

release from the SR (sarcoplasmic reticulum) Ca2+ stores. In smooth muscle

2+ tissues, Ca release from the SR can occur via the activation of IP3 receptors

(IP3R) or via ryanodine receptor (RyR) activation. Interestingly, however,

2+ although the activation of either RyR or IP3R leads to the release of internal Ca

stores, they have been demonstrated to have opposing effects of NFAT

activation.

As was shown in Figure 7, the activation of IP3R, and the subsequent

release of SR Ca2+, has been shown to be a fundamental part of the NFAT

activation pathway in immune cells. In smooth muscle tissues as well, the

2+ release of SR Ca via activation of IP3R has also been shown to be crucial for

NFAT activation. Exposure of native cerebral vascular arteries to UTP, a Gq/11-

coupled receptor agonist that activates PLC and leads to the generation of IP3, causes a significant increase in NFATc3 nuclear localization (Gomez et al.,

2002). However, this effect is blocked completely by pre-treatment of the arteries with the SERCA (smooth endoplasmic reticulum Ca2+ ATPase) inhibitor

thapsigargin, a drug that prevents the refilling of SR Ca2+ stores. Similarly, co-

treatment of UTP with the IP3R antagonists xestospongin or 2-APB also

abolishes the effects of UTP on NFATc3 nuclear accumulation, indicating the

2+ Ca release from the SR via IP3R is indispensable to NFATc3 nuclear

translocation in cerebral VSM.

In contrast to the dependence of NFATc3 nuclear translocation on the

activation of IP3R, the activation of RyRs was found to have an inhibitory effect

on the UTP-induced nuclear accumulation of NFATc3 in cerebral arteries

(Gomez et al., 2002). Likewise, blocking RyRs and, thus, inhibiting the release of

SR Ca2+ via RyRs, has a potentiating effect on UTP-mediated NFATc3 nuclear

accumulation. These findings would seem to indicate that Ca2+ release from the

RyR was activating BK channels, an effect that, in vascular smooth muscle cells,

has been demonstrated to hyperpolarize the cell membrane and would be

expected to reduce the open probability of VDCC and, hence, reduce NFAT

activation. Thus, blocking Ca2+ spark activity would be predicted to increase Ca2+

influx via VDCC. However, when the membrane potential was clamped with 60

mM external K+, conditions in which the outward flux of K+ ions would be prohibited, the potentiating effects of RyR were still apparent. Thus, it appears that Ca2+ sparks may play some novel role in the regulation of NFAT activation that is not directly due to activation of BK channels.

Thus, in smooth muscle cells, the activation of NFAT appears to be influenced by both extracellular and intracellular Ca2+ pathways. NFAT nuclear

translocation is dependent upon the influx of extracellular Ca2+ via L-VDCC and

2+ upon the release of SR Ca through the activation of IP3R. In contrast, the

release of SR Ca2+ via RyR in the form of Ca2+ sparks has an inhibitory effect of

NFAT nuclear translocation, although the mechanistic basis of this effect remains

unclear.

JNK and NFAT nuclear export in SMC

Using JNK2-/- mice, Nelson and co-wokers (Gomez et al., 2003) have

shown that the subcellular distribution of NFATc3 is dependent upon JNK

activity. As was previously mentioned, HK (60 mM K+) does not induce NFATc3

nuclear accumulation in the isolated cerebral arteries of wild-type mice

(Stevenson et al., 2001; Gomez et al., 2002). However, JNK2-/- animals do

display a significant nuclear accumulation of NFATc3 in response to HK (Gomez

et al., 2003). This observation indicates that, in wild-type mice, HK is able to stimulate NFATc3 nuclear translocation, but JNK2 activity rapidly removes it from the nucleus. Interestingly, while HK fails to induce a net accumulation of NFATc3 in the wild-type animals, treatment with UTP does lead to a net nuclear accumulation of NFATc3 in these same animals. Analyses using antibodies that recognizes the active, phosphorylated form of JNK (p-JNK) revealed that treatment with UTP, in addition to stimulating NFAT nuclear accumulation via elevating intracellular Ca2+ levels, also suppresses the activity of JNK, i.e. there was less of the active p-JNK presence after UTP treatment. Thus, these findings suggest that certain stimuli, such as UTP, are able to trigger a net increase in

NFATc3 nuclear accumulation by stimulating the pathways that lead to NFATc3 nuclear import while simultaneously inhibiting the kinases (e.g. JNK2) that contribute to NFAT nuclear export. In addition, it also indicates that the failure of certain stimuli to induce NFAT nuclear accumulation (i.e. 60 mM K+) may actually reflect an inability of the stimuli to inhibit export mechanisms, rather than a failure to stimulate nuclear import mechanisms. This, in turn, contributes to a model of

NFAT regulation in which episodic stimulatory events are overlaid upon a constitutively active nuclear export mechanisms (Zhu & McKeon, 2000;

Stevenson et al., 2001).

NFAT and Pressure

One of the more recent physiological factors that has been found to

contribute to the regulation of NFAT activity is intraluminal pressure. Using

cannulated cerebral vascular arteries, Gonzalez Bosc et al. (2004) found that, under physiologically relevant intraluminal pressures (~100 mm Hg), NFATc3

displays a prodominantly nuclear distribution. However, the nuclear localization

of NFAT under these conditions is blocked if the endothelium is denuded, and is

abrogated by blockade of either nitric-oxide synthase or PKG activity, indicating

that NO signaling is an essential component of the pressure-induced NFAT

activation. The fact that treatment with 60mM K+ alone is not a sufficient stimulus

for NFATc3 nuclear accumulation, but exogenous administration of NO combined

with 60 mM K+ does result in NFATc3 nuclear localization, lends further support

for a role for NO signaling in the regulation of NFAT in vascular smooth muscle.

These findings suggest that NO signaling is involved in the regulation of

NFAT nuclear export mechanisms in cerebral vascular arterial smooth muscle. In

wild-type animals, blockade of PKG leads to a significant decrease in the degree

of NFATc3 nuclear staining. However, PKG inhibition has no effect on the

accumulation of nuclear NFATc3 in arteries from JNK2 knock-out mice under the

same conditions. This indicates that, in vascular smooth muscle, NFATc3 is localized to the nucleus under physiological pressure, and that this nuclear

accumulation is dependent upon the inactivation of JNK2 activity through a

NO/cGMP/PKG-mediated pathway.

The role of NFAT in smooth muscle

Murphy and colleagues (Boss et al., 1998) were the first to characterize

NFAT expression and activity in smooth muscle cells. They found that cultured

VSMC expressed the NFATc1 and NFATc2 isoforms. Under basal conditions,

NFATc1 was found primarily in the cytosol, while NFATc2 was found to be localized to the nuclei of these same cells. Although the SMC-specific targets of

NFAT activity were not studied, they showed that Io/PMA treatment could drive the activity of an NFAT-responsive promoter-reporter construct, as could treatment with the smooth muscle mitogen PDGF.

Much of the early research on NFAT in smooth muscle focused on the

Ca2+ requirements for activation (Stevenson et al., 2001; Gomez et al., 2002;

Gomez et al., 2003). These studies laid the groundwork for an understanding of the activation requirements of NFAT in smooth muscle tissues. More recent work has focused on exploring the functional role of NFAT in smooth muscle tissues and can be broken up into two broad (and somewhat overlapping) categories: (1)

NFAT and the modulation of smooth muscle phenotype and (2) NFAT and SMC contractility.

NFAT and SMC phenotypic modulation

Smooth muscle cells, even in the mature, fully differentiated state, are typified by a degree of phenotypic plasticity and, upon the proper stimulus, can

modulate from a quiescent, contractile phenotype to a proliferative, non-

contractile phenotype (Owens, 1995; Sobue et al., 1999; Kumar & Owens, 2003).

In culture, SMCs can be "forced" to express either a differentiated or dedifferentiated phenotype, most often by modulating the amount of serum in the media. Cells maintained in media containing low (1%) levels of serum media maintain a differentiated phenotype, characterized by the expression of various

SMC-associated proteins such as SM MHC or SM α-actin, while higher levels of

serum (10%) induce a dedifferentiated, proliferative phenotype in which the

expression of these contractile proteins is typical lost or reduced. This property

has been used extensively by many researchers to explore the potential role of

NFAT in the phenotype expression patterns of various SMC tissues.

In smooth muscle tissue, GATA-6 and NFATc1 cooperatively regulate the expression of SM myosin heavy chain (Wada et al., 2002). The interaction between NFATc1 and GATA-6, as determined by co-immunoprecipitation experiments, was dependent upon the zinc-finger domain on GATA, as was previously reported for the interaction of GATA-4 and NFATc4 in cardiac tissues

(Molkentin et al., 1998). GATA-6 and NFATc1 were able to synergistically transactivate a reporter plasmid containing a 1346bp segment of the SM-MHC promoter region. Exposure of the cultured VSMC to 1% serum induced a differentiated phenotype and resulted in the translocation of NFATc1 to the nuclei of the differentiated VSMCs. In addition, this differentiation also led to the transactivation of the SM-MHC reporter, which was blocked by the presence of

CsA and FK506. These observations indicate that NFAT is essential to the

transcriptional upregulation of SM-MHC in response to stimuli that trigger

expression of the contractile phenotype.

Similarly, it has been reported that NFAT activity is an integral part of the

gene expression patterns associated with the differentiated phenotype of cultured visceral smooth muscle cells. Ohkawa et al. (2003) found that dedifferentiated, cultured SMC could be induced to develop a differentiated phenotype by culturing in media containing IGF-1 (insulin-like growth factor-1). Adding CsA or

FK506 reversed the modulation, converting the spindle-shaped, differentiated cells into cells that exhibited fibroblastic or dedifferentiated morphology. The differentiated SMC exhibited nuclear localization of calcineurin, while in dedifferentiated cells, CN was found localized to the cytosol. CsA and FK506 treatment also led to a downregulation of the SMC markers caldesmon (CaD) and α1- integrin in dose- and time-dependent fashion. Likewise, blocking CN activation with CAIN (a natural CN inhibitor) reduced the activity of a CaD and

α1- integrin reporter. Thus, these observations also support a role for NFAT in the maintenance of the differentiated, contractile phenotype.

Lamaziere and coworkers (Larrieu et al., 2005) also found that NFAT activation was essential to the expression of the differentiated SMC phenotype.

Culturing SMC in serum-free media for up to 21 days led to the shift from a fibroblastic morphology to a more classical, spindle-shaped SMC morphology. In addition, the levels of SM MHC increased over this same time frame. However,

culturing these cells in the presence of CsA blocked the increase in SM MHC expression. In the dedifferentiated, proliferative state, NFATc1 was localized to the cytosol. However, induction of the differentiated phenotype led to a significant increase in the levels of NFATc1 nuclear accumulation. These finding suggest that the development of the differentiated phenotype is accompanied by the increase in the expression of SM MCH concomitant with an increase in NFATc1 nuclear accumulation, and that blockade of NFAT activation with CsA is sufficient to prevent differentiation.

However, there have also been reports that NFAT activation is associated with the expression of the proliferative, rather than differentiated, phenotype.

Rao and colleagues (Yellaturu et al., 2002) report that, in cultured rat thoracic arterial SMC, stimulation with the smooth muscle mitogen PDGF led to NFATc1 translocation to nucleus. PDGF treatment also stimulated cellular proliferation as measured by H3-thymidine uptake, and this proliferation was blocked by co- incubation with CsA. VIVIT, an NFAT competitor peptide, also inhibited DNA synthesis and proliferation, indicating the NFAT activation was essential to the proliferative growth of these cells. Although NFAc1, NFATc2 and NFATc3 were found to be expressed in these cells, only NFATc1 was found in the nucleus.

Thus, although there is lack of uniformity as to whether NFAT activation is associated with the proliferative or differentiated phenotype, it does seem likely that NFAT does, indeed, play a role in the phenotypic modulation. It is possible that the contrasting results (i.e. NFAT supporting the proliferative vs.

differentiated phenotypes) may be isoform-specific in nature. However, given that

the NFATc1 isoform was implicated in both the proliferative (Yellaturu et al.,

2002) and differentiated (Wada et al., 2002; Larrieu et al., 2005) phenotypes, this explanation seems unlikely. It is possible that the nature of the stimulus used to induce the differentiated or proliferative phenotype may affect the outcome. For instance, when phenotype is manipulated by changes in serum levels (Wada et al., 2002; Larrieu et al., 2005), NFAT activation is associated with the differentiated phenotype. However, when mitogens such as PDGF are used,

NFAT activation is associated with the proliferative phenotype (Yellaturu et al.,

2002). One possible explanation is that differential stimulus paradigm activate different signaling cascade and, ultimately lead to the activation of different sets of potential NFAT transcriptional cofactors. In this paradigm, it is the presence of specific NFAT cofactors that would dictate the phenotypic outcome, rather than the outcome being influenced by NFAT activity alone.

NFAT and SMC contractility

Recent works indicate that NFAT plays a central role in the maintenance of smooth muscle contractility. NFAT activity, as mentioned previously, has been associated with maintenance of the differentiated, contractile phenotype in several studies (Wada et al., 2002; Gonzalez Bosc et al., 2004; Larrieu et al.,

2005). NFAT has been implicated in the regulation of contractile proteins such as

SM MHC (Wada et al., 2002; Gonzalez Bosc et al., 2004; Larrieu et al., 2005) and caldesmon and α-1 integrin (Ohkawa et al., 2003).

Recently, Santana and colleagues (Amberg et al., 2004) have demonstrated that NFATc3 can also regulate the expression of ion channels that contribute to the membrane potential, and therefore contractile status, of smooth muscle tissues. Administration of ANGII led to reduction in KV currents in mice,

and in mRNA for KV2.1. Co-administration of CsA reduced the downregulation of

KV2.1, and mice expressing a constitutively active form of NFATc3 recapitulated

these effects in the absence of ANGII. In addition, when NFATc3-/- mice were

subject to ANGII treatment, they did NOT show any changes in KV current or

KV2.1 channel expression. These data indicate that NFATc3 is responsible for

downregulating the expression of KV2.1 in cerebral vascular SMC. Given that KV

channels play a central role in the maintenance of the resting membrane

potential and afterhyperpolarization of SMC, these findings also indicate that

NFAT must also be central to smooth muscle contractility and function.

In the Chapters to follow, I will present data that lend further support for a

role for NFAT in the regulation of SM contractility through its role in regulating the

expression of both ion channel and contractile proteins.

HYPOTHESES

The central role of Ca2+ in dictating the contractile state of smooth muscle,

as has been discussed in this literature review, is well-established. According to

this classic excitation-contraction coupling (E-C) model (Wray et al., 2005),

excitation of the muscle cell leads to depolarization and Ca2+ entry through

VDCC. This elevates the intracellular Ca2+ pool and, ultimately, leads to muscle

contraction (Figure 9). However, it is also widely recognized that this elevation in

intracellular Ca2+ can, in addition to activating contraction (E-C coupling), also

modulate the transcriptional environment of the cell through the activation of

Ca2+-sensitive transcription factors (E-T coupling) (Hill-Eubanks et al., 2003;

Barlow et al., 2006; Wamhoff et al., 2006). Thus, the dynamic Ca2+ environment of the cell dictates not only its instantaneous contractile status by engaging the

contractile apparatus (contraction) or opening BK channels (relaxation), but can

also modulate the long-term transcriptional changes in the expression of

contractile proteins and ion channels that, in turn, modulate contractility.

This thesis examines the role of the Ca2+-sensitive transcription factor

NFAT in the regulation of smooth muscle contractility. Using a wide range of

tools, from physiological examinations of UBSM contractility to molecular

analysis of the expression of ion channels, we have sought to investigate two

primary hypotheses are investigated.

Figure 9. Integration of E-C and E-T coupling pathways. Extracellular Ca2+ entering the cell through L-VDCC can stimulate both contraction (E-C coupling) and transcriptional pathways (E-T coupling). Data presented in this thesis indicates that activation of the NFAT pathway can lead to changes in the expression of α-actin (Chapter 2), as well as BK and KV channels (Chapter 3).

Hypothesis 1: NFAT contributes to the contractile state of smooth muscle

via its role in regulating the expression of α-actin.

The smooth muscle contractile phenotype is characterized by the

expression of a subset of proteins that are associated with the contractile

process. Several of these proteins, including myosin heavy chain, caldesmon,

and α-integrin, have been reported to be targets of NFAT transcriptional activity,

which strongly suggests that NFAT is a crucial regulator of smooth muscle

phenotypic expression.

We have identified a conserved NFAT binding site located within the first

intronic region of the SM α-actin gene that overlaps with a CArG box that has

previously been demonstrated to be essential to SM α-actin expression. Here we provide evidence that NFAT and SRF cooperatively regulate the expression of α-

actin at this composite binding site. Mutations to either the SRF- or NFAT- conserved sequences abolished reporter activity, indicating that the activity of both transcription factors was required to drive expression. In cells transfected with a luciferase reporter containing these conserved elements, stimulation of the

NFAT pathway with Io/PMA lead to a ~4 fold increase in reporter activity. Using co-immunoprecipitation assays, we found that NFATc3 and SRF formed a complex in solution via co-immunoprecipitation, and this interaction was

facilitated by the presence of an oligonucleotide contained the conserved

NFAT/SRF site. Finally, inhibition of the NFAT pathway using FK506 and CsA

resulted in a reduction in the expression of SM α-actin in cultured smooth muscle

cells. These findings represent the first reports directly linking NFAT activity to

the expression of SM α-actin and provide further evidence supporting a role for

NFAT activity in the maintenance of the smooth muscle contractile phenotype.

Hypothesis 2: NFAT activity contributes to the regulation of UBSM

contractility via modulation of the expression of ion channels.

Ca2+ is central to the contractile machinery of smooth muscle myocytes as

it is required to activate the contractile machinery. Given that the primary source of contractile Ca2+in smooth muscle tissues is supplied via L-VDCC, the overall contractile state of a smooth muscle myocyte will be dictated by the effect of factors that tend to hyperpolarize or to depolarize the cell as their sum effect will

determine the membrane potential of the cell and, therefore, the open probability

of L-VDCC .

In UBSM cells, current through K+ channels provides the bulk of hyperpolarizing current and the activity of these channels is crucial to regulating the contractile properties of these cells. Recently, it has been reported that

NFATc3 may regulate the expression of KV2.1 channels in both vascular smooth

muscle and in cardiac myocytes. In addition, NFATc3 has also been implicated in

the regulation of the β1-subunit of the BK channel. Given the prominent roles of

these ion channels in shaping the UBSM action potential, and in the regulation of

UBSM membrane potential, these findings suggest that NFATc3 may further

contribute to the regulation of smooth muscle contractility via the modulation of

KV and BK channel expression.

In the data presented in Chapter 3, we show that, in murine UBSM tissue,

NFATc3 regulates the expression of both KV2.1 and of the pore-forming α- subunit of the BK channel. UBSM from NFATc3-null mice display elevated TEA- sensitive KV currents, and this is supported at the molecular level by an

upregulation in the expression of mRNA for KV2.1. These mice simultaneously

display a decrease in IBTX-sensitive BK current and decreased BK channel

density, and decreased expression for the BK α-subunit. Despite the elevated KV

currrent, UBSM strips from NFATc3-null mice display enhanced contractility in

response to EFS than do UBSM strips from wild-type mice, which suggests that,

at least in response to EFS, the downregulation of BK current plays a more

significant role than does the elevated KV current. These findings lend further

support for a role for NFAT in the maintenance of smooth muscle contractility

through the regulation of BK and KV channels.

CHAPTER 2: NUCLEAR FACTOR OF ACTIVATED T CELLS AND SERUM

RESPONSE FACTOR COOPERATIVELY REGULATE THE ACTIVITY OF

AN α-ACTIN INTRONIC ENHANCER

Nuclear Factor of Activated T Cells and Serum Response Factor Cooperatively Regulate the Activity of an α-Actin Intronic Enhancer*

L. V. Gonzalez Bosc‡, J. J. Layne‡, D.C. Hill-Eubanks, M. T. Nelson.

Dept. Pharmacology, College of Medicine, University of Vermont, Burlington, VT,

US.

‡This authors contributed equally to this work.

Running title: An α-actin intronic element confers NFAT responsiveness.

To whom correspondence should be addressed:

Dept. of Pharmacology, University of Vermont,

89 Beaumont Ave.

Burlington, VT 05405, USA.

Tel: (802) 656-2500; Fax: (802) 656-4523;

E-mail: [email protected]

ABSTRACT

Expression of α-actin in smooth muscle cells (SMCs) is regulated, in part, by an intronic serum response factor (SRF)-binding CArG element. We have

identified a conserved nuclear factor of activated T cells (NFAT) binding site that

overlaps this CArG box and tested the hypothesis that this site plays a previously

unrecognized role in regulating α-actin expression. A reporter construct prepared

using a 56-bp region of the mouse α-actin first intron containing SRF, NFAT, and

AP-1 sites (SNAP) acted as an enhancer element in the context of a minimal

thymidine kinase promoter. Basal reporter activity following expression in SMCs

was robust and sensitive to the calcineurin-NFAT pathway inhibitors cyclosporin

A and FK506. Mutating either the NFAT or SRF binding site essentially abolished

reporter activity, suggesting that both NFAT and SRF binding are required. Basal

activity in non-smooth muscle HEK293 cells was SRF-dependent but NFAT-

independent and ~8-fold lower than that in SMCs. Activation of NFAT in HEK293

cells induced an ~4-fold increase in activity that was dependent on the integrity of

both NFAT and SRF binding sites. NFATc3·SRF complex formation,

demonstrated by co-immunoprecipitation, was facilitated by the presence of

SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased -

actin expression in cultured SMCs, suggesting that the molecular interaction of

NFAT and SRF at SNAP may be physiologically relevant. These data provide the

first evidence that NFAT and SRF may interact to cooperatively regulate SMC-

specific gene expression and support a role for NFAT in the phenotypic maintenance of smooth muscle.

INTRODUCTION

Smooth muscles cells (SMCs), unlike terminally differentiated cardiac and

skeletal myocytes, are phenotypically dynamic and maintain their differentiated

phenotype through the regulated expression of a repertoire of smooth muscle-

specific genes (Owens, 1995). This phenotypic flexibility is consistent with the

physiological demands placed on vascular tissue and may underlie changes in

smooth muscle structure and function that accompany pathological processes,

such as those that occur in hypertension and atherosclerosis (Pauletto et al.,

1994; Gorski & Walsh, 2003; Owens et al., 2004).

Serum response factor (SRF), a member of the MADS (MCM1, Agamous,

Deficiens, SRF) family of transcription factors (Shore & Sharrocks, 1995) is

central to the regulation of a large subset of SMC-specific genes (Owens, 1995,

1998; Kumar & Owens, 2003). Almost without exception, important regulatory

regions of these SMC-specific genes contain CArG boxes (CC[A/T]6GG) that are

indispensable for expression in smooth muscle (Kumar & Owens, 2003). One motif that is common to many of these genes is a pair of CArG boxes, designated

A and B, in the proximal upstream promoter region of the gene. The integrity of

each of these elements, as well as conservation of their spacing and orientation, is required for efficient SMC expression (Kumar & Owens, 2003). In addition to

proximal CArG boxes, important CArG box-containing regulatory elements have

been identified in intronic regions of SMC-specific genes, most notably within the

first intron of genes for smooth muscle (SM) myosin heavy chain (MHC) and SM

α-actin (Nakano et al., 1991; Shimizu et al., 1995; Madsen et al., 1998; Mack &

Owens, 1999; Manabe & Owens, 2001b, a; Kumar & Owens, 2003).

SRF transcriptional activity may be modulated by additional cofactors,

some of which form complexes with SRF-CArG DNA, whereas others act to

enhance SRF binding to CArG boxes without forming a detectable ternary complex (Treisman, 1995; Cahill et al., 1996; Kumar & Owens, 2003). Although

SRF activity is critically important in the regulation of SMC differentiation and

SMC marker gene expression, CArG boxes alone are not sufficient to direct

SMC-specific gene expression; additional cis-elements and trans-acting factors

are required (reviewed in (Kumar & Owens, 2003). Mechanisms that exist in

parallel with the SRF-CArG pathway, as well as SRF-intersecting mechanisms,

contribute to SMC phenotypic modulation and maintenance.

One of the more recent entries in the smooth muscle phenotypic

maintenance derby is the calcium (Ca2+)-dependent transcription factor nuclear

factor of activated T cells (NFAT), which regulates the expression of genes in a

diverse array of immune and non-immune cells and is closely linked to developmental processes that involve modulation of cellular phenotypes (Rao et al., 1997; Chin et al., 1998; Ho et al., 1998; Ranger et al., 1998; Delling et al.,

2000; Graef et al., 2001a; Graef et al., 2001b; Horsley & Pavlath, 2002; Schulz &

Yutzey, 2004). NFAT also contributes to pathological processes, as exemplified

by its role in the etiology of pathological, but not exercise-induced, cardiac

hypertrophy (Molkentin et al., 1998; Wilkins et al., 2002; Wilkins et al., 2004).

NFAT activation is regulated primarily through control of its subcellular

localization (Flanagan et al., 1991; Crabtree & Clipstone, 1994; Rao et al., 1997).

In response to Ca2+-elevating stimuli, NFAT is dephosphorylated at multiple N-

terminal phosphoserines by the Ca2+-dependent phosphatase, calcineurin, which

promotes NFAT nuclear translocation and DNA binding competence (Okamura et

al., 2000; Neal & Clipstone, 2001). NFAT binds DNA with very low affinity in the

absence of a cofactor, and in general, formation of an NFAT·cofactor complex is required for significant NFAT-mediated transcriptional activity. The NFAT family

consists of four members (NFAT1/c2, NFAT2/c1, NFAT3/c4, and NFAT4/c3) that

share the property of Ca2+-dependent nuclear translocation and a fifth member,

NFAT5, which is Ca2+-independent and shares limited homology with the other

family members.

NFAT activity has been shown to contribute to the maintenance of the

differentiated smooth muscle phenotype through regulation of 1 integrin and

caldesmon expression (Ohkawa et al., 2003) and has been implicated in

regulating the expression of the SM MHC gene (Wada et al., 2002). More

recently, NFAT has been proposed to regulate the expression of the KV2.1

voltage-dependent potassium channel in SMCs and thereby regulate arterial smooth muscle excitability (Amberg et al., 2004). A number of transcription

factors that play a role in regulating SMC-specific gene expression are known to

act as NFAT cofactors, including Egr-1, AP-1, YY1, the MADS family member

MEF2, and members of the GATA family (Kumar & Owens, 2003) and the references therein). Using intact cerebral artery preparations, we have recently

found that the NFATc3 isoform exhibits nitric oxide/protein kinase G-dependent

nuclear localization in vascular smooth muscle cells in response to a

normotensive pressure stimulus (Gonzalez Bosc et al., 2004). The implication is that NFATc3 is constitutively nuclear and potentially active under normal

physiological pressure and may thereby provide a link to the postulated role for

protein kinase G in the phenotypic maintenance of differentiated smooth muscle

(Lincoln et al., 2001).

Mack and Owens (Mack & Owens, 1999) have demonstrated that in the

context of a construct containing 5’ CArG boxes A and B, exon 1, and intron 1, a double mutation in the α-actin intronic CArG box that disrupts SRF binding also disrupts normal SMC-specific expression, decreasing reporter activity in vitro and

in vivo.By sequence analysis, we have identified a highly conserved NFAT

binding site that overlaps this intronic SRF binding site. Intriguingly, the CArG box

mutations that had been shown to disrupt SMC-specific expression also resulted

in disruption of the overlapping NFAT site. The individual contributions of SRF

and NFAT to enhancer activity have not been experimentally tested. Here we

show that a 56-bp α-actin intronic element that contains conserved SRF, NFAT

and AP-1 binding sites is capable of acting as an enhancer element, increasing the basal activity of a minimal promoter-reporter construct in both SMCs and

HEK293 cells and conferring responsiveness to NFAT-activating stimuli in

HEK293 cells. Mutations that independently disrupt either the NFAT site or the

SRF binding site are each capable of essentially eliminating basal activity in

SMCs and inducible activity in HEK293 cells, suggesting that both SRF and

NFAT are required for enhancer activity under these conditions. In support of the

idea that SRF and NFAT cooperate to mediate enhancer activity, we found that

both SRF and NFAT bind simultaneously to the overlapping CArG box/NFAT site

in this conserved intronic regulatory element under conditions that are associated

with enhancer activity. Finally, in vitro expression of α-actin in SMCs was

decreased by inhibiting NFAT activation, suggesting that the molecular interaction of NFAT and SRF at the intronic enhancer element may be

physiologically relevant.

EXPERIMENTAL PROCEDURES

Cell Culture—The rat A7r5 aortic smooth muscle cell line and an HEK293 human

embryonic kidney-derived cell line (BD Biosciences) were grown in Dulbecco’s

modified Eagle’s medium (Invitrogen) containing 10% fetal bovine serum and penicillin-streptomycin (10 units/ml; Invitrogen). G418 (2 mg/ml) was included in

cultures of this reverse tetracycline transactivator (rtTA)-expressing HEK293

derivative (used in experiments involving co-transfection of a Tet operator-EGFP-

NFATc3 construct) to maintain stable rtTA expression. The cultures were

maintained in a humidified incubator at 37 °C and 5% CO2.

Construction of Plasmids—A 56-bp region of the α-actin first intron

(corresponding to nucleotides 1035–1090 in GenBankTM accession number

U63129 [GenBank] ) containing a previously identified SRF-binding CArG box, an

overlapping NFAT binding motif, and an AP-1 binding site, designated SNAP

(SRF/NFAT/AP-1), was used as a starting point in the design of reporter

constructs (see Fig. 1). A double-stranded oligonucleotide covering this region

was prepared by combining equimolar amounts of single-stranded sense and

antisense oligonucleotides (IDX Technologies), heat denaturing (95 °C, 5 min),

and allowing them to slowly anneal at room temperature. SNAP oligonucleotides,

synthesized to contain NheI sites at each end (61 bp, total length), were cloned

into an NheI site present in the multi-cloning region of a luciferase reporter

plasmid containing a minimal thymidine kinase (TK) promoter (pTAL; BD

Biosciences). After transforming competent Escherichia coli (Invitrogen) and

preparing DNA minipreps from isolated colonies (Qiagen), individual clones were

screened by restriction analysis to determine the number of inserted copies and

then sequenced to determine the orientation of each insert. Reporter constructs containing mutations in the NFAT (AGGTTT), SRF (AATAATTAGG), or AP-1

(TCATACA) binding sites were similarly prepared.

Full-length NFATc3 was prepared from a plasmid (pSH205A) containing

an alternatively spliced murine NFATc3 cDNA (37) kindly provided by Dr. Gerald

Crabtree. The N terminus of native NFATc3 was amplified from mouse smooth

muscle total RNA by reverse transcription-PCR and ligated into an XbaI site

defining the 5’ end of the NFATc3 cDNA. A Tet-operated EGFP-NFATc3 fusion protein expression plasmid (pTetOP-EGFP-NFATc3-HGHpA) was prepared by

cloning full-length NFATc3 into a pTetOP-EGFP-HGHpA plasmid constructed

from pTetOP-HGHpA (containing a Tet operator and human growth hormone

poly(A) tail) (38) and pEGFP-C3 (Clontech).

Transient Transfection and Luciferase Assay—HEK293 cells were transfected

with TK-luciferase reporter constructs containing one, two, or three tandem

copies of the SNAP element in the all-forward or all-reverse orientation using the

FuGENE 6 transfection reagent (3:1 ratio of transfection reagent (µl)/total plasmid

DNA (µg)) according to the manufacturer’s protocol (Roche Applied Science).

Luciferase activity in cell lysates was determined using the Luciferase Assay

System according the manufacturer’s protocol (Promega) and expressed relative to empty pTAL vector controls. Luciferase activity was normalized to protein

concentration.

A7r5 cells were co-transfected with wild-type or mutant SNAP reporter

constructs or pTAL empty vector and pRL-TK (Renilla expression plasmid;

Promega) using Superfect transfection reagent (10 µl reagent/2 µg total

DNA/well) according to the manufacturer’s protocol (Qiagen). Luciferase and

Renilla activity were simultaneously detected (Dual-Luciferase Reporter Assay

System; Promega); Renilla activity was used to normalize luciferase activity.

In some experiments, exogenously expressed EGFP-NFATc3 was used to monitor NFAT subcellular localization and transfection efficiency. For

experiments employing HEK293 cells, an HEK293 derivative stably expressing rtTA was co-transfected with TetOP-EGFP-NFATc3 and cultured in the presence

of doxocyclin (2 µg/ml) to induce EGFP-NFATc3 expression. A7r5 cells were also

co-transfected with an rtTA expression plasmid, but were otherwise treated the

same as HEK293 cells.

Whole-cell Extracts—After trypsinization, cells were washed three times in PBS, resuspended in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1

mM dithiothreitol, and 1% Nonidet P-40) containing protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 9 µg/ml leupeptin, and 10 µg/ml

pepstatin), and incubated for 30 min on ice. Lysates were cleared by

centrifugation at 14,000 rpm for 5 min at 4 °C, and the supernatants were

assayed for protein content using the Bradford assay with bovine serum albumin

as the protein standard. Aliquots were stored at –80 °C prior to use. Whole-cell

extracts, containing a mixture of phosphorylated (primarily cytoplasmic) and

dephosphorylated (primarily nuclear) NFAT, are a convenient source of material

for DNA binding assays (below) because only the calcineurin-activated,

dephosphorylated form exhibits significant DNA binding competence.

Avidin Biotin Conjugated DNA Binding (ABCD) Assay—Binding of SRF and/or

NFAT to the SNAP element was determined using cellular extracts and biotin-

conjugated nucleotide probes/streptavidin-coated agarose beads in a modification of the DNA-protein binding assay developed previously by Wu and co-workers (39, 40). For each binding assay, whole-cell extracts containing 500

µg of protein were incubated with 400 ng of biotinylated oligonucleotide probes in

binding buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% Nonidet P-

40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 9

µg/ml leupeptin, 10 µg/ml pepstatin, and 10 mM sodium molybdate) for 30 min on ice. Streptavidin-conjugated agarose beads (Pierce) were added (25 µl of a 50%

slurry), and the suspensions were placed on a gyrating shaker platform at 4 °C

for 1–3 h. The beads were pelleted by centrifugation at 9000 x g (4 °C), washed

three times in binding buffer, and eluted in 2x SDS loading buffer for SDS-PAGE

and Western analysis.

Co-immunoprecipitation—Whole-cell lysates from HEK293 cells (500 µg total

protein content/sample, treated as described in the text) were incubated with 500

ng of mouse anti-NFATc3-agarose conjugate (Santa Cruz Biotechnology) in

DNA-protein binding buffer for 24 h at 4 °C with constant rocking in the presence

or absence of unlabeled SNAP oligonucleotide. The beads were pelleted by

centrifugation (2000 x g at 4 °C for 5 min), and the pellet was washed three times

in a buffer consisting of 10 mM Hepes, pH 7.5, 150 mM NaCl, and 0.1% Nonidet

P-40. After the third wash, the pellet was resuspended in SDS-loading buffer and

boiled for 5 min at 95 °C. The proteins were separated by SDS-PAGE,

transferred to nitrocellulose, and probed for SRF or NFATc3 by Western blotting.

The absence of nonspecific binding to NFATc3-agarose beads was confirmed by

stripping and reprobing for glyceraldehyde-3-phosphate dehydrogenase (primary

antibody from Chemicon International; 1:1000 dilution for 1 h at room

temperature; data not shown).

Western Blot Analysis—Protein samples were separated by SDS-PAGE using

7.5% acrylamide gels (Bio-Rad) in standard Laemmli buffer and transferred to

nitrocellulose membranes (Bio-Rad). After blocking for 1 h at room temperature in

Tris-buffered saline containing 5% nonfat milk and 0.1% Tween 20, blots were

exposed to primary antibodies (diluted in blocking solution) overnight at 4 °C or

for 1 h at room temperature. The primary antibodies used were mouse

monoclonal NFATc3 (1:200; Santa Cruz Biotechnology) and rabbit polyclonal

SRF (1:4000; Santa Cruz Biotechnology). After multiple washes in Tris-buffered saline, horseradish peroxidase-conjugated secondary antibody (from Santa Cruz

Biotechnology (1:10,000) or Pierce (1:2000) in blocking solution) was applied for

1 h at room temperature. Bands were visualized using standard or high sensitivity

chemiluminescence detection systems (Pierce Supersignal West Dura or Femto,

respectively).

Immunofluorescence—HEK293 and A7r5 cells were grown in an 8-well Lab-Tek

chamber slide (Nalge Nunc). Cells were fixed with 4% formaldehyde in PBS for

15 min, permeabilized in 0.1% Triton in PBS for 10 min, and then blocked in 3% donkey serum in PBS for 1 h. Primary antibody (rabbit polyclonal anti-NFATc3,

1:100; Santa Cruz Biotechnology) was prepared in 0.1% gelatin in PBS and

applied overnight at 4 °C. Secondary antibody (anti-rabbit Cy5, 1:500; Jackson

Immunoresearch Laboratories) was prepared in 0.1% gelatin in PBS and applied

for 1 h at room temperature. Nuclei were stained using SYTOX green (1:5000 in

PBS).

Cells were examined at x40 magnification using a Bio-Rad 1000 laser

scanning confocal microscope. NFAT and nuclear staining were detected by

sequentially monitoring the Cy5 and SYTOX fluorescence using excitation

wavelengths of 650 and 488 nm and emission wavelengths of 670 and 520 nm,

respectively. Specificity of immune staining was confirmed by the absence of

fluorescence in cells incubated with secondary antibody alone. For scoring of

NFAT-positive nuclei, multiple fields for each replicate of each experimental

condition were imaged and counted by two independent observers under double-

blind conditions with the aid of Metamorph software (Universal Imaging Corp).

The software was programmed so that individual pixels within a given image

would appear white if co-localization of the green nucleic acid stain and the Cy5-

NFAT stain occurred. Thus, for quantification purposes, a cell was considered

positive if co-localization (white) was distributed throughout the nucleus, whereas

a cell was considered negative if no co-localization (green only) or only isolated

co-localization was observed.

In-cell Western Analysis—SM α-actin expression in SMCs was quantified using an infrared-based in-cell Western assay system as described by the

manufacturer (Li-Cor Biosciences). Briefly, confluent cultures of A7r5 cells were

trypsinized and seeded into 96-well plates (70% confluence). After 48 h in 1%

fetal bovine serum, cells were treated as described in the text, washed once with

PBS, fixed with 4% formaldehyde in PBS for 15 min at room temperature, and

blocked (Aqua Block with 0.1% Tween 20). Primary antibody (mouse monoclonal

anti-SM α-actin (1:250; Biogenex) or mouse monoclonal anti-α-actin (1:200;

Sigma)) was applied overnight at 4 °C, followed by secondary antibody (goat anti-

mouse IRDye 800 (1:800 dilution in Aqua Block with 0.1% Tween 20; Rockland)) for 1 h. TOTO-3 (1:1000; Molecular Probes) was used to counterstain DNA.

Infrared fluorescence was monitored using an Odyssey Imager (700 nm for

TOTO-3 and 800 nm for IRDye 800). The intensity of both channels was set at

7.5, with a focus setting of 3 mm. Integrated intensity of SM α-actin or β-actin

(housekeeping protein) stain was normalized to the integrated intensity of DNA

stain. Because of the ease and accuracy with which multiple individual replicates

can be independently analyzed, this approach is especially useful where

quantitation is the goal.

RESULTS

A CArG Box-containing Region of the Mouse α-Actin First Intron Acts as an

NFAT- and SRF-dependent Enhancer Element—We have identified a putative

NFAT binding site in the α-actin first intron that overlaps a functionally important

SRF-binding CArG box (Mack & Owens, 1999). The sequence of this SRF-NFAT

binding site-containing region is remarkably conserved among rodent, human,

and chicken α-actin and includes a completely conserved AP-1 site at a position

24 bp downstream of the NFAT site (Fig. 1). To test these putative binding sites

for potential enhancer activity, an oligonucleotide encompassing this mouse -

actin first intronic region containing SRF, NFAT, and AP-1 binding sites (SNAP)

was cloned into a luciferase reporter plasmid upstream of a minimal TK promoter.

Constructs containing one, two, or three tandem copies of SNAP in either forward or reverse orientation were tested in HEK293 cells and SMCs for luciferase

expression. The minimal TK promoter-luciferase construct containing three

tandem copies of the insert in the reverse orientation showed the highest reporter

activity of those constructs tested (data not shown) and was used in subsequent

experiments. The wild-type reporter construct (SNAP-Luc) and variants

containing mutated SRF (SmutNAP-Luc), NFAT (SNmutAP-Luc), or AP-1 (SNAPmut-

Luc) binding sites are depicted schematically in Fig. 2A.

Fig. 2, B and C, shows luciferase activity, normalized to the minimal TK

promoter-containing (empty) pTAL vector, in SNAP-Luc-transfected SMCs and

HEK293 cells, respectively. In both cell lines, the SNAP element promoted a significant increase in reporter activity under basal (unstimulated) conditions, demonstrating that this small region of the mouse α-actin first intron is capable of

acting as a functional enhancer element. Inhibition of the calcineurin/NFAT

pathway with FK506 (1 µM) and CsA (1 µM) significantly decreased reporter

activity in SNAP-Luc-transfected SMCs (Fig. 2B), suggesting NFAT involvement.

To determine the relative contribution of SRF, NFAT, and/or AP-1 sites to the

enhanced reporter activity, HEK293 cells and SMCs were transfected with SNAP-

Luc constructs containing mutations at each of these sites, designed such that

mutations in one binding element were independent of each of the other binding

elements. Mutating either the NFAT site (SNmutAPLuc) or the SRF binding site

(SmutNAP-Luc) almost completely abolished basal enhancer activity in SMCs,

suggesting that both NFAT and SRF binding sites were required for this activity

(Fig. 2B). Mutating the AP-1 binding site (SNAPmut-Luc) partially decreased basal

activity in SMCs (Fig. 2B), suggesting that full activity may require formation of an

SRF·NFAT·AP-1 complex. In contrast, only SRF binding appears to be required

for basal activity in HEK293 cells because a mutation in the CArG box alone

(SmutNAP-Luc) abrogated enhancer activity, whereas a construct containing a

mutated NFAT site (SNmutAP-Luc) or a mutated AP-1 site (SNAPmut-Luc) retained

full basal activity (Fig. 2C).

NFAT/AP-1 Pathway Stimulation Increases SNAP Enhancer Activity in HEK293

Cells—Concurrent treatment with the Ca2+ ionophore, ionomycin (Io), and

phorbol 12-myristate 13-acetate acetate (PMA) is a stimulus paradigm that is

commonly used to simultaneously activate NFAT and NFAT cofactor pathways

(Liu et al., 1991; Chen et al., 1998; Macian et al., 2001). Dephosphorylation by the Ca2+-dependent phosphatase, calcineurin, serves to promote both NFAT nuclear accumulation and DNA binding (Neal & Clipstone, 2001). Stimulation of

SNAP-Luc-transfected HEK293 cells with Io (1 µM) and PMA (100 nM) induced

an ~4-fold increase in luciferase activity over unstimulated conditions (Fig. 3).

This inducible luciferase activity was significantly inhibited by pre-treatment with

the calcineurin inhibitors CsA and FK506, suggesting that dephosphorylation-

dependent translocation of NFAT from the cytosol to the nucleus contributes to the observed increase in activity. The Io/PMA-induced increase in reporter

activity, like the basal enhancer activity in SMCs, was dependent on the integrity

of both SRF and NFAT binding sites, as indicated by the virtual absence of

inducible activity in HEK293 cells transfected with reporter constructs containing

mutations in the NFAT (SNmutAP-Luc) or SRF (SmutNAP-Luc) sites. Similar to

results obtained under basal conditions in SMCs, a mutation in the AP-1 site

(SNAPmut-Luc) partially but significantly reduced the response to Io/PMA

stimulation in HEK293 cells. Interestingly, the high basal activity of the SNAP-Luc

construct in SMCs (~8-fold higher than that in similarly transfected HEK293 cells)

cannot be further enhanced by treatment with Io/PMA (data not shown; see

“Discussion”).

NFATc3 and SRF Simultaneously Bind to the SNAP Enhancer Element—

Functional effects of individual site mutations on SNAP enhancer activity strongly suggest that NFAT and SRF bind simultaneously at overlapping SRF and NFAT

sites within the α-actin intronic regulatory element under study. To determine

whether this was the case, we performed ABCD assays to pull down SNAP-

associated proteins from cellular extracts, followed by Western analysis. Fig. 4A

shows a representative ABCD assay employing whole-cell lysates from non-

stimulated and Io/PMA-stimulated HEK293 cells. Consistent with the functional

data, only SRF was bound to the oligonucleotide probe under non-stimulated conditions (Fig. 4A, lane 1). Under stimulated conditions, both SRF and NFATc3 were present in the DNA·protein complex (Fig. 4A, lane 2). Thus, the overlapping

CArG box/NFAT site in the α-actin first intron can bind both NFATc3 and SRF in

response to an NFAT-activating stimulus.

In competition assays employing either HEK293 or SMC whole-cell

lysates, SRF binding to biotinylated SNAP was abrogated in the presence of an

excess of unlabeled wild-type SNAP oligonucleotides. Unlabeled probes

containing a scrambled sequence or a mutation in the SRF binding site

(SmutNAP), however, failed to compete for SRF binding, demonstrating that the observed binding is sequence-specific (Fig. 4, B and C). To rule out the

possibility that mutations in the adjacent NFAT or AP-1 sites might disrupt SRF

binding, even though they do not disrupt the CArG site itself, we performed

competition binding assays using unlabeled SNAP containing mutated NFAT

(SNmutAP) or AP-1 (SNAPmut) binding sites. In each case, an excess of unlabeled

oligonucleotides was able to prevent SRF binding to the labeled probe, indicating

that an intact CArG box alone is sufficient to bind SRF.

These experiments do not rule out the formal possibility that NFATc3 and

SRF are binding individually to separate populations of probe molecules rather

than binding simultaneously to the same probe. To address this, we

immunoprecipitated NFATc3 in the presence or absence of unlabeled SNAP

oligonucleotide and probed for associated SRF by Western analysis (Fig. 5).

Under standard detection conditions (Fig. 5A), a strong SRF band is evident in

the presence of the SNAP probe (lane 1), whereas no SRF band is evident in the

absence of SNAP (scrambled oligonucleotide, lane 2; no oligonucleotide, lane 3).

By increasing the gain (i.e. using a higher sensitivity chemiluminescence

detection system), we are able to show that SRF is present in NFATc3

immunoprecipitates in the absence of SNAP, albeit to a lesser extent (Fig. 5B).

These results indicate that SNAP acts as a sequence-specific scaffold to facilitate

NFATc3 and SRF interactions, but they also suggest that NFATc3 and SRF may

interact in solution.

NFATc3 Exhibits Constitutive Nuclear Localization in A7r5 Cells but Not HEK293

Cells—In general, NFAT is localized to the cytosol of unstimulated cells.

However, in the A7r5 cells used in this study, we found that in an overwhelming

majority of cells (95.33 ± 2.53%, n = 3 images (81 cells)), endogenous NFATc3 is constitutively localized to the nucleus under basal conditions (Fig. 6A, right

panel). Inhibition of calcineurin activity by treatment with CsA and FK506 (15 min) induced a substantial redistribution of nuclear NFATc3 to the cytoplasm (NFATc3-

positive nuclei = 27.40 ± 7.40%, n = 3 images (43 cells); p < 0.001 versus basal).

Overexpressed EGFP-NFATc3 also exhibits nuclear localization in SMCs in the

absence of an NFAT-activating stimulus (Fig. 6B, right panel). This distribution is strikingly different from the primarily cytosolic and perinuclear localization of both

endogenous (Fig. 6A, left panel) and exogenously expressed (Fig. 6B, left panel)

NFATc3 in HEK293 cells. These results are consistent with both the elevated

basal activity of SNAP-Luc in SMCs and the requirement for an NFAT-activating

stimulus for maximal reporter activity in HEK293 cells.

Calcineurin/NFAT Pathway Inhibition Reduces α-Actin Expression in Smooth

Muscle Cells—The fact that NFAT and SRF can simultaneously bind to a

CArG/NFAT site corresponding to a region of the α-actin intron known to be

important for regulating SMC-specific α-actin expression (Shimizu et al., 1995;

Mack & Owens, 1999) suggests that NFAT cooperates with SRF in the regulation

of α-actin expression. To confirm that the calcineurin/NFAT pathway is involved

in regulating the expression of SM α-actin, we treated confluent, serum-deprived

A7r5 SMCs with the calcineurin inhibitors FK506 and CsA, and we measured changes in SM α-actin protein expression by in-cell Western analysis (see

“Experimental Procedures”). Results are expressed relative to β-actin, and expression of both proteins was normalized to DNA content. Fig. 7 shows that inhibition of NFAT activation significantly decreased SM α-actin expression, reducing protein levels by nearly 50% compared with untreated controls. These results suggest that the NFAT-dependent regulation of SNAP enhancer activity, described above, is relevant to the regulation of α-actin expression.

DISCUSSION

We have found that a 56-bp region of the mouse SM α-actin first intron

that contains a highly conserved SRF/NFAT/AP-1 (SNAP) composite binding site

sequence is capable of acting as an enhancer element, increasing reporter

activity in the context of a minimal TK promoter. These results are consistent with

a previous report showing that a larger (180-bp) segment of the corresponding

region of the human α-actin first intron, cloned upstream or downstream of a

minimal SV40 promoter, exhibits enhancer activity (Nakano et al., 1991) and

further support more recent results showing that the corresponding intronic region

of the rat α-actin gene is required for robust in vitro and in vivo reporter expression in the context of a construct that includes 5’-regulatory elements and

the first exon (Mack & Owens, 1999). More importantly, our results indicate that

the previously unrecognized NFAT site and the overlapping SRF-binding CArG element are both absolutely required for the elevated basal activity in SMCs and

induced activity in HEK293 cells. The SNAP region simultaneously binds both

SRF and NFAT in HEK293 cells under conditions in which activity is induced but

binds only SRF in the absence of stimulation, providing the first evidence that

NFAT and SRF may interact to cooperatively regulate gene expression. Our

results also show that inhibition of the calcineurin/NFAT pathway decreases α-

actin expression in SMCs, consistent with a role for NFAT in the regulation of α-

actin transcription.

In HEK293 cells transfected with the SNAP-Luc construct, reporter

expression in the absence of stimulation was significantly increased over that in

cells transfected with a control luciferase reporter plasmid containing only a

minimal TK promoter. The basal level of expression in HEK293 cells, however,

was ~8-fold lower than that in SNAP-Luc-transfected SMCs, consistent with a

potential SMC-selective role for this element. Basal expression in HEK293 cells was sensitive to disruption of the CArG box but unaffected by mutations in the

NFAT site or the AP-1 site. This is in sharp contrast to Io/PMA-stimulated

HEK293 cells (or unstimulated SMCs), in which mutations in NFAT or SRF sites

were equally effective in abrogating reporter activity and a mutation to the AP-1

site partially inhibited activity. Thus, in non-smooth muscle HEK293 cells, SRF alone appears capable of mediating a low basal level of transcription from the

SNAP enhancer element, whereas the induced activity in these cells and the much higher basal activity in SMCs require both NFAT and SRF.

Interestingly, in SMCs, stimulation of the calcineurin/NFAT pathway with

Io/PMA failed to increase the reporter activity of the SNAP-Luc construct beyond the already elevated basal level. The absence of inducible activity in SNAP-Luc-

transfected SMCs, as well as the profound difference in basal enhancer activity

between SMCs and HEK293 cells, may be partially accounted for by differences

in the subcellular distribution of the NFATc3 isoform between these two cell

types. Although NFAT is normally presumed to localize to the cytosol of

unstimulated cells, we found that a preponderance of endogenous as well as

exogenously expressed NFATc3 is constitutively localized to the nucleus of A7r5 cells. In HEK293 cells, both endogenous and exogenously expressed NFATc3 is

localized to the cytosol under basal conditions. Thus, a virtual absence of nuclear

NFATc3 in unstimulated HEK293 cells is associated with low level, NFAT-

independent SNAP enhancer activity. Stimulation of HEK293 cells with Io/PMA

increases nuclear NFATc3 (data not shown) and SNAP enhancer activity. In

SMCs, in which NFATc3 is constitutively nuclear, SNAP activity is high, NFAT- dependent, and unresponsive to Io/PMA treatment. Although these results do not necessarily indicate that SNAP preferentially binds the NFATc3 isoform, they are

consistent with the interpretation that sufficient nuclear NFAT in the form of

NFATc3 is present in SMCs to drive maximal enhancer activity under basal

conditions.

The constitutively nuclear NFATc3 observed in SMCs is redistributed to the cytosol following inhibition of calcineurin with FK506 and CsA (Fig. 6C). This

result suggests that NFAT import mechanisms may be constitutively elevated in these cultured SMCs, an observation that could be explained by elevated

calcineurin activity. Alternatively, mechanisms that act to promote nuclear export may be less active under basal conditions. Recent work from our laboratory has

shown that regulation of NFATc3 nuclear export may be central to the activation

state of NFAT in smooth muscle (reviewed in (Hill-Eubanks et al., 2003). In unpressurized arteries, basal NFATc3 nuclear export is high as a result of

elevated JNK2-mediated export; stimuli that induce a net nuclear accumulation of

NFATc3 are those that not only elevate intracellular Ca2+ but also result in a

decrease in JNK2-mediated export (Gomez et al., 2003). In response to a

normotensive pressure stimulus, JNK2 activity in native smooth muscle is

reduced via a nitric oxide/protein kinase G-dependent pathway, and NFATc3 is

predominantly localized to the nucleus (Gonzalez Bosc et al., 2004). In this

respect, A7r5 may exist in a state similar to that of pressurized arteries, with

reduced JNK2-mediated export activity and constitutively nuclear NFATc3.

SMCs are known to express higher levels of SRF than other cell types (Tuil et al.,

1990; Chang et al., 2001), so it is conceivable that the difference between the levels of basal and induced enhancer activity between SMCs and HEK293 cells

may also reflect limiting SRF levels in HEK293 cells. Although HEK293 cells do

contain sufficient SRF to drive low basal levels of enhancer activity and to detect in a complex with the SNAP element, it may be that higher levels of SRF are

required for efficient recruitment of NFAT and maximal enhancer activity.

Our reporter assay data showing that basal SNAP enhancer activity in

SMCs and induced activity in HEK293 cells are dependent on both intact SRF

and NFAT sites suggest a cooperative mechanism of action between SRF and

NFAT. This idea is supported by the binding of both NFATc3 and SRF to the

SNAP element under conditions in which the NFAT pathway is activated in

HEK293 cells. The presence of SRF in NFATc3 immunoprecipitates provides

compelling evidence that SRF and NFATc3 interact. The fact that associated SRF is increased in the presence of SNAP is most consistent with the interpretation

that simultaneous binding of SRF and NFATc3 to their respective sites facilitates

NFATc3/SRF interactions. SRF binding alone is insufficient to promote enhancer

activity under NFAT stimulated conditions because a mutation at the NFAT

binding site does not substantially inhibit SRF binding to the overlapping CArG

box but almost completely abrogates induced reporter activity.

SRF, a member of the MADS family of transcription factors (Shore &

Sharrocks, 1995), has been shown to bind to the REL homology domain of

nuclear factor-κB (Franzoso et al., 1996) to enhance nuclear factor-κB

transcriptional activity. NFAT, which also contains a REL homology domain, has

been previously shown to functionally interact with the MADS family member

Mef2 (Chin et al., 1998). This is the first report, however, to suggest that NFAT

and SRF may interact with one another to function as transcriptional co-

activators. The SRF and NFAT domains that may be responsible for mediating

protein-protein interactions in the putative SRF·NFAT complex, as well as the role that the individual NFAT and/or SRF transactivation domains may play in

mediating interactions with the transcriptional machinery, are currently unknown.

Our data also suggest that AP-1, which is a common NFAT cofactor (Macian et

al., 2001) that has also been implicated in SRF signaling (Paradis et al., 1996), contributes to NFAT/SRF enhancer efficacy, although the molecular details of this involvement remain to be defined.

SRF has been previously shown to interact with other transcription factors,

such as ETS domain-containing transcription factors (e.g. Elk-1), the

homeodomain proteins Barx1b and Nkx3.2, and the zinc finger protein GATA6

(Kumar & Owens, 2003). In smooth muscle, the transcription factor myocardin

has been shown to regulate SRF-dependent transcription of a –2.6/+2.8-kb α- actin promoter/intronic construct through interactions that exhibit a combinatorial dependence on CArG elements, primarily involving CArG B and CArG A boxes

(Yoshida et al., 2003). The apparent preferential interaction of myocardin with proximal promoter CArG boxes suggests that NFAT action at the intronic SNAP element may operate in parallel with myocardin activity, with both pathways

required for full transcriptional activity. Considered in the context of the

demonstrated Ca2+ dependence of smooth muscle differentiation marker gene

expression (Wamhoff et al., 2004), it is possible that the NFAT pathway may

contribute to the Ca2+ sensitivity component of SMC gene expression, serving to integrate Ca2+ signals at regulatory elements of smooth muscle-specific genes,

including α-actin.

In addition to the molecular evidence supporting a role for NFAT in the

regulation of α-actin expression, this study shows that inhibition of the

calcineurin/NFAT pathway in cultured rat SMCs decreases α-actin protein expression. This result is in accord with a recent report showing that blockade of

calcineurin/NFAT in primary cultures of human vascular smooth muscle is associated with a reduction in both α-actin and SM MHC protein expression

(Amberg et al., 2004). Another recent study has also implicated NFAT in the

regulation of α1 integrin and caldesmon expression in cultured visceral SMCs,

showing that calcineurin activity is required for the expression of these smooth

muscle markers and maintenance of the contractile smooth muscle phenotype

(Ohkawa et al., 2003). The proposed interactions between NFAT and SRF at the

α-actin intronic composite binding site may provide a molecular framework to

help explain the apparent NFAT dependence of α-actin expression in SMCs, but it appears unlikely that this interaction can be invoked as a general mechanism to

account for the NFAT dependence of other smooth muscle-specific genes. For

example, although the regulation of SM MHC expression in smooth muscle

exhibits similarities to that of α-actin regulation in that dual 5’ CArG boxes and an

intronic CArG box are all required for smooth muscle-specific expression, neither

the 5’ nor intronic CArG box is associated with NFAT binding sites. Instead, NFAT

appears to regulate SM MHC expression through an association with GATA at

unidentified sites in the 5’-flanking sequence of the SM MHC gene (Wada et al.,

2002). The single 5’-upstream CArG box of α1 integrin that is required for

expression in differentiated SMCs (Obata et al., 1997) likewise lacks an associated NFAT site. In addition to the conserved intronic NFAT site

characterized here, the first intron of α-actin is rich in potential NFAT binding sites, many of which are phylogenetically conserved and might be expected to contribute to NFAT-sensitive expression. To the extent that NFAT is involved in

regulating the expression of SMC-specific genes, it is likely that it does so

through multiple mechanisms involving interactions with different cofactors.

Acknowledgements- We gratefully acknowledge Dr. Mercedes Rincon and Dr.

Anthony Morielli for helpful discussions. We also thank Dr. Kathleen Martin for advice on the ABCD assay and Drs. Gerald Crabtree and Prabir Ray for providing plasmids used in preparation of the pTetOP-EGFP-NFATc3-HGHpA construct.

Figure 1. Region of the mouse α-actin gene containing 5' CArG boxes A and B, exon 1, and intron 1. The intronic SNAP element used in these studies is composed of an SRF binding element (CArG box), an overlapping NFAT binding site, and an AP-1 binding site shown in red, blue, and green, respectively. The CArG box and NFAT site are identical in mouse and rat; nucleotides in capital letters are completely conserved among chicken, mouse, and human.

100

Figure 2. A CArG box-containing region of the mouse α-actin first intron

acts as an NFAT- and SRF-dependent enhancer under basal conditions. A, reporter constructs containing three tandem copies (depicted in forward orientation) of the SM α-actin first intronic enhancer element (SNAP) used in transfection experiments: wild-type (SNAP-Luc), NFAT-mutated (SNmutAP-Luc),

SRF-mutated (SmutNAP), or AP-1-mutated (SNAPmut). B, A7r5 SMCs were

transfected with the SNAP-Luc construct or its mutants, as described under

"Experimental Procedures." Activity is expressed relative to the empty pTAL

vector, which contains a minimal thymidine kinase promoter-luciferase

expression cassette (TK-Luc). A subgroup of cells transfected with SNAP-Luc

(wild-type) was pre-treated with 1 µM FK506 and 1 µM CsA. *, p < 0.001 versus

all; n = 6–9. C, HEK293 cells were transfected with the SNAP-Luc wild-type and

mutant constructs, as described. Luciferase activity was normalized to reporter

activity of cells transfected with empty vector. *, p < 0.01 versus all; n = 6.

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102

Figure 3. NFAT/AP-1 pathway stimulation increases SNAP reporter activity

in HEK293 cells. HEK293 cells were transfected with wild-type SNAP-luciferase

construct (SNAP-Luc) or constructs containing mutations in NFAT (SNmutAP-

Luc), SRF (SmutNAP-Luc), or AP-1 (SNAPmut-Luc) sites and incubated for 24 h

with or without 1 µM Io and 100 nM PMA. HEK293 cells transfected with a

minimal TK-Luc vector (pTAL/no insert) served as a control. Luciferase activity of

each construct under stimulated conditions is expressed relative to reporter

activity under non-stimulated conditions. Also shown is Io/PMA-stimulated SNAP-

Luc activity in the presence of 1 µM FK506 and 1 µM CsA expressed relative to

unstimulated SNAP-Luc without FK506/CsA. *, p < 0.01 versus no insert +

Io/PMA, FK506/CsA + Io/PMA, SNmutAP + Io/PMA, SmutNAP + Io/PMA, and

SNAPmut + Io/PMA; #, p < 0.05 versus no insert + Io/PMA. n = 4–10.

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104

Figure 4. ABCD assay showing NFAT and SRF binding to SNAP. A, whole- cell lysates (500 µg of protein) from untreated HEK293 cells (Basal) or cells treated for 1 h with 1 µM ionomycin and 100 nM PMA (I/PMA) were incubated for

30 min in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of biotin- labeled SNAP (Biotin-SNAPwt) probe. Streptavidin-agarose-conjugated beads were added and incubated for 2 h. Pellet eluted was separated by SDS-PAGE and blotted for NFATc3 or SRF. Lanes 5 and 6 contain 30 µg of whole-cell extract. B, whole-cell lysates (500 µg of protein) from Io/PMA-treated HEK293 cells were pre-incubated for 30 min with non-labeled probe containing wild-type

(SNAPwt), NFAT-mutated (SNmutAP), SRF-mutated (SmutNAP), AP-1-mutated

(SNAPmut), or scrambled sequences (lanes 4–8). Lane 9 contains 30 µg of whole- cell extract. C, A7r5 whole-cell lysates (500 µg of protein) were pre-incubated for

30 min with non-labeled probe containing wild-type, mutated, or scrambled sequences (lanes 3–7). Lane 8 corresponds to 30 µg of whole-cell extract.

Representative experiments of n = 3.

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106

Figure 5. SNAP facilitates co-immunoprecipitation of SRF and NFAT.

NFATc3 was immunoprecipitated from whole-cell lysates (500 µg of protein) of Io

(1 µM)/PMA (100 nM)-treated (1 h) HEK293 cells in the presence or absence of

SNAP followed by Western blotting for SRF using standard sensitivity (A) or high sensitivity (B) chemiluminescence detection systems (see "Experimental

Procedures"). In C, Western blotting of NFATc3 immunoprecipitates for NFATc3 demonstrates uniform loading and immunoprecipitation efficacy. Lane 1, SNAP oligonucleotide; lane 2, scrambled sequence oligonucleotide; lane 3, no oligonucleotide.

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108

Figure 6. Differential subcellular localization of NFATc3 between HEK293

and A7r5 cells. A, representative immunofluorescence confocal microscopic

images of endogenous NFATc3 in HEK293 and A7r5 cells. White pixels

represent co-localization of NFATc3 (red) and nuclei (SYTOX green staining).

Scale bar represents 50 µm. B, EGFP fluorescence in EGFP-NFATc3- transfected HEK293 and A7r5 cells monitored at 37 °C using 440DF20/480DF30

excitation/emission filters (Lambda 10-2; Sutter Instruments, Novato, CA) with

the aid of METAFLUOR 4.01 analysis software (Universal Imaging, Media, PA).

EGFP-NFATc3 exhibits both nuclear and cytosolic localization in SMCs; HEK293

cells show only cytosolic/perinuclear fluorescence. C, representative images of

an A7r5 cell showing EGFP-NFATc3 nuclear expression before and 15 min after

treatment with 1 µM FK506 and 1 µM CsA at 37 °C. Inset images correspond to

endogenous NFATc3 expression detected by immunofluorescence in A7r5 cells

under the same treatment conditions.

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110

Figure 7. Calcineurin/NFAT pathway inhibition reduces α-actin expression

in aorta-derived smooth muscle cells (A7r5). Cells were seeded in 96-well

plates and grown in Dulbecco's modified Eagle's medium/10% fetal bovine

serum. After 24 h, medium was changed to Dulbecco's modified Eagle's

medium/1% fetal bovine serum in the absence (Control) or presence of 1 µM

FK506 and 1 µM CsA (FK + CsA) for 48 h. SM α-actin and α-actin were detected by in-cell Western blot (Odyssey Technology). Results are expressed as the ratio of integrated α-or β-actin staining intensity to integrated intensity of DNA staining.

*, p < 0.0001 (n = 48). NS, non-significant (n = 18).

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REFERENCES

Amberg GC, Rossow CF, Navedo MF & Santana LF. (2004). NFATc3 regulates Kv2.1 expression in arterial smooth muscle. J Biol Chem 279, 47326- 47334.

Cahill MA, Janknecht R & Nordheim A. (1996). Signalling pathways: jack of all cascades. Curr Biol 6, 16-19.

Chang PS, Li L, McAnally J & Olson EN. (2001). Muscle specificity encoded by specific serum response factor-binding sites. J Biol Chem 276, 17206- 17212.

Chen L, Glover JN, Hogan PG, Rao A & Harrison SC. (1998). Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature 392, 42-48.

Chin ER, Olson EN, Richardson JA, Yang Q, Humphries C, Shelton JM, Wu H, Zhu W, Bassel-Duby R & Williams RS. (1998). A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes Dev 12, 2499-2509.

Crabtree GR & Clipstone NA. (1994). Signal transmission between the plasma membrane and nucleus of T lymphocytes. Annu Rev Biochem 63, 1045- 1083.

Delling U, Tureckova J, Lim HW, De Windt LJ, Rotwein P & Molkentin JD. (2000). A calcineurin-NFATc3-dependent pathway regulates skeletal muscle differentiation and slow myosin heavy-chain expression. Mol Cell Biol 20, 6600-6611.

Flanagan WM, Corthesy B, Bram RJ & Crabtree GR. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352, 803-807.

Franzoso G, Carlson L, Brown K, Daucher MB, Bressler P & Siebenlist U. (1996). Activation of the serum response factor by p65/NF-kappaB. Embo J 15, 3403-3412.

Gomez MF, Bosc LV, Stevenson AS, Wilkerson MK, Hill-Eubanks DC & Nelson MT. (2003). Constitutively elevated nuclear export activity opposes Ca2+- dependent NFATc3 nuclear accumulation in vascular smooth muscle: role of JNK2 and Crm-1. J Biol Chem 278, 46847-46853.

113

Gonzalez Bosc LV, Wilkerson MK, Bradley KN, Eckman DM, Hill-Eubanks DC & Nelson MT. (2004). Intraluminal pressure is a stimulus for NFATc3 nuclear accumulation: role of calcium, endothelium-derived nitric oxide, and cGMP-dependent protein kinase. J Biol Chem 279, 10702-10709.

Gorski DH & Walsh K. (2003). Control of vascular cell differentiation by homeobox transcription factors. Trends Cardiovasc Med 13, 213-220.

Graef IA, Chen F, Chen L, Kuo A & Crabtree GR. (2001a). Signals transduced by Ca(2+)/calcineurin and NFATc3/c4 pattern the developing vasculature. Cell 105, 863-875.

Graef IA, Chen F & Crabtree GR. (2001b). NFAT signaling in vertebrate development. Curr Opin Genet Dev 11, 505-512.

Hill-Eubanks DC, Gomez MF, Stevenson AS & Nelson MT. (2003). NFAT regulation in smooth muscle. Trends Cardiovasc Med 13, 56-62.

Ho IC, Kim JH, Rooney JW, Spiegelman BM & Glimcher LH. (1998). A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis. Proc Natl Acad Sci U S A 95, 15537- 15541.

Horsley V & Pavlath GK. (2002). NFAT: ubiquitous regulator of cell differentiation and adaptation. J Cell Biol 156, 771-774.

Kumar MS & Owens GK. (2003). Combinatorial control of smooth muscle- specific gene expression. Arterioscler Thromb Vasc Biol 23, 737-747.

Lincoln TM, Dey N & Sellak H. (2001). Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol 91, 1421-1430.

Liu J, Farmer JD, Jr., Lane WS, Friedman J, Weissman I & Schreiber SL. (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP- FK506 complexes. Cell 66, 807-815.

Macian F, Lopez-Rodriguez C & Rao A. (2001). Partners in transcription: NFAT and AP-1. Oncogene 20, 2476-2489.

Mack CP & Owens GK. (1999). Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions. Circ Res 84, 852-861.

114

Madsen CS, Regan CP, Hungerford JE, White SL, Manabe I & Owens GK. (1998). Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence. Circ Res 82, 908-917.

Manabe I & Owens GK. (2001a). CArG elements control smooth muscle subtype-specific expression of smooth muscle myosin in vivo. J Clin Invest 107, 823-834.

Manabe I & Owens GK. (2001b). The smooth muscle myosin heavy chain gene exhibits smooth muscle subtype-selective modular regulation in vivo. J Biol Chem 276, 39076-39087.

Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR & Olson EN. (1998). A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215-228.

Nakano Y, Nishihara T, Sasayama S, Miwa T, Kamada S & Kakunaga T. (1991). Transcriptional regulatory elements in the 5' upstream and first intron regions of the human smooth muscle (aortic type) alpha-actin-encoding gene. Gene 99, 285-289.

Neal JW & Clipstone NA. (2001). Glycogen synthase kinase-3 inhibits the DNA binding activity of NFATc. J Biol Chem 276, 3666-3673.

Obata H, Hayashi K, Nishida W, Momiyama T, Uchida A, Ochi T & Sobue K. (1997). Smooth muscle cell phenotype-dependent transcriptional regulation of the alpha1 integrin gene. J Biol Chem 272, 26643-26651.

Ohkawa Y, Hayashi K & Sobue K. (2003). Calcineurin-mediated pathway involved in the differentiated phenotype of smooth muscle cells. Biochem Biophys Res Commun 301, 78-83.

Okamura H, Aramburu J, Garcia-Rodriguez C, Viola JP, Raghavan A, Tahiliani M, Zhang X, Qin J, Hogan PG & Rao A. (2000). Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity. Mol Cell 6, 539-550.

Owens GK. (1995). Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75, 487-517.

Owens GK. (1998). Molecular control of vascular smooth muscle cell differentiation. Acta Physiol Scand 164, 623-635. 115

Owens GK, Kumar MS & Wamhoff BR. (2004). Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84, 767-801.

Paradis P, MacLellan WR, Belaguli NS, Schwartz RJ & Schneider MD. (1996). Serum response factor mediates AP-1-dependent induction of the skeletal alpha-actin promoter in ventricular myocytes. J Biol Chem 271, 10827- 10833.

Pauletto P, Sarzani R, Rappelli A, Chiavegato A, Pessina AC & Sartore S. (1994). Differentiation and growth of vascular smooth muscle cells in experimental hypertension. Am J Hypertens 7, 661-674.

Ranger AM, Oukka M, Rengarajan J & Glimcher LH. (1998). Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development. Immunity 9, 627-635.

Rao A, Luo C & Hogan PG. (1997). Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15, 707-747.

Schulz RA & Yutzey KE. (2004). Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development. Dev Biol 266, 1-16.

Shimizu RT, Blank RS, Jervis R, Lawrenz-Smith SC & Owens GK. (1995). The smooth muscle alpha-actin gene promoter is differentially regulated in smooth muscle versus non-smooth muscle cells. J Biol Chem 270, 7631- 7643.

Shore P & Sharrocks AD. (1995). The MADS-box family of transcription factors. Eur J Biochem 229, 1-13.

Treisman R. (1995). Journey to the surface of the cell: Fos regulation and the SRE. Embo J 14, 4905-4913.

Tuil D, Clergue N, Montarras D, Pinset C, Kahn A & Phan-Dinh-Tuy F. (1990). CC Ar GG boxes, cis-acting elements with a dual specificity. Muscle- specific transcriptional activation and serum responsiveness. J Mol Biol 213, 677-686.

Wada H, Hasegawa K, Morimoto T, Kakita T, Yanazume T, Abe M & Sasayama S. (2002). Calcineurin-GATA-6 pathway is involved in smooth muscle- specific transcription. J Cell Biol 156, 983-991.

116

Wamhoff BR, Bowles DK, McDonald OG, Sinha S, Somlyo AP, Somlyo AV & Owens GK. (2004). L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism. Circ Res 95, 406-414.

Wilkins BJ, Dai YS, Bueno OF, Parsons SA, Xu J, Plank DM, Jones F, Kimball TR & Molkentin JD. (2004). Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy. Circ Res 94, 110- 118.

Wilkins BJ, De Windt LJ, Bueno OF, Braz JC, Glascock BJ, Kimball TF & Molkentin JD. (2002). Targeted disruption of NFATc3, but not NFATc4, reveals an intrinsic defect in calcineurin-mediated cardiac hypertrophic growth. Mol Cell Biol 22, 7603-7613.

Yoshida T, Sinha S, Dandre F, Wamhoff BR, Hoofnagle MH, Kremer BE, Wang DZ, Olson EN & Owens GK. (2003). Myocardin is a key regulator of CArG- dependent transcription of multiple smooth muscle marker genes. Circ Res 92, 856-864.

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CHAPTER 3: NFATC3 REGULATES BK CHANNEL FUNCTION IN

MURINE URINARY BLADDER SMOOTH MUSCLE

NFATc3 Regulates BK Channel Function in

Murine Urinary Bladder Smooth Muscle

Layne JJ, *Werner ME , Hill-Eubanks DC, and Nelson MT

Department of Pharmacology, College of Medicine, University of Vermont,

Burlington, Vermont 05405, USA

*Division of Cardiovascular and Endocrine Science, School of Medicine,

University of Manchester, Manchester, UK

Running Head: NFATc3 regulates K+ channel expression

Key Words: BK, KV, NFAT, contractility, urinary bladder

Correspondence: Mark T. Nelson, Ph.D.

Department of Pharmacology

College of Medicine, University of Vermont

Burlington, VT 05405

Tel: (802) 656-2500

Fax: (802) 656-4523

Email: [email protected]

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ABSTRACT

The nuclear factor of activated T-cells (NFAT) is a calcium (Ca2+)-dependent transcription factor that has been reported to regulate the expression of smooth muscle contractile proteins and ion channels. Here we report that large conductance Ca2+-sensitive potassium (K+) (BK) channels and voltage-gated K+

(KV) channels may be regulatory targets of NFATc3 in urinary bladder smooth muscle (UBSM). UBSM myocytes from NFATc3-null mice displayed a reduction in iberiotoxin (IBTX)-sensitive BK currents (IBK), a decrease in mRNA for the pore-forming α-subunit of the BK channel, and a reduction in the BK channel density compared to myocytes from wild-type mice. TEA-sensitive KV currents

(IKv) were elevated in UBSM myocytes from NFATc3-null mice, as was mRNA for the Shab family member, KV2.1. Despite the upregulation in KV current, bladder strips from NFATc3-null mice displayed an elevated contractile response to electrical field stimulation (EFS) relative to strips from wild-type mice, but this difference was abrogated in the presence of the BK channel blocker IBTX. These results support a role for the transcription factor NFATc3 in regulating UBSM contractility primarily through a downregulation of BK channel activity.

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INTRODUCTION

Urinary bladder smooth muscle (UBSM) exhibits spontaneous phasic

contractions that are coordinated by neuronal activity to generate the force

necessary to expel urine. These contractions are initiated by bursts of action

potentials that individually consist of a depolarizing upstroke followed by a

repolarizing phase and afterhyperpolarization (Brading, 1992; Hashitani et al.,

2001; Hashitani & Brading, 2003b). Calcium (Ca2+) influx through L-type voltage-

gated Ca2+ channels (L-VDCC) mediates the upstroke of the action potential

(Heppner et al., 1997; Buckner et al., 2002; Hashitani & Brading, 2003b).

Potassium (K+) efflux through two types of voltage-sensitive K+ channels, large-

2+ + + conductance Ca -sensitive K (BK) channels and voltage-gated K (KV), channels contributes to the repolarization phase of the action potential (Heppner et al., 1997; Hashitani & Brading, 2003b, a; Thorneloe & Nelson, 2003). In addition to their role in repolarization, KV channels, as well as small conductance,

Ca2+-sensitive K+ (SK) channels, likely play a role in the afterhyperpolarization

(Hashitani & Brading, 2003b; Thorneloe & Nelson, 2003).

BK channels in UBSM are composed of four pore-forming α-subunits and

accessory β1-subunits (Garcia-Calvo et al., 1994; Knaus et al., 1994; Thorneloe

& Nelson, 2005). The β1-subunit increases the apparent voltage- and Ca2+-

sensitivity of the BK α-subunits (Brenner et al., 2000; Cox & Aldrich, 2000;

Petkov et al., 2001). Block of BK channels by the scorpion toxins iberiotoxin

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(IBTX) or charybdotoxin (CTX) increases the duration and peak amplitude of

UBSM action potentials (Kaczorowski et al., 1996; Heppner et al., 1997),

resulting in an increase in the force of muscle contraction (Herrera et al., 2000;

Imai et al., 2001; Hashitani & Brading, 2003b, a). BK currents are absent in isolated UBSM myocytes from mice in which the pore-forming α-subunit of the

BK channel has been deleted (Slo-/-) (Meredith et al., 2004). These mice display an increase in the frequency and amplitude of spontaneous phasic contractions, as well as a significantly elevated contractile response to EFS (~5-fold increase over wild-type at 20 Hz stimulation) and to direct muscarinic stimulation with carbachol (Thorneloe et al., 2005; Werner et al., 2007) Slo-/- mice exhibit significantly elevated bladder pressures, an increase in the oscillations of the intravesicular pressure, reduced capacity, and a reduced volume per void

(Thorneloe et al., 2005), which suggests that a decrease in BK channel activity may contribute to the symptoms associated with urinary bladder dysfunction.

Like BK channels, KV channels are comprised of four α-subunits arranged

radially around a central pore (Jan & Jan, 1997; Yi et al., 2001; Yellen, 2002).

The pore-forming α-subunits are encoded by a large family of genes that can

combine to form channels with a broad range of biophysical and pharmacological

properties. KV α-subunits from the KV1 - 4 subfamilies can form functional homo-

or heteromultimeric complexes, while the electrically-silent KV family members

(KV 5-11) cannot form functional channels alone but, rather, co-assemble with

other KV family proteins to make ion channels with unique conductances (Post et

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al., 1996; Salinas et al., 1997a; Salinas et al., 1997b; Kramer et al., 1998;

Martens et al., 1999; Thorneloe & Nelson, 2003).

We have previously evaluated the biophysical, pharmacological, and molecular properties of the murine UBSM KV current (Thorneloe & Nelson, 2003).

The insensitivity of the mouse UBSM KV current to block by 4-aminopyridine (4-

AP) indicates that KV1 or KV4 subunits, which are known to be blocked by

submillimolar concentrations of 4-AP (Fiset et al., 1997; Nerbonne, 2000), do not

contribute to UBSM KV currents. However, the murine UBSM KV current is

blocked by tetraethylammonium chloride (TEA) with an IC50 of 5.2 mM, which is

consistent with the properties of KV2 and KV3 subunits (Nerbonne, 2000) . RT-

PCR analysis of UBSM tissue, combined with biophysical comparisons of the

murine UBSM KV current to values reported in the literature for other KV channel

complexes, suggests that the UBSM KV channel is most likely a heteromultimeric

assemblage of KV2.1 subunits combined with KV5.1 and/or KV6.1 subunits

(Thorneloe & Nelson, 2003). This stands in contrast to the KV currents of

vascular smooth muscle, which are carried primarily by channels composed of

members of the 4-AP-sensitive KV1 subfamily (Yuan et al., 1998; Thorneloe et al., 2001).

The activity of both BK and KV channels is subject to short term modulation

by protein kinase activity (Robertson et al., 1993; Schubert et al., 1996; Schubert

et al., 1999; Schubert & Nelson, 2001; Ledoux et al., 2006). However, it has also been recently reported that the expression of these channels can be modulated

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by Ca2+-dependent transcription factors, providing a link between K+ channel

expression and alterations in Ca2+ signaling patterns. Specifically, the

transcription factor NFAT (nuclear factor of activated T cells) has been reported

to regulate the expression of KV2.1 channels in vascular smooth muscle tissue

and in cardiac myocytes (Amberg et al., 2004; Rossow et al., 2004). In addition, it has been recently reported that NFAT also regulates the expression of the modulatory β1-subunit of the BK channel (Nieves-Cintron et al., 2006).

NFAT is a Ca2+-sensitive transcription factor that integrates Ca2+ signals with

the activity of other signaling pathways, converting the information encoded in

Ca2+ events into transcriptional responses (Rao et al., 1997; Crabtree, 1999;

Crabtree & Olson, 2002; Hill-Eubanks et al., 2003; Hogan et al., 2003). NFAT

activity is controlled primarily through regulation of its subcellular localization

(Flanagan et al., 1991; Crabtree & Clipstone, 1994; Zhu & McKeon, 2000). Under

non-stimulated conditions, the four Ca2+-sensitive members of the NFAT family

(NFATc1-c4) are cytosolic phosphoproteins. Elevations in intracellular Ca2+ promote the nuclear localization of NFAT by activating the Ca2+- and calmodulin- dependent protein phosphatase calcineurin, which dephosphorylates N-terminal

NFAT serine residues, inducing a conformational change that exposes nuclear localization signals (Clipstone & Crabtree, 1992; Jain et al., 1992; Beals et al.,

1997; Rao et al., 1997; Okamura et al., 2000; Crabtree & Olson, 2002). Once in

the nucleus, NFAT acts as a transcriptional co-regulator, associating with

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cofactors at composite regulatory elements to modulate the transcription of target

genes (Rao et al., 1997; Crabtree & Olson, 2002; Im & Rao, 2004).

K+ channel expression and activity is a crucial factor in determining the

contractile status of smooth muscle including UBSM. The fact that KV channels

are putative regulatory targets of NFAT would, in turn, imply that NFAT

transcriptional activity may modulate UBSM contractility. There are currently no

reports of NFAT activity in UBSM, although stimulation of the calcineurin/NFAT

pathway in UBSM myocytes by overexpressing a constitutively active form of

calcineurin induces a hypertrophic phenotype (Nozaki et al., 2003), suggesting a

possible role for NFAT activity in UBSM tissues.

The goal of the present study was to examine the role of NFAT in regulating

UBSM contractility. We have found that murine UBSM expresses several NFAT

isoforms (NFATc1, NFATc3, and NFATc4), and have chosen to focus on

NFATc3 since it has been previously reported to modulate the expression of K+

channels and α-actin in smooth muscle (Amberg et al., 2004; Gonzalez Bosc et al., 2005; Nieves-Cintron et al., 2006). UBSM strips from NFATc3-null mice exhibit increased force generation in response to EFS as compared to strips from wild-type mice. We attribute the increased contractility of the UBSM from

NFATc3-null mice to a downregulation in BK channel expression and activity, which would lead to increased excitability and contractility. Therefore, NFATc3 may play important role in the regulation of UBSM contractility through regulation of the expression of BK channels. Furthermore, these results suggest that UBSM

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disorders, which are characterized by alterations in Ca2+ regulation, might reflect

NFAT-mediated alterations in BK channel function.

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METHODS

Isolation of myocytes. Adult mice were euthanized with an overdose of

pentobarbital, followed by exsanguination according to a protocol approved by the University of Vermont Office of Animal Care and Management. NFATc3-null

mice, originally supplied by Dr. Laurie Glimcher, and the wild-type background

strain mice (Balb/C) were used in all experiments. The bladders were removed

and placed into an ice-cold Ca2+-free dissection solution (DS; 55 mM NaCl, 5.6

mM KCl, 2 mM MgCl2, 80 mM sodium glutamate, 10mM glucose; buffered to pH

7.3 with 10 mM Hepes). After the external fatty and connective tissues were

removed, the bladders were cut open, rinsed free of urine, and the urothelial

layer was carefully peeled away. The detrusor was cut into small sections

(approximately 2 mm x 2 mm) and digested for 20-30 min in DS containing 1

mg/mL Papain (Worthington, Lakewood, NJ, USA) with 1mg/mL dithioerythreitol

(Sigma, St. Louis, MO, USA), rinsed briefly with DS, and then further digested in

DS containing 1 mg/mL collagenase type II (Sigma, St. Louis, MO, USA) with

100 µm CaCl2 for 6-10 min. Both digestions were performed at 37°C. The

digested tissue sections were gently triturated using a fire-polished glass Pasteur

pipette to release individual UBSM myocytes.

Electrophysiology. Whole cell currents were recorded using the perforated- patch configuration of the whole-cell patch clamp technique. Aliquots of

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dissociated myocytes were transferred to a 0.5 mL electrophysiology chamber.

After 15-20 min, the cells were thoroughly rinsed in a bath solution consisting of

134 mM NaCl, 6 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM

Hepes, pH 7.4. The pipette solution consisted of 110 mM potassium aspartate,

10 mM NaCl, 30 mM KCl, 1 mM MgCl2, 0.05 mM EGTA, and 10 mM Hepes pH

7.2. Amphotericin, initially solubilized in DMSO, was added to the pipette solution

to a final concentration of 200 µg/mL. Iberiotoxin (IBTX; 100 nM final

concentration) and tetraethylammonium chloride (TEA; 5 mM final concentration)

were both purchased from Sigma and solubilized in the bath solution.

Single channel BK currents were recorded in excised inside-out patches

using the patch-clamp technique. The patches were exposed to a 10 µM buffered

Ca2+ solution. Single channel currents were recorded for to 2-5 min at potentials

of -40 to + 40 mV at 22° C using pipettes fire polished to a final tip resistance of approximately 4 - 6 MΩ. The bath and pipette solution consisted of 140 mM KCl,

1.8 mM MgCl2, 1 mM 5-hydroxyethylene-diaminetriacetic acid (HEDTA) and 0.13

mM CaCl2 (10 µM free), adjusted to pH 7.2 with NaOH. Data were analyzed using pCLAMP software (Axon Instruments). The number of channels per patch

was determined at a holding potential of + 60 mV. The open probability (Po) was

determined by dividing the NPo, derived using the pCLAMP software, by the

number of channels in the respective patch.

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UBSM contractility studies. Bladders were harvested and rinsed, and the urothelium was removed, as detailed above. Detrusor tissue strips (1 mm x 3 mm), cut longitudinally from the dome to the bladder base, were suspended in jacketed waters baths (7 ml volume, 37°C) in an aerated bath solution (119 mM

NaCl, 4.7 mM KCl, 24 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM

MgSO4, 0.023 mM EDTA, and 11 mM glucose). Contractility was measured after

pre-stretching strips to 5 mN of tension for 45 min using a myograph (MyoMED,

MED Associates, Inc.). The electrical field stimulation protocol consisted of

sequential pulses from 0.5 to 50 Hz (20 volt pulse amplitude, 0.2 ms pulse width,

2 sec pulse duration) at 3 minute intervals.

Immunohistochemistry and nuclear localization assay. Dissociated myocytes

were placed into 8-well plates and the cells were allowed to adhere for 1 hour at

37°C. The media was then replaced with growth media consisting of Dulbecco's

modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Invitrogen,

Carlsbad, CA, USA) and penicillin-streptomycin (10 units/mL, Invitrogen). The

plates were maintained at 37°C, 5% CO2 for 24 hours, at which time the cells

were approximately 60% confluent. Stimulation with platelet-derived growth

factor (PDGF; 20 ng/mL) or endothelin (ET-1; 100 nM), or pretreatment with

FK506 and CsA (1 µM each) was performed as detailed in the Results section.

Immunostaining and quantitation of NFAT nuclear localization was performed as

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detailed previously (Gonzalez Bosc et al., 2005). All NFAT antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Ca, USA).

Semi-quantitative RT-PCR. Approximately 100 mg sections of mouse urinary

bladder detrusor muscle, denuded of urothelium, were homogenized in TRIZOL

reagent (Sigma) using motorized homogenizer and the RNA was isolated as per

the manufacturer's protocol. The cDNA was generated using the Superscript

First-Strand cDNA kit (Invitrogen) according to the manufacturer's protocol.

Primers for KV2.1 (forward: 5'-TGGACAT CGTGGTGGAGAA-3', reverse: 5'-

CAGATACTCTGATCCCGAG-3') were described previously (Thorneloe and

Nelson, 2003). Primers for the α- and β-subunits of the BK channel (GenBank

accession numbers L16912 and AF020711, respectively) were as follows: α-BK

forward: 5'-AGTCTGCATCTTTGGGGATG-3', reverse: 5'-

TGTCCATTCCAGGAGGTGT-3'; β-BK forward: 5'-

TTCTGCACCTCAAGTCAACG-3', reverse: 5'-GACAGCGTCATTGCAGG TAA-

3'. RT-PCR was performed using the Fail-Safe PCR kit (Epicenter) with Amplitaq

+ Gold DNA polymerase. For semi-quantitative analysis of K channel expression,

PCR reactions were terminated at a point empirically determined to lie within the

logarithmic phase (33 cycles for KV2.1 and the α- and β-BK subunits) and expressed relative to the housekeeping gene, β-actin (23 cycles). Densitometry of bands in ethidium bromide-stained agarose gels was determined using the

Quantity One imaging analysis software (Bio-Rad).

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Statistical analyses. The summary data are presented as means ± S.E.M. unless indicated otherwise. Statistical significance was determined using

Student's t-test (unpaired or paired) with a P value less than 0.05 being considered statistically significant.

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RESULTS

Expression and stimulation of NFAT nuclear localization in urinary bladder smooth muscle. We have previously reported that NFATc3 and NFATc4 are expressed in cerebral vascular and ileal smooth muscle (Stevenson et al., 2001;

Gomez et al., 2002). In primary cultured UBSM myocytes, immunohistochemistry reveals expression of NFATc1, NFATc3, and NFATc4 (Figure 1A). Similarly, RT-

PCR analysis of fresh UBSM detrusor tissue indicates that NFATc1, NFATc3, and NFATc4 are present (Figure 1B). There was no evidence for NFATc2 expression by either method, a finding that is consistent with previously published reports on the absence of NFATc2 expression in native smooth muscle

(Stevenson et al., 2001; Gomez et al., 2002).

The smooth muscle mitogens PDGF and ET-1 are able to stimulate NFATc3 nuclear translocation in ileal and cerebral vascular smooth muscle tissues

(Stevenson et al., 2001; Gomez et al., 2002) and have been suggested to play a role in the physiology and pathophysiology of the urinary bladder (Saenz de

Tejada et al., 1992; Khan et al., 1999; Adam et al., 2003). To determine if these growth factors are able to activate NFATc3 in UBSM, we treated primary cultured

UBSM myocytes with either ET-1 (100 nM) or PDGF (20 ng/mL) for 30 min and quantified NFATc3 nuclear accumulation using confocal microscopy. Exposure to either ET-1 or PDGF led to a significant increase in NFATc3 nuclear accumulation (p < 0.05; 3.3 ± 0.6 and 3.4 ± 0.5 fold increase over media alone

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for ET-1 and PDGF, respectively; Figure 1C). ET-1- and PDGF-dependent

nuclear translocation was blocked by pre-incubation of the cells with the

calcineurin inhibitors FK506 and CsA (1 μM each). Thus, NFAT is expressed in

intact and cultured UBSM tissue and nuclear translocation of NFATc3 can be

induced by physiologically relevant agents.

UBSM strips from NFATc3-null mice exhibit enhanced contractility. We have

previously reported that the NFATc3 isoform is expressed in vascular and ileal

smooth muscle (Stevenson et al., 2001; Gomez et al., 2002) and regulates the

expression of the contractile protein smooth muscle α-actin (Gonzalez Bosc et

al., 2005). NFATc3 has also been implicated in the regulation of both BK and KV

channels in vascular smooth muscle (Amberg et al., 2004; Nieves-Cintron et al.,

2006), suggesting that the NFATc3 isoform may be an important factor in the regulation of smooth muscle function. To determine if NFATc3 modulates the contractility of UBSM, we compared the force generated by UBSM strips taken from NFATc3-null and wild-type mice in response to EFS.

Representative myograph recordings obtained from UBSM strips from wild- type and NFATc3-null mice are presented in Figure 2A, and the averaged force- frequency plot is presented in Figure 2B. UBSM strips from NFATc3-null mice generated more force than strips from wild-type mice at all stimulation frequencies greater than 5 Hz (n = 16 strips from 3 animals each; p < 0.05). At 20

Hz, the mean force generated by NFATc3-null UBSM strips was approximately

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1.8-fold that of strips from wild-type animals. Direct membrane potential

depolarization of UBSM strips with 60 mM K+ in the bath did not result in a

statistically significant difference in the contractile response between wild-type

and NFATc3-null animals (p > 0.05; 10.6 ± 2.9 mN for wild-type and 8.5 ± 1.5 mN

for NFATc3-null mice; n = 13), suggesting that the contractile response to

membrane potential depolarization and activation of L-type voltage dependent

Ca2+ channels (L-VDCC) are unchanged in NFATc3-null mice. The enhanced contractile response to EFS of UBSM strips taken from NFATc3-null mice

suggests that the NFATc3 isoform contributes to the regulation of detrusor

smooth muscle contractility, but not directly through L-VDCCs.

UBSM strips from NFATc3-null mice exhibit reduced sensitivity to the BK

channel blocker, IBTX. Muscle contractions are dependent upon the

concentration of cytosolic Ca2+.The overall contractility of UBSM tissues will be

determined by the net contribution of factors that tend to increase Ca2+ influx,

which is mediated principally by L-VDCCs, and those factors that hyperpolarize

the cell membrane. In UBSM, much of this hyperpolarizing current is due to K+

current through BK and KV channels (Heppner et al., 1997; Thorneloe & Nelson,

2003). Therefore, we focused on identifying potential changes in the activity and/or expression of BK and KV channels as factors mediating the enhanced

contractility of the NFATc3-null UBSM strips.

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BK channels play a central role in the repolarization of the UBSM action

potential and have been shown to figure prominently in the contractility of the

bladder detrusor (Heppner et al., 1997). Blocking BK channels with the scorpion

venom iberiotoxin (IBTX) increases the contractility of UBSM strips by shifting the

force-frequency relationship to the left and by increasing the maximum force

generated in response to muscarinic or purinergic stimulation, or to EFS

(Heppner et al., 1997; Herrera et al., 2001; Petkov et al., 2001; Thorneloe et al.,

2005; Werner et al., 2006). To determine whether a decrease in BK channel

activity might account for the increased contractility of bladder strips from

NFATc3-null mice, we evaluated the effects of IBTX on the EFS-induced

contractions of UBSM strips from NFATc3-null and wild-type mice.

Representative recordings obtained before and after the application of IBTX are presented in Figure 3A. IBTX increased the amplitude of EFS-induced contractions in UBSM strips from both wild-type and NFATc3-null mice. However,

IBTX treatment has a greater effect on the contractile response of UBSM strips taken from wild-type mice than on strips from NFATc3-null mice. For example, at

5 Hz the contraction amplitude in the wild-type UBSM strips was increased 2.2 ±

0.07 fold by treatment with IBTX, but was increased only 1.5 ± 0.02 fold in the

NFATc3-null mice (n = 6; p < 0.05). The decreased responsiveness of NFATc3- null UBSM strips to IBTX treatment suggests that BK channel activity is downregulated in these animals, which may account for the increase contractility previously noted in UBSM strips from NFATc3-null mice. In support of this, the

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force-frequency relationships of the NFATc3-null and wild-type mice are not

significantly different when BK channels are blocked with IBTX (Figure 3B;

compare with Figure 2B). These results indicate that the enhanced contractile

response of UBSM strips from NFATc3-null mice is due to a reduction in BK

channel activity.

BK currents are reduced in UBSM myocytes from NFATc3-null mice. UBSM

strips from NFATc3-null mice displayed a reduced sensitivity to IBTX, which we

interpreted as reflecting a decrease in the activity or expression of BK channels.

To explore this possibility, we compared whole cell currents elicited by 10 mV

voltage steps from -70 to + 70 mV from myocytes obtained from NFATc3-null and

wild-type mice. Currents were recorded before and after the application of IBTX

+ to selectively block BK channels. An evaluation of the total K currents (IK) recorded before the application of IBTX revealed no significant difference in either end-pulse or tail currents between the myocytes isolated from wild-type or

NFATc3-null mice. Representative recordings are presented in Figure 4A and averaged I-V plots for the end-pulse and tail currents are presented in Figure 4B.

Although there was no significant difference in the total K+ currents, the BK

current, i.e. IBTX-sensitive current, was significantly reduced in UBSM cells from

NFATc3-null mice (representative recordings in Figures 5A & B). As shown in

Figure 5C, the IBK was significantly lower in UBSM myocytes from NFATc3-null animals at all membrane potentials more positive than -20 mV (p < 0.05). At +40

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mV, the IBK was 4.1 ± 0.8 pA/pF (n = 9) for wild-type myocytes, but was only 1.2

± 0.6 pA/pF (n = 10) for NFATc3-null myocytes, a ~70% reduction in IBK. Thus,

+ despite the similarity in total K currents (IK), BK currents are reduced in

myocytes isolated from NFATc3-null mice. The downregulation of BK currents in

UBSM myocytes from NFATc3-null mice is consistent with the contractility data

and supports the hypothesis that the enhanced contractile response of UBSM

strips taken from these mice is due to a reduction of BK channel activity.

BK channel expression is downregulated in NFATc3-null mice. The UBSM

BK channel is composed of a pore-forming α-subunit and a smooth muscle- specific regulatory β1-subunit, the presence of which increases the apparent sensitivity of the BK channel to internal Ca2+ levels and voltage (Brenner et al.,

2000; Cox & Aldrich, 2000; Petkov et al., 2001). It is possible that expression of both the pore-forming α-subunit and the modulatory β1-subunits are altered in the NFATc3-null mice. Therefore, we used semi-quantitative RT-PCR to quantify the expression levels of mRNA for both the BK α- and BK β1-subunits

(representative agarose gel in Figure 6A). There was no significant difference in the expression of mRNA for the β1-subunit between wild-type and NFATc3-null mice (p>0.05; Figure 6B). However, the expression of mRNA for the pore-forming

α-subunit was 26% lower in UBSM from NFATc3-null mice than in wild-type mice

(α-subunit : β1-subunit ratio of 0.99 ± 0.04 in wild-type mice vs. 0.71 ± 0.02 in

NFATc3-null mice p<0.05; n = 3; Figure 6C). Thus, the reduction in BK currents

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in UBSM myocytes from NFATc3-null mice is supported at the molecular level by

a decrease in the expression of the pore-forming α-subunit of the BK channel.

A reduction in the expression of the pore-forming α-subunit of the BK

channel should not affect the channel open probability (Po), but should lead to a

reduction in the channel density, i.e. there should be fewer channels per patch. In

contrast, a loss of β1-subunit expression should dramatically lower the single

channel Po (Brenner et al., 2000). To examine this possibility, we evaluated

single BK channel currents measured in excised inside-out patches to determine

if there was any difference in the open probabilities of BK channels isolated from

wild-type or NFATc3-null UBSM myocytes (Figure 7A). All recordings were made in symmetrical 140 mM K+ and with intracellular 10 µM Ca2+. There was no

significant difference in the Po at negative (-40 mV) or positive (+ 40 mV)

membrane potentials (Figure 7B; p>0.05; n = 9). However, the BK channel

density was significantly lower (Figure 7C; p<0.05; n = 9) in the excised patches

isolated from NFATc3-null myocytes (1.3 ± 0.7 channels per patch) than for the

wild-type UBSM myocytes (2.6 ± 1.3 channels per patch). These data indicate

that the reduction in the whole-cell IBK noted previously (Figure 5C) is due to a

downregulation in BK channel density.

UBSM myocytes isolated from NFATc3-null mice have increased KV currents. Our observation that the total IK in wild-type and NFATc3-null mice was similar (Figure 4B), despite a significant decrease in IBK in the NFATc3-null mice

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+ (Figure 5C), was initially puzzling. However, the total outward K current (IK) in

UBSM is comprised of currents through multiple families of K+ channels

(Heppner et al., 1997; Imai et al., 2001; Buckner et al., 2002; Herrera & Nelson,

2002), so it was conceivable that an upregulation in another class of K+ channels

might compensate for the reduction in BK current in the NFATc3-null mice. While

our findings thus far suggest that BK channels may be a target of NFATc3 transcriptional regulation, there are reports that NFATc3 may be a transcriptional

regulator of KV channel expression (Amberg et al., 2004; Rossow et al., 2004).

Thus, we investigated the possibility that an upregulation of KV channel activity in

UBSM myocytes from NFATc3-null mice might compensate for the decrease in

IBK and account for the observed similarities in total IK.

The wild-type murine UBSM KV current is insensitive to block by 4-AP, but is

subject to block by TEA with an IC50 of 5.2mM (Thorneloe & Nelson, 2003). Thus,

to determine if the KV component (IKV) of IK was altered in UBSM myocytes from

NFATc3-null mice, we evaluated whole-cell currents elicited by 10 mV membrane

potential steps before and after the application of 5 mM TEA (representative

voltage steps in Figures 8A and B for wild-type and NFATc3-null myocytes,

respectively). All recordings were also made in the presence of IBTX to block BK

channels. As shown in the Figure 8C, the TEA-sensitive current (IKV), defined as

the current blocked by the addition of 5 mM TEA in the presence of IBTX, is

significantly larger in the NFATc3-null UBSM myocytes at all membrane

potentials more positive than -20 mV (p < 0.05; Fig. 4B). At +40 mV, the IKV was

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5.40 ± 1.31 pA/pF for the NFATc3-null mice and only 2.18 ± 0.32 pA/pF for the

wild-type myocytes (n = 7). These results indicate that the similarity in total K+ currents (IK) between UBSM myocytes from NFATc3-null and wild-type mice,

despite the significantly reduced BK current (IBK) in the NFATc3-null myocytes, is

likely due to a compensatory upregulation in TEA-sensitive KV currents (IKV).

Biophysical and molecular evidence for increased KV2.1 expression in

NFATc3-null mice. The wild-type mouse UBSM KV channel is most likely a

heteromultimeric assemblage composed of pore-forming KV2.1 subunits

combined with KV5.1 and/or KV6 subunits (Thorneloe & Nelson, 2003). The

simplest explanation for the observed increase in KV current in the NFATc3-null

mice would be an upregulation of the native KV channel complexes, without any

change in subunit composition. However, it is also possible that the changes in

KV activity may be due to alterations in the subunit composition of the channels.

To distinguish between these two possibilities, we compared some of the

biophysical and pharmacological properties of the KV currents of isolated myocytes of wild-type and NFATc3-null mice. There was no significant difference in the steady-state activation curves of currents from myocytes isolated from

NFATc3-null or wild-type mice (Figure 9A). The slope factors (k) of the activation curves for the wild-type (n = 5) and NFATc3-null mice (n = 7) were 16.31 ± 3.3 and 17.6 ± 4.1, respectively (p > 0.05). Similarly, comparison of the activation time constants (Figure 9B) also revealed no significant difference between UBSM

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myocytes from NFATc3-null and wild-type mice (p < 0.05). In addition, neither the

wild-type nor the NFATc3-null UBSM tissues strips responded to treatment with

4-aminopyridine (4-AP; data not shown), indicating that a change in the

expression of 4-AP sensitive KV family members (KV1 and KV4), did not

contribute to the difference between the wild-type and NFATc3-null responses.

Thus, the biophysical similarities and the lack of responsiveness to 4-AP suggest

that the upregulation in KV current in the NFATc3-null myocytes is likely due to

increased expression of the native channel complex rather than the expression of

an alternative channel assemblage.

To determine if the upregulation in KV current was supported at the molecular

level, we performed semi-quantitative RT-PCR to compare the levels of mRNA

for KV2.1, which has previously been reported to be regulated by NFATc3

(Amberg et al., 2004; Rossow et al., 2004), in UBSM from wild-type and NFATc3-

null mice (representative ethidium bromide-stained agarose gel in Figure 9C).

Consistent with the increase in KV current, UBSM from NFATc3-null mice exhibited a 40% increase in the expression of mRNA for KV2.1 relative to that

from the wild-type mice (p < 0.05; n = 3 animals each; Figure 9D). Thus, these

data suggest that the similarity in total IK between NFATc3-null and wild-type

mice, despite a downregulated IBK in the NFATc3-null mice, is due to a compensatory upregulation in IKV, supported at the molecular level by an increase in the expression of mRNA for KV2.1

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DISCUSSION

In this study, we have found that NFATc3 plays a role in the regulation of

UBSM contractility by regulating the expression of BK and KV channels. UBSM

tissue strips from NFATc3-null mice generate more force in response to EFS

compared to strips from wild-type mice. We attribute this enhanced contractility to

a reduction in BK channel activity. This interpretation is supported at the

molecular level by a reduction in BK α-subunit expression in the NFATc3-null

UBSM myocytes, and at the electrophysiological level by a significant reduction

+ in BK current (IBK) and in the BK channel density. However, the total K current

(IK) is similar in both NFATc3-null and wild-type mice, an observation that can be

explained by a compensatory upregulation in KV current (IKV) in the NFATc3-null

mice that is supported at the molecular level by an increase in the expression of

mRNA for the KV2.1 subunit, a major component of the mouse UBSM KV channel complex.

Our observation that the net contractility in the NFATc3-null mice is increased despite the presence of an enhanced KV current suggests that, at least

in response to EFS, the downregulation of BK currents plays a more significant

role than the upregulation of KV currents. Presumably, this is due to the prominent role of the BK channel in the repolarization phase of the action potential (Heppner et al., 1997; Herrera et al., 2000; Herrera & Nelson, 2002), where a small decrease in BK channel activity could lead to a relatively large

142

increase in force. Furthermore, the observation that the force-frequency

relationships in UBSM strips from NFATc3-null and WT mice were identical in the

presence of the BK channel blocker IBTX suggests that modulations of other

channel types (e.g. KV) do not impact EFS-induced contractions as significantly

as do the change in BK channel activity. Although it is conceivable that changes

in the activity or expression of voltage-dependent Ca2+ channels (L-VDCC) in the

NFATc3-null mice could lead to an increase in Ca2+ influx and, hence, greater

contractile response, we did not find a significant difference in the amplitude of

the contractile response to K+ (60 mM)-mediated depolarization between UBSM

muscle strips isolated from NFATc3-null and wild-type mice, suggesting that L-

VDCC expression was unchanged.

Several families of K+ channels are expressed in UBSM including

+ 2+ + voltage-dependent K channels (KV), large-conductance Ca -activated K channels (BK), small-conductance Ca2+-activated K+ channels (SK), and ATP-

+ dependent K channels (KATP). In the absence of a channel opener, KATP channels contribute little, if anything, to UBSM contractility and it has been shown that glibenclamide does not have a significant effect on UBSM contractile activity

(Imai et al., 2001). Furthermore, KATP channel activity is independent of voltage,

and therefore would not contribute to the voltage-dependent K+ current. In contrast, blocking BK, SK, or KV channels leads to significant changes in phasic

contraction amplitude and/or frequency (Heppner et al., 1997; Herrera & Nelson,

2002; Hashitani & Brading, 2003a, b). The absence of a significant difference in

143

the total IK between the NFATc3-null and wild-type mice seems to be explained by the reciprocal changes in BK and KV channel expression and activity that we

have described. Quantitatively, our data would support this hypothesis given that,

at +40 mV, the BK current was reduced by ~3 pA/pF, while the KV current was elevated by approximately the same amount. However, we cannot rule out the possibility that alterations to SK channel expression or activity, which we did not examine, may have contributed as well, as these channels have been previously demonstrated to also play a role in the contractility of UBSM tissues (Creed et al.,

1983; Herrera & Nelson, 2002; Hashitani & Brading, 2003b; Herrera et al., 2005).

Our observation that KV currents and KV2.1 channel expression are

upregulated in UBSM from NFATc3-null mice is consistent with previous reports.

In rats, sustained administration of angiotensin II (ANG II) leads to a reduction in

TEA-sensitive KV currents and in mRNA for KV2.1 in cerebral arteries (Amberg et

al., 2004). This downregulation is blocked by the calcineurin/NFAT inhibitor

cyclosporine A (CsA), and is also abrogated in NFATc3-null mice. Similarly,

Santana and colleagues (Rossow et al., 2004) found that after surgically-induced

myocardial infarction (MI), TEA-sensitive KV currents and KV2.1 mRNA levels are reduced in cardiac tissue from mice. Administration of CsA prevented the reduction in KV currents post-MI, NFATc3-null mice did not exhibit the decrease

in KV currents post-MI, and the expression of a constitutively active form of

NFATc3 recapitulated these changes in KV current in the absence of MI. Thus,

these previous reports strongly suggest that NFATc3 mediates the

144

downregulation of KV2.1 channel expression and activity, similar to what we have reported in this current study.

We have found evidence for the downregulation of basal BK channel

expression and activity in UBSM from NFATc3-null mice at multiple levels,

including a reduction in BK α-subunit expression and channel density, a

reduction in IBTX-sensitive BK current, and a reduced responsiveness of UBSM

muscle strips to IBTX treatment. Recently, Santana and colleagues (Nieves-

Cintron et al., 2006) have reported that NFATc3 downregulates the expression of

the β1-subunit of the BK channel in the arterial smooth muscle. In our current

work, we found no evidence at the molecular level for a downregulation of β1- subunit expression in the NFATc3-null urinary bladders. This may reflect tissue- specific differences in NFATc3 regulatory patterns.

Sequence analysis of putative 5' upstream regulatory regions of the KV2.1 gene from mouse, human, and rat genomes reveals 15 potential NFAT consensus binding sites (-GGAAA-) within 3600 base pairs of the transcriptional initiation site. Analysis of the promoter regions for the human and mouse BK channel α-subunit reveals eight potential NFAT binding sites within 2600 base pairs of the initiation site, two of which are conserved in both the human and mouse genome. Thus, both the KV2.1 and the BK α-subunit genes could

potentially be targets of NFAT regulation. In theory, NFATc3 might directly

regulate the expression of both genes. However, this possibility seems unlikely

since the opposing influences of these two transcriptional events create an

145

outcome that is physiologically contradictory. More likely, NFATc3 is directly

involved in regulating either KV2.1 or the BK α-subunit, but not both. In this scenario, the complimentary change in the non-NFATc3 regulated gene might constitute an example of developmental compensation that would serve to maintain membrane potential within a normal physiological range. Although it has been previously proposed that NFATc3 may directly downregulate KV2.1

(Amberg et al., 2004; Rossow et al., 2004), neither our data nor the results of

these previous studies definitively rule out the possibility that the changes in

KV2.1 expression are an indirect consequence of NFATc3 action on an

alternative target. Additional experimental approaches - including the use of

promoter-reporter constructs or chromatin binding assays to establish functional

significance of putative NFAT binding sites, or temporally controlled disruptions

of NFATc3 using siRNA/antisense or dominant negative approaches to

determine immediate and secondary consequences of NFAT inhibition - will be

required to deconvolute the underlying molecular events.

In conclusion, our data indicates that that the transcription factor NFATc3

plays a role in regulating the contractility of UBSM tissues through the regulation

of the expression of BK and KV channels. We have found that the NFATc1, c3,

and c4 isoforms are expressed in UBSM tissues and nuclear translocation of

NFATc3 is stimulated by physiologically relevant mitogens (PDGF and ET-1).

UBSM tissue strips from NFATc3-null mice are more contractile than strips from

wild-type mice, and this enhanced contractility is attributable to a decrease in the

146

expression and activity of BK channels. The total K+ currents in NFATc3-null and wild-type mice are statistically similar, an observation due, at least in part, to a concomitant upregulation in KV current and KV2.1 channel expression. It is unclear presently if the BK channel or/and the KV2.1 channel are both direct targets of NFATc3 regulation, or if the expression of one is directly regulated by

NFATc3 activity and the reciprocal change in the other channel is a compensatory event.

Acknowledgements. We would like to gratefully thank Dr. Adrian Bonev for his advice and guidance on the single-channel recordings and Dr. Laurie Glimcher for initially providing the NFATc3-null mice used in these experiments. We also thank Dr. Stephen Straub for his insightful and helpful discussions. This research was funded by Training Program in the Molecular Basis of Cardiovascular

Disease NIH T32 HL007944 and grants from the NIH (DK-5R01DK053832 and

1RO1DK065947).

147

Figure 1. NFAT expression in murine UBSM cultured cells and tissue. A.

Immunostaining of primary cultures of UBSM myocytes indicates that murine

UBSM expresses the NFATc1, NFATc3, and NFATc4 isoforms. NFAT

expression is shown in red, nuclear staining in green, and regions of NFAT

nuclear localization in white. Some nuclei exhibit NFATc3 nuclear localization

under these basal conditions. B. RNA extracted from UBSM tissues was probed

for presence of mRNA for NFATc1-c4 using RT-PCR. RNA isolated from thymus

and heart tissue was used as a positive control. NFATc2 was not detected by

either method. C. The extent of NFAT nuclear localization in response to

stimulation with ET-1 or PDGF was determined using confocal microscopy.

Treatment with either mitogen lead to a significant increase in NFATc3 nuclear translocation as compared to controls (*p < 0.05; c = number of cells analyzed).

Treatment with FK506 and CsA (1 µM each) prior to the addition of the PDGF or

ET-1 blocked NFATc3 nuclear translocation.

148

149

Figure 2. UBSM strips from NFATc3-null mice are more contractile than

strips from wild-type mice. A. Representative myograph recordings obtained

from UBSM tissue strips from wild-type and NFATc3-null mice subject to

electrical field stimulation from 1- 50 Hz. B. UBSM tissues strips from NFATc3- null mice exhibited a greater contractile response than UBSM strips from wild- type mice at all frequencies of stimulation greater than 5 Hz (*p < 0.05; n = 3 animals and 16 strips each).

150

151

Figure 3. UBSM strips from NFATc3-null mice display a reduced responsiveness to IBTX. A. UBSM strips from wild-type and NFATc3-null mice were stimulated with EFS (1 - 50 Hz), exposed to IBTX (100 nM), and then re- stimulated with EFS. B. In the presence of IBTX, there is no significant difference

(p>0.05) in the amplitude of EFS-induced contractions of UBSM strips from wild- type and NFATc3-null mice (compare with Figure 2B).

152

153

Figure 4. UBSM myocytes from wild-type and NFATc3-null mice have

+ similar total K currents (IK). A. Representative whole-cell current recordings elicited by 10 mV membrane potential steps from freshly isolated UBSM myocytes from wild-type and NFATc3-null mice. B. Analyses of the resulting end- pulse and tail currents reveals no significant difference between the myocytes isolated from wild-type () and NFATc3-null mice ({) (p > 0.05).

154

155

Figure 5. UBSM myocytes from NFATc3-null mice have reduced BK

currents (IBK). A and B. Representative whole-cell current recordings elicited by

10 mV membrane potential steps from freshly isolated UBSM myocytes from

wild-type (A) and NFATc3-null mice (B) before and after the addition of IBTX to

block BK channels. C. UBSM myocytes obtained from NFATc3-null mice ({) display reduced IBTX-sensitive BK currents (IBK) as compared to myocytes

obtained from control mice () (*p < 0.05).

156

157

Figure 6. Reduced expression of the BK α-subunit in UBSM from NFATc3-

null mice. A. Representative electrophoresis gel of semi-quantitative RT-PCR

analysis of RNA isolated from wild-type and NFATc3-null mice probed for β-actin,

BK α-subunit, and BK β-subunits. B. There was no difference in the expression

of mRNA for the β1-subunit (relative to β-actin) in UBSM from wild-type and

NFATc3-null mice. C. UBSM myocytes isolated from NFATc3-null mice express significantly lower levels of the pore-forming BK α-subunit (relative to β1-subunit levels) (*p < 0.05; n = 3 animals each).

158

159

Figure 7. Single channel recordings and BK channel density from wild-type

and NFATc3-null myocytes. A. Representative single channel recordings from

excised patches held at -40 and +40 mV. Arrows indicate closed state. B. There

was no significant difference in open probabilities (Po) of excised BK channels

isolated from wild-type or NFATc3-null UBSM myocytes. C. Current density is

significantly reduced in UBSM myocytes from NFATc3-null mice (p<0.05; n = 9; error bars represent means ± S.D.).

160

161

Figure 8. UBSM myocytes from NFATc3-null mice have elevated KV currents (IKV). A and B. Representative whole-cell current recordings elicited by

10 mV membrane potential steps from freshly isolated UBSM myocytes from wild-type (A) and NFATc3-null mice (B) before and after the addition of TEA to block KV channels. All cells were also pretreated with IBTX to block BK channels.

C. Analysis of the averaged difference currents from wild-type () and NFATc3- null mice ({) after treatment with 5 mM TEA indicates that UBSM myocytes from

NFATc3-null mice display significantly elevated TEA-sensitive KV currents (IKV)

(*p < 0.05).

162

163

Figure 9. Molecular and electrophysiological evidence for elevated KV2.1 expression in NFATc3-null mice. A. There was no significant difference in the steady-state activation curves between UBSM myocytes obtained from wild-type and NFATc3-null mice (p > 0.05), or in the activation time constants (p < 0.05; mean ± S.E.M.) (B). C. UBSM tissue from wild-type type and NFATc3-null mice was probed for expression of mRNA for KV2.1 and β-actin. D. Comparison of the

averaged ratios of KV2.1:β-actin in UBSM tissues indicates that the UBSM from

NFATc3-null mice exhibit significantly more mRNA for KV2.1 than do UBSM

tissues from wild-type mice (D) (*p < 0.05; n = 3 animals each).

164

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References

Adam RM, Roth JA, Cheng HL, Rice DC, Khoury J, Bauer SB, Peters CA & Freeman MR. (2003). Signaling through PI3K/Akt mediates stretch and PDGF-BB-dependent DNA synthesis in bladder smooth muscle cells. J Urol 169, 2388-2393.

Amberg GC, Rossow CF, Navedo MF & Santana LF. (2004). NFATc3 regulates Kv2.1 expression in arterial smooth muscle. J Biol Chem 279, 47326- 47334.

Beals CR, Clipstone NA, Ho SN & Crabtree GR. (1997). Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction. Genes Dev 11, 824-834.

Brading AF. (1992). Ion channels and control of contractile activity in urinary bladder smooth muscle. Jpn J Pharmacol 58 Suppl 2, 120P-127P.

Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT & Aldrich RW. (2000). Vasoregulation by the beta1 subunit of the calcium-activated potassium channel. Nature 407, 870-876.

Buckner SA, Milicic I, Daza AV, Coghlan MJ & Gopalakrishnan M. (2002). Spontaneous phasic activity of the pig urinary bladder smooth muscle: characteristics and sensitivity to potassium channel modulators. Br J Pharmacol 135, 639-648.

Clipstone NA & Crabtree GR. (1992). Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357, 695-697.

Cox DH & Aldrich RW. (2000). Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity. J Gen Physiol 116, 411-432.

Crabtree GR. (1999). Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96, 611-614.

Crabtree GR & Clipstone NA. (1994). Signal transmission between the plasma membrane and nucleus of T lymphocytes. Annu Rev Biochem 63, 1045- 1083.

Crabtree GR & Olson EN. (2002). NFAT signaling: choreographing the social lives of cells. Cell 109 Suppl, S67-79. 166

Creed KE, Ishikawa S & Ito Y. (1983). Electrical and mechanical activity recorded from rabbit urinary bladder in response to nerve stimulation. J Physiol 338, 149-164.

Fiset C, Clark RB, Shimoni Y & Giles WR. (1997). Shal-type channels contribute to the Ca2+-independent transient outward K+ current in rat ventricle. J Physiol 500 ( Pt 1), 51-64.

Flanagan WM, Corthesy B, Bram RJ & Crabtree GR. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352, 803-807.

Garcia-Calvo M, Knaus HG, McManus OB, Giangiacomo KM, Kaczorowski GJ & Garcia ML. (1994). Purification and reconstitution of the high-conductance, calcium-activated potassium channel from tracheal smooth muscle. J Biol Chem 269, 676-682.

Gomez MF, Stevenson AS, Bonev AD, Hill-Eubanks DC & Nelson MT. (2002). Opposing actions of inositol 1,4,5-trisphosphate and ryanodine receptors on nuclear factor of activated T-cells regulation in smooth muscle. J Biol Chem 277, 37756-37764.

Gonzalez Bosc LV, Layne JJ, Nelson MT & Hill-Eubanks DC. (2005). Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an alpha-actin intronic enhancer. J Biol Chem 280, 26113-26120.

Hashitani H & Brading AF. (2003a). Electrical properties of detrusor smooth muscles from the pig and human urinary bladder. Br J Pharmacol 140, 146-158.

Hashitani H & Brading AF. (2003b). Ionic basis for the regulation of spontaneous excitation in detrusor smooth muscle cells of the guinea-pig urinary bladder. Br J Pharmacol 140, 159-169.

Hashitani H, Fukuta H, Takano H, Klemm MF & Suzuki H. (2001). Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder. J Physiol 530, 273-286.

Heppner TJ, Bonev AD & Nelson MT. (1997). Ca(2+)-activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle. Am J Physiol 273, C110-117.

167

Herrera GM, Etherton B, Nausch B & Nelson MT. (2005). Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle. Am J Physiol Regul Integr Comp Physiol 289, R402-R409.

Herrera GM, Heppner TJ & Nelson MT. (2000). Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. Am J Physiol Regul Integr Comp Physiol 279, R60-68.

Herrera GM, Heppner TJ & Nelson MT. (2001). Voltage dependence of the coupling of Ca(2+) sparks to BK(Ca) channels in urinary bladder smooth muscle. Am J Physiol Cell Physiol 280, C481-490.

Herrera GM & Nelson MT. (2002). Differential regulation of SK and BK channels by Ca(2+) signals from Ca(2+) channels and ryanodine receptors in guinea-pig urinary bladder myocytes. J Physiol 541, 483-492.

Hill-Eubanks DC, Gomez MF, Stevenson AS & Nelson MT. (2003). NFAT regulation in smooth muscle. Trends Cardiovasc Med 13, 56-62.

Hogan PG, Chen L, Nardone J & Rao A. (2003). Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17, 2205-2232.

Im SH & Rao A. (2004). Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling. Mol Cells 18, 1-9.

Imai T, Okamoto T, Yamamoto Y, Tanaka H, Koike K, Shigenobu K & Tanaka Y. (2001). Effects of different types of K+ channel modulators on the spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle. Acta Physiol Scand 173, 323-333.

Jain J, McCaffrey PG, Valge-Archer VE & Rao A. (1992). Nuclear factor of activated T cells contains Fos and Jun. Nature 356, 801-804.

Jan LY & Jan YN. (1997). Voltage-gated and inwardly rectifying potassium channels. J Physiol 505 ( Pt 2), 267-282.

Kaczorowski GJ, Knaus HG, Leonard RJ, McManus OB & Garcia ML. (1996). High-conductance calcium-activated potassium channels; structure, pharmacology, and function. J Bioenerg Biomembr 28, 255-267.

Khan MA, Dashwood MR, Thompson CS, Mumtaz FH, Mikhailidis DP & Morgan RJ. (1999). Up-regulation of endothelin (ET(A) and ET(B)) receptors and

168

down-regulation of nitric oxide synthase in the detrusor of a rabbit model of partial bladder outlet obstruction. Urol Res 27, 445-453.

Knaus HG, Garcia-Calvo M, Kaczorowski GJ & Garcia ML. (1994). Subunit composition of the high conductance calcium-activated potassium channel from smooth muscle, a representative of the mSlo and slowpoke family of potassium channels. J Biol Chem 269, 3921-3924.

Kramer JW, Post MA, Brown AM & Kirsch GE. (1998). Modulation of potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1 alpha-subunits. Am J Physiol 274, C1501-1510.

Ledoux J, Werner ME, Brayden JE & Nelson MT. (2006). Calcium-activated potassium channels and the regulation of vascular tone. Physiology (Bethesda) 21, 69-78.

Martens JR, Kwak YG & Tamkun MM. (1999). Modulation of Kv channel alpha/beta subunit interactions. Trends Cardiovasc Med 9, 253-258.

Meredith AL, Thorneloe KS, Werner ME, Nelson MT & Aldrich RW. (2004). Overactive bladder and incontinence in the absence of the BK large conductance Ca2+-activated K+ channel. J Biol Chem 279, 36746-36752.

Nerbonne JM. (2000). Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol 525 Pt 2, 285-298.

Nieves-Cintron M, Amberg GC, Nichols CB, Molkentin JD & Santana LF. (2006). Activation of NFATC3 down-regulates the beta 1 subunit of large conductance, calcium-activated K+ channels in arterial smooth muscle and contributes to hypertension. J Biol Chem.

Nozaki K, Tomizawa K, Yokoyama T, Kumon H & Matsui H. (2003). Calcineurin mediates bladder smooth muscle hypertrophy after bladder outlet obstruction. J Urol 170, 2077-2081.

Petkov GV, Bonev AD, Heppner TJ, Brenner R, Aldrich RW & Nelson MT. (2001). Beta1-subunit of the Ca2+-activated K+ channel regulates

169

contractile activity of mouse urinary bladder smooth muscle. J Physiol 537, 443-452.

Post MA, Kirsch GE & Brown AM. (1996). Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current. FEBS Lett 399, 177-182.

Rao A, Luo C & Hogan PG. (1997). Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15, 707-747.

Robertson BE, Schubert R, Hescheler J & Nelson MT. (1993). cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am J Physiol 265, C299-303.

Rossow CF, Minami E, Chase EG, Murry CE & Santana LF. (2004). NFATc3- induced reductions in voltage-gated K+ currents after myocardial infarction. Circ Res 94, 1340-1350.

Saenz de Tejada I, Mueller JD, de Las Morenas A, Machado M, Moreland RB, Krane RJ, Wolfe HJ & Traish AM. (1992). Endothelin in the urinary bladder. I. Synthesis of endothelin-1 by epithelia, muscle and fibroblasts suggests autocrine and paracrine cellular regulation. J Urol 148, 1290- 1298.

Salinas M, de Weille J, Guillemare E, Lazdunski M & Hugnot JP. (1997a). Modes of regulation of shab K+ channel activity by the Kv8.1 subunit. J Biol Chem 272, 8774-8780.

Salinas M, Duprat F, Heurteaux C, Hugnot JP & Lazdunski M. (1997b). New modulatory alpha subunits for mammalian Shab K+ channels. J Biol Chem 272, 24371-24379.

Schubert R & Nelson MT. (2001). Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 22, 505-512.

Schubert R, Noack T & Serebryakov VN. (1999). Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells. Am J Physiol 276, C648-658.

Schubert R, Serebryakov VN, Engel H & Hopp HH. (1996). Iloprost activates KCa channels of vascular smooth muscle cells: role of cAMP-dependent protein kinase. Am J Physiol 271, C1203-1211.

170

Stevenson AS, Gomez MF, Hill-Eubanks DC & Nelson MT. (2001). NFAT4 movement in native smooth muscle. A role for differential Ca(2+) signaling. J Biol Chem 276, 15018-15024.

Thorneloe KS, Chen TT, Kerr PM, Grier EF, Horowitz B, Cole WC & Walsh MP. (2001). Molecular composition of 4-aminopyridine-sensitive voltage-gated K(+) channels of vascular smooth muscle. Circ Res 89, 1030-1037.

Thorneloe KS, Meredith AL, Knorn AM, Aldrich RW & Nelson MT. (2005). Urodynamic properties and neurotransmitter dependence of urinary bladder contractility in the BK channel deletion model of overactive bladder. Am J Physiol Renal Physiol 289, F604-610.

Thorneloe KS & Nelson MT. (2003). Properties and molecular basis of the mouse urinary bladder voltage-gated K+ current. J Physiol 549, 65-74.

Thorneloe KS & Nelson MT. (2005). Ion channels in smooth muscle: regulators of intracellular calcium and contractility. Can J Physiol Pharmacol 83, 215- 242.

Werner ME, Knorn AM, Meredith AL, Aldrich RW & Nelson MT. (2007). Frequency encoding of cholinergic- and purinergic-mediated signaling to mouse urinary bladder smooth muscle: modulation by BK channels. Am J Physiol Regul Integr Comp Physiol 292, R616-624.

Yellen G. (2002). The voltage-gated potassium channels and their relatives. Nature 419, 35-42.

Yi BA, Minor DL, Jr., Lin YF, Jan YN & Jan LY. (2001). Controlling potassium channel activities: Interplay between the membrane and intracellular factors. Proc Natl Acad Sci U S A 98, 11016-11023.

Yuan XJ, Wang J, Juhaszova M, Golovina VA & Rubin LJ. (1998). Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells. Am J Physiol 274, L621-635.

Zhu J & McKeon F. (2000). Nucleocytoplasmic shuttling and the control of NF-AT signaling. Cell Mol Life Sci 57, 411-420.

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CHAPTER 4: SYNOPSIS AND FUTURE DIRECTIONS

SYNOPSIS

Urinary bladder function can be compromised by any of a number of complications, but is most typically associated with bladder outlet obstruction due to enlargement of the prostate (cite). Despite the extensive research that has been performed on the functional and morphological changes that occur in the urinary bladder smooth muscle wall subsequent to outlet obstruction, very little is known regarding the molecular mechanisms that underlie these physiological changes. However, it is currently believed that many of the symptoms that are associated with bladder outlet obstruction, i.e. urinary incontinence and overactive bladder, may be due to changes in the contractile and electrical properties of the urinary bladder smooth muscle wall (cite). Changes in the contractile protein expression (cite), ion channel activity (cite), and Ca2+-handling

(cite) have been reported to be downstream effects of outlet obstruction. Given the fact that its activity is controlled by intracellular Ca2+ levels and that it has previously been associated with alterations in the expression of contractile proteins (cite) and ion channels (cite), NFAT is a potential mediator of some of the physiological changes which occur in the bladder subsequent to outlet obstruction. In addition, the established role of NFAT as a mediator of cardiac hypertrophy (cite), a physiological response that may be considered analogous to bladder hypertrophy, lends additional support for NFAT in the bladder hypertrophic response. However, there is currently a dearth of information

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regarding the expression and functional role of NFAT in the urinary bladder.

Thus, the focus of this dissertation was to explore the role of NFAT in mediating

the contractility of the urinary bladder.

In Chapter 1, we explored the role of NFAT as a mediator of contractile protein expression in smooth muscle tissues. Using a reporter construct containing a overlapping CArG/NFAT binding element, we determined that NFAT and SRF, another prominent transcription factor that has been implicated in the regulation of many smooth muscle-specific proteins (cite), cooperatively regulate the expression of smooth muscle α-actin. The fact that mutations to either the

SRF- or NFAT-binding sites abolished reporter activity indicated that both transcription factors are required for SM α-actin expression. This is the first report of SRF and NFAT cooperatively regulating expression of a S-specific gene.

In addition, our observation that blocking the calcineurin/NFAT pathways using

FK506/CsA led to a reduction α-actin expression in smooth muscle cells indicates that NFAT activity may be physiologically relevant to normal SM contractile protein expression.

The focus of our experiments which we present in Chapter 2 of this dissertation focus on the functional role(s) of NFAT in urinary bladder smooth muscle. There are currently no published reports on the activity or expression of

NFAT in UBSM tissues. Thus, our findings, which we have submitted for

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publications, are novel observation in this field. Using a combination of RT-PCR

and immunohistochemistry, we have identified 3 NFAT isoforms (NFATc1, c3,

c4) in native and cultured UBSM. We were able to stimulate nuclear translocation

of NFATc3 using PDGF and ET-1, two smooth muscle mitogens that have

previously been shown to activate NFAT in smooth muscle (cite) and, in addition,

may play a role in the bladder hypertrophic response (cite). Smooth muscle strips

from NFATc3-null mice displayed an enhanced contractile response to electrical

field stimulation as compared to strips from wild-type mice.

Electrophysiological analyses revealed that IBTX-sensitive BK currents

were downregulated in the UBSM myocytes from NFATc3-null mice, and this

downregulation of BK current was supported at the molecular level by a reduction

in the expression of mRNA for the pore-forming α-subunit, although levels of the

β-subunit, which increase the sensitivity of the channels to voltage and Ca2+, were not altered. Furthermore, we used excise inside-out patches to obtain single-channel recording that showed that there was no change in the voltage- dependent activity of the NFATc3-null myocyte BK channels, also indicating that

β-subunit expression was not altered. However, we did find a decrease in BK channel density in myocytes from the NFATc3-null mice; which is consistent with a downregulation of the pore-forming α-subunit. A recent publication (cite) has indicated that NFAT may also play a role in the regulation of the BK β-subunit in vascular smooth muscle tissue. However, our data did not find any change in β-

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subunit expression in the UBSM of NFATc3-null mice.

Interestingly, the TEA-sensitive KV currents were upregulated in the

NFATc3-null mice by approximately the same amount (~3pa/pf) as the BK currents were downregulated. This appears to be an adaptive response of the mice, although it is unclear which was done in response to which, i.e. did the changes in changes in BK channel activity occur in response to changes in KV activity, or vice-versa? Indeed, at this point it is unclear if either ion channel is even a direct genetic target of NFATc3. It is quite possible that the changes in BK and KV channel expression were both responses to a third, unidentified target of

NFAT activity. However, we did identify potential NFAT binding sites in both promoters, but we have not performed reporter assays using constructs derived from the respective promoter regions to determine whether or not they are NFAT- responsive.

In conclusion, in this dissertation we have shown that NFAT does play a role in the regulation of smooth muscle contractility via its role in modulating the expression of smooth muscle contractile proteins (α-actin) and ion channels (BK and KV). This lays the foundation for future experiments to determine the role of

NFAT in the bladder hypertrophic response to outlet obstruction, which will be discussed in the next section.

176

FUTURE DIRECTIONS

The role of NFAT in BK and KV channel expression

Are the BK α-subunit and KV2.1 targets of NFAT transcriptional activity?

Although the data presented in Chapter 2 indicate that there are significant

alterations in BK and KV channel expression in UBSM from NFATc3-null mice,

we have yet to determine if these changes are directly mediated by NFATc3. To

determine whether the BK α- and KV2.1-subunits are regulatory targets of

NFATc3, we will construct reporters using the regions of the respective promoters that contain conserved NFAT recognition sites. The responsiveness to these reporters will be tested using classical Ca2+-elevating stimuli (ET-1, PDGF) that have previously been shown to activate NFAT nuclear translocation in smooth muscle (cite). Blockade of reporter activity using FK506 and CsA will lend further evidence in support, or rejection, of a role for NFAT in the expression of these two ion channel proteins.

Considering the fact that BK channel expression was downregulated in the

NFATc3-cull mice while KV2.1 channel expression was upregulated, our findings

suggest that NFATc3 normally upregulates BK channel expression and

downregulates KV2.1 channel expression. Although NFAT transcriptional activity

is most often associated with an increase in the expression of the target gene

(cite), there is precedence for NFAT also acting to downregulate expression

(cite). Since our findings suggest that NFATc3 downregulates KV2.1 expression,

177

one might conjecture that transfection of the KV2.1 reporter plasmid into the SMC

line would lead to a basal level of expression that would, in the presence of

NFAT-activating stimuli, then be decreased.

In vivo analysis of the role of NFAT in the expression of KV2.1 and the BK α-

subunit

While the previous set of experiments will determine if KV2.1 and the α-

subunit are transcriptional targets of NFAT activity in cultured SMC, it would also

be insightful to determine if the findings were also applicable in vivo. We will use a transgenic dominant-negative NFAT mouse (NFATdn) to determine the time- course of the changes in BK and KV2.1 channel expression in live animals. NFAT activity in these mice can be "turned off" by feeding the mice dietary doxycycline.

IBTX-sensitive BK and TEA-sensitive KV currents will be recorded from isolated

UBSM myocytes derived from NFATdn mice before (time 0) and at various time

points (24 hour intervals) after doxycycline treatment to determine the onset and

progression of the changes in ion channel expression and activity. In addition to

determining the in vivo functional relevance of NFAT in BK and KV channel

expression, these data should also help to clarify whether the changes in BK and

KV channel expression occur simultaneously, or if changes to one ion channel

(e.g. BK) trigger a compensatory up- or downregulation in the activity of the other

(i.e. KV) ion channel.

178

Are specific NFAT isoforms responsible for BK/KV channel transcriptional

activity?

Lastly, we will use antisense RNA or siRNA to selectively determine

exactly which NFAT isoforms are transcriptionally active in regards to BK and

KV2.1 channel expression. Since we have shown that only NFATc1, c3, and c4 are present in UBSM tissues, we will design siRNA probes that we will use to

selectively inhibit expression of each of these isoforms (individually). After

blocking the NFAT isoform, we will monitor the changes (if any) if the IBTX-

sensitive BK current and TEA-sensitive KV currents using patch-clamp analysis. It

is quite possible that there is some redundancy in the ability of the NFAT

isoforms to affect BK or KV channel expression, so we will also perform experiments in which multiple subsets of NFAT (e.g. NFATc1 and NFATc3) will be blocked at the same time.

The role of NFAT in the bladder hypertrophic response to outlet obstruction

Does NFAT activity play a role in the bladder response to partial bladder outlet

obstruction?

In the first set of experiments, we will attempt to determine if NFAT activity

plays a role in the bladder hypertrophic response that is initiated by partial

bladder outlet obstruction. Wild-type mice will be subject to surgically-induced

obstruction of the urethra. A subset of animals will be maintained on a diet

179

including the calcineurin/NFAT pathways inhibitor cyclosporine A (CsA). At daily time intervals, mice will be sacrificed and their bladders analyzed for various changes induced by the outlet obstruction. We will then compare the changes induced by the outlet obstruction between the normal diet animals and those maintained on dietary CsA to determine if blocking NFAT activation affected the outcome of the obstruction. Some of the parameters to be analyzed include:

1. Bladder : body mass ratio (wet weight)

2. Contractile response to electrical field stimulation (myograph)

3. Nuclear localization of NFAT isoforms (immmunostaining)

4. BK and KV currents (electrophysiology)

5. BK channel density (excised patch recordings)

6. Expression of smooth muscle proteins by Western Blot (e.g. α-actin, SM

myosin heavy chain, caldesmon)

These experiments can then be repeated using the NFATc3 mice which we have used in the experiments presented in this dissertation to determine the functional contribution of that specific isoform.

180

COMPREHENSIVE BIBLIOGRAPHY

Amberg GC, Rossow CF, Navedo MF & Santana LF. (2004). NFATc3 regulates Kv2.1 expression in arterial smooth muscle. J Biol Chem 279, 47326- 47334.

Andersson KE & Arner A. (2004). Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev 84, 935-986.

Austin JC, Chacko SK, DiSanto M, Canning DA & Zderic SA. (2004). A male murine model of partial bladder outlet obstruction reveals changes in detrusor morphology, contractility and Myosin isoform expression. J Urol 172, 1524-1528.

Avni O, Lee D, Macian F, Szabo SJ, Glimcher LH & Rao A. (2002). T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nat Immunol 3, 643-651.

Baeuerle PA. (1991). The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta 1072, 63-80.

Barlow CA, Rose P, Pulver-Kaste RA & Lounsbury KM. (2006). Excitation- transcription coupling in smooth muscle. J Physiol 570, 59-64.

Baumann G, Zenke G, Wenger R, Hiestand P, Quesniaux V, Andersen E & Schreier MH. (1992). Molecular mechanisms of immunosuppression. J Autoimmun 5 Suppl A, 67-72.

Beals CR, Sheridan CM, Turck CW, Gardner P & Crabtree GR. (1997). Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science 275, 1930-1934.

Beard NA, Sakowska MM, Dulhunty AF & Laver DR. (2002). Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels. Biophys J 82, 310-320.

181

Belaguli NS, Schildmeyer LA & Schwartz RJ. (1997). Organization and myogenic restricted expression of the murine serum response factor gene. A role for autoregulation. J Biol Chem 272, 18222-18231.

Benham CD & Bolton TB. (1986). Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. J Physiol 381, 385-406.

Bert AG, Burrows J, Hawwari A, Vadas MA & Cockerill PN. (2000). Reconstitution of T cell-specific transcription directed by composite NFAT/Oct elements. J Immunol 165, 5646-5655.

Bodor J, Bodorova J & Gress RE. (2000). Suppression of T cell function: a potential role for transcriptional repressor ICER. J Leukoc Biol 67, 774- 779.

Bond M & Somlyo AV. (1982). Dense bodies and actin polarity in vertebrate smooth muscle. J Cell Biol 95, 403-413.

Boss V, Abbott KL, Wang XF, Pavlath GK & Murphy TJ. (1998). The cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) proteins are expressed in vascular smooth muscle cells. Differential localization of NFAT isoforms and induction of NFAT-mediated transcription by phospholipase C-coupled cell surface receptors. J Biol Chem 273, 19664- 19671.

Bower KE, Zeller RW, Wachsman W, Martinez T & McGuire KL. (2002). Correlation of transcriptional repression by p21(SNFT) with changes in DNA.NF-AT complex interactions. J Biol Chem 277, 34967-34977.

Brading AF. (1992). Ion channels and control of contractile activity in urinary bladder smooth muscle. Jpn J Pharmacol 58 Suppl 2, 120P-127P.

Brading AF & Williams JH. (1990). Contractile responses of smooth muscle strips from rat and guinea-pig urinary bladder to transmural stimulation: effects of atropine and alpha,beta-methylene ATP. Br J Pharmacol 99, 493-498.

Bremel RD. (1974). Myosin linked calcium regulation in vertebrate smooth muscle. Nature 252, 405-407.

182

Brindle PK & Montminy MR. (1992). The CREB family of transcription activators. Curr Opin Genet Dev 2, 199-204.

Burkhard FC, Lemack GE, Zimmern PE, Lin VK & McConnell JD. (2001). Contractile protein expression in bladder smooth muscle is a marker of phenotypic modulation after outlet obstruction in the rabbit model. J Urol 165, 963-967.

Burnstock G. (2006). Purinergic signalling. Br J Pharmacol 147 Suppl 1, S172- 181.

Cahill MA, Janknecht R & Nordheim A. (1996). Signalling pathways: jack of all cascades. Curr Biol 6, 16-19.

Cartin L, Lounsbury KM & Nelson MT. (2000). Coupling of Ca(2+) to CREB activation and gene expression in intact cerebral arteries from mouse : roles of ryanodine receptors and voltage-dependent Ca(2+) channels. Circ Res 86, 760-767.

Chang PS, Li L, McAnally J & Olson EN. (2001). Muscle specificity encoded by specific serum response factor-binding sites. J Biol Chem 276, 17206- 17212.

Chen J, Kitchen CM, Streb JW & Miano JM. (2002). Myocardin: a component of a molecular switch for smooth muscle differentiation. J Mol Cell Cardiol 34, 1345-1356.

Chen L, Glover JN, Hogan PG, Rao A & Harrison SC. (1998). Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature 392, 42-48.

Chen L, Oakley MG, Glover JN, Jain J, Dervan PB, Hogan PG, Rao A & Verdine GL. (1995). Only one of the two DNA-bound orientations of AP-1 found in solution cooperates with NFATp. Curr Biol 5, 882-889.

Chiavegato A, Scatena M, Roelofs M, Ferrarese P, Pauletto P, Passerini-Glazel G, Pagano F & Sartore S. (1993). Cytoskeletal and cytocontractile protein composition of smooth muscle cells in developing and obstructed rabbit bladder. Exp Cell Res 207, 310-320. 183

Ching LL, Williams AJ & Sitsapesan R. (2000). Evidence for Ca(2+) activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex. Circ Res 87, 201-206.

Chow CW, Dong C, Flavell RA & Davis RJ. (2000). c-Jun NH(2)-terminal kinase inhibits targeting of the protein phosphatase calcineurin to NFATc1. Mol Cell Biol 20, 5227-5234.

Chytil M & Verdine GL. (1996). The Rel family of eukaryotic transcription factors. Curr Opin Struct Biol 6, 91-100.

Clipstone NA & Crabtree GR. (1992). Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357, 695-697.

Cohen PT, Chen MX & Armstrong CG. (1996). Novel protein phosphatases that may participate in cell signaling. Adv Pharmacol 36, 67-89.

Collier ML, Ji G, Wang Y & Kotlikoff MI. (2000). Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release. J Gen Physiol 115, 653-662.

Cox DH & Aldrich RW. (2000). Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity. J Gen Physiol 116, 411-432.

Crabtree GR & Clipstone NA. (1994). Signal transmission between the plasma membrane and nucleus of T lymphocytes. Annu Rev Biochem 63, 1045- 1083.

Crabtree GR. (1999). Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96, 611-614.

Crabtree GR & Olson EN. (2002). NFAT signaling: choreographing the social lives of cells. Cell 109 Suppl, S67-79.

Creed KE, Ishikawa S & Ito Y. (1983). Electrical and mechanical activity recorded from rabbit urinary bladder in response to nerve stimulation. J Physiol 338, 149-164.

184

Croissant JD, Kim JH, Eichele G, Goering L, Lough J, Prywes R & Schwartz RJ. (1996). Avian serum response factor expression restricted primarily to muscle cell lineages is required for alpha-actin gene transcription. Dev Biol 177, 250-264.

Du KL, Ip HS, Li J, Chen M, Dandre F, Yu W, Lu MM, Owens GK & Parmacek MS. (2003). Myocardin is a critical serum response factor cofactor in the transcriptional program regulating smooth muscle cell differentiation. Mol Cell Biol 23, 2425-2437.

Fill M & Copello JA. (2002). Ryanodine receptor calcium release channels. Physiol Rev 82, 893-922.

Fiset C, Clark RB, Shimoni Y & Giles WR. (1997). Shal-type channels contribute to the Ca2+-independent transient outward K+ current in rat ventricle. J Physiol 500 ( Pt 1), 51-64.

Flanagan WM, Corthesy B, Bram RJ & Crabtree GR. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352, 803-807.

Franzoso G, Carlson L, Brown K, Daucher MB, Bressler P & Siebenlist U. (1996). Activation of the serum response factor by p65/NF-kappaB. Embo J 15, 3403-3412.

Furstenau U, Schwaninger M, Blume R, Jendrusch EM & Knepel W. (1999). Characterization of a novel calcium response element in the glucagon gene. J Biol Chem 274, 5851-5860.

Ghoniem GM, Regnier CH, Biancani P, Johnson L & Susset JG. (1986). Effect of vesical outlet obstruction on detrusor contractility and passive properties in rabbits. J Urol 135, 1284-1289.

185

Ghosh G, van Duyne G, Ghosh S & Sigler PB. (1995). Structure of NF-kappa B p50 homodimer bound to a kappa B site. Nature 373, 303-310.

Gollasch M & Nelson MT. (1997). Voltage-dependent Ca2+ channels in arterial smooth muscle cells. Kidney Blood Press Res 20, 355-371.

Gorski DH & Walsh K. (2003). Control of vascular cell differentiation by homeobox transcription factors. Trends Cardiovasc Med 13, 213-220.

Gosling JA, Kung LS, Dixon JS, Horan P, Whitbeck C & Levin RM. (2000). Correlation between the structure and function of the rabbit urinary bladder following partial outlet obstruction. J Urol 163, 1349-1356.

Graef IA, Chen F, Chen L, Kuo A & Crabtree GR. (2001a). Signals transduced by Ca(2+)/calcineurin and NFATc3/c4 pattern the developing vasculature. Cell 105, 863-875.

Graef IA, Chen F & Crabtree GR. (2001b). NFAT signaling in vertebrate development. Curr Opin Genet Dev 11, 505-512.

Graef IA, Mermelstein PG, Stankunas K, Neilson JR, Deisseroth K, Tsien RW & Crabtree GR. (1999). L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401, 703-708.

186

Hashitani H & Brading AF. (2003a). Electrical properties of detrusor smooth muscles from the pig and human urinary bladder. Br J Pharmacol 140, 146-158.

Hashitani H & Brading AF. (2003b). Ionic basis for the regulation of spontaneous excitation in detrusor smooth muscle cells of the guinea-pig urinary bladder. Br J Pharmacol 140, 159-169.

Hashitani H, Fukuta H, Takano H, Klemm MF & Suzuki H. (2001). Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder. J Physiol 530, 273-286.

Hathaway DR, March KL, Lash JA, Adam LP & Wilensky RL. (1991). Vascular smooth muscle. A review of the molecular basis of contractility. Circulation 83, 382-390.

Hautmann MB, Thompson MM, Swartz EA, Olson EN & Owens GK. (1997). Angiotensin II-induced stimulation of smooth muscle alpha-actin expression by serum response factor and the homeodomain transcription factor MHox. Circ Res 81, 600-610.

Heppner TJ, Bonev AD & Nelson MT. (1997). Ca(2+)-activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle. Am J Physiol 273, C110-117.

Heppner TJ, Bonev AD & Nelson MT. (2005). Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle. J Physiol 564, 201-212.

Herrera GM, Heppner TJ & Nelson MT. (2000). Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. Am J Physiol Regul Integr Comp Physiol 279, R60-68.

Herrera GM, Heppner TJ & Nelson MT. (2001). Voltage dependence of the coupling of Ca(2+) sparks to BK(Ca) channels in urinary bladder smooth muscle. Am J Physiol Cell Physiol 280, C481-490.

187

Hewett TE, Martin AF & Paul RJ. (1993). Correlations between myosin heavy chain isoforms and mechanical parameters in rat myometrium. J Physiol 460, 351-364.

Hill-Eubanks DC, Gomez MF, Stevenson AS & Nelson MT. (2003). NFAT regulation in smooth muscle. Trends Cardiovasc Med 13, 56-62.

Ho IC, Hodge MR, Rooney JW & Glimcher LH. (1996). The proto-oncogene c- maf is responsible for tissue-specific expression of interleukin-4. Cell 85, 973-983.

Ho S, Timmerman L, Northrop J & Crabtree GR. (1994). Cloning and characterization of NF-ATc and NF-ATp: the cytoplasmic components of NF-AT. Adv Exp Med Biol 365, 167-173.

Hogan PG, Chen L, Nardone J & Rao A. (2003). Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17, 2205-2232.

Horsley V & Pavlath GK. (2002). NFAT: ubiquitous regulator of cell differentiation and adaptation. J Cell Biol 156, 771-774.

Hu CM, Jang SY, Fanzo JC & Pernis AB. (2002). Modulation of T cell cytokine production by interferon regulatory factor-4. J Biol Chem 277, 49238- 49246.

Huxley AF & Niedergerke R. (1954). Structural changes in muscle during contraction; interference microscopy of living muscle fibres. Nature 173, 971-973.

Huxley H & Hanson J. (1954). Changes in the cross-striations of muscle during contraction and stretch and their structural interpretation. Nature 173, 973- 976.

188

Im SH & Rao A. (2004). Activation and deactivation of gene expression by Ca2+/calcineurin-NFAT-mediated signaling. Mol Cells 18, 1-9.

Isenberg G, Wendt-Gallitelli MF & Ganitkevich V. (1992). Contribution of Ca(2+)- induced Ca2+ release to depolarization-induced Ca2+ transients of myocytes from guinea-pig urinary bladder myocytes. Jpn J Pharmacol 58 Suppl 2, 81P-86P.

Jaggar JH, Porter VA, Lederer WJ & Nelson MT. (2000). Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 278, C235-256.

Jaggar JH, Wellman GC, Heppner TJ, Porter VA, Perez GJ, Gollasch M, Kleppisch T, Rubart M, Stevenson AS, Lederer WJ, Knot HJ, Bonev AD & Nelson MT. (1998). Ca2+ channels, ryanodine receptors and Ca(2+)- activated K+ channels: a functional unit for regulating arterial tone. Acta Physiol Scand 164, 577-587.

Jain J, Burgeon E, Badalian TM, Hogan PG & Rao A. (1995). A similar DNA- binding motif in NFAT family proteins and the Rel homology region. J Biol Chem 270, 4138-4145.

Jain J, McCaffrey PG, Valge-Archer VE & Rao A. (1992). Nuclear factor of activated T cells contains Fos and Jun. Nature 356, 801-804.

Jan LY & Jan YN. (1997). Voltage-gated and inwardly rectifying potassium channels. J Physiol 505 ( Pt 2), 267-282.

Jin L, Sliz P, Chen L, Macian F, Rao A, Hogan PG & Harrison SC. (2003). An asymmetric NFAT1 dimer on a pseudo-palindromic kappa B-like DNA site. Nat Struct Biol 10, 807-811.

Kaczorowski GJ, Knaus HG, Leonard RJ, McManus OB & Garcia ML. (1996). High-conductance calcium-activated potassium channels; structure, pharmacology, and function. J Bioenerg Biomembr 28, 255-267.

Khan MA, Dashwood MR, Thompson CS, Mumtaz FH, Mikhailidis DP & Morgan RJ. (1999). Up-regulation of endothelin (ET(A) and ET(B)) receptors and down-regulation of nitric oxide synthase in the detrusor of a rabbit model of partial bladder outlet obstruction. Urol Res 27, 445-453. 189

Kim JC, Yoon JY, Seo SI, Hwang TK & Park YH. (2000). Effects of partial bladder outlet obstruction and its relief on types I and III collagen and detrusor contractility in the rat. Neurourol Urodyn 19, 29-42.

Kim S, Ip HS, Lu MM, Clendenin C & Parmacek MS. (1997). A serum response factor-dependent transcriptional regulatory program identifies distinct smooth muscle cell sublineages. Mol Cell Biol 17, 2266-2278.

Kim YS, Wang Z, Levin RM & Chacko S. (1994). Alterations in the expression of the beta-cytoplasmic and the gamma-smooth muscle actins in hypertrophied urinary bladder smooth muscle. Mol Cell Biochem 131, 115- 124.

Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW & et al. (1995). Crystal structures of human calcineurin and the human FKBP12-FK506- calcineurin complex. Nature 378, 641-644.

Klee CB, Ren H & Wang X. (1998). Regulation of the calmodulin-stimulated protein phosphatase, calcineurin. J Biol Chem 273, 13367-13370

Knot HJ & Nelson MT. (1998). Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure. J Physiol 508 ( Pt 1), 199-209.

Kramer JW, Post MA, Brown AM & Kirsch GE. (1998). Modulation of potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1 alpha-subunits. Am J Physiol 274, C1501-1510.

Kumar MS & Owens GK. (2003). Combinatorial control of smooth muscle- specific gene expression. Arterioscler Thromb Vasc Biol 23, 737-747.

Larrieu D, Thiebaud P, Duplaa C, Sibon I, Theze N & Lamaziere JM. (2005). Activation of the Ca(2+)/calcineurin/NFAT2 pathway controls smooth muscle cell differentiation. Exp Cell Res 310, 166-175.

190

Ledoux J, Werner ME, Brayden JE & Nelson MT. (2006). Calcium-activated potassium channels and the regulation of vascular tone. Physiology (Bethesda) 21, 69-78.

Levin RM, Longhurst PA, Monson FC, Kato K & Wein AJ. (1990). Effect of bladder outlet obstruction on the morphology, physiology, and pharmacology of the bladder. Prostate Suppl 3, 9-26.

Levin RM, Wein AJ, Buttyan R, Monson FC & Longhurst PA. (1994). Update on bladder smooth-muscle physiology. World J Urol 12, 226-232.

Li L, Miano JM, Cserjesi P & Olson EN. (1996). SM22 alpha, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis. Circ Res 78, 188-195.

Liu J, Farmer JD, Jr., Lane WS, Friedman J, Weissman I & Schreiber SL. (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP- FK506 complexes. Cell 66, 807-815.

Lopez-Rodriguez C, Aramburu J, Jin L, Rakeman AS, Michino M & Rao A. (2001). Bridging the NFAT and NF-kappaB families: NFAT5 dimerization regulates cytokine gene transcription in response to osmotic stress. Immunity 15, 47-58.

Luo C, Burgeon E & Rao A. (1996). Mechanisms of transactivation by nuclear factor of activated T cells-1. J Exp Med 184, 141-147.

Martens JR, Kwak YG & Tamkun MM. (1999). Modulation of Kv channel alpha/beta subunit interactions. Trends Cardiovasc Med 9, 253-258.

Macian F, Lopez-Rodriguez C & Rao A. (2001). Partners in transcription: NFAT and AP-1. Oncogene 20, 2476-2489.

Mack CP & Owens GK. (1999). Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions. Circ Res 84, 852-861. 191

Mack CP, Somlyo AV, Hautmann M, Somlyo AP & Owens GK. (2001). Smooth muscle differentiation marker gene expression is regulated by RhoA- mediated actin polymerization. J Biol Chem 276, 341-347.

Mack CP, Thompson MM, Lawrenz-Smith S & Owens GK. (2000). Smooth muscle alpha-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum response factor-containing activation complex. Circ Res 86, 221-232.

Malkowicz SB, Wein AJ, Elbadawi A, Van Arsdalen K, Ruggieri MR & Levin RM. (1986). Acute biochemical and functional alterations in the partially obstructed rabbit urinary bladder. J Urol 136, 1324-1329.

Malmqvist U, Arner A & Uvelius B. (1991a). Contractile and cytoskeletal proteins in smooth muscle during hypertrophy and its reversal. Am J Physiol 260, C1085-1093.

Malmqvist U, Arner A & Uvelius B. (1991b). Cytoskeletal and contractile proteins in detrusor smooth muscle from bladders with outlet obstruction--a comparative study in rat and man. Scand J Urol Nephrol 25, 261-267.

Manabe I & Owens GK. (2001a). CArG elements control smooth muscle subtype-specific expression of smooth muscle myosin in vivo. J Clin Invest 107, 823-834.

Manabe I & Owens GK. (2001b). The smooth muscle myosin heavy chain gene exhibits smooth muscle subtype-selective modular regulation in vivo. J Biol Chem 276, 39076-39087.

Meiss RA. (1995). Contractile Properties of Muscle Cells. In Medical Physiology, ed. Rhoades RA & Tanner GA. Little, Brown and Company.

Meissner JD, Umeda PK, Chang KC, Gros G & Scheibe RJ. (2007). Activation of the beta myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/NFATc1 pathway. J Cell Physiol 211, 138-148.

Mericskay M, Li Z & Paulin D. (1999). Transcriptional regulation of the desmin and SM22 genes in vascular smooth muscle cells. Curr Top Pathol 93, 7- 17. 192

Miano JM. (2003). Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol 35, 577-593.

Miano JM, Carlson MJ, Spencer JA & Misra RP. (2000). Serum response factor- dependent regulation of the smooth muscle calponin gene. J Biol Chem 275, 9814-9822.

Miyakawa H, Woo SK, Dahl SC, Handler JS & Kwon HM. (1999). Tonicity- responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci U S A 96, 2538-2542.

Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR & Olson EN. (1998). A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215-228.

Musaro A, McCullagh KJ, Naya FJ, Olson EN & Rosenthal N. (1999). IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature 400, 581-585.

Nagai R, Kuro-o M, Babij P & Periasamy M. (1989). Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis. J Biol Chem 264, 9734-9737.

Neal JW & Clipstone NA. (2001). Glycogen synthase kinase-3 inhibits the DNA binding activity of NFATc. J Biol Chem 276, 3666-3673.

Negulescu PA, Shastri N & Cahalan MD. (1994). Intracellular calcium dependence of gene expression in single T lymphocytes. Proc Natl Acad Sci U S A 91, 2873-2877.

Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ & Lederer WJ. (1995). Relaxation of arterial smooth muscle by calcium sparks. Science 270, 633-637.

Nerbonne JM. (2000). Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol 525 Pt 2, 285-298.

193

Northrop JP, Ho SN, Chen L, Thomas DJ, Timmerman LA, Nolan GP, Admon A & Crabtree GR. (1994). NF-AT components define a family of transcription factors targeted in T-cell activation. Nature 369, 497-502.

Nozaki K, Tomizawa K, Yokoyama T, Kumon H & Matsui H. (2003). Calcineurin mediates bladder smooth muscle hypertrophy after bladder outlet obstruction. J Urol 170, 2077-2081.

O'Reilly BA, Kosaka AH, Knight GF, Chang TK, Ford AP, Rymer JM, Popert R, Burnstock G & McMahon SB. (2002). P2X receptors and their role in female idiopathic detrusor instability. J Urol 167, 157-164.

Ohkawa Y, Hayashi K & Sobue K. (2003). Calcineurin-mediated pathway involved in the differentiated phenotype of smooth muscle cells. Biochem Biophys Res Commun 301, 78-83.

Owens GK. (1995). Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75, 487-517.

Owens GK. (1998). Molecular control of vascular smooth muscle cell differentiation. Acta Physiol Scand 164, 623-635.

Owens GK, Kumar MS & Wamhoff BR. (2004). Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84, 767-801.

Palea S, Artibani W, Ostardo E, Trist DG & Pietra C. (1993). Evidence for purinergic neurotransmission in human urinary bladder affected by interstitial cystitis. J Urol 150, 2007-2012. 194

Park J, Takeuchi A & Sharma S. (1996). Characterization of a new isoform of the NFAT (nuclear factor of activated T cells) gene family member NFATc. J Biol Chem 271, 20914-20921.

Pauletto P, Sarzani R, Rappelli A, Chiavegato A, Pessina AC & Sartore S. (1994). Differentiation and growth of vascular smooth muscle cells in experimental hypertension. Am J Hypertens 7, 661-674.

Petkov GV, Bonev AD, Heppner TJ, Brenner R, Aldrich RW & Nelson MT. (2001). Beta1-subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle. J Physiol 537, 443-452.

Pfitzer G. (2001). Invited review: regulation of myosin phosphorylation in smooth muscle. J Appl Physiol 91, 497-503.

Post MA, Kirsch GE & Brown AM. (1996). Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current. FEBS Lett 399, 177-182.

Quayle JM, Nelson MT & Standen NB. (1997). ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol Rev 77, 1165- 1232.

Ranger AM, Oukka M, Rengarajan J & Glimcher LH. (1998). Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development. Immunity 9, 627-635.

Rao A, Luo C & Hogan PG. (1997). Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15, 707-747.

Rengarajan J, Mowen KA, McBride KD, Smith ED, Singh H & Glimcher LH. (2002). Interferon regulatory factor 4 (IRF4) interacts with NFATc2 to modulate interleukin 4 gene expression. J Exp Med 195, 1003-1012.

Robertson B. (1997). The real life of voltage-gated K+ channels: more than model behaviour. Trends Pharmacol Sci 18, 474-483.

195

Robertson BE, Schubert R, Hescheler J & Nelson MT. (1993). cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am J Physiol 265, C299-303.

Rooney JW, Sun YL, Glimcher LH & Hoey T. (1995). Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation. Mol Cell Biol 15, 6299-6310.

Rossow CF, Minami E, Chase EG, Murry CE & Santana LF. (2004). NFATc3- induced reductions in voltage-gated K+ currents after myocardial infarction. Circ Res 94, 1340-1350.

Ruff VA & Leach KL. (1995). Direct demonstration of NFATp dephosphorylation and nuclear localization in activated HT-2 cells using a specific NFATp polyclonal antibody. J Biol Chem 270, 22602-22607.

Sakurada S, Takuwa N, Sugimoto N, Wang Y, Seto M, Sasaki Y & Takuwa Y. (2003). Ca2+-dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction. Circ Res 93, 548-556.

Salinas M, de Weille J, Guillemare E, Lazdunski M & Hugnot JP. (1997a). Modes of regulation of shab K+ channel activity by the Kv8.1 subunit. J Biol Chem 272, 8774-8780.

Salinas M, Duprat F, Heurteaux C, Hugnot JP & Lazdunski M. (1997b). New modulatory alpha subunits for mammalian Shab K+ channels. J Biol Chem 272, 24371-24379.

Schmitz ML, dos Santos Silva MA, Altmann H, Czisch M, Holak TA & Baeuerle PA. (1994). Structural and functional analysis of the NF-kappa B p65 C terminus. An acidic and modular transactivation domain with the potential to adopt an alpha-helical conformation. J Biol Chem 269, 25613-25620.

Schubert R & Nelson MT. (2001). Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 22, 505-512.

196

Schubert R, Noack T & Serebryakov VN. (1999). Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells. Am J Physiol 276, C648-658.

Schubert R, Serebryakov VN, Engel H & Hopp HH. (1996). Iloprost activates KCa channels of vascular smooth muscle cells: role of cAMP-dependent protein kinase. Am J Physiol 271, C1203-1211.

Schulz RA & Yutzey KE. (2004). Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development. Dev Biol 266, 1-16.

Sen R & Baltimore D. (1986). Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705-716.

Shaw KT, Ho AM, Raghavan A, Kim J, Jain J, Park J, Sharma S, Rao A & Hogan PG. (1995). Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells. Proc Natl Acad Sci U S A 92, 11205-11209.

Shaw JP, Utz PJ, Durand DB, Toole JJ, Emmel EA & Crabtree GR. (1988). Identification of a putative regulator of early T cell activation genes. Science 241, 202-205.

Shibasaki F, Price ER, Milan D & McKeon F. (1996). Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF- AT4. Nature 382, 370-373.

Sibley GN. (1984). A comparison of spontaneous and nerve-mediated activity in bladder muscle from man, pig and rabbit. J Physiol 354, 431-443.

Sigal NH & Dumont FJ. (1992). Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction. Annu Rev Immunol 10, 519-560.

Sjuve R, Haase H, Morano I, Uvelius B & Arner A. (1996). Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder. J Cell Biochem 63, 86-93.

197

Small JV & North AJ. (1995). Architecture of the Smooth Muscle cell. In The Vascular Smooth Muscle Cell Molecular and Biological Responses to the Extracellular Matrix, ed. RP SSaM. Academic Press, San Diego.

Sobue K, Hayashi K & Nishida W. (1999). Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation. Mol Cell Biochem 190, 105-118.

Stemmer PM & Klee CB. (1994). Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B. Biochemistry 33, 6859-6866.

Stevenson AS, Gomez MF, Hill-Eubanks DC & Nelson MT. (2001). NFAT4 movement in native smooth muscle. A role for differential Ca(2+) signaling. J Biol Chem 276, 15018-15024.

Stroud JC, Lopez-Rodriguez C, Rao A & Chen L. (2002). Structure of a TonEBP- DNA complex reveals DNA encircled by a transcription factor. Nat Struct Biol 9, 90-94.

Su X, Stein R, Stanton MC, Zderic S & Moreland RS. (2003). Effect of partial outlet obstruction on rabbit urinary bladder smooth muscle function. Am J Physiol Renal Physiol 284, F644-652.

Sui GP, Wu C & Fry CH. (2001). Inward calcium currents in cultured and freshly isolated detrusor muscle cells: evidence of a T-type calcium current. J Urol 165, 621-626.

Sweeney HL, Rosenfeld SS, Brown F, Faust L, Smith J, Xing J, Stein LA & Sellers JR. (1998). Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket. J Biol Chem 273, 6262-6270.

Shore P & Sharrocks AD. (1995). The MADS-box family of transcription factors. Eur J Biochem 229, 1-13.

Tekgul S, Yoshino K, Bagli D, Carr MC, Mitchell ME & Yao LY. (1996). Collagen types I and III localization by in situ hybridization and immunohistochemistry in the partially obstructed young rabbit bladder. J Urol 156, 582-586.

Timmerman LA, Clipstone NA, Ho SN, Northrop JP & Crabtree GR. (1996). Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression. Nature 383, 837-840.

198

Thorneloe KS & Nelson MT. (2003). Properties and molecular basis of the mouse urinary bladder voltage-gated K+ current. J Physiol 549, 65-74.

Thorneloe KS & Nelson MT. (2005). Ion channels in smooth muscle: regulators of intracellular calcium and contractility. Can J Physiol Pharmacol 83, 215- 242.

Thrower EC, Hagar RE & Ehrlich BE. (2001). Regulation of Ins(1,4,5)P3 receptor isoforms by endogenous modulators. Trends Pharmacol Sci 22, 580-586.

Treisman R. (1985). Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5' element and c-fos 3' sequences. Cell 42, 889-902.

Treisman R. (1995). Journey to the surface of the cell: Fos regulation and the SRE. Embo J 14, 4905-4913.

Uvelius B & Mattiasson A. (1984). Collagen content in the rat urinary bladder subjected to infravesical outflow obstruction. J Urol 132, 587-590.

Wada H, Hasegawa K, Morimoto T, Kakita T, Yanazume T, Abe M & Sasayama S. (2002). Calcineurin-GATA-6 pathway is involved in smooth muscle- specific transcription. J Cell Biol 156, 983-991.

199

Wamhoff BR, Bowles DK & Owens GK. (2006). Excitation-transcription coupling in arterial smooth muscle. Circ Res 98, 868-878.

Wang D, Chang PS, Wang Z, Sutherland L, Richardson JA, Small E, Krieg PA & Olson EN. (2001). Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell 105, 851-862.

Wegener JW, Schulla V, Lee TS, Koller A, Feil S, Feil R, Kleppisch T, Klugbauer N, Moosmang S, Welling A & Hofmann F. (2004). An essential role of Cav1.2 L-type calcium channel for urinary bladder function. Faseb J 18, 1159-1161.

Wellman GC & Nelson MT. (2003). Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca2+-sensitive ion channels. Cell Calcium 34, 211-229.

Wiederrecht G, Lam E, Hung S, Martin M & Sigal N. (1993). The mechanism of action of FK-506 and cyclosporin A. Ann N Y Acad Sci 696, 9-19.

Williams DA & Fay FS. (1986). Calcium transients and resting levels in isolated smooth muscle cells as monitored with quin 2. Am J Physiol 250, C779- 791.

Wray S, Burdyga T & Noble K. (2005). Calcium signalling in smooth muscle. Cell Calcium 38, 397-407.

200

Yellaturu CR, Ghosh SK, Rao RK, Jennings LK, Hassid A & Rao GN. (2002). A potential role for nuclear factor of activated T-cells in receptor tyrosine kinase and G-protein-coupled receptor agonist-induced cell proliferation. Biochem J 368, 183-190.

Yellen G. (2002). The voltage-gated potassium channels and their relatives. Nature 419, 35-42.

Yi BA, Minor DL, Jr., Lin YF, Jan YN & Jan LY. (2001). Controlling potassium channel activities: Interplay between the membrane and intracellular factors. Proc Natl Acad Sci U S A 98, 11016-11023.

Youn HD, Chatila TA & Liu JO. (2000). Integration of calcineurin and MEF2 signals by the coactivator p300 during T-cell apoptosis. Embo J 19, 4323- 4331.

Yuan XJ, Wang J, Juhaszova M, Golovina VA & Rubin LJ. (1998). Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells. Am J Physiol 274, L621-635.

Zhu J & McKeon F. (1999). NF-AT activation requires suppression of Crm1- dependent export by calcineurin. Nature 398, 256-260.

Zhu J & McKeon F. (2000). Nucleocytoplasmic shuttling and the control of NF-AT signaling. Cell Mol Life Sci 57, 411-420.

Zhu J, Shibasaki F, Price R, Guillemot JC, Yano T, Dotsch V, Wagner G, Ferrara P & McKeon F. (1998). Intramolecular masking of nuclear import signal on NF-AT4 by casein kinase I and MEKK1. Cell 93, 851-861.

201