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ASPECTS OF THE TRANSCRIPTIONAL AND TRANSLATIONAL REGULATION OF 1

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of

Philosophy in the Graduate School of The Ohio State University

By

Shawn M. Pierson, B.S.

******

The Ohio State University

2005

Dissertation Committee

Professor Anthony Young, Adviser Approved by:

Assoc. Professor John Bauer

Assoc. Professor Dale Hoyt ______Adviser Professor Michael Ostrowski College of Pharmacy

ABSTRACT

The nitric oxide (NOS) catalyze the formation of nitric oxide (NO) and from the reactants and oxygen. Three isoforms of the have been discovered and designated NOS1, NOS2 and NOS3. NO produced by

NOS1 plays a role in a variety of physiological processes including learning and memory, bronchial relaxation and GI motility. Excessive production of NO can be toxic to and is associated with various pathophysiological conditions. This dissertation explores the regulation of the encoding for NOS1 in order to gain insights into its normal and abnormal regulation.

Using PC12 cells, we studied the effect the peptide pituitary derived adenylate cyclase activating peptide (PACAP38), a 38 peptide hormone, had on endogenous NOS1 expression. We showed that PACAP led to an increase in both

NOS1 mRNA and . Further, it was demonstrated that the transcript upregulated was a shortened transcript driven by a promoter located in what is normally exon 2. . PACAP38 is able to activate a variety of second messenger pathways when binding to its receptors. Among these are the protein A (PKA) and protein kinase C (PKC) pathways. Treatment of PC12 cells with forskolin, a drug that activates the PKA pathway led to a mild increase in NOS1 mRNA and protein

- ii - that was smaller than that observed in cells treated with PACAP. When cells were treated with both forskolin and TPA, a drug that activates PKC, the increases in NOS1 protein and mRNA were similar to that observed with PACAP. Thus PACAP may have to activate both pathways to fully activate NOS1 expression.

We next looked at the effect of PACAP38 had on the rat and human exon 2 promoters. We showed both of these promoters were stimulated by PACAP and required the activation of multiple pathways for full activation.

We next looked at the effect of PACAP38 on another human NOS1 promoter, the

5’2(1F) promoter. We demonstrated that the activation of the cAMP-PKA pathway was totally responsible for activation of this promoter by PACAP38. We then mapped the region of 5’2 necessary for PACAP stimulation to a 230 region just upstream of the 5’2 alternate first exon and that a single CRE site within this region was essential for PACAP38 stimulation.

Lastly, we cloned and characterized two novel promoters of NOS1. The first promoter drives the expression of a NOS1 transcript containing a novel alternate first exon discovered by Greg Hartt working in our lab. This exon was designated 5’3 by us and 1D by others. The second promoter drives the expression of a transcript containing a second alternate first exon discovered by Dr Hartt and designated 5’4 by us and 1C by others.

iii

ACKNOWLEDGMENTS

I would like to thank my adviser, Tony Young, for his advice and encouragement in completing this project, for giving me the freedom to explore my own ideas and for pushing me when necessary.

I would like to thank my family for their support in this endeavor. Even when they did not understand why I was doing this, they were always supportive.

I thank my labmates past and present for their helpful discussions and for showing me how to use the equipment. In the beginning, Wei-Kang Chen, Terrie Rife and Greg Hartt were very helpful. I am grateful to Linda Zhang for showing me the tricks of cloning and to Deyu Zhang for his help with my first western blots. Kunyi

Wu was quite helpful with the animal work and this is greatly appreciated as was Hua

Wei’s assistance with the real time PCR.

I would also like to thank the students, staff and faculty of the division of pharmacology, College of Pharmacy for their help and friendship.

Finally, I want to thank the faculty, staff and students of the Center for

Molecular Neurobiology. Whenever I needed to try a new technique, I could always find someone who had performed it before somewhere in the center and they were always willing to help (and sometimes even loan me equipment and supplies).

This work was supported by a grant from the National Institutes of Health.

iv

VITA

September 1, 1971...... Born- Columbus, OH

1994...... B.S. Pharmacy, The Ohio State University

1994-1997...... Staff Pharmacist Childrens Hospital Columbus, OH

1997-present...... Graduate Teaching and Research Associate, The Ohio State University

FIELDS OF STUDY

Major Field: Pharmacy

v

TABLE OF CONTENTS

Abstract...... ii

Acknowledgments...... iv

Vita...... v

List of figures...... viii

List of Tables...... x

Chapters:

1. Introduction...... 1

The chemistry of nitric oxide...... 1 The production of nitric oxide...... 5 The NOS1 gene...... 7 The NOS1 protein...... 12 Physiological roles of NO produced by NOS1...... 17 Roles of NOS1 in disease...... 21 An introduction to PACAP...... 24

2. NOS1 Regulation by PACAP...... 39

Introduction...... 39 Materials and Methods...... 41 Results...... 52 Conclusions and discussion...... 73 Chapter references...... 81

3. Effects of PACAP on various NOS1 promoters...... 84

Introduction...... 84 Materials and Methods...... 87 Results...... 93 Conclusions and Discussion...... 107

vi Chapter references...... 112

4. Cloning and Characterization of 2 Novel Promoters of NOS1...... 113

Introduction...... 113 Materials and Methods...... 115 Results...... 126 Conclusions and discussion...... 135 Chapter references...... 144

List of References...... 146

vii

LIST OF FIGURES

Figure Page

1.1 Structure and biologically important reactions of NO...... 4

1.2 Reaction catalyzed by the Nitric Oxide Synthases (NOS)...... 6

1.3 Genomic map of the human NOS1 alternate first exons...... 9

1.4 Map of the NOS1 protein domains...... 14

1.5 Some of the Signal transduction Pathways Activated by PACAP...... 27

2.1 Time Course of NOS1 protein expression in PC12 cells treated with PACAP...... 53

2.2 Time Course of NOS1 protein expression in PC12 cells treated with forskolin...... 54

2.3 NOS Assay using extracts from PC12 cells treated with NGF and forskolin...... 55

2.4 Time course of NOS1 expression in PC12 cells treated with forskolin and TPA...... 57

2.5 Effect of TPA on NOS1 protein expression...... 58

2.6 Immunoprecipitation of NOS1 from PC12 cells...... 59

2.7 Real time PCR for NOS1 in PC12 cells ...... 61

2.8 Real time PCR for NOS1 in PC12 cells treated with PACAP38...... 63

2.9 Half life of NOS1 in forskolin, forskolin + TPA and PACAP treated PC12 cells...... 66

2.10 Diagram of RT-PCR assay...... 68

viii 2.11 RT-PCR assays for NOS1 in forskolin and TPA treated PC12 cells...... 69

2.12 RT-PCR for NOS1 in NGF and PACAP treated PC12 cells...... 71

3.1 Sequence alignment of mouse, rat and human exon 2 5’UTR...... 86

3.2 Primers used in the construction of 1613-1842CREmut...... 92

3.3 Inhibition of forskolin and PACAP upregulation of luciferase by H89...... 94

3.4 Mutation of CRE sites attenuates PACAP and forskolin mediated luciferase upregulation...... 95

3.5 Stimulation of the Human E2 promoter by TPA and forskolin...... 97

3.6 PACAP stimulates the rat E2 promoter...... 98

3.7 PACAP38 Stimulates 5’15’2 Driven Luciferase expression in PC12 cells...... 100

3.8 The 5’2 promoter is responsible for PACAP38 stimulated increases in luciferase activity...... 101

3.9 Mapping of the NOS1 5’2 Promoter...... 103

3.10 H89 Inhibits Luciferase upregulation due to PACAP38 treatment...... 105

3.11 Mutation of CRE site inhibits upregulation of luciferase expression by PACAP38...... 106

4.1 Map of the lambda clone containing 5’3(1D) and 5’4(1C)...... 127

4.2 Sequence of 2.6KB insert...... 129

4.3 Luciferase assays using HeLa extracts transfected with various 5’3 and 5’4 constructs...... 131

4.4 Luciferase assays using cell extracts of cells transiently transfected with 5’3(1D) deletion constructs...... 133

4.5 Luciferase assays using PC12 cell extracts...... 134

4.6 Partial genomic map of the human NOS1 gene...... 138

ix

List of Tables

Table Page

2.1 Antibodies used in this chapter...... 48

2.2 Sequences of PCR primers used in this chapter...... 51

4.1 PCR primers utilized in chapter 4...... 125

4.2 Alternate first exons of the Human NOS1 gene...... 137

x

CHAPTER 1

INTRODUCTION

Since the Nobel Prize winning discovery that the gas nitric oxide (NO) is actively produced by mammalian endothelial cells and plays an important role in the regulation of pressure and in immune function, a large volume of research has amassed on the production and functions of this small signaling molecule. (Ignarro et al. 1987) This dissertation describes various aspects of the transcriptional and translational regulation of the gene encoding one of that catalyzes the production of NO, 1 (NOS1). In particular, the effect of the pituitary derived adenylate cyclase activated peptide (PACAP) on NOS1 expression is explored. This first chapter gives a brief introduction to NO and the nitric oxide synthases. It includes sections on the chemistry of NO, its production, its physiological roles and its possible roles in disease. It also gives a brief introduction to PACAP, its physiological roles and signaling pathways activated by it.

The chemistry of nitric oxide

Nitric oxide is a gaseous free radical (see figure 1). As with all free radicals, it is rather reactive and has a very short half-life. In blood, this half-life has been

1 estimated to be as little as 1.8 milliseconds. (Liu et al. 1998) As could be deduced based on its chemical structure, NO participates in a variety of biologically important reactions. It is able to react with the iron moiety of , with thiol groups of cysteine residues and with both molecular oxygen and the radical (Wink et al. 2000; Gow et al. 2001). The reactions with molecular oxygen and superoxide result in the formation of a variety of reactive nitrogen oxygen species (RNOS) (see figure 1b). These species can then undergo destructive reactions with lipids, DNA and . (Gow et al. 2001)

The reaction of nitric oxide with the iron moiety of has a variety of biological implications. Reaction of NO with the heme constituent of , for example, results in its activation and the production of cyclic GMP. This is one of the major pathways activated by nitric oxide and is the pathway by which NO dilates blood vessels. (Ignarro et al. 1995) The iron-heme centers of a variety of other enzymes also react with and thus are regulated by nitric oxide. For example, the cytochrome P450 enzymes, a group of enzymes important in the of various drugs, are down regulated by nitric oxide. (Khatsenko et al.

1993) Finally, NO is able to decrease its own activity by binding to the cytochrome P-

450 like domain. (Griscavage et al. 1995) The above are just representative examples and any iron or heme containing enzyme could potentially be regulated by NO.

Finally, NO is able to react with heme irons with or without bound oxygen. For this reason, reactions with hemoglobin (and ) are probably the major method of

NO ‘inactivation’ (Lancaster 1994).

2 The reaction of NO with thiol groups such as those of cysteine results in the formation of S-nitrosothiol (see figure 1B) (Gow et al. 2001). It appears that this reaction is specific for certain cysteine residues and that this modification affects the activity of the protein. For example, p21Ras, a small , is activated when it is

S-nitrosylated while Jun N-terminal kinase (JNK) is deactivated when it is S- nitrosylated. (Lander et al. 1996; Park et al. 2000)

In addition to modifying proteins, the NO will react with oxygen or superoxide to form a variety of reactive nitrogen oxygen species (RNOS). These species can then react with DNA, lipids and proteins damaging them. This is a major mechanism by which aberrant expression of the that produce NO contribute to disease. The major RNOS formed include N2O3, ONOO-, NO- and NO2 (see figure

1A). (Wink et al. 2000)

N2O3 has been implicated in a variety of adverse reactions including S- nitrososation (described above) and the deamination of nucleic acids which can result in DNA strand breakage (Wink et al. 2000). The other three reactive oxygen nitrogen species are strong oxidants. (ONOO-) in particular is a very potent oxidant that can cause lipid peroxidation, DNA strand breakage and thiol oxidation

(Ducrocq et al. 1999).

In conclusion, nitric oxide is a gaseous free radical capable of taking part in a variety of relevant biological reactions. Some of these reactions are important for its signaling properties while others can be detrimental to cells. NO reacts with iron and iron-heme moieties, with thiol with oxygen and with the superoxide radical. The

3

A. Chemical Structure of Nitric Oxide

B. Important Reactions

1). NO + Fe(III) Fe(III)-NO

- 2). NO + Fe(II)-O2 Fe(III) + NO3

3). NO + R-SH R-SNO(H)

- - 4). NO + O2 ONOO

5). 4NO + O2 2N2O3

Figure 1.1: Structure and biologically important reactions of NO. Figure 1A shows the chemical structure of Nitric oxide. Note the unpaired electron in red. Figure 1B shows some of the important reactions that nitric oxide undergoes in vivo including reactions with Iron, Thiol groups, Oxygen and superoxide

4 reactions with iron are often associated with its second messenger functions. The thiol reactions may also play a role in signaling. The reactions of NO with oxygen and superoxide result in the formation of a variety of RNOS, some of which can be damaging to the cell.

The production of nitric oxide.

Nitric oxide is produced by the oxidation of the guanidino nitrogen of L- arginine by molecular oxygen. L-citrulline is produced as a byproduct of this reaction

(see figure 2) (Marletta et al. 1988; Palmer et al. 1988; Bredt et al. 1989). Three have been identified that catalyze this reaction and have been designated nitric oxide synthase (NOS) 1, NOS2 and NOS3. These enzymes require a variety of cofactors including NADPH, FAD, FMN, , and . As this paper concentrates on the regulation of NOS1, a brief description of the nitric oxide synthases will be followed by a more in depth look at what is known about

NOS1.

Nitric oxide synthase 1 (NOS1), also known as nNOS or neuronal NOS, was first isolated from rat brain. (Bredt et al. 1990) Since its discovery, NOS1 has been found in a variety of other tissues including , kidney, heart lung and penis.(Burnett et al. 1993; Wang et al. 1999) As one may expect from its wide distribution, it is believed to play a role in a variety of physiological processes. This enzyme is constitutively expressed in some cells but has been found to be induced under some circumstances such as in models of ischemic .

5

NH 2 NH2

+ C NH 2 C O

CH2 + O 2 CH 2 + NO NADPH FMN CH 2 CH2 FAD Ca+2/ CH 2 CH2 Calmodulin - + BH4 + - H 3 N CH COO H3N CH COO

Figure 1.2: Reaction catalyzed by the Nitric Oxide Synthases (NOS). The guanodino nitrogen is oxidized by molecular oxygen forming NO and citrulline. These enzymes require a variety of co-factors including NADPH, FMN, FAD, Calmodulin, and tetrahydrobiopterin (BH4)

6 NOS2 (also known as iNOS or inducible NOS) was first identified in activated . (Marletta et al. 1988; Stuehr et al. 1991) When its production is induced by various , this enzyme produces large amounts of NO. This release of NO plays an important role in the destruction of invading pathogens. Like all of the isoforms of NOS, a -calmodulin is required, but unlike the other 2 isoforms, NOS2 binds this cofactor with such affinity that it is active at all normal physiological calcium concentrations. (Stuehr et al. 1991)

NOS3 was first identified in endothelial cells, but had since been found in a variety of different including the brain. (Pollock et al. 1991) The NO produced by this enzyme plays a major role in the regulation of vascular tone and thus blood pressure.(Ignarro et al. 1995) As with NOS1, this enzyme was first thought to be constitutively expressed (and is in some tissue), but has since been found to be inducible under certain circumstances. (Forstermann 2000) Also like NOS1, NOS3 requires a calcium-calmodulin cofactor and is activated by increases in cytosolic calcium.

The NOS1 Gene

NOS1 is encoded by a large and complex gene. Using fluorescent in-situ hybridization, it has been found to reside on the long arm of 12

(12q24.2). (Xu et al. 1993) It consists of 29 exons and 28 introns and the entire gene spans over 250 Kb. The open reading frame of this gene is 4302 base pairs and encodes for a 1434 amino acid protein.(Ignarro et al. 1995) Translation normally begins in exon 2 and terminates in exon 28. The NOS1 gene gives rise to a large

7 number of mRNA transcripts.(Xie et al. 1995; Wang et al. 1997; Wang et al. 1999;

Wang et al. 1999) These transcripts differ at the 5’ untranslated region (UTR), the 3’

UTR and a there are a few variations that occur within the coding regions of the gene.

Adding to the complexity of this gene is the fact that multiple promoters drive its transcription.

The NOS1 gene gives rise to a multiple transcripts that differ in their 5’ untranslated regions. In the human, 12 alternate first exons have been identified and designated 1A – 1L and range in size from 67 base pairs to 523 base pairs (Xie et al.

1995; Wang et al. 1999; Boissel et al. 2003; Hartt 2003). In some cases, it has been clearly demonstrated using reporter gene assays that separable promoters drive the expression of these alternate transcripts. This is the case with alternate first exons 1F,

1G and 1D. (Xie et al. 1995) In other cases, separable promoters can be inferred based on the genomic position of the alternate first exon. This is probably the case with exons 1A, 1E and 1L. Transcripts with alternate exons 1H-1K have yet to be shown to have separable promoters. It is quite possible that these different transcripts share some cis elements while some are unique. As translation predominantly begins in exon 2, these alternate transcripts give rise to the same protein. In the rat, a total of 5 different alternate exons have been discovered. (Lee et al. 1997; Oberbaumer et al.

1998) At present, 3 alternate first exons have been identified in the mouse. (Brenman et al. 1997) As with the human gene, these alternate first exons do not change the protein for which the transcript encodes (though it may well affect the efficiency of translation). In addition to the above, there appears to be a promoter in exon 2 that

8

Exon 1G Exon 1H Exon 1C Exon 1F Exon 1I Exon 1B Exon 1D Exon 1J/1K Exon 1A Exon 1E Exon 1L Exon 2

NOS1 Partial genomic map 130000 bp

Figure 1.3: Genomic map of the human NOS1 alternate first exons. Above is a scale partial genomic map of showing the relative location of the known 12 alternate first exons and the common exon 2 of the human NOS1 gene. Alternate exon nomenclature utilized in the map is that proposed by Forsterman et al.

9 gives rise to a truncated transcript in mouse, rat, and human (Sasaki et al. 2000) (and

Chen and Young, unpublished observations).

Given that the RNA diversity in the 5’ UTR does not affect the protein expressed, one has to ask why there is so much variety. One explanation is that differential expression of these alternate transcripts is one way the expression of

NOS1 is temporally and spatially regulated. There is some evidence to support this hypothesis. Using RACE cloning, it has been shown that, in humans, different alternate first exons were preferentially expressed in certain tissue.(Wang et al. 1999)

In rat, an exon expressed only in kidney has been identified as well as one expressed predominantly in skeletal muscle (Lee et al. 1997; Oberbaumer et al. 1998). Of course, other NOS1 transcripts containing alternate first exons have a wide tissue distribution. Temporal regulation of the transcript has also been demonstrated. In rat, an alternate first exon (1B) expressed only in embryonic tissue has been observed, while in human tissue, an alternate exon specifically excluded from fetal tissue has been observed (Hartt and Young, unpublished observations).

In addition to those transcripts differing at the 5’ UTR described above, a variety of other transcripts arise from the NOS gene. Among these are NOS1β and

NOS1γ. These alternate transcripts were first identified in transgenic mice in which a targeted disruption of exon 2 of the NOS1 gene was carried out. A small residual amount of NOS1 activity was detected in these mice. Ultimately, two alternate transcripts lacking exon 2 were detected. NOS1β consists of the mouse alternate exon

1A spliced to exon3 while NOS1γ consists of mouse exon 1B spliced directly to exon

10 3. Unlike the variations in the 5’ UTR, the NOS1β and NOS1γ alternate transcripts produce truncated proteins. Translation of the NOS1β transcript begins at a CTG codon located within exon 1A and encodes for a 136kD protein. While this protein apparently has full catalytic activity, it lacks the PZD domain encoded by exon 2.

This domain is responsible for the localization of NOS1 to the post synaptic density

(PSD) in a ; therefore it is likely that the NOS1β protein has a different intracellular distribution than its full length counterpart. Translation of the NOS1γ begins at an ATG codon located in exon 5 and it encodes for a 125 kD protein. This protein lacks significant catalytic activity but this it may possibly play a regulatory role (Eliasson et al. 1997).

NOS1µ is another alternate transcript that has been characterized in rodents.

This alternate transcript contains a 102 bp insertion between exons 16 and 17, encoding a, protein that is 34 amino acids larger that normal NOS1. This protein has full catalytic activity and is found only in skeletal and cardiac muscle. In humans, this alternate transcript has been detected by rt-PCR but the expression of the protein has yet to be confirmed (Eliasson et al. 1997; Lin et al. 1998).

Some alternate transcripts exist that have deletions of various exons. An example of this is nNOS2. This transcript has a 105 base pair deletion corresponding to exons 9 and 10 of full length NOS1. This results in a non-functional protein.

Despite this, this alternate transcript appears to be developmentally regulated. The physiological significance of these observations has yet to be determined. (Iwasaki et al. 1999)

11 Finally, a variety of alternative transcripts are specifically found in the testis.

Using Rapid Amplification of cDNA ends (RACE), a PCR method utilized to detect and clone the ends (5’ or 3’ depending on the method used), Wang and colleagues discovered a variety of alternate transcripts specific to the testis that are truncated at the 5’ end terminus. These transcripts all encoded for a protein that was truncated at the amino terminus but appeared to be functional, though it lacked the PDZ domain of full length NOS1. (Wang et al. 1997) Recent studies of the promoter driving the expression of one of these alternate transcripts have shown that it specifically drives the expression of this transcript in the Leydig cells of the testis. (Wang et al. 2002)

In conclusion, the human NOS1 gene is a large and complex gene. This gene, which encompasses approximately 250kb of chromosome 12, gives rise to a multitude of transcripts and multiple promoters drive its transcription. Differential regulation of these transcripts helps to explain the mechanisms by which NOS1 is spatially and temporally regulated.

The NOS1 Protein

Full length NOS1 protein consists of 1434 amino acids and has a molecular weight of approximately 155kD. This enzyme must dimerize in order to be active and requires a variety of co-factors such as FMN, FAD, calmodulin, tetrahydrobiopterin, and NADPH. The protein also has an associated heme moiety that binds to the domain of the enzyme.

12 The domain structure of the NOS1 enzyme has been mapped and is shown in figure 3 below. It appears that the NOS1 enzyme consists of 2 functional domains that operate independently of one another. (Sheta et al. 1994) The N-terminal domain serves as an oxygenase. This domain binds the heme moiety, which in turn binds the oxygen. The arginine is also found in this domain. The C-terminal domain is the domain. This domain interacts with and acquires electrons from NADPH. These electrons are then ‘passed’ through the FAD and FMN cofactors that also bind to the reductase domain. The electrons are then passed to the iron bound to the heme. Once the iron is reduced (to FeII), it can bind the oxygen necessary for the reaction to occur. As mentioned above, a calcium/calmodulin binding region is also present and lies between the two functional domains. Calmodulin binding is required for the enzyme to be active and this only occurs when calcium concentrations are elevated.

Enzymatic activity of NOS1 is regulated at the post-translational level. The primary regulator of NOS1 activity at this level is calcium. As mentioned above, a calmodulin cofactor is required for activity and this cofactor only binds at higher than normal calcium concentrations. Phosphorylation is another mechanism by which the

NOS1 protein is regulated. Finally, as NOS1 has a heme/iron cofactor that is required for the binding of oxygen, it is possible that NO itself may act as a negative feedback regulator of NOS1

It has been shown that phosphorylation of serine (847) by various calmodulin dependant decreases NOS1 activity. This attenuation of enzyme activity is

13

Heme CaM FMN FAD NADPH NH2 COOH Oxygenase domain Reductase domain

Figure 1.4: Map of the NOS1 protein domains. Above is a map of the NOS1 enzyme. The enzyme consists of 2 functional domains, a reductase domain and an Oxygenase domain. The reductase domain binds the NADPH, FAD, FMN and calmodulin (CaM) cofactors. The Oxygenase domain binds the Heme, arginine and tetrahydrobiopterin (BH4) cofactors.

14 apparently due to a decrease in the affinity of NOS1 for calmodulin. (Hayashi et al.

1999) It is interesting to note the activity of these kinases is also calcium dependent.

Thus, an increase in the concentration of calcium both turns on the enzyme and limits its activity. Of course, the phosphorylation of serine 847 is reversible. Several phosphatases have been implicated in this process including calcineurin and protein phosphatase 2A. (Rameau et al. 2003; Rameau et al. 2004) (Komeima et al. 2001)

It also appears that NOS1 is phosphorylated on a different serine and threonine residues by protein kinase C (PKC). (Nakane et al. 1991; Bredt et al. 1992) The physiological effect of this phosphorylation is not clear. In-vitro, phosphorylation by

PKC leads to a slight increase in NOS1 activity (Nakane et al. 1991) On the other hand, when cells stably transfected with NOS1 are treated with TPA, a drug that activates PKC, there is a decrease in NOS activity. (Bredt et al. 1992)

Protein Kinase A can also phosphorylate NOS1 (Brune et al. 1991; Bredt et al.

1992; Dinerman et al. 1994). The effects of phosphorylation by PKA are also not clear. Phosphorylation of NOS1 by PKA in vitro does not effect the enzymes activity

(Brune et al. 1991; Bredt et al. 1992). In some studies, treatment of HEK-293 cells stably transfected with NOS1 with drugs that stimulate cAMP did not effect NOS1 activity(Bredt et al. 1992). Others showed a decrease in activity using this paradigm

(Dinerman et al. 1994). Of course, phosphorylation may also lead to changes in cellular localization, but this remains to be evaluated.

Protein kinase G (PKG), a cyclic GMP dependent protein kinase, is also able to phosphorylate NOS1. This kinase appears to phosphorylate NOS1 at the same site (or

15 sites) as PKA (Dinerman et al. 1994) Phosphorylation by PKG appears to decrease the activity of the enzyme. This could be of some importance as cGMP levels increase due to increased levels of NO. (NO activates soluble guanylate cyclase) Thus phosphorylation of NOS1 by PKG could act as a negative feedback mechanism preventing overproduction of NO. (Dinerman et al. 1994)

Finally, NO itself may act as a negative regulator of NOS1. This was first hypothesized by Ignarro and colleagues when they noticed that the formation of NO and citrulline was not linear with respect to time. (Rogers et al. 1992; Griscavage et al. 1995) They suspected this was due to inhibition of the enzyme by one of the products of the reaction (NO or citrulline). As the addition of oxyhemoglobin caused the production of citrulline to be linear, it was suspected that NO was inhibiting the

NOS1 enzyme. To test this hypothesis, crude cytosolic extracts of rat cerebellum were treated with either NO, NO donors or citrulline. In the NO and NO donor treated extracts, NOS1 enzyme activity was reduced. This effect was reversed by the addition of oxyhemoglobin, which scavenges NO. Similar results were obtained when purified

NOS1 was used. The mechanism of this appeared to be an interaction of NO with the

Heme moiety associated with NOS1. (Griscavage et al. 1995). Whether NO plays a role in the regulation of NOS1 in vivo is unclear though as at least one report utilizing a neuroblastoma cell line showed an increase in NOS1 in intact cells treated with NO donors (Hu et al. 1995). This contrasts with the results of experiments using cell lysates and purified NOS1.

16 To summarize, the NOS1 protein is one of three enzymes that catalyzes the formation of NO from the reactants oxygen and arginine. This enzyme is requires a large number of cofactors including NADPH, FAD, FMN, calmodulin, tetrahydrobiopterin and heme. This enzyme is also highly regulated. Calcium regulates this enzyme as elevated calcium levels are required for the calmodulin cofactor to bind to the enzyme. NOS1 is also phosphorylated by various kinases including the calmodulin kinases, protein kinase A, protein kinase C and protein kinase G. Finally, NO itself reduces the activity of NOS1.

Physiological roles of NO Produced by NOS1

NO produced by NOS1 has been implicated in a variety of normal physiological functions. Included among these are long term potentiation (LTP) in neurons, sexual function, pituitary hormone release, and regulation of the release of other such as . In addition, NO is also a in the GI tract and in the bronchi (Belvisi et al. 1992; Takahashi 2003). In this section, some of the more common physiological roles of NO produced by NOS1 are described.

Long-term potentiation (LTP) can be defined as ‘a persistent increase in synaptic strength that can be rapidly induced by brief neural activity’. (Zigmond et al.

1999). LTP is believed to be a cellular mechanism of learning and memory. During the formation of LTP, changes occur in both the presynaptic and postsynaptic neurons.

For this reason, it has been a hypothesized that the postsynaptic neuron releases some substance that triggers the changes observed in the presynaptic neuron. Typical

17 neurotransmitters only ‘transmit’ in one direction. NO, on the other hand, can diffuse across membranes and exert its affect on surrounding neurons including those

‘upstream’ of the neuron producing the NO. This is termed retrograde transmission and there is a considerable amount of evidence that NO acts as a retrograde messenger in LTP. A variety of studies have shown that NOS antagonists reduce the formation of LTP in hippocampal slices. Further, hemoglobin, a scavenger of NO, also inhibits

LTP formation in hippocampal slices. (O'Dell et al. 1991; Schuman et al. 1991; Haley et al. 1992) Utilizing a hippocampal cell culture system, Hawkins and colleagues have shown that the intracellular injection of a membrane impermeant NO scavenger

(oxymyoglobin) to either the presynaptic neuron or the postsynaptic neuron inhibited

LTP. In contrast, the injection of a NOS inhibitor was only able to inhibit LTP when it was injected into the post-synaptic neuron. This data implies that NO is produced in the post-synaptic neuron, then diffuses to the pre-synaptic neuron and exerts its effects. (Arancio et al. 1996)

The above data provides convincing evidence that NO plays an important role in LTP. There is conflicting data as to which isoform of nitric oxide synthase is responsible for the production of NO in LTP. It appears as both NOS3 and NOS1 play a role in the production of NO during LTP at least in the hippocampus. Evidence to support this includes the fact that both NOS1 and NOS3 are present in the hippocampus and that both isoforms must be disrupted in order for LTP to be impaired. (Son et al. 1996)

18 There is large body of evidence implying that NO produced by NOS1 is an important inhibitory neurotransmitter in the GI tract. It has been shown that nitric oxide synthase inhibitors hinder the relaxation caused by stimulation of non-adrenergic non-cholinergic (NANC) neurons. Further, isolated dog ileocolonic junction in tissue bath releases NO from nerves of the myenteric plexus when electrically stimulated. (Bult et al. 1990) Using similar techniques NO also has been shown to be released upon stimulation of nerves in the fundus and is important in relaxation of the smooth muscle of the stomach. (Boeckxstaens et al. 1991) Since these initial studies, NO has been found to be an important inhibitory neurotransmitter in a wide variety of GI structures. (Takahashi 2003)

Studies indicate that the NOS1 isoform appears to be the isoform responsible for NO production in the enteric . For example, utilizing NADPH diaphorase staining, NOS1 has been localized to the enteric nervous system in rat.

(Aimi et al. 1993) Also supporting this is the observation that in mice with a targeted disruption of the NOS1 gene, the stomachs are enlarged and there is associated hypertrophy of the circular muscle. (Huang et al. 1993) Finally, NANC mediated lower esophageal sphincter relaxation is impaired in NOS1 knockout mice while

NOS3 knockout mice were not impaired. (Kim et al. 1999)

In addition to being important in NANC nerve mediated GI relaxation, NO produced by NOS1 appears to be essential for NANC nerve mediated bronchial relaxation in humans. Utilizing isolated human tracheal segments; Belvisi and co- workers showed that the NOS inhibitor L-NAME inhibited the relaxation normally

19 observed with electrical stimulation. This inhibition was reversed by L-arginine. This indicated that NO was a neurotransmitter in neural mediated bronchial relaxation.

(Belvisi et al. 1992) Evidence that NOS1 is the source of the NO involved in this neural mediated bronchial relaxation comes from experiments using trachea from

NOS1 knockout mice. In the trachea from these mice, relaxation due to electrical stimulation was diminished as compared to wild type mice. (Hasaneen et al. 2003).

NO produce by NOS1 plays a vital role in penile erection. Evidence of this includes the observation that non-selective NOS and selective NOS1 antagonists inhibit penile erection in rats. (Spiess et al. 1996) Like long term potentiation, there is still some confusion as to which isoform of NOS is involved in penile erection and quite likely NOS1 and NOS3 both play a role. NOS1 is localized to nerves of the penis while NOS3 is present in the vessels of the corpus cavernosa. (Burnett et al.

1993) One model that has been suggested is that NOS1 is required for initiation of penile erection while NOS3 is required for maintenance of the erection. (Hurt et al.

2002) Evidence of the importance of NOS1 has come from experiments using NOS1 knockout mice. Although these mice are able to reproduce normally, their ability to get an erection in response to nerve stimulation is diminished. In contrast, NOS3 knockout mice are unaffected. Further, , a drug that inhibits phosphodiesterase type 5, thus enhancing the NO/ cGMP pathway, is unable to reverse this deficiency. This indicates that NOS1 is required to make the NO that stimulates this pathway (Cashen et al. 2002). Further evidence of the involvement of NOS1 comes from experiments using 7-nitroindazole. This drug is selective for NOS1 in

20 vivo and was able to inhibit erections due to nerve stimulation in the rat as doses that did not increase blood pressure (and thus presumably were not inhibiting the NOS3 isoform). (Spiess et al. 1996)

NO also plays a role in the regulation of serotonin release. Male mice with a targeted disruption of the NOS1 gene are abnormally aggressive. (Nelson et al. 1995)

As the serotonin system plays a role in aggression, it was hypothesized that the aggressiveness in these mice may be caused by an abnormality in the serotonin system. Nelson and colleagues tested this hypothesis using NOS (-/-) mice. They showed decreased serotonin turnover in these mice. Additionally, they demonstrated that treatment of these mice with a serotonin precursor increased serotonin turnover and decreased aggression while treatment with an inhibitor of serotonin production increased aggression in wild type mice but not in NOS1 knockout mice. (Chiavegatto et al. 2001) NO also appears to affect the release of serotonin in the hypothalamus, the raphe nuclei and frontal cortex of the rat. (Smith et al. 2000)

Roles of NOS1 in Disease

As described above, the NO produced by NOS1 participates in a variety of physiological functions. Despite this, NO is a free radical and there is evidence that when produced inappropriately, NO participates in a variety of disease processes.

Aberrant NO production has been implicated in ischemic stroke, various neurodegenerative diseases, , and many others.

21 Ischemic stroke is the occlusion of a vessel that blocks blood flow to a portion of the brain. Stroke can be modeled in animals by occlusion of the middle cerebral artery. This is often called the middle cerebral artery occlusion model (MCAO). This occlusion can be permanent, modeling ischemia or transient, modeling ischemia and reperfusion.

There is considerable evidence that NO produced by NOS1 plays a role in the neuronal death associated with ischemic stroke. For example, Zhang and colleagues, using the MCAO model of stroke in rats, showed that NOS1 was up-regulated in neurons. The number of NOS1 positive neurons, and the amount of NOS1 mRNA increased shortly after occlusion and this increase in NOS1 expression preceded morphological changes. (Zhang et al. 1994) Further support for the involvement of

NOS1 in stroke came from experiments using NOS1 knockout mice. Occlusion of the middle cerebral artery in these mice resulted in smaller infarct areas than those observed in wild type littermates. (Huang et al. 1994) Further, there was less functional damage in the NOS1 (-/-) mice. (Huang et al. 1994) Similar results were observed in models ischemia/ reperfusion. (Hara et al. 1996) Finally, treatment of rats with selective NOS1 inhibitors (but not non-selective NOS inhibitors) results in reduced infarct areas in a model of ischemia/ reperfusion. (Escott et al. 1998)

Exposure of neurons to relatively large concentrations of glutamate or N- methyl-D-aspartate (NMDA) results in cell death. This is termed and is believed to be one mechanism of cell death in ischemic stroke. NO may play a role in excitotoxicity. The exact role NO plays in excitotoxicity, if any, is rather

22 controversial. Some researchers have found that NOS inhibitors and NO scavengers protect cultured neurons from the effect of large doses of NMDA while others see little or no decrease in cell death. (Dawson et al. 1991; Hewett et al. 1993) In vivo data is also not clear. One study showed that injection of 7-nitroindazole (7-NI) decreased the lesion size in the of rats who received an intrastriatal injection of NMDA. (Schulz et al. 1995) A second study directly contradicts this, finding that

7-NI does not protect the striatum from NMDA toxicity. (Loschmann et al. 1995)

NO produced by NOS1 is also implicated in neurodegenerative diseases.

Parkinson’s disease is a neurodegenerative disease characterized by the death of neurons in the substantia nigra and is associated clinically with a variety of movement disorders (Bannister 1992). A large body of research implicates aberrant NOS1 expression in Parkinson’s disease (PD). It has been found that NOS1 is upregulated in the neutrophils and basal ganglia of PD patients (Gatto et al. 2000) Further evidence of NOS1 involvement comes form experiments carried out using the MPTP model of

Parkinson’s disease. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that reproduces many of the effects of PD in experimental animals.

(Muramatsu et al. 2002) In mice treated with MPTP, NOS1 was significantly upregulated in the substantia nigra. (Muramatsu et al. 2003) Using baboons, Ferrante and colleagues showed a marked increase in nitrotyrosine immunoreactivity.

Nitrotyrosine is a marker for increased levels of NO. (Ferrante et al. 1999) Finally, administration of the relatively selective NOS1 antagonist 7-nitroindazole protects mice and baboons administered MPTP. (Hantraye et al. 1996; Watanabe et al. 2004)

23 Alzheimers disease is the most common cause of senile (Feldmann

1994). Though less compelling than that for Parkinson’s disease, there is some evidence for aberrant expression of NOS1 in Alzheimers disease. For example it has been shown that the amount of NOS1 present in various neuronal subtypes increases as the severity of Alzheimer’s increases and that there are morphological changes in other NOS1 producing neurons. (Fernandez-Vizarra et al. 2004) NOS1 has also been found uniquely in reactive astrocytes in patients with Alzheimer’s disease. (Simic et al. 2000) It has been shown that the increase in the nitric oxide synthase results in an increase nitrotyrosine immunostaining. (Luth et al. 2002) As the nitration of is usually carried out by peroxynitrite, a product of the reaction of NO and superoxide, this increased staining is indicative of increased . (Luth et al. 2002)

In addition to stroke and the neurodegenerative disorders, NOS1 has been implicated in a variety of other disease processes. For example, a polymorphism of

NOS1 has a higher occurrence in asthmatics. (Grasemann et al. 2000) Also, NOS1 is altered in a variety of GI disorders.(Takahashi 2003) NOS1 has been implicated in a variety of other pathologies and a complete review is beyond the scope of this paper.

An Introduction to PACAP

PACAP is a peptide hormone that was first isolated from sheep hypothalamus based on its ability to increase cAMP levels in rat anterior pituitary cells (Miyata et al.

1989). Full length PACAP consists of 38 amino acids but contains an internal cleavage site that gives rise to a 27 amino acid peptide. This 27 amino acid peptide also has biological activity. This peptide appears to be highly conserved among many

24 different organisms (Vaudry et al. 2000). The peptide, like many peptide hormones, is translated as part of a larger protein and is then cleaved from this protein before being secreted from the cell (Ohkubo et al. 1992) .

PACAP receptors have been divided into two major classes based on their affinity for PACAP and vasoactive intestinal peptide (VIP). Both classes of receptors are 7 transmembrane G-protein coupled receptors. The first class of receptors, called type I, have a 1000 fold higher affinity for PACAP38 (and 27) than for VIP. The second class of PACAP receptors, designated as type II receptors, have about the same affinity for PACAP as they do for VIP. At present only one subtype of type I receptor has been identified. This receptor is named PAC1. Multiple splice variants of this receptor exist and these variants can stimulate different signal transduction pathways when bound to ligand. There are 2 subtypes of type II receptors and they have been designated VPAC1 and VPAC2 (Arimura et al. 1995; Vaudry et al. 2000). The only receptor that has been found in PC12 cells, the model system used in our studies, is the

PAC1 receptor. (Onoue et al. 2002)

PACAP appears to play a role in a wide variety of physiological functions.(Vaudry et al. 2000) Further, it appears to have many overlapping functions with NO produced by NOS1. For example, both PACAP and NO play a role in neural mediated bronchodilation (Belvisi et al. 1992; Kinhult et al. 2000). These two molecules also may play role in eliciting the release of leutenizing hormone (LH) from the anterior pituitary. (Garrel et al. 2002) Additionally, PACAP and NO produced by

NOS1 both play a role in learning and memory (Sauvage et al. 2000). The overlapping

25 functions of PACAP and NO produced by NOS1 and the fact that in certain model systems PACAP effects the transcription of NOS1 makes the study of their interaction quite important in understanding the regulation of these functions and may have important therapeutic implications as well.

When PACAP binds to one of its receptors, a variety of different signal transduction pathways can be stimulated. As one could deduce from the name, the adenylate cyclase – PKA pathway is one of the pathways that can be activated (Miyata et al. 1989). As with all typical G protein couples receptors, when PACAP binds to its receptor, a conformational change causes the GDP bound by the associated G-protein to be replaced by a GTP. This results in the dissociation of the Gα subunit from the

Gβγ subunit. The Gα subunit then activated the enzyme adenylate cyclase, which in turn activates protein kinase (PKA). PKA is then able to phosphorylate a variety of downstream effectors such as the cyclic AMP response element binding protein

(CREB) or mitogen activated kinase kinase kinase (MAPKKK) (Vaudry et al. 2000;

Zhou et al. 2002).

Four splice variants of the PAC1 receptor can also activate the protein kinase C

(PKC) pathway (Spengler et al. 1993). In this pathway, the (PLC) enzyme is activated and this enzyme then cleaves a particular membrane lipid, phosphatidylinositol- 4,5- bisphosphate (PIP2), into two second messengers, diacylglycerol (DAG) and inositol- 1,4,5 –triphosphate (IP3). DAG then activates

26

Figure 1.5: Some of the Signal transduction Pathways Activated by PACAP. The above figure shows some of the signal transduction pathways activated when PACAP binds to one of its receptors. Included among these is the cAMP-PKA pathway which then leads to phosphorylation of CREB and/ or MAPKKK. The PKC pathway can also be activated. Further, calcium mobilization can occur via the L-Type calcium stores or via internal stores by activation of the IP3 channels. This figure was adapted from Vaudry et al. Pharmacological Reviews Vol. 52 no 2 (2000) pg 269-324

27 PKC, which, like PKA, is able to phosphorylate multiple downstream signaling moleculesIP3 also has signaling activity. This molecule binds to internal calcium channels causing them to open and releasing calcium from internal stores (Bourne

1998).

Activation of the PACAP receptor can cause entry of calcium through the L- type calcium channel. A splice variant of the PAC1 receptor exists that is unable to stimulate cAMP production or activate the PKC pathway. Instead this splice variant activates the L-type calcium channel allowing for calcium entry (Chatterjee et al.

1996). Additionally, other splice variants can increase calcium entry through l-type calcium channels in a PKA dependant manner (Rawlings et al. 1993).

Finally,PACAP38 has also been shown to activate both the TRK-A and the

TRK-B receptors. TRK-A is a receptor tyrosine kinase that normally binds and is stimulated by the neurotrophin nerve growth factor (NGF). Lee et. al. have shown that in PC12 cells, PACAP is able to activate this receptor in an NGF independent manner.

TRK B is a tyrosine kinase normally activated by the neurotrophin brain derived neurotrophic factor (BDNF). It has also been shown that, in hippocampal neurons,

PACAP38 leads to phosphorylation and dimerization of the TRK-B receptor (Lee et al. 2002).

Conclusion

To summarize, NOS1 is a large, complex enzyme that gives rise to multiple transcripts. It encodes for a protein that catalyzes the formation of NO from the reactants oxygen and arginine and requires a large number of cofactors. (Bredt et al.

28 1990) NO produced by NOS1 is involved in a large number of important physiological processes ranging form long term potentiation in the CNS to regulation of GI motility.

(Son et al. 1996; Takahashi 2003). Aberrant expression of NOS1 may be involved in pathological processes as well. (Ignarro et al. 1995) Because of the importance of this gene in and disease, it is important to have a better understanding of its regulation. Further, as the regulation of this gene is quite complex, lessons learned from the study of this gene may well be applied in the study of others.

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38

CHAPTER 2

PACAP38 STIMULATES NOS1 GENE EXPRESSION

Introduction

As previously mentioned, NOS1 was originally described as being constitutively expressed and indeed it is constitutively expressed in some cells

(Ignarro et al. 1995). In other circumstances though, the expression of NOS1 has been shown to be dynamically regulated. For example, in a rat model of focal ischemia, both NOS1 mRNA and protein have been shown to be upregulated. (Zhang et al.

1994) Nerve growth factor (NGF) has also been shown to induce NOS1 under certain circumstances (Sheehy et al. 1997; Rife et al. 2000). Interestingly, in rat primary pituitary cultures the hormone pituitary adenylate cyclase activating peptide (PACAP) has shown the ability to upregulate NOS1 (Garrel et al. 2002).

This chapter explores the mechanism by which effects PACAP effects NOS1 gene expression. We found that when PC12 cells were treated with forskolin, a drug that activates all known forms of adenylate cyclase, modest increases in NOS1 protein and mRNA were observed. PACAP, on the other hand, led to significant increases in

39 NOS1 mRNA and protein levels. This implied that PACAP was doing more than just activating adenylate cyclase.

As described in the previous chapter, PACAP38 binding activates both the

PKA and PKC pathways. It also can stimulate calcium influx through L-type calcium channels and calcium release from internal stores. We hypothesized that PACAP must activate both the PKA and PKC pathways for NOS1 to be upregulated. We tested this hypothesis by treating PC12 cells with TPA, a drug that activates PKC and forskolin, a drug that activates adenylate cyclase. We found that the magnitude of NOS1 upregulation observed in cells treated with this combination was similar to that observed in cells treated with PACAP38. The time courses of the protein upregulation and the mRNA upregulation observed in cells treated with TPA and forskolin were slightly different than those observed in cells treated with PACAP. This could reflect different rates of inactivation of the drugs utilized in this study or could indicate that additional signal transduction pathways are involved.

As we believed that PACAP’s ability to increase expression of the NOS1 gene was dependant on its ability to activate the both the PKA and PKC pathways, we next wanted to determine what role TPA was playing in the observed upregulation of

NOS1 when cells were treated with both TPA and forskolin. As TPA alone caused no increase in NOS1 protein levels, we needed to determine if TPA, when combined with forskolin, was causing an increase in protein synthesis or decreasing NOS1 protein degradation. We showed, using metabolic labeling experiments followed by immunoblots that TPA, when combined with forskolin, stimulates an increase in

40 protein synthesis over cells treated with forskolin alone. Further, we showed that TPA appears to act at the transcriptional level to increase RNA levels which were subsequently translated resulting in the observed increase in protein levels.

We also evaluated the effect of PACAP on NOS1 mRNA expression. As expected, PACAP treatment caused an increase in NOS1 mRNA that was greater in magnitude and duration than that observed with forskolin alone. This supported the notion that PACAP had to activate multiple signal transduction pathways in order to stimulate NOS1 production. Treatment of PC12 cells with forskolin and TPA resulted in increases in NOS1 mRNA of similar magnitude as that observed in cells treated with PACAP but NOS1 mRNA remained elevated for a longer period of time in cells treated with PACAP.

Materials and Methods

Cell culture: PC12 cells were grown in Dulbecco’s modified eagle medium with glucose 4500mg/ml (DMEM-HG) supplemented with 5% horse , 5% fetal bovine serum, penicillin (100Units/ml) and streptomycin (100ug/ml). They were maintained in a 37 degree incubator with 5% CO2, 95% room air. Media and serum were obtained from Irvine Scientific (Irvine, CA) while penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA)

PACAP treatment: PC12 cells were seeded at 1 x 106 cells per well on 6 well tissue culture plates obtained from Corning (Corning, NY) in 2.5ml DMEM-HG with supplements as described above. The cells were then returned to the incubator for 16-

20 hours. PACAP38 was then added to the cells at a final concentration of 100nM

41 (25ul of a 10uM stock as added to each well). The cells were then returned to the incubator for various amounts of time before being harvested as described below.

PACAP38 was obtained from Phoenix Pharmaceuticals (Belmont, CA)

Forskolin and TPA treatment: PC12 cells were seeded at 1 x 106 cells per well on 6 well plates in DMEM-HG with supplements as described above. The cells were then returned to the incubator for 16-20 hours. Forskolin, TPA or both were then added at final concentrations of 10uM and 200nM respectively. Cells were then harvested and protein extracted as described below at various time points after drug addition. TPA and forskolin were both obtained from Sigma (St Louis, MO) and stored at -20 degrees C as stock solutions in DMSO.

NGF treatment: PC12 cells were seeded at 3x104 cells per well on a 12 well plate in 1.5 ml DMEM-HG supplemented with 5% FBS, 5% HS, penicillin

100units/ml and streptomycin 100ug/ml. Cells were then incubated for 16-20 hours before addition of NGF. After incubation, one half of the media was removed from each well and replaced with media containing NGF 200ng/ml resulting in a final NGF concentration of 100ng/ml. One half of the media was removed and replaced with fresh media containing NGF on days 4 and 6. Cells were harvested on day 7 and protein crude protein extracts were prepared as described below. Murine NGF was obtained from Austral Biologicals (San Ramon, CA) and was stored as a 100ug/ml stock in water at –20 degrees C. When cells were treated with both forskolin and

NGF, forskolin was added to the wells at a final concentration of 10uM on day 5 of

NGF treatment.

42 Protein extract preparation: At various time points after drug addition, the cells were washed once in ice cold PBS then harvested in the same buffer. The cells were then centrifuged 14000xG for 5 minutes in a micro centrifuge. The supernatant was removed and the cells were re-suspended in 250ul NOS assay buffer A (50mM

Tris-HCl ph 7.4, 1mM EDTA, and phenylmethysulfonyl fluoride (PMSF) 100mg/L) and the cells were disrupted using a microtip sonicator. The extracts were then stored at -80C until use.

NOS Assays: The NOS assay was carried out using the following protocol

(Hevel et al. 1994). Briefly, a reaction mix consisting of 50ul 2X buffer C (100mM

Hepes pH 7.4, 2.5mM CaCl2, 20ug/ml calmodulin, 2mM dithiotriotal, 0.02M sodium acetate, 6uM tetrahydrobiopterin, 2uM FAD and 2uM FMN), 2.3ul of 14C labeled arginine (300mCi/mmol, obtained from Amersham), and 23ul of water was set up.

Fifty micrograms of protein was then added to the reaction mix and it was incubated at

37 degrees C for at least 20 minutes. The reaction was then stopped by adding 1ml of a stop solution buffer consisting of 20mM Hepes pH 5.5 and 2 mM EDTA.

The reactant arginine was then separated from the citrulline produced by the reaction using an AG-50WX8 prefilled chromatography column from Bio-Rad

(Hercules, CA). The column was converted to its sodium form and pre equilibrated with 5ml stop solution prior to use. The separation was carried out by applying the reaction mix to the column. The bed volume (750ul) was discarded and the column was washed twice with 2ml of stop solution. Four milliliters of eluate was collected

43 and mixed. The amount of radiolabeled citrulline present was then measured using a liquid scintillation counter manufacture by Packard (Downers Grove, IL).

Immunoblots: Immunoblots were carried out using a standard protocol

(Sambrook et al. 2001). Briefly, 50ug of protein was mixed with 1/5 volume of 5X loading buffer ( per well was loaded onto a 5% stacking, 7.5 resolving polyacrylamide gel. The proteins were then separated using electrophoresis at 95 volts for approximately 3 hours (until the 40KD marker was about ¾ of the way down the gel).

The separated proteins were then blotted onto a nitrocellulose membrane

(Amersham) using a semi dry blotting apparatus from Bio-Rad (Hercules, CA) using the following method. The gel, membrane, 2 pieces of extra thick blotting paper and 1 piece of whatman paper were soaked in immunoblot transfer buffer (48mM Tris,

39mM glycine, 20% methanol, and 0.375gm/L SDS) for 30 minutes. The components were then stacked on the anode in the following order (from anode to cathode): 1 piece extra thick blotting paper, the nitrocellulose membrane, the gel, 1 piece regular whatman paper, and one piece extra thick blotting paper. Air bubbles were removed by rolling a test tube across the stack after the addition of each layer. The apparatus was then assembled per the manufacturer’s instructions and the proteins blotted using for35- 45 minutes at 25 volts and 300mA.

After blotting was complete, nonspecific binding was blocked by placing the membrane was placed in buffered saline with 0.1% tween 20 (PBS-T) and

5% powdered and incubated with gentle shaking overnight at 4 degrees C. After blocking, the membrane was sealed in a bag with 10ml of PBS-T with 5% powdered

44 milk and the primary anti-NOS1 antibody diluted 1:400 (see table 1 for a list of all antibodies used in this chapter). This was then incubated on a rotator/shaker for 2 hours at room temperature. The membrane was then washed with PBS-T three times

(ten minutes per wash) and sealed in a bag with 10ml PBS-T + 5% powdered milk and the secondary antibody, a goat anti rabbit IGG antibody conjugated to the horse peroxidase enzyme (HRP), diluted 1:4000. This bag was then incubated at room temperature for 1.5 hours. The membrane was then removed from the bag and washed three times with PBS-T (10 minutes per wash). The proteins were then detected using an ECL detection system from Amersham per the manufacturer’s instructions.

Briefly, solution 1 and 2 were mixed in equal volumes and spread evenly on the membrane. After one minute, the ECL solution was removed and the membrane was wrapped in plastic wrap and exposed to X-ray film for 1-3 minutes.

Immunoprecipitation: PC12 cells were seeded at a concentration of 2x106 cells per well on six well plates as described above in DMEM-HG supplemented with penicillin, streptomycin, FBS and horse serum and as described above. After overnight incubation, the cells were treated with DMSO (vehicle) 0.1%, Forskolin

10uM, TPA 200nM or both forskolin and TPA for 6 hours. During drug treatment, media was prepared by adding 1.4mCi of a cysteine/methionine labeling mix from

ICN () was added to 14ml DMEM-HG with supplements but lacking methionine. The old media was then removed from the cells and replaced with the new media containing the 35S labeled cysteine/methionine. The drugs were then re-added to the cells and they were returned to the incubator for 1.5 hours. In the delayed TPA

45 experiments, TPA and forskolin were added to the wells that had been initially treated only with forskolin.

After incubation, the cells were lysed using the following protocol. Media was removed (and saved) and the cells were washed with ice cold PBS. The cells were then lysed by pipetting with 200ul NP-40 lysis buffer (1% NP-40, 10% glycerol,

20mM Tris pH 8, 137mM NaCl, 1mM MgCl2, 0.5mM EDTA and 10mM sodium pyrophosphate). The Lysate was then centrifuged at 14000x G for 5 minutes and the supernatant was then placed in a fresh tube. Anti-NOS1 antibody was then added to the lysate (4ul per 200ul supernatant) and this mix was incubated at 4 degrees C overnight on a rotating mixer.

After incubation, 20ul of protein A-sepharose beads diluted 1:1 in NP-40 lysis buffer was added to the cell lysate and this mix was incubated on a rotating mixer for at least 1 hour. The mix was then centrifuged briefly and the supernatant was removed and saved for TCA precipitation. The protein A-sepharose beads were then washed with NP-40 lysis buffer three times. After the last wash, all supernatant was removed and 40ul of SDS sample buffer (50% glycerol, 0.3125M tris pH 6.8, 25% beta mercaptoethanol, 10% SDS in water) was added to the beads and this was boiled for 5 minutes. The mixture was then centrifuged briefly and the supernatant was loaded on a 5% stacking, 7.5% resolving polyacrylamide gel with SDS. The proteins were then separated based on size by electrophoresis.

Once electrophoresis was complete, the gel was for 2 hours in 50% methanol,

10% acetic acid in water. The fixed gel was then soaked in En3Hance autoradiography

46 enhancer (PerkinElmer Life Sciences) for 1 hour then washed with water for 30 minutes. After drying the gel on a gel dryer for 1.25 hours at 60 degrees C, the gel was wrapped in plastic wrap and exposed to x-ray film.

TCA precipitation of the cell lysates was carried out according to the following protocol (Hockfield et al. 1993). Briefly, Twenty microliters of cell extract was mixed with 1ul of 2.5mg/ml sodium deoxycholate and 20ul of 10% trichloroacetic acid

(TCA). This reaction mix was incubated on ice for 30 minutes then centrifuged at

1500x G for 15 minutes in a microcentrifuge. The supernatant was removed and the remaining pellet was re-suspended in scintiverse BD and the amount of 35S incorporation was evaluated using a scintillation counter. An equal amount of radioactivity was then mixed with loading buffer and separated on a 5% stacking 7.5% resolving gel as described above. This gel was then fixed, soaked in En3Hance, dried and exposed to x-ray film as described above.

RNA extraction: RNA was isolated using Trizol ® (Invitrogen) per the manufacturer’s instructions. Media was removed from cells and trizol was added directly to the plate (1ml per well for 6 well plates, 0.5 ml per well for 12 well plates).

Trizol was then pipetted up and down 15-20 times and placed in a microcentrifuge tube then incubated for 3-5 minutes. Chloroform was added to the trizol (0.2ml per ml trizol) and the tube was mixed well. After the aqueous and organic phases were allowed to separate for 2 minutes, the tubes were centrifuged at 12000x g for fifteen minutes. The upper aqueous phase was then placed in a fresh microcentrifuge tube and the RNA was then precipitated by the addition of isopropranol (0.5ml per ml of

47

Antigen Type Source Vendor Dilution animal NOS1 polyclonal rabbit Santa Cruz 1:400 polyclonal Rabbit Sigma 1: 250 Rabbit IGG polyclonal goat Santa Cruz 1:4000 (HRP conjucate)

Table 2.1: Antibodies used in this chapter. The above table lists all of the antibodies used in this chapter and the sources and working dilutions of these antibodies.

48 trizol used). After a ten-minute incubation at room temperature, the RNA was pelleted by centrifugation at 12000x g for 10 minutes then washed with 75% ethanol. After a final centrifugation at 7500x g for 5 min, the ethanol was removed and the RNA was allowed to dry briefly then re-suspended in RNAse free water. The concentration was obtained spectrophotometrically using a microplate reader (Molecular Devices) and stored at –80 degrees C until use.

Reverse transcriptase: Five micrograms of total RNA and 200ng random primer (Invitrogen) was added to a total of 12ul RNase free water and the mixture was then incubated at 70 degrees C for 10 minutes. The mixture was then cooled on ice and briefly centrifuged. Four microliters 5x first strand buffer, 2ul 0.1M DTT, and 1ul

10mM each dNTP mix were added to the reaction and it was incubated at room temperature for 10 minutes. The reaction was then incubated at 42 degrees C for 2 minutes prior to the addition of 1ul superscript II ® reverse transcriptase (Invitrogen).

The reaction was then incubated at 42 degrees C for 50 minutes. In order to inactivate the reverse transcriptase, the reaction mix was then incubated at 70 degrees C for

15minutes. The reaction mix was then diluted with 180ul RNase free water and stored at –20 degrees C until use.

PCR: A 50ul PCR mix consisting of 1x PCR buffer, 0.2mM each dNTP,

2.5mM MgCl2, 0-5% DMSO (depending on primers used, see table 1), 2.5 IU Taq polymerase (Invitrogen), and 0.5uM forward and reverse primers was set up on ice for each treatment group. Ten microliters of the above RT reaction was then added to each PCR mix. The PCR was carried out in a Bio-Rad I-Cycler using the following

49 conditions: 95 degrees C for 5 minutes for 1 cycle followed by 95 degrees C for 30 seconds, 55 degrees C for 30 seconds, and 72 degrees C for 60 seconds for 27 cycles.

The reaction was then incubated at 72 degrees C for 10 minutes then stored at 4 degrees C until amplicons were evaluated using gel electrophoresis.

Image and statistical analysis: Image analysis was carried out using ImageJ, a freeware Java based image analysis program available from the National Institutes of

Health (Rasband 2004). Graphs were created using Microsoft Excel and Graphpad

Prism software while statistical analysis was performed using Graphpad Prism.

Real Time PCR: Real time PCR was carried out using an I-Cycler real time

PCR system (Bio-Rad) and a Quantitect SYBR ® Green RT-PCR kit (Qiagen) per the manufacturer’s instructions. Briefly, RNA was isolated from cells using Trizol ® as described above. The RNA concentration was obtained spectrophotometrically using a microplate reader (Molecular Devices) then diluted to a concentration of 40ng/ul with RNase free water. A reaction mix consisting of 80ng (2ul) total RNA, 0.25 ul RT mix, 12.5 ul SYBR mix, 0.5 uM forward primer, 0.5 uM reverse primer and 7.75 ul

RNase free water (total reaction volume was 25 ul) was set up in triplicate for each sample. The reverse transcriptase reaction was then carried out at 50 degrees C for 30 minutes. The reverse transcriptase enzyme was then inactivated by incubation at 95 degrees C for 15 minutes. The following PCR conditions were then used: 94 degrees

C for 15 seconds, 55 degrees C for 30 seconds, then 72 degrees C for 30 seconds for a total of 45 cycles. After PCR was complete a melt curve was obtained to ascertain the specificity of the PCR. Absolute starting quantities were obtained using a standard

50 Table 2A: Primers Used to Clone the Rat Exon 2 UTR

Primer Name Sequence (5’ to 3’)

RatNOS1E2fwd3 5’ CGGATCCAGGTAGAAGCCCCTATGTCA 3’ BamHI RatNOS1E2rev3 5’ GAGATCTTGTCCTTCAGAAGACTAAGC 3’ Bgl II

Table 2B: Primers used in Real time PCR (designed by Hua Wei)

Primer Name Sequence (5’ to 3’)

Rat nNOS exon3 fwd 5’ AGAAGGAACAGTCCCCTACCTC 3’

Rat nNOS exon3 rev 5’ GTTTCCAGTGTGCTCTTCAGGT 3’

Table 2C: Primers used for RT-PCR analysis

Primer Name Sequence (5’to 3’)

RatNOS1E2fwd-1 5’ TCCTGACCTGTTGCTTAGGG 3’

RatNOS1E2fwd-2 5’ TTTGGGGTTCAGCAGATCCA 3’

RatNOS1E3rev-1 5’ AGCTTTGTGCGATTTGCCAT 3’

Table 2.2: Sequences of PCR primers used in this chapter: The above table shows the sequences of the primers used throughout this chapter. Table 1A shows the primers used to construct the RatNOS1E2-pXP2 construct. Note the incorporated restriction sites in bold. Table 1B shows the primers used for real time PCR analysis. Hua Wei designed these primers. Table 1C shows the sequences of primers used to analyze the full length and short NOS1 transcripts by RT-PCR

51 curve obtained by carrying out the PCR using known quantities of a plasmid containing the rat NOS1 cDNA obtained from Dr. Solomon Snyder.

Results

We determined that PACAP treatment enhanced the levels of NOS1 present in

PC12 cells. At various times after treatment with PACAP38 the cells were harvested and crude protein extracts prepared and used in western blots. The relative amounts of

NOS1 protein present were obtained by image analysis. PACAP increased the expression of NOS1. As shown in figure 2.1, a twofold increase in NOS1 expression is seen as early as 12 hours and 8 fold increases observed at 24 and 48 hours. It should be noted that occasionally of NOS1 was not detected in untreated cells precluding and estimate of the fold increase. However, in all cases, PACAP treatment led to a marked induction of NOS1 protein

We questioned whether activation of adenylate cyclase could account for the effect of PACAP on NOS1 protein levels. To test this PC12, cells were treated with forskolin, a drug that activates adenylate cyclase, and then an immunoblot was carried out using crude protein extracts made from these cells. As one can see from figure 2-

2, treatment with forskolin led to only a twofold increase in NOS1 protein levels. As shown in figure 2-3, forskolin also failed to produce a significant induction of NOS enzyme activity in PC12 cells.

These data suggest that activation of the PKA pathway might not be sufficient to explain the effects of PACAP38. Therefore, we questioned whether activation of

52 A. PACAP treatment Markers Unt. 6Hr 12hr 24hr 48hr fsk +TPA 24hr

NOS 1

B.

9 8 7 6 5 4 3 2 1 0 Relative Expression (arbitrary units) units) (arbitrary Expression Relative Unt 6 hr 12 hr 24 hr 48 hr

Figure 2.1: Time Course of NOS1 protein expression in PC12 cells treated with PACAP. Figure A is an immunoblot for NOS1 using extracts from PC12 cells treated with PACAP for 6, 12, 24 or 48 hours. There was a 2 fold increase at 12 hours and an 8 fold increase at 24 and 48 hours. Figure B is a quantification of the above immunoblot. Quantification of the blot was carried out using the gel analysis function in ImageJ. As a control to allow for quantitative comparison between blots, cells extracts from PC12 cells treated with both forskolin and TPA for 24 hours were included on all of the blots

53

A.

Forskolin 10uM treatment Unt. DMSO 6 hr 12 hr 24 hr 48 hr Fsk +TPA 24hr

B.

2.5

2

1.5

1

0.5

0

Relative Expression (arbitrary units) units) (arbitrary Expression Relative r h hr 6 12 hr 24 48 hr treated SO 24 M Un D

Figure 2.2: Time Course of NOS1 protein expression in PC12 cells treated with forskolin. Figure A is an immunoblot for NOS1 using extracts from PC12 cells treated with forskolin for 6, 12, 24 or 48 hours. Also included to allow for quantitative comparison between blots was extract from cells treated with forskolin and TPA for 24 hours. There was a 2 fold increase at 24 and 48 hours, far less than that seen with PACAP treatment. Figure B is a quantification of the above immunoblot. Quantification of the blot was carried out using the gel analysis function in ImageJ (NIH) and graph was created using Excel (Microsoft). In

54

25000

20000 * 15000

DPM 10000

5000

0 lin F lin ted o o tract a MSO k NG k tre D rs rs n o o U F F No Ex + F G N

Treatment

Figure 2.3: NOS Assay using extracts from PC12 cells treated with NGF and forskolin. The above figure shows the results of assays for nitric oxide synthase activity using extracts from PC12 cells treated with NGF, forskolin or both. Note that, as expected, treatment of PC12 cells with NGF led to an increase in NOS activity in PC12 cells while treatment with forskolin (for 48 hours) did not. When cells pre- treated with NGF for 5 days were treated with forskolin for 48 hours, the increase in NOS activity was greater that that observed in cells treated with NGF alone (P = 0.0026)

55 the PKC pathway contributed to the induction of NOS1. To test this, we treated PC12 cells with TPA, a direct activator of PKC as well as forskolin and assayed for NOS1 protein by immunoblotting. The induction of NOS1 after treatment with forskolin and

TPA was greater than that observed in cells treated with forskolin alone (figure 2-2) and similar to that seen in cells treated with PACAP (figure 2-1). In the sample experiment shown in figure 2-4, treatment with TPA and forskolin resulted in a 1.8 fold increase at 12 hours, a 2.8 fold increase at 24 hours and a 4.8 fold increase at 48 hours. As above, the amount of NOS1 detected varied between experiments but the relative trends remained constant. Although TPA enhanced the effect of forskolin, treatment of the cells with TPA alone had no effect on NOS1 protein levels. NOS1 was either barely detectable or not detectable at all in untreated cells or in TPA treated cells when standard blotting conditions were used (see figure 2-5C). If ECL hyperfilm

(Amersham) was used and extra blotting time allowed, basal NOS1 could be detected and no increase in NOS1 levels was seen in cells treated with TPA (see figure 2-5A and 2.5B). Thus our results are consistent with the notion that PACAP activation of both the PKA and PKC pathways mediates the induction of the NOS1 protein

We next wanted to determine if new NOS1 protein synthesis occurred in cells treated with forskolin and TPA. To test this, PC12 cells were first treated with the drugs, then metabolically labeled with 35S-methionine and 35S cysteine. NOS1 was then immunoprecipitated and separated by SDS-PAGE. The incorporation of label into NOS1 was evaluated using autoradiography. A typical result is shown in figure

2-6A. Labeled NOS1 protein is not detected in control cells, in cells treated with

56 A. Forskolin + TPA Unt. DMSO 6 hr 12 hr 24 hr 48 hr

NOS1

6

n 5

4

3

2

(Arbitrary units) . (Arbitrary 1 Relative Expressio 0 4 2 hr 6 T + ntreated F U DMSO F+T 12hr F+T 24hr F+T 48hr Treatment

Figure 2.4: Time course of NOS1 expression in PC12 cells treated with forskolin and TPA. Figure A is an immunoblot for NOS1 using extracts from PC12 cells treated with forskolin (10uM) and TPA (200nM) (labeled F+T) for 6, 12, 24 or 48 hours. The lower band in NOS1, the upper band is believed to be and artifact as it is too large to be NOS1 and is its intensity is not affected by drug treatment. There was a 1.8 fold increase at 12 hrs, a 2.8 fold increase at 24 hours and a 4.8 fold increase seen at 48 hours. Figure B is a quantification of the above immunoblot. Quantification of the blot was carried out using the gel analysis function in ImageJ (NIH) and graph was created using Excel (Microsoft).

57 A. Unt. TPA 6 hr 12 hr 24hr 48 hr T+F 24hr

B.

1.4

1.2 n 1

0.8 0.6

0.4

Relative expressio 0.2

0 Unt. TPA 6hr TPA 12hr TPA 24hr TPA 48hr Treatment

C.

T+F48 TPA 6hr 12hr 24hr 48hr

NOS1

Actin

Figure 2.5 Effect of TPA on NOS1 protein expression. Figure A shows the results of an immunoblot for NOS1 using extracts from PC12 cells treated with TPA. No change in protein levels was detected. Note that enhanced blotting times and the use of ECL hyperfilm (Amersham) were required to detect NOS1 in TPA treated cells. Figure B is a quantification of the blot shown in A. Image analysis was performed using Image J software. Figure C is a second immunoblot carried out using extracts from PC12 cells treated with TPA. In this case, standard Kodak X-OMAT-AR film was used and the blotting time was shorter (35 minutes). Note that NOS1 was not detectable at any treatment times under these conditions. 58 A.

Unt. DMSO TPA Fsk T+F 1 T+F 2

B. Unt. DMSO fsk F+T F+dT F+dT

Figure 2.6: Immunoprecipitation of NOS1 from PC12 cells. Figure A: This figure shows the result of an immunoprecipitation of NOS1 using extracts from PC12 cells treated with DMSO, forskolin or forskolin and TPA. After 6 hours treatment, the cells were metabolically labeled with 35S methionine and cysteine. After 1.5 hours, NOS1 was immunoprecipitated and separated using SDS-PAGE. The gel was then fixed, dried and exposed to x-ray film. Note that TPA+ forskolin treatment led to a significant increase in NOS1 protein production while forskolin treatment was less effective in increasing NOS1 translation. Figure B: This figure shows a similar IP experiment but, in two of the treatment groups, the addition of TPA was delayed until 15 minutes prior to the addition of the radiolabeled methionine and cysteine. The amount of new NOS1 produced was elevated in the cells treated with TPA and forskolin for 6 hours but when TPA addition was delayed (labeled F+dt), the amount of nascent NOS1 decreased to levels similar to that observed when cells were treated with forskolin alone.

59 vehicle (DMSO) or in cells treated with TPA. In contrast, labeled NOS1 is readily detectable in PC12 cells treated with both forskolin and TPA while smaller levels if labeled NOS1 is detected in cells treated with forskolin alone. These results show that the increased levels of NOS1 protein observed by immunoblotting are accompanied by new protein synthesis

We questioned whether TPA was exerting its influence by directly and rapidly stimulating NOS1 translation. To test this, cells were treated with forskolin to elevate

NOS1 mRNA (see figure 2-7) and then treated with or without TPA during a 1.5 hour period of labeling with radiolabeled cysteine and methionine. We reasoned that if an increase in new protein synthesis was observed when TPA was present only during the labeling period, then TPA was likely acting directly at the translational level. If, on the other hand, the presence of TPA was required for the entire incubation period, this would imply that TPA was not directly effecting translation. The results showed that if the addition of TPA was delayed until just prior to cell labeling, the amount of new

NOS1 protein produced was significantly reduced and was actually similar in magnitude to cells treated with forskolin alone. A typical result is shown in figure 2-

6B. Thus TPA does not directly activate NOS1 translation

Using real time PCR, we next examined the effect that PACAP38, TPA, forskolin and the combination of TPA and forskolin had on NOS1 mRNA levels.

Treatment of the cells with TPA had no effect on the levels of NOS1 mRNA present at any of the time points evaluated (see figure 2.7). Treatment of PC12 cells with

PACAP, on the other hand, had a significant effect on NOS1 accumulation (see figure

60

Figure 2.7: Real time PCR of NOS1 in PC12 cells. This figure shows the results of a real time PCR experiment in which the amount of NOS1 present in PC12 cells treated with forskolin, TPA or both was evaluated. Figure A shows the raw data for the standard curve made using known quantities of the rat NOS1 cDNA. The X axis is PCR cycle and the Y axis is (corrected for background). Figure B shows the standard curve plotting the starting concentration of NOS1 cDNA vs. the threshold cycle (this is the PCR cycle in which the amount of fluorescence present exceeded a threshold value based on the background fluorescence.) Figure C shows the raw data from the real time RT-PCR experiment using 80ng RNA from PC12 cells treated with the forskolin, TPA, both, or the vehicle DMSO. Figure D shows a melt curve indicating that an amplicon of only one size, represented by the peak at 85°C was produced by the RT-PCR. Figure E shows the calculated starting quantities of RNA present in PC12 cells treated with forskolin, TPA or both. Starting quantities were calculated using the standard curve shown in A. Note that the expression of NOS1 in cells treated with forskolin + TPA was much greater than that observed in cells treated with forskolin alone. DMSO (the vehicle) and TPA had no effect on NOS expression at any of the time points tested.

61 A B

C D

E.

3.0×10 -4

2.5×10 -4 DMSO 2.0×10 -4 Forskolin TPA 1.5×10 -4 FSK + TPA

1.0×10 -4 ng/80ng RNA

5.0×10 -5

0 0 3 6 9 12 15 18 21 24 27 Hours

62 A B.

C. D.

E.

5.5×10 -4 5.0×10 -4 Untreated 4.5×10 -4 DMSO 4.0×10 -4 PACAP38 3.5×10 -4 Forskolin 3.0×10 -4 T+F 2.5×10 -4 2.0×10 -4 1.5×10 -4 ng/80ng total RNA 1.0×10 -4 5.0×10 -5 0 0 3 6 9 12 15 18 21 24 Hours

Figure 2.8: Real time PCR for NOS1 in PC12 cells treated with PACAP38. This figure shows the results of a real time PCR experiment evaluating the amount of NOS1 present in PC12 cells treated with PACAP, forskolin, TPA + forskolin or vehicle. It was carried out as described in methods and figure 2-6. Figure A is raw data from the standard curve. Figure B is the standard curve. Figure C is the raw data from the real time PCR using RNA from PC12 cells treated as indicated. Figure D is the melt curve. Figure E is a graph showing the calculated starting quantities of NOS1 present in PC12 cells treated with PACAP38, forskolin TPA or both TPA and forskolin. 63 2.8). After 3 hours treatment, PACAP increased NOS1 mRNA levels in PC12 cells approximately 8.5(+/- 0.68) fold. NOS1 RNA levels peaked in PACAP38 treated

PC12 cells at 6 hours with a 19.4 (+/-1.6) fold increase over baseline and remained elevated at 12 and 24 hours (17.3 +/- 4.55 and 12.9 +/- 0.58 fold increase respectively).

Treatment of PC12 cells with forskolin alone led to an increase in NOS1 mRNA that was considerably less than that observed with PACAP38 (see figure 2.8).

The apex of NOS1 mRNA upregulation due to forskolin treatment occurred at the 3- hour time point and the resulting mRNA levels were 3.25 (+/- 0.79) times greater than that found in untreated controls. NOS1 levels remained elevated 6 and 12 hours after the addition of forskolin and had returned to basal levels by 24 hours. Thus the forskolin mediated induction of NOS1 mRNA was smaller and lasted for a shorter period of time than that mediated by PACAP38.

Since a combination of TPA and forskolin mimics the effect of PACAP on accumulation of NOS1 protein, it was hypothesized that stimulating both of these pathways would also mimic the effects PACAP had on NOS1 mRNA levels. To test this hypothesis, we treated PC12 cells with both TPA and forskolin. The combination of drugs led to an increase in NOS1 mRNA greater than that of forskolin alone (see figures 2.7E and 2.8E). The induction observed after three hours treatment was 7.08

(+/- 1.6) fold. After 6 hours of treatment, induction peaked at 10.4 (+/-3.6) fold and remained elevated at 12 and 24 hours. The effect forskolin and TPA had on the time course of the rising phase of the accumulation of NOS1 was somewhat variable. In

64 general, the peak levels of NOS1 mRNA were similar after treatment with either

PACAP or forskolin and TPA. The affect of PACAP38 was more prolonged relative to the effect of forskolin and TPA

We questioned whether the synergy between TPA and forskolin and/or the differences observed after treatment with PACAP relative to forskolin and TPA reflected differences in RNA turnover. To test this possibility, the half-life of the

NOS1 mRNA was measured in cells treated with forskolin, the combination of forskolin and TPA or PACAP38 using an inhibitor of transcription, actinomycin D. In general, the NOS1 mRNA lifetimes appear similar within experimental error in all cases. The half-life of NOS1 mRNA in forskolin treated cells was found to be approximately 17 hours. In PC12 cells treated with both forskolin and TPA, the

NOS1 mRNA half-life is approximately 13 hours (see figure 2-9). Thus, TPA treatment does not stabilize NOS1 mRNA. The half life of NOS1 in PACAP treated cell is approximately 10 hours. Thus the prolonged lifetime of NOS1 mRNA in

PACAP treated cells does not reflect enhanced RNA stability. Several attempts were made to measure the half life of NOS1 mRNA in untreated PC12 cells but they were unsuccessful. This may be due to the relatively low levels of NOS1 present in untreated cells.

NOS1 expression is controlled by multiple promoters and expression driven by these promoters results in a variety of transcripts (Xie et al. 1995; Wang et al. 1999;

Boissel et al. 2003). In addition to transcripts containing one of five alternate first exons known to exist in rats, a shorter transcript driven from a promoter located within

65

Figure 2.9: Half Life of NOS1 in Forskolin, Forskolin + TPA and PACAP treated PC12 cells. Figure A: The graph on the left shows the absolute amounts of NOS1 mRNA as measured by real time PCR at various time points after forskolin plus TPA addition and subsequent actinomycin D addition. The graph on the right shows the same data graphed as the natural log of the RNA amt/ 80ng total RNA versus time. Half life as calculated using the equation using the slope of the second graph (0.693/ slope) was approximately 13 hours. Figure B shows the same data obtained from cells treated with forskolin alone. The calculated half life for NOS1 mRNA in these cells was about 17 hours. Figure C shows the same data obtained from cells treated with PACAP38. The calculated half life in PACAP38 treated cells is approximately 10 hours

66 A. Forskolin and TPA

1.5×10 -4 -7.5

-8.5

1.0×10 -4 -9.5

-10.5

-11.5 T1/2 =13hrs 5.0×10 -5

Ln [ng/80ng RNA] -12.5 ng/80ng total RNA -13.5

0 0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28 Hours After Actinomycin addition Hours Actinomycin D addition

B. Forskolin

1.5×10 -4 -8

-9

1.0×10 -4 -10

-11 T1/2 =17hrs

5.0×10 -5 -12 ng/80ng RNA Ln ng/80ng RNA -13

0 -14 0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28 Hours Hours After actinomycin addition

Actinomycin Added

C. PACAP

3.5×10 -4 -7

3.0×10 -4 -8

2.5×10 -4 -9

2.0×10 -4 -10

-4 1.5×10 -11 T1/2 = 10hrs -4

ng/80ng mRNA 1.0×10 -12 Ln[ng/80ng RNA] 5.0×10 -5 -13

0 -14 0 4 8 12 16 20 24 28 32 0 4 8 12 16 20 24 28 Hours time after actinomycin added actinomycin addition

67

ATG

Exon 1X Exon 2 Exon3 Forward primer 1 Forward primer 2 Common reverse primer

Putative transcript start

Figure 2.10: Diagram of RT-PCR Assay. The above figure is a schematic showing the PCR carried out to determine whether the NOS1 transcript upregulated by PACAP, Forskolin or Forskolin and TPA contained one of the alternate first exons or instead if it was the shorter transcript in which transcription starts in exon 2. PCR was carried out using 1 of 2 different forward primers (1 and 2 above) and a common reverse primer. The first forward primer lay in exon 2 but was outside the putative transcription start site and thus should amplify transcripts that contain any one of the 3 alternate first exons known to exist in the rat while the second forward primer lay inside the ATG translation start site and thus would amplify transcripts that contained a first exon and those that started in exon 2.

68

A. Unt TPA FSK T+F brain Unt. TPA FSK T+F Brain

Forward primer 1 Forward primer 2

GAPDH

B. Forward primer 1 Forward primer 2 7

6 7

5 6

4 5

3 4

2 3 relative expression 1 2 Relative Intensity Relative 0 1 untreated TPA Forskolin T+F 0 Treatment Untreated TPA Forskolin F+T

Figure 2.11: RT-PCR Assays for NOS1 in Forskolin and TPA treated PC12 cells. Figure A shows RT-PCR for NOS1 using RNA from PC12 cells treated with forskolin, TPA or both for 4 hours. In the figure on the right of the PCR was carried out using a forward primer from exon 2 that lay outside of the putative transcription start site of exon 2 promoter driven transcript and a common reverse primer and should therefore only amplify transcripts that have one of the alternate first exons. In the figure on the left the forward primer was inside the ATG translation start site and should amplify all major transcripts of NOS1. Note that the amount of exon 1 containing transcript did not significantly change due to forskolin or forskolin + TPA treatment but the total amount of NOS1 mRNA did increase due to these treatments. This would imply that the promoter responsible for this upregulation is the promoter located in exon 2.

69 exon 2 exists. This corresponds to a promoter that has been shown to exist in mice and appears to exist in humans as well (Sasaki et al. 2000). To determine which promoters were functioning in cells treated with PACAP and/or forskolin plus TPA, an rt-PCR based assay was utilized (see figure 2-9). Two forward primers initiating within exon 2 were designed. One of these primers lay upstream of the putative exon

2 transcript start site and would amplify only those transcripts containing a first exon.

This primer was designated forward primer 1. The second primer lay just inside the

ATG translation start site and would amplify both exon 1 containing transcripts and those that begin within exon 2. This primer was designated forward primer 2. The resulting PCR product formed using forward primer 2 was increased by 5-fold when using RNA template from PC12 cells treated with forskolin and TPA for 4 hours as compared to template from untreated cells. Four hour treatment with forskolin alone resulted in a 3.5 fold increase in the transcript. On the other hand, neither treatment led to an increase in transcripts that could be used as a template for forward primer

1(see figure 2-11).

As a control for exon 1 containing transcripts, PC12 cells were treated with

NGF for 7 days. This treatment has been shown to upregulate a NOS1 transcript that contains the rat 1A alternate first exon (Rife 1999). Indeed, treatment of PC12 cells with NGF led to a 4-fold upregulation of a first exon-containing transcript (see figure

2-12). PACAP treatment alone did not induce production of a first exon containing transcript. The combination of NGF and PACAP led to a similar upregulation of a

70

Figure 2.12: RT-PCR for NOS1 in NGF and PACAP treated PC12 cells: A). This image is from a representative experiment. It shows the amplicons obtained from rt-PCR for NOS1 using RNA obtained from PC12 cells treated with NGF, PACAP or both. The upper panel shows PCR products obtained using a forward primer that lay outside the putative transcription start site for the transcript driven by the exon 2 promoter and therefore should only amplify transcripts that contain an alternate first exon. The second panel shows PCR products obtained using a forward primer that lay just inside the ATG translation start site and should amplify transcripts containing a first exon and those that begin in exon 2. The untreated one group is from PC12 cells incubated for 1 week with no drug added as a control for NGF treatment while the unt- 2 group was a group incubated for 2 days as a control for the PACAP treated group. B). The graphs are a quantification of the above pictures obtained using ImageJ software then normalized to GAPDH and then the unt-1 group. Note that in an exon 1 containing transcript was upregulated by NGF while only a shorter transcript was upregulated by PACAP.

71 A. Unt. NGF unt-2 PAC N+P rat brain

Forward primer 1

Unt-1 NGF Unt-2 PAC N+P rat brain

Forward primer 2

Unt. NGF Unt2 PAC N+P rat brain

GAPDH

B.

Forward Primer 1

6

5

4

3

2

Relative expression 1

0 Unt. 1 NGF Unt 2 Pacap NGF+Pac treatment

Forward Primer 2

6

5

4

3

2

Relative expression 1

0 Unt. 1 NGF Unt 2 Pacap NGF+Pac Treatment

72 first exon containing transcript as NGF alone. Thus PACAP does not appear to induce exon 1 containing NOS1 transcripts. In sharp contrast, PACAP treatment does increase NOS1 mRNA levels measured using primer 2. Thus the exon 2 promoter is implicated in both the action of PACAP and of TPA plus forskolin.

Conclusions and Discussion

In this study, we first determined that NOS1 was induced by PACAP 38 in rat

PC12 cells, enhancing both NOS1 protein and mRNA levels.. Our data contrast sharply with previous results of others. Onoue and colleagues have shown that PC12 cells treated with PACAP38 for 12 hours produce about 20% less nitrite, a stable metabolite of NO. This presumably indicates lower NOS activity in these cells.

Using semi quantitative rt-PCR, they showed a slight decrease in NOS1 mRNA after 6 hours of PACAP treatment (Onoue et al. 2002). One possible explanation for this is that PC12 cells possessed by one lab may well differ from that used by another. In fact, PC12 cells from different sources differ in their adherence and in the presence of short processes. Therefore, it is a formal possibility that PACAP stimulates different pathways in our version of PC12 cells relative to the version used by Onoue et al. In

SK-N-SH cells, a human neuroblastoma cell line, we have preliminary results that indicate TPA treatment diminishes NOS1, If, in the PC12 cell variant used by Onoue et al, PACAP predominantly activated the PKC pathway while in our variant both the

PKC and PKA pathways were activated, this might explain the disparate results.

Regardless of the results of others, the extensive data shown in this chapter

73 demonstrate that PACAP treatment increases NOS1 protein and mRNA in the PC12 cells used for this study.

The induction of NOS1 by PACAP treatment was much greater than that observed in cells that were treated with forskolin, a drug that activates all known adenylate cyclases. Forskolin treatment results in a less than 2 fold increase in NOS1 protein and does not affect NOS activity. PACAP38 treatment led to as much as an 8 fold increase in NOS1 protein levels. This result indicates that PACAP38 is doing more that merely stimulating the cAMP-PKA pathway. As previously mentioned,

PACAP38 is able to stimulate multiple signal transduction pathways, such as the PKC pathway. We hypothesized that PACAP must stimulate both of these pathways in order to stimulate NOS1 expression. As a test of this hypothesis, we treated PC12 cells with forskolin and TPA, a drug that activates PKC. Treatment of PC12 cells with both of these agents increased NOS1 protein more than treatment with forskolin alone but was still slightly effective less than treatment with PACAP38. NOS1 protein levels in cells treated with PACAP38 peak at 24 hours and remain elevated at 48 hours. NOS1 levels in cells in cells treated with TPA plus forskolin, on the other hand, reach their highest levels at 48 hours. As the drug doses used are sufficient to cause a maximal response, the observed differences in the protein expression profile are unlikely to be a dose effect. Different rates and mechanisms of inactivation and or receptor desensitization may explain some of these time-course differences. This will be discussed in more detail later

74 As the combination of TPA and forskolin led to increases in NOS1 protein expression greater than that observed in cells treated with forskolin alone and TPA alone does not affect NOS1 mRNA levels, we wanted to determine if TPA acted at the transcription or translation (or both). To test for effects on translation, PC12 cells were treated with forskolin for 6 hours to increase NOS1 mRNA levels. These cells were then treated with or without TPA for 1.5 hours. During this period, these cells were incubated with 35S methionine and cysteine. NOS1 was then immunoprecipitated. These results were then compared with those obtained by pre- treating with forskolin and TPA for 6 hours followed by a 1.5 hour labeling period. If

TPA was present only during the 1.5 hour labeling period, the amount of newly synthesized NOS1 protein was similar to that seen in cells treated with forskolin alone.

This amount of newly synthesized (labeled) NOS1 was less that seen in cells treated with both TPA and forskolin for 6 hours prior to labeling. If TPA was acting to directly stimulate protein translation, it would be expected that addition of TPA during the 1.5 hour labeling should enhance new NOS1 synthesis. However, if TPA was only acting to increase the NOS1 mRNA pool, a long incubation period might be necessary.

Thus, our data suggest that TPA is acting to regulate NOS1 mRNA levels and not directly influencing NOS1 translation.

We also evaluated the effect the various drug regimens had on NOS1 mRNA levels. A significant increase in NOS1 mRNA was observed in cells treated with

PACAP. Treatment of PC12 cells with forskolin alone, on the other hand, resulted in a considerably smaller increase in NOS1 mRNA. This provided additional evidence

75 that PACAP was stimulating multiple signal transduction pathways. When PC12 cells were treated with both forskolin and TPA, activating both the PKA and PKC pathways, the peak levels of NOS1 mRNA was similar to that seen in cells treated with PACAP. However, the lifetimes of the increases in NOS1 mRNA were different.

The increase in NOS1 mRNA in cells treated with PACAP was more sustained and remained elevated at 12 and 24 hours. When cells were treated with TPA and forskolin, NOS1 levels peaked at 6 hours then fell rapidly to a level that by 24 hours was considerably lower than the levels seen in cells treated with PACAP

mRNA levels are controlled both by the rate of synthesis and by the rate of degradation. Thus, in principle, the different drug treatment regimens might alter turnover of NOS1 mRNA. However, the half-life of NOS1 mRNA is similar in cells treated with forskolin or with forskolin and TPA. Thus TPA mediated message stabilization does not appear to contribute to the increase in NOS1 mRNA.

Furthermore, the half life of NOS1 mRNA in PACAP treated cells is also similar to that observed in cells treated with forskolin or TPA plus forskolin. Thus message stabilization per se does not appear to be the mechanism leading to prolonged elevation of NOS1 mRNA in PACAP treated cells. Our favored model is that all of the drugs effect NOS1 transcription and that subtle differences in desensitization rates and durations of action produce differences in the lifetime of the NOS1 mRNA.

Desensitization refers to a decreased response of cells secondary to prolonged exposure to drugs or hormones. With homologous desensitization, only response to the hormone/ drug is affected. With heterologous desensitization, treatment with one

76 drug causes desensitization to other hormones or drugs as well. Whether forskolin causes homologous desensitization varies among systems. In some cases, desensitization of adenylate cyclase occurs (Seamon et al. 1986) . In other cases, sensitization actually occurs (el Jamali et al. 1996). The duration of action of forskolin is effected by other factors as well. Included among these are increased phosphdiesterase synthesis or activity and active removal of the drug from the cell

(Seamon et al. 1986) Finally, prolonged treatment with forskolin can lead to heterologous desensitization, inhibiting the effect of other hormones or drugs that activate adenylate cyclase (el Jamali et al. 1996). This could include any autocrine or paracrine factors released from the cells that activate adenylate cyclase.

TPA causes both heterologous and homologous desensitization. It does this by binding to and initially activating PKC and then triggering its degradation (Ballester et al. 1985; Fabbro et al. 1986). In fact, pretreatment of cells with TPA has become an accepted method of depleting cells of PKC (McArdle et al. 1989). Thus, desensitization is a major determinant of TPA’s duration of action.

PACAP treatment also leads to rapid desensitization, but only to itself

(homologous desensitization) (Dautzenberg et al. 2001; Niewiadomski et al. 2002).

This desensitization is also probably a major determinant of PACAP’s duration of action but degradation of the peptide and other cellular compensatory mechanisms may influence it as well. To summarize, TPA, forskolin and PACAP38 all have different pharmacokinetic profiles resulting from different mechanisms of inactivation or desensitization. These different profiles are apt to contribute to the different NOS1

77 expression profiles observed even if PACAP38 is acting through these two pathways to stimulate NOS1 expression.

It is also possible that the differences in NOS1 gene expression observed reflect additional activities of PACAP that are not mediated by PKA or PKC. PACAP has been shown to activate a variety of other pathways including the MAPKKK pathway and the TRK A receptor pathways (Lee et al. 2002). PACAP38 can also stimulate calcium influx via the L- type calcium channels (Chatterjee et al. 1996).

The rat NOS1 gene gives rise to multiple transcripts and much of the heterogeneity observed is at the 5’ terminus (Xie et al. 1995; Wang et al. 1999; Boissel et al. 2003). In addition to the 5 alternate first exons that have been identified in the rat, a shorter transcript in which transcription begins in exon 2 and whose expression is driven by a promoter found in the 5’ UTR also likely exists. Based on the results of the RT-PCR assay shown above (see figure 2-11) it can be concluded that the shorter transcript in which the first exon is actually what is designated exon 2 in a full length transcript is the predominant mRNA upregulated by PACAP38 treatment.

The ability of PACAP38 to regulate NOS1 level could have significant physiological implications. PACAP38 and nitric oxide produced by NOS1 are both involved in learning and memory, bronchial dilation and in regulation of leutenizing hormone release (Belvisi et al. 1992; Kinhult et al. 2000; Sauvage et al. 2000) (Garrel et al. 2002). Based on these overlapping functions, one could hypothesize that

PACAP38 may modify NOS1 expression in these areas as a mechanism of regulating these functions. Some evidence for this exists as PACAP has been shown to effect

78 NOS1 expression in rat pituitary cells (Garrel et al. 2002). The interaction between

PACAP38 and NOS1 may turn out to be important in a variety of physiological functions Further, the mechanisms of NOS1 regulation identified in this chapter (and in the next chapter) may be important in other cell types that express NOS1. Even cells that don’t express either type of PACAP receptor will possess multiple other receptors capable of increasing cAMP and/or PKC levels as these are universal signaling pathways.

PC12 cells are derived from a rat pheochromocytoma, a tumor of the adrenal gland (Greene et al. 1976). Although these cells are not peripheral neurons, they do have some similarities with peripheral nerves. PC12 cells are derived from the neural crest, as are peripheral nerves. PC12 cells make and store neurotransmitters

(predominantly norepineprine) and release them upon depolarization. Further, when this cell line is treated with NGF, it develops processes and takes on a neuronal phenotype. Based on these characteristics, this cell line may be a decent model for studying the effects of PACAP on gene expression in peripheral nerves.

In conclusion, the studies in this chapter established that our PC12 model could be used to study the effect of PACAP38 on NOS1 expression. We showed that

PACAP38 increased NOS1 mRNA and protein levels in PC12 cells and that the magnitude of this upregulation was greater than that observed in cells treated with forskolin. This result implied that PACAP must be activating more than just the cAMP-PKA pathway in these cells. Treatment of cells with drugs that activated both the cAMP-PKA and the PKC pathways (forskolin and TPA) lead to an increase in the

79 magnitude of the observed NOS1 upregulation over that seen when cells were treated with only forskolin. The activation of both of these pathways by PACAP may partially explain how it is stimulating NOS1 production. As NO produced by NOS1 and PACAP are involved in many of the same physiological processes, one could speculate that regulation of NOS1 by PACAP may regulate these processes (Belvisi et al. 1992; Kinhult et al. 2000; Sauvage et al. 2000) (Garrel et al. 2002).

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Sauvage, M., P. Brabet, et al. (2000). "Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks." Brain research Molecular brain research 84(1-2): 79-89. 82 Seamon, K. B. and J. W. Daly (1986). "Forskolin: its biological and chemical properties." Adv Cyclic Nucleotide Protein Phosphorylation Res 20: 1-150.

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Wang, Y., D. C. Newton, et al. (1999). "RNA Diversity Has Profound Effects on the Translation of Neuronal Nitric Oxide Synthase." Proceedings of the National Academy of Science USA 96(21): 12150-12155.

Xie, J., P. Roddy, et al. (1995). "Two Closely Linked But Separable Promoters For Human Neuronal Nitric Oxide Synthase Gene Transcription." Proceedings of the National Academy of Science USA 92: 1242-1246.

Zhang, Z. G., M. Chopp, et al. (1994). "Upregulation of Neuronal Nitric Oxide synthase and mRNA, and Selective Sparing of Nitric Oxide Synthase Containing Neurons after Focal Ischemia in Rat." Brain Research 654: 85-95.

83

CHAPTER 3

PACAP38 STIMULATES MULTIPLE PROMOTERS OF NOS1

Introduction

In the previous chapter, using a rat PC12 cell model, we demonstrated that

PACAP38 was able to stimulate NOS1 expression. Further, expression of the transcript upregulated by PACAP38 appeared to be driven by a promoter within exon

2 (designated the E2 promoter). In this chapter, we followed up on these previous results by examining showing that PACAP activated the human E2 promoter. We also show that a corresponding promoter exists within exon 2 of that rat and that this promoter is also regulated by PACAP. Finally, the effect PACAP had on the human

5’2 promoter is also examined

Previously, Wei-Kang Chen and Hua Wei established the existence of the E2 promoter in humans and mapped the regions of this promoter necessary for activation by forskolin. They showed that mutation of 3 putative CRE sites in the E2 promoter abrogated its ability to drive expression of a reporter gene in PC12 cells treated with forskolin. These same mutations, on the other hand, only partially block the ability of

PACAP treatment to stimulate luciferase expression driven by this promoter. These

84 data support the hypothesis that PACAP must stimulate multiple pathways in order to fully activate NOS1 by way of its E2 promoter

The human and rat E2 promoters share one region of significant homology (see figure 3-1). Within this region, there are 3 conserved putative CRE sites. For this reason, we postulated that the rat exon 2 5’UTR would be able to drive the expression of a reporter gene and would be responsive to PACAP38. To test this, the 5’ untranslated region of rat exon 2 was positioned upstream of the luciferase gene and this construct was transfected into PC12 cells. Data described in this chapter show that this promoter is able to drive expression of the reporter gene in PC12 cells and is stimulated by PACAP38.

We also show in this chapter that an additional NOS1 promoter is responsive to PACAP38. Terrie Rife, working in our lab, has previously shown that when stably transfected into PC12 cells, the expression of a luciferase reporter gene driven by the

5’1-5’2 (1G-1F) promoter complex was inducible by forskolin. (Rife 1999). As one of the actions of PACAP is to increase cAMP levels, we hypothesized that PACAP would be able to stimulate the expression of a reporter gene driven by these promoters.

In order to test this possibility, a construct containing the 5’15’2 (1F-1G) promoter complex was transfected into PC12 cells. The cells were then treated with PACAP38 and the amount of luciferase present was determined. We found that the 5’15’2 (1F-

1G) complex is capable of driving luciferase expression in PC12 cells and that this expression is indeed induced by PACAP38 treatment.

85 A.

Rat: 102 cttagggacacatcccgttgctgcccctgacgtctgcctggtcaaccttgacttcctttg 161 ||||||||||| |||| || | | |||||||||||||||||||| | |||||||| | Hum: 190 cttagggacacgtcccaccgcctctcttgacgtctgcctggtcaaccatcacttccttag 249

Rat: 162 agagtaaggaagggggcggggacacgttgaaatcatgccacccaaggccgaatcggaatg 221 ||| |||||| | ||||| || |||||||||||||| | || | | | ||| Hum: 250 agaataaggagagaggcggatgca---ggaaatcatgccaccgacgggccaccagccatg 306

Rat: 222 agcagatgacgccaagttgacgtcaaagacagag 255 || | |||||| || ||||||||||||||||| hum: 307 agtgggtgacgctgagctgacgtcaaagacagag 340

B

Rat: 4 tctgacaagctggtgaccaagatgcccagagactagaccctatgcttgtgagtcacagtc 63 |||| |||||||||||||||||||||||||| |||| | ||| ||||| ||||||||| Mouse: 32 tctgtcaagctggtgaccaagatgcccagagcctagtttccatgtttgtgcgtcacagtc 91

Rat: 64 atcagacacggcaaacctccagtcttcctgacctgttgcttagggacacatcccgttgct 123 |||||||||||||| ||||||||||||||||||||||||||||||||||| ||| | || Mouse: 92 atcagacacggcaagcctccagtcttcctgacctgttgcttagggacacaacccatcact 151

Rat: 124 gcccctgacgtctgcctggtcaaccttgacttcctttgagagtaaggaagggggcgggga 183 | ||||||||||||||||||||||||||||||||| |||| |||||||||||||||| | Mouse: 152 gttcctgacgtctgcctggtcaaccttgacttccttagagaataaggaagggggcgggaa 211

Rat: 184 cacgttgaaatcatgccacccaaggccgaatcggaatgagcagatgacgccaagttgacg 243 ||| ||||||||||||||||||||||||| ||||||||| ||||| |||||||||| Mouse: 212 cac-atgaaatcatgccacccaaggccgaacaacaatgagcaggtgacgtcaagttgacg 270

Rat: 244 tcaaagacagaggcgacagaaactctgcagccagctcttgcccccgaggagctcaggttc 303 |||||||||||||||||||||||||||||||| | ||||| ||| ||| ||||||||||| Mouse: 271 tcaaagacagaggcgacagaaactctgcagccggttcttg-cccggagtagctcaggttc 329

Rat: 304 ctgcaggagtcattttagctt---agtcttctgaaggacacagatacc 348 ||| |||||| | || |||| ||||||||||||||||||||||| Mouse: 330 ctgtgggagtcgtcttggcttggaggtcttctgaaggacacagatacc 377

Figure 3.1: Sequence alignment of mouse, rat and human exon 2 5’UTR. Figure A shows a sequence alignment of the 5’untranslated region (UTR) of exon 2 of the rat with the human 5’ UTR. Only one 153 base pair region shows significant homology. Within this region, 3 putative CRE sites are conserved. Figure B shows sequence alignment of the 5’ UTRs of rat and mouse exon 2. The rat and mouse have significant homology (88%) throughout the entire region and the same CRE sites are conserved between rat and human are conserved between rat and mouse. Note that only the 5 base core regions as identified by the Match program are highlighted. 86 We have also identified the regions of this promoter that are necessary for the observed induction. We transfected a variety of deletion constructs into PC12 cells then treated them with PACAP38. We first showed that PACAP38 stimulates the 5’2 promoter. The region of this promoter responsible for this induction maps to a 230 base pair region just upstream of the corresponding alternate first exon. The basal luciferase expression driven by this region is considerably less than the full 5’2 (1F) region but the magnitude of the upregulation by PACAP is unchanged

PACAP38 can activate a variety of signal transduction pathways. One of these is the cAMP-PKA pathway. It seems likely that activation of this pathway is at least partially responsible for the observed increase in luciferase levels. (Vaudry et al.

2000). To test this, we attempted to block the activation of the 5’2 promoter using a

PKA inhibitor (H-89). It was found that this inhibitor is able to completely block the increase in luciferase activity observed in transfected PC12 cells treated with

PACAP38. We next examined the sequence of the truncated 5’2 promoter and identified a single putative CRE/ATF binding site. Mutation of this site abolished the ability of PACAP to stimulate an increase in luciferase activity. Therefore PACAP is able to stimulate expression of luciferase driven by the NOS1 5’2 promoter by activating the cAMP-PKA pathway via a CRE/ATF binding site located within this promoter.

Materials and methods

Cell culture: PC12 cells were grown in Dulbecco’s modified eagle medium with glucose 4500mg/ml (DMEM-HG) supplemented with 5% horse serum, 5% fetal

87 bovine serum, penicillin (100Units/ml) and streptomycin (100ug/ml). They were maintained in a 37 degree incubator with 5% CO2, 95% room air. Media and serum were obtained from Irvine Scientific (Irvine, CA) while penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA).

PACAP and H89 treatment: PC12 cells were transfected as described above.

The cells were then incubated in 2.5ml DMEM-HG plus supplements on a 6 well plate. After incubation, 25ul of a 10uM stock solution of PACAP38 in DMEM (with no serum or supplements) was added to each well. This resulted in a final PACAP38 concentration of 100nM. At various times after PACAP addition, cells were harvested and protein extracted as described. PACAP 38 was obtained form Phoenix

Pharmaceuticals (Belmont, CA). When the PKA inhibitor H89 was added, 5 ul of a

10mM stock solution in DMSO was added to each well. This resulted in a final H89 concentration of 20uM.

Forskolin treatment: PC12 cells were seeded at 2x106 cells per well as described above. After incubation for 16-20 hours, 2.5ul of a 10mM stock solution of forskolin in DMSO was added to each well. This resulted in a final concentration of

10uM. When H89 was added to the cells, it was added as described above. Forskolin was obtained for Sigma (St Louis, MO)

Transient transfections were carried out using lipofectamine and plus reagent

(Invitrogen) per the manufacturers instructions. PC12 cells were seeded on 6 well plates at a density of 6 x 105 cells per well using DMEM-HG with serum and as described above. The cells were then incubated at 37 degree C in 5%

88 CO2, 95% room air for 20-24 hours. Forty minutes prior to transfection, the media was changed to DMEM-HG with no serum or antibiotics. A transfection mix consisting of 3 mcg of the construct to be transfected, 9 micrograms carrier DNA

(pBluescript SK +), 600 microliters DMEM-HG (without additives), and 48 microliters Plus reagent was set up. After a 15 minute incubation at room temperature, 600 microliters DMEM-HG and 33 microliters lipofectamine was added to the mix. After incubation for 15 minutes at room temperature, 4.8ml DMEM was added to the mix. The media was then removed from the cells to be transfected and replaced with 1ml of the transfection mix. The cells were then returned to the incubator for three hours. The transfection mix was then removed and replaced with

DMEM-HG with serum and antibiotics. Cells were then returned to the incubator overnight prior to drug addition.

Cell extracts were obtained 24 hours after drug addition using the following protocol. Cells were washed with ice cold PBS then lysed using a lysis buffer consisting of 1% Triton X-100, 25mM Gly-Gly (pH 7.8), 15mM MgSO4, 4mM

EGTA, and 1mM DTT. Lysates were then centrifuges at 14000G for 5 minutes then stored at –80 degrees C until use.

Luciferase assays were performed using the following protocol (Sambrook et al. 2001). Fifty microliters of cell extract was added to 350 microliters reaction buffer consisting of 25mM gly-gly buffer (pH 7.8), 5mM ATP, and 15 mM MgSO4. The reaction was briefly mixed and place in a luminometer that injected 100 microliters of

1 mM luciferin solution into the tube. The amount of light emitted was then counted

89 for a ten seconds and was reported as relative light units (RLU). These RLU readings were then normalized to protein concentration, which was obtained using the Bio-Rad protein assay, a modification of the Bradford protein assay, per the manufacturer’s instructions. Data was reported as RLU/ 50ug protein.

The rat exon 5’ UTR was cloned using a PCR based strategy. Forward and reverse primers were designed that flanked the region of interest (see table 1 for all primer sequences used in this chapter). To facilitate subcloning, a unique BamHI restriction site was incorporated into the forward primer while a Bgl II site was incorporated into the reverse primer. A 50ul reaction mix consisting of 10ul first strand from rat brain, 1x PCR buffer, 0.2mM each dNTP, 0.5uM forward primer,

0.5uM reverse primer, 2.5mM MgCl2, 10% DMSO, and 0.5ul Taq polymerase was set up. The PCR mix was then incubated at 95 degrees C for 5 minutes followed by thirty cycles of 95 degrees for 30 seconds, 52 degrees C for 30 seconds, and 72 degrees C for 40 seconds. The mix was then incubated at 72 degrees C for 10 minutes. The amplicons were evaluated on an agarose gel. The mix containing 10% DMSO had a single band of the correct size and thus was cloned into the pCR 2.1 vector using the original TA cloning kit (Invitrogen) per the manufacturer’s instructions. Clones were evaluated first by digestion with Bgl II and Bam HI then by sequencing. The E2 region was then liberated from pCR 2.1 by digestion with BamH1 and Bgl II and subcloned into pXP2. The sequence of the forward primer used is 5’

CGGATCCAGGTAGAAGCCCCTATGTCA 3 and of the reverse primer is 5’

GAGATCTTGTCCTTCAGAAGACTAAGC 3’

90 CRE site mutation: The putative CRE site in the 1613-1842 region of the

NOS1 5’2 promoter was mutated using a two step PCR based protocol (Sambrook et al. 2001). Briefly, two PCR reactions were set up using 1613-1842-Luc as the template. The first reaction was set up using a forward primer from just upstream of the promoter region and a reverse primer that spanned the desired mutation site and contained the desired mutations. The second reaction used the same template. The forward primer in this reaction was the reverse complement of the reverse primer used in the first reaction while the reverse primer was from a region of the vector just outside of the promoter region. Figure 3-2 shows all the sequence of the primers used in the construction of this mutant. The amplicons were then separated on a 1.5% agarose gel using electrophoresis and the bands were isolated using a QIAquick gel extraction kit (Qiagen) per the manufacturer’s instructions. A third PCR reaction was then set up using 10ng of the amplicon from the first reaction and 10ng of the band from the second reaction as template. The forward primer used was the forward primer from reaction 1 above and the reverse primer was the reverse primer from reaction 2 above. The third PCR was then evaluated using gel electrophoresis and the desired band was then cloned into the pCR2.1 vector using the original TA Cloning

Kit (Invitrogen) per the manufacturer’s instructions. Plasmid DNA from four of the resultant colonies was prepared using a Mini prep kit from Qiagen and the clones were then sequenced (sequencing was carried out by the OSU Rightmire DNA sequencing

Facility). The mutated promoter region was then subcloned into the KPN and Bgl II sites of the luciferase vector pXP2.

91

A.

Primer Sequence (5’ to 3’) pXP2 fwd GCTCAGATCCAAGCTTGTCG’ pXP2 rev AACAGTACCGGAATGCCAAG 5’2CREmut-fwd CCAGTCGGGTTGGACTGAACTGCTAATTCGTTTC 5’2CREmut-rev GAAACGAATTAGCAGTTCAGTCCAACCCGACTGG

B. 5’ CCAGTCGGGTTGGACGTCACTGCTAATTCGTTTC 3’ | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 5’ CCAGTCGGGTTGGACTGAACTGCTAATTCGTTTC 3’

Figure 3.2: Primers used in the construction of 1613-1842CREmut. Figure A shows the sequences of the primers used in the construction of the CRE mutant construct. Figure B shows the mutations made. The top strand is the wild type and the bottom strand is the mutant. The core region of the putative CRE site is in bold.

92 Results

Summary of results with the human E2 promoter

To evaluate the response of the human E2 promoter to PACAP, Wei-Kang

Chen in our lab cloned the 5’ UTR of human NOS1 exon 2 upstream of the luciferase reporter gene in the vector pXP2. Wei-Kang Chen and Hua Wei in our lab then transfected this construct into PC12 cells and assayed for forskolin and/or PACAP inducibility of the reporter. Luciferase expression is enhanced by both drugs.

PACAP38 results in an increase in luciferase expression of 9.9(+/- 4.7, n=4) fold while forskolin results in an increase of 8.5(+/- 3.6, n=4) fold.

It was hypothesized that H89, a drug that blocks PKA, would prevent PACAP from upregulating luciferase expression driven by the exon 2 promoter. Hua Wei tested this hypothesis by transfecting PC12 cells with the construct containing the human promoter then treating the cells with H89 and either forskolin or PACAP. As shown in figure 3-3, H89 diminishes both the forskolin and PACAP mediated induction of luciferase in a dose dependent manner. The induction by PACAP appears to be partially resistant to H89 treatment.

Four putative CRE sites were identified within the human E2 promoter. Three of these sites are conserved in mice and rats. Mutations of the 2 sites proximal to the translation start site abrogate basal expression levels and reduce forskolin mediated induction to levels seen in cells transfected with the promoterless vector. These same mutations partially diminish PACAP mediated induction of luciferase expression.

93 80 70 60 50 40 30 20 RLU/ug protein 10 0

Plasmids: pXP2 pXP2E2wt Forskolin: - + + + PACAP: - + + + 10µMH89: - + + 20µMH89: - + + Fold Induction: 1x 13.8x 8.2x 5.6x 17x 10.3x 7.4x

Figure 3.3: Inhibition of forskolin and PACAP upregulation of luciferase by H89. This figure shows the results of a transient transfection experiment in which a construct containing the luciferase reporter gene driven by the human exon 2 promoter was transfected into PC12 cells. Twenty-four hours after transfection, the cells were treated with the PKA inhibitor H89 (10 or 20uM) and either forskolin (10uM)or PACAP38 (100nM) for 20 hours. Note that H89 diminished both forskolin and PACAP mediated increases in luciferase expression. Note that PACAP increased luciferase levels to a larger extent than forskolin and seemed slightly more resistant to H89 inhibition. This experiment was performed by Hua Wei.

94 5000

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2000 RLU/ug protein RLU/ug 1000

0

pXP2 E2 WT Mut1 Mut2 Mut123 Basal activity(%): 100 36 27 21 Fold Induction(forskolin): 7.2x 4.8x 4.2x 3.6x Fold Induction(PACAP): 7.55x 8x 6.4x 5.5x

Figure 3.4: Mutation of CRE sites Attenuates PACAP and forskolin mediated luciferase upregulation. The above figure shows the results of an experiment in which constructs containing the luciferase reporter gene driven by the E2 promoter region with or without mutations to various CRE sites were transfected into PC12 cells. Twenty-four hours after transfection, these cells were treated with PACAP38 (100nM) or forskolin (10uM) for 21 hours and luciferase activities evaluated as described above. The mutation of either of the 2 CRE sites proximal to the translation start site or mutation of all 3 conserved sites inhibited both forskolin and PACAP mediated increases in luciferase expression. Note that the mutated constructs were stimulated by PACAP to a greater extent than by forskolin. This supports the idea that PACAP stimulation of NOS1 via the E2 promoter requires the stimulation of multiple signal transduction pathways. This experiment was performed by Hua We

95 Typical results can be seen in figure 3-4. Mutant constructs were built by and transfections carried out by Wei-Kang Chen and Hua Wei working in our lab.

As PACAP has been shown to stimulate both the PKA and PKC pathways, we tested the effect that TPA and forskolin had on human E2 promoter driven expression of luciferase in our transient transfection model. Wei Kang Chen, working in our lab, transfected the human E2 construct into PC12 cells then treated these cells with forskolin, TPA or both. Figure 3-5 shows a typical result. While forskolin alone led to a 6.7 fold increase in luciferase activity, treatment with both forskolin and TPA led to an 11.6 fold increase.

Summary of results with the rat E2 promoter

The human exon 2 promoter and 5’ untranslated region of exon 2 of the rat share a single region of significant homology and three of the four putative CRE sites found in the human are retained in the rat. Based on this significant homology and the data described in chapter 2, we hypothesized that the exon 2 region of the rat NOS1 gene also contained a promoter. We further believed that this promoter would be responsive to PACAP. To test this, the 5’UTR of exon 2 of rat NOS1 was cloned upstream of the luciferase gene and this construct was transfected into PC12 cells.

The cells were then treated with PACAP38 and luciferase activity levels were evaluated. A typical result is shown in figure 3-6. The basal luciferase activity was

251 (+/- 50, n=3) fold higher than that observed in cells transfected with the empty vector. Luciferase expression was induced 8.5(+/-1.8, n=3) fold in these cells

96 6000

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1000

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pXP E2(Hum)-Luc DMSO (+) (-) (-) (-) (+) (-) (-) (-) TPA (-) (+) (-) (+) (-) (+) (-) (+) FSK (-) (-) (+) (+) (-) (-) (+) (+) Fold Induction 1 1.2 3.4 5.3 1 1.3 6.7 11.6

Figure 3.5: Stimulation of the Human E2 promoter by TPA and forskolin. The above figure shows the result of an experiment in which a construct containing the luciferase reporter gene driven by the human NOS1 exon 2 promoter was transfected into PC12 cells and the cells were treated with forskolin (10uM), TPA (200nM) or both. After 12 hours, cell extracts were prepared and assayed for luciferase activity. Note when these cells were treated with both TPA and forskolin, the luciferase activity present in extracts was considerably higher than that observed in cell extracts from cells treated with either agent alone. This experiment was performed by Wei-Kang Chen

97 400000

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50000

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pXP2 Rat E2 Human E2

PACAP (-) (+) (-) (+) (-) (+) Fold Induction 4.6 8.9 7.5

Figure 3.6: PACAP stimulates the rat E2 promoter. This figure shows the result of an experiment in which a constructs containing either the human or rat E2 promoters cloned upstream of the luciferase gene were transfected into PC12 cells. Twenty four hours after transfection, these cells were treated with PACAP(100nM) for 20 hours. The luciferase levels were then determined using luciferase assays as described in the methods. Both the human and rat E2 promoters were stimulated by PACAP.

98 by PACAP38 treatment. Both the basal levels and induction by PACAP38 were similar to that observed with the human promoter (also shown in figure 3-6).

Summary of results obtained with the 5’15’2 promoter complex

In order to determine if PACAP38 stimulated the 5’1 or 5’2 NOS1 promoters, a plasmid containing the 5’1 and 5’2 promoters cloned upstream of the luciferase reporter gene was transfected into PC12 cells (the plasmid was designated pNOS 4.3-

Luc and was constructed by Dr Jinling Xie). The cells were treated with PACAP38 for 24 hours and protein extracts from these cells were then assayed for luciferase activity. Results from a typical experiment are shown in figure 3-7. The basal luciferase activity in cells transfected with this construct were 58.4 (+/- 6.3, n=2) fold higher than that observed in cells transfected with the empty vector. When cells transfected with the pNOS4.3-luc vector were treated with PACAP38, luciferase activity is increased 4.2 (+/- 0.1, n=2) fold. The luciferase activity levels in cells transfected with the empty vector also increase slightly (2.9 +/- 0.2 fold, n=12) when they were treated with PACAP38.

The pNOS4.3-luc vector actually contains 2 separate promoters (Xie et al.

1995). We tested one of these promoters, the 5’2 (1F) promoter, to see if it responded to PACAP38 treatment. Cells transfected with the construct containing this promoter upstream of the luciferase gene have a basal luciferase activity about one half of that observed in cells transfected with the p4.3NOS-Luc construct. When these cells are treated with PACAP38, luciferase activity is increased by 9.5 (+/- 1.0, n=3) fold over untreated controls. Results from a typical experiment are shown in figure 3.8. Since

99

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Treatment group

Figure 3.7: PACAP38 Stimulates 5’15’2 Driven Luciferase expression in PC12 cells. The above figure shows results from a representative experiment. A luciferase expression vector in which luciferase expression is driven by the NOS1 5’1 and 5’2 promoters (or the vector alone) was transfected into PC12 cells. Twenty four hours after transfection, the cells were treated with PACAP38 (100nm). After 20-24 hours crude protein extracts were prepared and used in luciferase assays as described in the methods section. Note that basal luciferase levels for the promoter driven vector were significantly higher than the promoterless vector and the that luciferase levels increased 4.3 fold when the cells were treated with PACAP38

100

A.

1842+pac

1842

4.3+ pacap

4.3

pXP+pac

pXP

0 250000 500000 750000 1000000 1250000

RLU/50ug

B.

4.3NOS (4.3)

1-1842 (1842)(5’2 promoter)

Figure 3.8: The 5’2 promoter is responsive to PACAP38 luciferase activity. Figure A: PC12 cells were transfected with constructs containing the luciferase reporter gene driven by the 5’2 promoter, or both the 5’1 and 5’2 promoters. Twenty- four hours later, the cells were then treated with PACAP38 and after 24 hours crude protein extracts were prepared and then assayed for luciferase activity. Note that in cells transfected with the 4.3NOS construct that contains both the 5’1 and 5’2 promoters and in cells transfected with the construct containing just the 5’2 promoter, basal luciferase activity was greater than that found in cells transfected with the promoterless vector. The luciferase activity was inducible by PACAP38 in these cells as well. 101 the 5’2(1F) promoter was responsive to PACAP38, we determined the minimal

5’2(1F) necessary for this inducibility. A series of constructs containing progressively shorter pieces of the 5’2 promoter cloned upstream of the luciferase gene were transfected into PC12 cells (these constructs were built by Dr Jinling Xie and Dr.

Terrie Rife). The cells were then treated with or without PACAP38 and cell extracts prepared and luciferase activity assayed. Typical results are shown in figure 3-9. All of the constructs with the exception of the one had about the similar basal luciferase activity. Although the basal luciferase level in cells transfected with the shortest construct p1613-1842-Luc diminished, it was still 26 (+/-6.9, n=7) fold higher than that found in cells transfected with the promoterless luciferase vector. All of the constructs, respond similarly to PACAP38 treatment. The 1-1842Luc construct was induced 9.5 (+/-1.0, n=3) fold. The 1195-1842-Luc construct was induced 9.4(+/- 2.4, n=2) fold, the 1470-1842 construct was induced 7.4 (+/- 1.7, n=2) fold and the 1613-

1842 construct was induced 9.0 (+/- 0.5, n=2) fold.

To test whether the cAMP-PKA pathway mediated the PACAP activation of the 5’2 promoter, we tested whether the increase was prevented by the PKA inhibitor

H89. Cells transfected with the 1613-1842-Luc construct were treated with PACAP with or without H89. H89 almost completely blocked PACAP38 mediated induction.

Results from a typical experiment are shown in figure 3-10. It should also be noted that H89 inhibited the induction observed in cells transfected with the promoterless vector secondary to PACAP treatment.

102 550000 500000 450000 400000 350000 300000 250000

RLu/50ug 200000 150000 100000 50000 0 pXP 1-1842 1195-1842 1470-1842 1613-1842 Pacap (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) Fold Induction 2.7 7.7 7.0 5.7 7.8

Construct

B

1-1842

1195-1842

1470-1842

1613-1842

Figure 3.9: Mapping of the NOS1 5’2 Promoter. In order to determine the region of the 5’2 promoter responsible for the increase in luciferase expression in observed in response to PACAP treatment, PC12 cells were transfected with luciferase constructs containing progressively shorter pieces of the 5’2 promoter. These cells were then treated with PACAP and extracts were used in luciferase assays. Figure A shows typical results. Note that in cells transfected shortest construct used (1613-1842) the basal expression was reduced but this construct was still inducible. Figure B: This schematic shows the relative sizes and positions of the promoter regions used to drive luciferase expression.

103 A search of the 1613-1842 region of the NOS1 5’2 promoter using the Match transcription factor binding site search program identified a single putative CRE site.

It was hypothesized that this site was important in mediating the response to

PACAP38. To test this, the core region of this putative site was mutated and cloned upstream of the luciferase reporter gene. The construct was then transfected into

PC12 cells. When these cells were treated with PACAP, the increase in luciferase activity levels observed was attenuated as compared to that observed in cells transfected with the wild type construct. The induction for the mutant construct was

2.9 (+/- 0.2, n=2) fold while the wild type construct was induced 9.0 fold (+/- 0.5, n=9). Is should be noted that the upregulation of luciferase in cells transfected with the mutant construct was similar to that observed in cells transfected with the promoterless vector. Not surprisingly, the response of the mutant constructs to forskolin was also attenuated. Luciferase activity was induced 3.0 (+/- 0.2, n=2) fold in cells transfected with the mutant construct while a 9.7 (+/- 0.3) induction was observed in cells transfected with the wild type vector. These increases in luciferase levels observed in cells treated with forskolin were quite similar to that observed in cells treated with PACAP38. Typical results are shown in figure 3-11.

104 125000

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0 Construct pXP1 1613-1842 PACAP (-) (+) (+) (+) (-) (+) (-) (-) (+) (+) H89 (-) (-) (+) (-) (-) (-) (+) (-) (+) (-) DMSO (-) (-) (-) (+) (-) (-) (-) (+) (-) (+) Fold Induction 2.54 1.21 2.32 6.88 1.09 0.94 1.26 5.28

Construct and Treatment

Figure 3.10: H89 Inhibits Luciferase upregulation due to PACAP38 treatment. PC12 cells were transfected with the 1613-1842-Luc construct. After 24 hours, the cells were treated with PACAP(100nM) and H89(20uM), a PKA inhibitor for 24 hours. Inhibition of PKA prevented the increase in luciferase activity normally observed when cells transfected with this construct were treated with PACAP

105

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RLU/50ug 4000 3000 2000 1000 0 Construct pXP2 1613-1842 1613-1842cremut PACAP (-) (+) (-) (-) (-) (+) (-) (-) (-) (+) (-) (-) FSK (-) (-) (-) (+) (-) (-) (-) (+) (-) (-) (-) (+) DMSO (-) (-) (+) (-) (-) (-) (+) (-) (-) (-) (+) (-) Rel. Exp 1 3.7 1.2 3.6 1 9.9 1.2 9.4 1 3.2 1.3 3.2

Construct and Treatment

Figure 3.11: Mutation of CRE site inhibits upregulation of Luciferase expression by PACAP38. The putative CRE site in the 1613-1842-Luc constructs was mutated and this new construct was transfected into PC12 cells. Twenty four hours after transfection, the cells were then treated with PACAP38 (100nM) or forskolin (10uM) for 24 hours. Protein extracts were then prepared and used in luciferase assays. The above graph shows typical results. Note that the basal expression level and the inducibility of the mutant construct are diminished considerably.

106 Conclusions and discussion

In this chapter, we first examined the effect that PACAP38 had on the human exon 2 promoter. Using transient transfection experiments in PC12 cells, it was demonstrated that the human E2 promoter was stimulated by both forskolin and

PACAP. The cAMP-PKA pathway is one of the pathways activated by PACAP

(Vaudry et al. 2000). For this reason, we tested to see if the PKA inhibitor H-89 was able to block this PACAP mediated increase of luciferase expression. We found that

H-89 partially blocked the increase in luciferase expression in a dose dependant manner. This implied that the activation of the cAMP-PKA pathway was partially responsible for the increase in luciferase expression caused by PACAP but other pathways were likely involved as well. This belief is consistent with the findings described in the last chapter.

An analysis of the sequence of the human exon 2 promoter led to the identification of 4 putative CRE sites. Three of these sites were conserved among the mouse, rat and human. Wei-Kang Chen then built constructs containing mutations in the core region of the first site, the second site or all three sites and transfected these mutants into PC12 cells. The ability of PACAP to stimulate these mutated promoters was diminished but not entirely blocked. Mutation of the third or fourth site alone had little effect on PACAP or forskolin mediated induction (data not shown) (Chen 2003).

This partial inhibition provided further evidence that though important in PACAP mediated stimulation of the human exon 2 promoter, the cAMP-PKA pathway is not the only pathway involved.

107 PACAP is able to activate the PKC pathway as well as the PKA pathway

(Martinez-Fuentes et al. 1998; Vaudry et al. 2000). For this reason, we hypothesized that if we treated transfected cells with TPA, a drug that activates PKC, and forskolin, we would get a larger increase in luciferase than seen in cells treated with forskolin alone. Indeed when cells were treated with both TPA and forskolin, the resulting increase in luciferase activity was greater than that observed in cells treated with either drug alone. This provides some evidence that PACAP may be activating both pathways in PC12 cells and this may be necessary to activate NOS1 by way of the exon 2 promoter

Based on the homology and the conserved CRE sites between the human, mouse and rat and the previous chapters data, we strongly suspected that there was a promoter in the 5’ untranslated region of rat exon 2. Further, we believed that PACAP would stimulate this promoter. To test this, a construct was built that contained this region upstream of the luciferase reporter gene. When this construct was transfected into PC12 cells, luciferase was expressed at levels far greater than background and this luciferase expression was stimulated by PACAP38 treatment. This allowed us to conclude that there was indeed a promoter located in this region and that it was stimulated by PACAP

When a construct containing the luciferase reporter gene under the control of the human 5’2 promoter is stably transfected in PC12 cells, luciferase expression is induced by forskolin. As PACAP is able to stimulate the cAMP-PKA pathway, it was hypothesized that PACAP would activate the human 5’2 promoter as well. Indeed,

108 PACAP can activate the 5’2(1F) promoter. Transient transfection experiments using constructs with progressively shorter pieces of the 5’2(1F) promoter allowed us to map the region responsible for this upregulation to a 230 base pair region just upstream of the 5’2(1F) exon.

We believed that activation of this promoter by PACAP was dependent at least partially on activation of the cAMP-PKA pathway. We first tested this hypothesis by attempting to block the increase in luciferase levels stimulated by PACAP treatment by adding H-89, an inhibitor of PKA, to the cells. When this inhibitor was added, the upregulation seen due to PACAP treatment was completely abrogated. This provided strong support that stimulation of the cAMP-PKA pathway played a role in activation of this promoter..

PKA has been shown to phosphorylate a variety of downstream effectors.

Included among these are CREB/ATF and MAPKKK (Vaudry et al. 2002). In order to determine which of these effectors was necessary for the observed increased in luciferase levels, the sequence of the region of the promoter necessary for PACAP mediated upregulation was scanned for putative CIS element binding sites using the

Match program. A single putative CRE binding site was found within the region.

When this site was mutated, the ability of PACAP38 to increase luciferase activity was decreased to a level similar to that observed in PC12 cells transfected with the empty vector. This provided strong evidence that this CRE/ATF site is important in the activation of this promoter. As mutation of the CRE site or incubation with H89 completely inhibited the ability of PACAP to stimulate this promoter, it appeared that

109 activation of the cAMP-PKA pathway was both necessary and sufficient for 5’2 (1F) activation. This contrasts with what was observed with the E2 promoter. The E2 promoter appeared to require the activation multiple pathways for full activation.

The 5’2(1F) alternate first exon has considerable homology with the rat 1A alternate first exon (Lee et al. 1997). The previous chapter showed that when PC12 cells were treated with PACAP38 the NOS1 transcript containing exon 1a was not upregulated. Instead the expression of a shorter transcript lacking any of the alternate first exons was augmented. On the surface this would seem to contradict what was observed here. It needs to be remembered though that the promoter utilized in this study was human and thus it is very possible that in humans this promoter responds to

PACAP while in rats the promoter driving the 1A transcript does not. Secondly, these were transient transfection experiments and thus the construct was not incorporated into the genome and was missing this level of regulation. One final possibility is that in PC12 cells, this 1A promoter is not responsive to PACAP while it may well be responsive in other cell types.

The fact that the promoterless vector is inducible by forskolin makes evaluation of the data in this chapter little more difficult. Though the absolute luciferase values remain relatively small in cells transfected with pXP2 and treated with forskolin or PACAP, the levels were increased about 2.9 fold. The source of this upregulation is unknown. There are likely CIS elements present in the vector that mediate this increase, one or more of these may lay within the luciferase gene itself.

One argument supporting this assertion is that when a promoterless retroviral vector

110 containing the luciferase reporter gene is transfected into PC12 cells and treated with forskolin or PACAP, a small (approximately 1.5 fold) increase in luciferase activity is observed (Chen 2003). While it would be best to have a promoterless vector that did not respond to the drugs utilized, the relatively low basal luciferase levels and the consistent levels of induction allowed us to account for this when analyzing the data.

In conclusion, in this chapter we transfected various constructs into PC12 cells in order to determine the effects that PACAP had on various NOS1 promoters. We showed that both the human and rat E2 promoters were responsive to PACAP and mutations of CRE sites within the human E2 promoter diminished (but did not completely wipe out) stimulation by PACAP. This implied that cAMP-PKA activation by PACAP was partially responsible for activation of this promoter.

Surprisingly, we also found that PACAP activated the human 5’2 promoter. Unlike the E2 promoter, this promoter appeared to depend solely on the activation of the cAMP-PKA pathway

111 Chapter 3 References:

Chen, W.-K. (2003). Analysis of Neural Gene Expression, Glutamine Synthetase and Nitric Oxide Synthase 1. Molecular, Cellular and Developmental Biology Program. Columbus, The Ohio State University: 174.

Lee, M. A., L. Cai, et al. (1997). "Tissue and Development Specific Expression of Multiple Alternatively Spliced Transcripts of Rat Neuronal Nitric Oxide Synthase." Journal of Clinical Investigation 100(6): 1507-1512.

Martinez-Fuentes, A. J., J. P. Castano, et al. (1998). "Pituitary adenylate cyclase- activating polypeptide (PACAP) 38 and PACAP27 activate common and distinct intracellular signaling pathways to stimulate growth hormone from porcine somatotropes." Endocrinology 139(12): 5116-24.

Rife, T. K. (1999). The Characterization of a Human Nitric Oxide Synthase 1 Promoter Complex. The Ohio State Biochemistry Program. Columbus, The Ohio State University: 163.

Sambrook, J. and D. W. Russell (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press.

Vaudry, D., B. J. Gonzalez, et al. (2000). "Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions." Pharmacological reviews 52(2): 269-324.

Vaudry, D., P. J. S. Stork, et al. (2002). "Signaling pathways for PC12 cell differentiation: making the right connections." Science 296(5573): 1648-9.

Xie, J., P. Roddy, et al. (1995). "Two Closely Linked But Separable Promoters For Human Neuronal Nitric Oxide Synthase Gene Transcription." Proceedings of the National Academy of Science USA 92: 1242-1246.

112

CHAPTER 4

CLONING AND CHARACTERIZATION OF 2 NOVEL PROMOTERS THAT

DRIVE THE EXPRESSION OF NITRIC OXIDE SYNTHASE 1

Introduction

Many genes make use of multiple promoters to Produce multiple transcripts.

Human NOS1 is one of these (Ayoubi et al. 1996). In 1994, Jinling Xie and Anthony

Young discovered that NOS1 gives rise to two different transcripts that have different first exons. As translation of NOS1 begins at an ATG located in exon 2, both of these transcripts give rise to the same protein (Xie et al. 1995). They also showed that, despite the proximity of these two first exons, separate promoters drive the expression of the two alternate transcripts. Subsequent to this discovery, Lee et al found three alternative first exons of NOS1 in the rat. These were designated 1A, 1B and 1C.

While 1A had significant homology with the human exons 5’1 and 5’2, exons 1B and

1C had no significant homology. It was hypothesized that homologues to these latter first exons existed in the human NOS1 gene. Greg Hartt, using rapid amplification of cDNA ends (5’ RACE) cloning method cloned these two alternative first exons using

RNA obtained from a human liposarcoma. These were designated 5’3 and 5’4 by us

113 and 1D and 1C respectively in the universal naming scheme described previously

(Hartt 2003) (Boissel et al. 2003).

Based on these previous discoveries, we chose to clone and characterize the promoter or promoters responsible for driving the expression of these alternate transcripts. In order to do this, a radiolabeled oligonucleotide encoding sequence derived from 5’3 was used to screen a human genomic DNA library. The sequence of this primer was 5’ ATTAGTGCCGCTGGCCTCTC 3’. A single lambda clone was isolated that contained both of the alternate first exons, an intervening intron of about

220 base pairs and approximately 2KB of upstream DNA (see figure 4-1). When this region of DNA was positioned upstream of luciferase, it was able to drive expression of the reporter in transiently transfected Hela and PC12 cells. In order to determine if two different promoters were responsible for the expression of these two transcripts, two different constructs were made. The first contained just the 5’4(1C) exon and its upstream region upstream of the luciferase reporter gene while the second contained the intron between 5’3(1D) and 5’4(1C) and a portion of the 5’3(1D) exon upstream of the luciferase reporter gene. Transient transfection of both of these constructs resulted in increases in luciferase activity in the extracts greater than that seen with the promoterless vector. This supports the idea that 2 different promoters drive expression of the alternate transcripts.

As the 5’3(1D) promoter was much stronger than the 5’4(1C) promoter, various deletion constructs were made of this promoter in hopes of identifying

114 necessary cis elements. A 187 bp region of this promoter was found to be sufficient drive expression of the luciferase gene in hela cells.

Materials and Methods:

DNA library screening: With the assistance of Mingyong Chen, a human genomic DNA library in the EMBL 3 bacteriophage vector obtained from Clontech

(Palo Alto, CA) was screened using a primer designed by Greg Hartt to hybridize to the alternate first exon 5’3(1D). The library was first titered by making serial dilutions in Lambda dilution buffer (100mM NaCl, 10mM MgCl2, 35mM Tris-HCl ph 7.5 and

0.01% gelatin). One hundred ul of each of these dilutions were then mixed with 200 ul E-Coli strain K-803 grown overnight in Luria Broth (LB) with 10mM MgSO4 and

0.2% maltose. After incubation for 15 minutes at 37° C, 3ml 0.7% agarose in LB at

60° C was added to the mix and this was then poured over a 90mm agar plate (1.2% agar in LB). The plates were then incubated overnight and plaques were counted on plates in which there were between 10 and 500 colonies. The titer was then determined by multiplying first by ten then by the dilution factor for the individual plate.

Once a titer was obtained, ten 150mm phage plates were prepared as follows.

The library was diluted to approximately 300000 PFU per ml in lambda dilution buffer. In ten different tubes, 100 ul of this dilution was mixed with 200 ul E-Coli and incubated for 15 minutes at 37° C. This mixes was then mixed with 7ml of 0.7% agarose in LB. Each of these tubes was then quickly and evenly poured over a 150mm agar plate. These plates were then incubate at 37° C overnight until plaques were just

115 beginning to make contact with one another. The plates were then stored at 4° C until use.

Once the phage plates were prepared, the plaque DNA was then fixed to a nylon membrane using the following procedure. A numbered precut Hybond-N nylon membrane from Amersham (Piscataway, NJ) was placed on the first plate and allowed to incubate for 1 minute. During the minute incubation, the plate and membrane were marked in an asymmetric manner using a hypodermic needle filled with India ink.

The membrane was then carefully removed from the plate and placed plaque side up on four pieces of whatman paper saturated with a denaturing solution (0.5M NaOH and 1.5M NaCl) for 7 minutes. The filter was then transferred to a stack of whatman paper saturated with a neutralization solution (0.5M Tris pH 7.4, 1.5M NaCl, 1mM

EDTA) for 3 minutes. The filter was then transferred to another stack of whatman paper soaked in neutralization solution. After three minutes, the filter was removed from the second neutralization stack and rinsed briefly in 2XSSC (0.3M NaCl, 0.03M trisodium citrate) and placed on a paper towel. While still damp, the DNA was permanently fixed to the nylon membrane using a Stratralinker (Stratagene, La Jolla,

CA) using the auto cross-link setting. The process was repeated for the other 9 plates.

The filters were then stored in aluminum foil at room temperature for until use.

The primer was labeled using an end labeling technique according to the following method. Five pMol of the oligonucleotide probe was added to 25 ul water,

5ul of 5X forward reaction buffer, 10 units (1ul) of T4 polynucleotide kinase

(Invitrogen), and 2.5ul gamma 32P ATP (10uCi/ul) (obtained from DuPont/NEN).

116 This reaction mix was then incubated at 37°C for 15 minutes. The T4 polynucleotide kinase was then inactivated by incubation at 65° C for ten minutes. The labeled probe was then separated from unincorporated nucleotide using a size exclusion column from Stratagene. Specific activity of the probe ranged from 108 – 109 CPM/ug.

Once the probe was labeled, the filters were then pre-hybridized for 2 hours at

42° C in a Prehybridization and hybridization (P&H) solution consisting of 6X SSPE pH 7.4, 5X Denhardt’s solution, 0.25% SDS and 0.1mg/ml denatured herring sperm

DNA. The labeled probe was then added to the bag and it was resealed and incubated at 42° C overnight. After incubation, the filters were carefully removed and washed in

2x SSC with 0.1% SDS at room temperature for 30 minutes. The filters were then washed 2 times with 0.2X SSC + 0.1% SDS for 30 minutes. The filters were then removed and allowed to dry then exposed to X-ray film. Filters were lined up and positive plaques were picked and placed in lambda dilution buffer.

In order to isolate a single positive clone, the above process was repeated using

90mm agar plates at a lower density until a single positive plaque could be picked.

Once a single pure plaque was obtained, The DNA from this phage clone was isolated for further analysis. A high titer stock was prepared from the isolated plaque by incubating it in 200ul lambda dilution buffer at 4° C overnight. This tube was then centrifuged at 8000X G for 2 minutes. The supernatant was then titered as described above. About 60000 PFU was mixed with 200 ul E-Coli K802 and plated on a 150 mm plate as described above. After 7 hours at 37° C, the plaques were nearly confluent. At this point, 10ml of lambda dilution buffer (with gelatin) was added to

117 the plate and incubated at 4° C overnight. This high titer stock was then titered as described above. DNA was then isolated from this high titer stock according to the following protocol. The lambda phage clone (2x106 PFU) and 2x109 E-Coli were mixed together and allowed to incubate at room temperature for 15 minutes. This mixture was then added to 100ml of NZCYM bacterial media and incubated at 37° C in a shaker incubator for 8 hours. Two milliliters of chloroform was then added to the culture and it was shaken vigorously for 30 minutes at 37° C. The culture was then centrifuged for 10 minutes in at 8000 rpm in a GSA rotor. The supernatant was then poured into a fresh tube and 1ml nuclease solution (0.5mg/ml DNase 1, 5mg/ml

RNase A, 30mM sodium acetate pH 6.8, 50% glycerol) was added and the mixture was incubated at 37° for 30 minutes. The phage was then precipitated by adding

5.8gm NaCl and 10gm polyethylene glycol to the supernatant and incubating on ice for at least 2 hours. This mixture was then centrifuged at 10000 rpm (at 4° C) in a

GSA rotor. The supernatant was removed and the phage was resuspended in 1ml of lambda dilution buffer. 500 ul of the above phage solution was then placed in each of

2 microcentrifuge tubes and 0.5ml chloroform was added to each tube. The tubes were vortexed then centrifuged for 2 minutes at 16000 x G in a microcentrifuge. The upper aqueous layers were then transferred to fresh tubes. Twenty ul 0.5M EDTA,

10ul 10% SDS, and 10 ul proteinase K (2.5mg/ml) were added to the mix. Protein was then removed by first extracting with and equal volume of phenol (x1) then with and equal volume of chloroform (x3). The DNA was then precipitated by addition of

118 1/10 volume of 5M NaCl and 2.1 volumes 95% ethanol and incubating at –80° C. The

DNA was then centrifuged and resuspended in TE.

Southern Blots: Southern blots were carried out using a modification of the procedure of E.M. Southern (Southern 1975). Briefly, digested DNA was electrophoresed on a 1% agarose gel until lower marker was about ¾ of the way down the gel. The DNA was then denatured by soaking in a denaturing solution consisting of 1.5M NaCl, 0.5M NaOH (3 30 minutes soaks were performed). The gel was then briefly rinsed in water before being soaked two times in a neutralization solution consisting of 1M Tris (pH 7.4) and 1.5M NaCl in water. The DNA was then blotted overnight onto a nylon membrane using 20X SSC (3M NaCl, 0.3M trisodium citrate pH 7.0) as transfer buffer. The DNA was then permanently linked to the membrane using a UV autocrosslinking device (Stratagene) as described above. This membrane was then probed with either an oligonucleotide probe or DNA fragment standard labeled with P32 dATP using standard molecular biology techniques (Sambrook et al.

2001). The membrane was then washed and exposed to x-ray film as described above.

Construction of 2.6BB-pXP2: A 2.6KB fragment of the above lambda clone that contained the region upstream of the newly identified alternative first exons was subcloned into pCR 2.1 using the a TA cloning kit (Invitrogen) using a PCR based method. The primers used had unique bgl 2 sites incorporated into them and are shown in table 1. As the region being amplified was around 2.6KB, a high fidelity

PFU polymerase was used to amplify this region. The fragment was then cloned into pCR 2.1, and subsequently subcloned into the bgl 2 site of pXP2.

119 Construction of 5’3-pXP2: A plasmid was constructed that contained the region between the 3’ end of the alternate exon designated 5’4(1C) and the beginning of the 5’3(1D) alternative first exon inserted upstream of the luciferase gene. A PCR based method was utilized in its construction. Primers were designed that flanked the regions of interest. The forward primer had a KPN 1 site added while the reverse primer had a Bgl 2 site added in order to facilitate subcloning into the luciferase vector. Using the 2.6BB-pXP2 plasmid as a template, the region of interest was amplified and cloned into the plasmid pCR2.1 using a TA cloning kit per the manufacturer’s instructions. This region was then subcloned into the KPN1 and Bgl 2 sites of pXP2.

Construction of other constructs: A similar strategy was used to build the other constructs used in this chapter. Primers were designed that flanked the region of interest. The forward primers had either a KPN1 or a SAL1 restriction site added while the reverse primer had a Bgl 2 site added. The amplified regions were first cloned into pCR 2.1 using the Original TA cloning kit (Invitrogen). They were then sub cloned into pXP2. See table 1 for the sequences of all primers used in this chapter. The shorter fragments (53D1, 53D2 and 53D3) were sequenced in order to verify that they were correct.

Cell culture: Hela cells were grown in Dulbecco’s modified eagle medium

(DMEM-LG) with glucose (1000mg/L) and pyruvate and supplemented with streptomycin (100 ug/ml), penicillin (100units/ml) and 10% fetal bovine serum. The cells were maintained in a 37° C incubator with 95% air and 5% carbon dioxide. The

120 media and fetal bovine serum were obtained from Irvine Scientific (Irvine, CA) while the penicillin/streptomycin stock solution was obtained from Invitrogen. Cells were split 1:5 every 4 days.

PC12 cells were grown in Dulbecco’s modified eagle medium with glucose

(4500mg/ml) (DMEM-HG) supplemented with 5% horse serum, 5% fetal bovine serum, penicillin (100Units/ml) and streptomycin (100ug/ml). They were maintained in a 37° C incubator with 5% CO2, 95% room air. Media and serum were obtained from Irvine Scientific (Irvine, CA) while penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA).

Hela cell transfections: Transfections of Hela cells were carried out by precipitation as follows: Hela cells grown on 100mm plates to approximately 80% confluence were used for these transfections. Four hours prior to transfection, the media was changed. 75ug construct and 75ug RSV beta-gal were added to 1.35ml water and of 150ul 2.5M CaCl2 was then added. 2xHBS (NaCl

280mM, 10mM KCl, 1.5mM sodium phosphate dibasic, 12mM dextrose, and 50mM

Hepes pH 7.05) was then added to the mix slowly while the solution was bubbled using a Pasteur pipette. This solution was then incubated at room temperature for 20 minutes and was then 1 ml was added to each plate to be transfected while the plate was gently swirled. The cells were then returned to incubator overnight. The next morning, the media was removed and 3ml of PBS with 15% glycerol was added to each plate. After one minute, the PBS/ glycerol was removed and replaced with fresh

121 medium. The cells were then placed into the incubator for at least 24 hours before the cells were harvested.

PC12 cell transfections: Transient transfections were carried out using lipofectamine and plus reagent (Invitrogen) per the manufacturers instructions. PC12 cells were seeded on 6 well plates at a density of 6 x 105 cells per well using DMEM-

HG with serum and antibiotics as described above. The cells were then incubated at

37° C in 5% CO2, 95% room air for 20-24 hours. Forty minutes prior to transfection, the media was changed to DMEM-HG with no serum or antibiotics. A transfection mix consisting of 3 ug of the construct to be transfected, 9 micrograms carrier DNA

(pBluescript SK +), 600 ul DMEM-HG (without additives), and 48 ul Plus reagent was set up. After a 15 minute incubation at room temperature, 600 ul DMEM-HG and

33 ul lipofectamine were added to the mix. After incubation for 15 minutes at room temperature, 4.8ml DMEM was added to the mix. The media was then removed from the cells to be transfected and replaced with 1ml of the transfection mix. The cells were then returned to the incubator for three hours. The transfection mix was then removed and replaced with DMEM-HG with serum and antibiotics. Cells were then returned to the incubator overnight.

Hela cell extract luciferase assays: Twenty-four hours after transfection, crude protein extracts were prepared using the following method. Cells were washed once in ice cold PBS, harvested in PBS using a rubber policeman, and centrifuged at

14000 x G for 5 minutes. The PBS was removed and the cells were resuspended with in 300ul 0.25M Tris-HCl (pH 7.8). Cells were then lysed using a tip sonicator

122 (Branson) and centrifuged at 14000 xG in a microcentrifuge and the supernatant was stored at –80° C until use. For luciferase assays, 50 ul of cell extract was mixed with

350ul of substrate 2 (25mM gly-gly buffer (pH 7.8), 5mM ATP, and 15 mM MgSO4) and placed into the luminometer which then injected 100ul of 1mM luciferin in water and read the light emitted from the tube in 10 seconds. Protein concentrations were obtained using the Bio Rad protein assay, a modification of the Bradford protein assay, per the manufacturer’s instructions.

In order to control for transfection efficiency, Hela cells were transfected with both the luciferase construct and a beta galactosidase expression vector. Beta galactosidase assays were carried out and luciferase values were normalized to beta galactosidase units. In order to perform the beta galactosidase assays a reaction mix consisting of 3ul 100 X Mg solution (0.1M MgCl2, 4.5M β-mercaptoethanol), 66ul o- nitrophenyl-β-D-galctopyranoside 4mg/ml solution, 100ul cell extract, and 0.1M

131ul 0.1M sodium phosphate (pH 7.5). The reaction mix was then incubated until a faint yellow color had developed (usually 30 minutes-1 hour). The reaction was then terminated by the addition of 500ul 1M Na2CO3. The OD at a wavelength of 420nm was then obtained using a spectrophotometer.

PC12 cell extracts and Luciferase assays: Cell extracts were obtained 24 hours after drug addition using the following protocol. Cells were washed with ice cold PBS then lysed using a lysis buffer consisting of 1% Triton X-100, 25mM Gly-

Gly (pH 7.8), 15mM MgSO4, 4mM EGTA, and 1mM DTT. Lysates were then centrifuged at 14000G for 5 minutes then stored at –80° C until use. Luciferase assays

123 were performed as described above except that luciferase activity was normalized to protein, and not to beta galactosidase units, as this method of transfection is more consistent and thus controlling for transfection efficiency is not necessary.

124 PCR Primer Sequence Use Name 2.6-1fwd GGAGATCTGTCGACGATCGTGCCACTGCA Construction of Bgl 2 2.6BB-pXP2 2.6-2 rev GGAGATCTAGGCCTCACCCTGGC Construction of Bgl 2 2.6BB-pXP2, 5’3-pXP2, 5’3 fwd-1 TGGGTACCACCTCTAAATGAAAGAAAG Construction of Kpn 1 5’3-pXP2 5’4 fwd-3 TCGTCGACCAGCCTGGGCAACAGAGC Construction of Sal 1 5’4-pXP2 5’4 Rev-2 TTAGATCTGGTGGCAGCAGCAGTGGC Construction of Bgl 2 5’4-pXP2 53d1-fwd TGGTACCTGCCCTTGTCTCTCCCAG Construction of Kpn 1 53D1-pXP2 53541.5Kb- TCGGTACCAAAGTGTGTGAGCCACC Construction of fwd Kpn 1 53541.5Kb-pXP2 53541.0Kb- GCGGTACCAGAGAGGTGGAGTAACTTGC Construction of Fwd Kpn 1 5354 1.0kb-pXP2 53d3-fwd GGTACCTCCCAACCCAGCAGAGC Construction of Kpn 1 53D3-pXP2 53d2-Fwd CGGTACCTGGTGTCAATTAAACC Construction of Kpn 1 53D2-pXP2

Table 4.1: PCR primers utilized in chapter 4. This table shows the PCR primers used in the construction of the various deletion constructs used in this chapter. The incorporated restriction sites are in bold and identified underneath the bold region.

125 Results

As a result of a library screen, a single lambda clone that contained the region of interest was isolated. Using restriction digests and southern blotting, two closely linked alternative first exons were found to reside on a 3.4 kb Xho 1 to Xba 1 fragment. This fragment itself was 1 kilobase from the short arm of the lambda clone.

Desired regions of this lambda clone were then subcloned in plasmids for sequencing and further study.

In order to determine if the region upstream of the two novel alternative first exons contained a promoter, a 2.6KB fragment containing all of 5’4, about 50 BP of

5’3 and 2 KB of upstream (5’) region was cloned into the luciferase reporter plasmid pXP2. This construct was named 2.6BB-pXP2. This construct was then transiently transfected into Hela Cells and luciferase levels were then evaluated. As shown in figure 2-3A, the luciferase activity detected in cells transfected with 2.6BB-pXP2 were about 142 (+/- 58, n=6) times that found in cells treated with the parent vector and were even considerably higher than levels found in cells transfected with a plasmid in which a known promoter of NOS1 (5’2/1F) was cloned upstream of the luciferase gene.

We next wanted to determine if the there was one promoter present in this region or if separate promoters drove transcription of the 5’3 and 5’4 transcripts. To do this, a plasmid was constructed in which the 5’3 region and the intron between 5’3 and 5’4 was cloned upstream of luciferase. This plasmid was named 5’3-pXP2. A

126 5'4 5'3

Xho1 Xho1 BamH1 BamH1 Xba1 Xho1 Xba1

Lambda NOS 17 12000 bp

Scale= 1000BP

Figure 4.1: Map of the lambda clone containing 5’3(1D) and 5’4(1C). The above map is a scale map of lambda NOS17. The map shows the location of the Xho1, BamH1 and Xba1 sites as well as the locations of 5’3(1D) and 5’4(1C). The bold black line shows the region subcloned into pXP2 as 2.6BB-pXP2

127 second plasmid was made that contained the 5’4(1C) exon and its upstream region and was called 5’4-pXP2. These two plasmids were then transiently transfected into Hela cells and the luciferase levels were evaluated. Cells transfected with the 5’3-pXP2 construct had luciferase levels that were 126 (+/- 30) fold higher than that observed in cells transfected with pXP2. The results from a typical experiment are shown in figure

4-3B. Cells transiently transfected with the 5’4-pXP2 construct had luciferase levels significantly lower than cells transfected with the 5’3-pXP2 construct but were still significantly (7.2 +/-2.0 fold, n=4) higher that that observed in cells transfected with the vector alone. Several constructs with less upstream sequence, but containing both

5’3 and 5’4 were also tested at this time. These constructs all had about the same activity as full-length 2.6BB as well as 5’3-pXP2.

As the 5’3(1D) promoter was able to drive expression of the luciferase gene in

Hela cells, we next wanted to determine which upstream regions were responsible for this. We hoped this would give us a clue to what transcription factors were important in NOS1 expression. In order to do this, a variety of deletion constructs were made in which progressively shorter pieces of the 5’3(1D) promoter/ exon region were cloned upstream of the luciferase gene. These constructs were then transiently transfected into Hela cells and the luciferase levels were evaluated using luciferase assays. All of the deletion constructs had about the same luciferase levels as the full length 5’3-pXP2 construct with the notable exception of the shortest one, which showed little ability to drive expression of the luciferase gene. The results of a typical experiment are shown in figure 4-4.

128

Figure 4-2: Sequence of 2.6KB insert. The above figure shows the DNA sequence of the 2.6KB region cloned into pXP2. This region contains all of the alternative first exon 5’4(1C) (shown in green), the intron between 5’3(1D) and 5’4(1C), and a portion of exon 5’3(1D) (shown in red). This construct also contains 2180 base pairs of upstream of both exons.

129 1 agatctgtcg acgatcgtgc cactgcactc cagcctgggc aacagagcga 51 gactccttct aaaaaaaatt atctttacta gagacagcca ccaccaccag 101 ggtgcagtca cctgagctta gagcccttcc cttgtcatct caaagcccca 151 ctctgtcccc caggcacctg ccctatgcct ggcttgggag ggccccatgg 201 ctaacagccc agccagctgc ttgctccctt ctcatctctg cttgacaagc 251 caggctggct gagtcacagg ctcagcttct cccaacccac gtggggtctg 301 ggtctatgcc agggggatga gcgcctgcca gagtgcccgc aagtcagacc 351 aaaaagagcc catgagcttc actgaggatc agacctaaga acattctgga 401 atcccatgct ctagcttgga gctcccagag ggccagatgg cagcagacag 451 gtatgtaatt ctaccagtga gaaaaccagg gcatccaatg tgttggggag 501 aacctgcatt gcctgagaat agtaatataa taattattat tgttatatgt 551 tgtcatggct aattattgct atttattgtc acggttaatt attaaagtgg 601 acagccatgt ctgcattggt cactgctata tccccaatgc ttagaagggt 651 acctggcaca taataggtgc tcagtgagtt cttctgtgga ataaatgcgt 701 gaacaacaac aataatagct acttctattt gctgagcaca taacacaagg 751 aattaatatt tatttttgtg ggggttttac atttgtctgt ctccaccaca 801 ggggcatgag tgactgactt tggttttttg gggttttttt tgtttgtgtg 851 tttgtttttt gagacggagt ttcactcttg ttgcccaggc tggagtgcaa 901 cggcatgttc ttggctcagt gcaacctcca cctctcaggt tcaagtgatt 951 ctcctgcctc agcctcttga gtagctggga ttacaggtgc ccaccaccat 1001 gtccggctaa tttttttgta tttttagtag agatagggtt tcaccatctt 1051 ggccaggctg gtctcgagct cctgtcctca ggtgatctgc ccgcctctgc 1101 ctcccaaagt gtgtgagcca ccgcacctgg ccaactgact gtttttacaa 1151 gaatattgtt cctgtaaaaa ttatatcccc agcacagtga cccatgtctc 1201 gtaggtgctc aataaatgtt gttgaataaa aaggcaggtg ctgagtatta 1251 tctcatttat tttcacgcag cccaggcagg gtgggagcgc ccgggagcac 1301 atttttagaa aggagaaatt gagactagag agtaaaatga cttctgaagg 1351 tcacgcagtt tgtgagactc aaatcagagc caggatctga accctgtgca 1401 cttgcggaac ctgtgtactc ctcactgctg agtcccgctg ctggcagtaa 1451 agacaaagaa cagggttggg ggcctttggc aagggctccc agggcagagt 1501 ggggctactt tggtactccc tcatcctgcc ctgtgaaccc aagtcacaaa 1551 gccttttcca tgtgatgtcc catttggggt tcacagcagc tgcagccaag 1601 cccagagagg tggagtaact tgctcaaggt cacacagcaa gttgggcaca 1651 ggttttctga ctctatgtcc agtgctcttt ctcctaaacc ccaaactgtg 1701 gctcaaacct ggagccctaa tataagcagg aacacatgtg agtcagggaa 1751 gaaggcttgt attttagcag tggactttac tttccctggc aacctgcccc 1801 aacacacctg gtggcaaatc cacgttccgt actttgctgt gtgactttga 1851 aaactggcat cccctctcta agcctcaatt tcctcatctc aaaaatgggg 1901 atgacattat ctatattgca ggtggccatg aaaatccctg actcaatgtc 1951 agatacagct gaatgtgtgt tctggctctt ctactgtgtg accttggaga 2001 agaagcttag cttctctgag tctccgtttc ctcatcttgc aaagtgggtt 2051 aataaatgcc caccttttgg ggacacatga gatttaagag gggacagtgt 2101 gggcaccatg cctggcctgg tgcatagtaa gtgtgcaata aaaggtagtt 2151 catgggatcc tttgtgactg ccgtgggtgg cctcactcag gatccggcgg 2201 gcacagcctg gtccctgcag acacagtgca ctcgatcctg gctgtcccca 2251 gccagctggg gggaaacctg agccaccctc ctcctgggca gacgctgcca 2301 ctgctgctgc cacctctaaa tgaaagaaag gtcagagcct ggggaagcca 2351 gggccatgcc atggaggcag ccggcaggag aggaagggag gggtggccgg 2401 tgactgcctg actgcccttg tctctcccag ggaagcctga actggcctgg 2451 tctgggtgca acccccctgc ccaaggcttg gcctcccaac ccagcagagc 2501 cgcctcccag cctgcccctg gggaggggcc acctggtgtc aattaaaccc 2551 cagtcgctcg gcctgcacca cgccaggcgg ctgattagtg ccgctggcct 2601 ctccagatgg ggagcactgt ctgagagggg gtgaccgcca ccatgccagg 2651 gtgaggccta gatct 130 A.

2.6BB

4.3-Luc Construct

pXP

0 100000 200000 300000 RLU/Bgal unit

B.

2.6bb-pXP2

2.0-pXP2 1.5-pXP2 1.0-pXP2

5'4-pXP2 5'3-pXP2

pXP2

Scale: 0 10000 20000 30000 40000 1000 BP RLU/Bgal Unit

Figure 4.3: Luciferase assays using HeLa cell extracts transfected with various 5’3 and 5’4 constructs. A.) Figure A above shows the results of a typical experiment in which HeLa cells were transiently transfected with the 2.6BB-pXP2 construct, a positive control (4.3-Luc) or with the empty pXP2 vector. The cell extracts were then assayed for luciferase content. Note that luciferase levels were several hundred fold higher in cells transfected with the 2.6BB-pXP2 construct. B). Figure B shows a typical experiment in which various deletion constructs containing either 5’3(1D), 5’4(1C) (and their associated 5’ regions) or both were transfected into HeLa cells and the extracts were then assayed for luciferase activity. Note that the 5’3(1D) exon and its associated upstream region were necessary and sufficient to drive luciferase expression in Hela cells.

131 HeLa cells are a human cervical cancer cell line and have an epithelial cell like appearance. As NOS1 is expressed in neurons, we wanted to know if the 2.6KB region containing the 5’3(1D) and 5’4(1C) alternate first exons and their associated promoter regions was capable of driving expression of luciferase in a different cell line, PC12 cells. Figure 4-5 shows typical result from an experiment in which the

2.6BB-pXP construct was transiently transfected into PC12 cells and luciferase carried out using the extracts from these cells. Cells transfected with the 2.6BB-pXP2 construct had activities that were 4 to 5 fold higher than that observed in cells transfected with the vector alone and were similar to that observed in cells transfected with a luciferase construct containing a known NOS1 promoter. Although luciferase levels were further increased by PACAP treatment, the fold induction was no greater than that observed with the promoterless vector hence it is not believed that this promoter is responsive to PACAP.

132 pXP

5’3 5'3-pXP2 (2312-2659) 5’3 5'3D1-pXP2 (2413-2659) 5’3 5'3d3-pXP2 2482-2659 5’3 5'3d2-pXP2 (2531-2659) 0 10000 20000 30000 RLu/BGal unit protein

Scale: 1 inch = 180 base pairs

B

cctcccaac ccagcagagc 2501 cgcctcccag cctgcccctg gggaggggcc acctggtgtc aattaaaccc 2551 cagtcgctcg gcctgcacca cgccaggcgg ctgattagtg ccgctggcct 2601 ctccagatgg ggagcactgt ctgagagggg gtgaccgcca ccatgccagg 2651 gtgaggccta gatct

Figure 4-4: Luciferase assays using cell extracts of cells transiently transfected with 5’3(1D) deletion constructs. A)The above figure is a representative experiment showing the results of luciferase assays carried out using Hela cell extracts transiently transfected with constructs containing progressively shorter pieces of the 5’3(1D) NOS1 promoter region. All deletion constructs except the shortest one were able to drive the expression of luciferase. B). This figure shows the sequence of the insert in 53d3-pXP2, the shortest construct capable of driving expression of luciferase in HeLa cells. The sequence in red is the exon (5’3/1D) while the green sequence represents intron/promoter region present in 53d3-pXP2 but absent in 53d2, which is incapable of driving expression of luciferase in Hela cells. Note that this sequence is GC rich.

133 pXP2

+ Pacap

2.6BB-pXP2

+ PACAP

0 10000 20000 30000 40000 RLu/50ug

Figure 4-5: Luciferase assays using PC12 cell extracts. The above figure shows the results of a typical experiment in which PC12 cells were transiently transfected with the 2.6BB-pXP2 construct. The cells were then treated with PACAP38 and the luciferase activity of the extracts was evaluated as described in the methods section. As one can see, the 2.6bb-pXP2 transfected cells had a higher basal luciferase level than cells transfected with the empty vector and the expression of luciferase was increased when cells were treated with PACAP38.

134 Discussion

This chapter described the cloning of two novel alternate first exons of the human NOS1 gene and their associated upstream regions from a human DNA library.

These alternate first exons, discovered using the 5’ RACE method by Greg Hartt in the lab were named 5’3 and 5’4 respectively (Hartt 2003). Several conclusions can be drawn from this study. First, it can be concluded that the 2.6 kilobase region that contained the two novel exons also contained at least one promoter that was able to drive the expression of a luciferase reporter gene in transiently transfected Hela and

PC12 cells. Using various deletion constructs in the Hela cell model, it was found that the region just upstream of the 5’3(1D) alternate first exon contained the cis elements required for the majority of the luciferase expression observed. There was no TATA box or CAAT box located in this promoter. As with many promoters lacking CAAT and TATA boxes, it is GC rich. A search for transcription factor binding sites using the Match ® program showed several putative GC boxes and SP1 sites. Although often associated with housekeeping genes, GC rich regions have actually been found in many different types of genes (Hapgood et al. 2001).

It also appeared as if a very weak promoter existed upstream of the 5’4(1C) exon. The luciferase levels observed in cells transfected with just this region were considerably less than that observed in cells transfected with a luciferase construct containing the 5’3(1D) region but were still approximately 7 fold higher than seen in cells transfected with the empty vector. This would then seem to imply that there are

2 separate promoters within this region. A transcription factor search of the region

135 just upstream of 5’4(1C) using Match ® shows that there is a putative TATA box in this region along with many other putative sites though no typical CAAT box is detected. It should be noted that the relatively low luciferase activities observed in cells transfected with the 5’4 constructs could be due to the lack of necessary transcription factors in the model system used or there could be necessary cis elements not contained within this cloned region. Finally, in humans, the transcript containing the 5’4(1C) exon does not represent a large percentage of the NOS1 transcript present and so it is not surprising that the promoter driving the expression of this transcript is considerably weaker than the 5’3(1D) promoter as transcript containing this exon is more abundant.(Wang et al. 1999)

As mentioned in the introduction, two alternate first exons had previously been discovered and they were designated 5’1(1G) and 5’2(1F). It was subsequently discovered that, despite their proximity, two separate promoters drove the expression of these alternate transcripts (Xie et al. 1995). One cannot help but notice the similarity between these 2 promoter ‘complexes’ that drive NOS1 expression.

The human NOS1 gene is a very complex gene that gives rise to multiple transcripts. Much of the differences between these transcripts lie as the 5’ end. In addition to the 4 alternate first exons identified in our lab (and described above), others have identified an additional eight (Xie et al. 1995; Wang et al. 1999; Boissel et al. 2003; Hartt 2003). It appears as though the common second exon is actually the first exon in some transcripts. This brings the total number of alternate first exons to

136 Young Marsden Forsterman Size Rat Mouse 1D 1A 523 1A 1B 100 5’4 1B 1C 150 Yes (1C) 5’3 1C 1D 111 Yes (1B) No 1E 1E 68 5’2 1F 1F 193 Yes (1A) Yes 5’1 1G 1G 67 Yes (1A) Yes 1H 1H 375 1I 172 1I 1J 97 AS 1K 89 1L 91 Exon 2 Exon 2 Exon 2 1213(human) Exon 2 Exon 2

Table 4.2: Alternate first exons of the Human NOS1 gene. The above table shows all of the alternate first exons of the human NOS1 gene and the different names they have been called. Also listed are the sizes of these exons and if they have any corresponding exon exists in the rat or mouse gene. Note that exon 2 is listed as a first exon as in some cases, the common exon 2 can be the first exon of the gene

137

Exon 1G Exon 1H Exon 1C Exon 1F Exon 1I Exon 1B Exon 1D Exon 1J/1K Exon 1A Exon 1E Exon 1L Exon 2

NOS1 Partial genomic map 130000 bp

Figure 4.6: Partial genomic map of the human NOS1 gene. Above is a scale partial genomic map of chromosome 12 showing the relative location of the known 12 alternate first exons and the common exon 2 of the human NOS1 gene. Alternate exon nomenclature utilized in the map is that proposed by Forsterman et al.

138 thirteen. Table 4-2 shows the known human alternate first exons, the different names that they have been called, their sizes and known homologues in rats and mice.

Forsterman et al have proposed the most complete naming system and so for clarity, I have used both naming schemes throughout this paper.

Although it appears as if these different transcripts all have a separate promoter, this has only been definitively shown for four of the alternate exons. Our lab has shown that 5’1(1G), 5’2(1F), 5’3(1D), and 5’4(1C) all have separate promoters

(Xie et al. 1995). With others, their location in the genome would require a separate promoter. This is the case for 1A, 1E and IL (see figure 2-6 for genomic map of all alternate first exons). The other clusters of alternate first exons may well share important cis elements. Of course, even though the individual promoters of 5’1(1G),

5’2(1F), 5’3(1D) and 5’4(1C) are each able to drive the expression of a reporter gene does not mean that they don’t share important upstream regulatory elements.

There has been much speculation as to the reason for the large amount of transcript diversity and large number of promoters involved in the expression of the human NOS1 gene. One possible reason for such diversity is that it provides a mechanism for the selective expression of NOS1 in particular cells or tissues. There is some evidence to support this. For example, Wang and Marsden demonstrated using

RACE cloning that certain transcripts are only present in certain tissues while others were widely distributed (Wang et al. 1999). Alternate exon 5’3(1D) and 5’4(1C), for example, are found in a variety of tissues including brain, skeletal muscle, kidneys,

139 lungs and heart. Exon 1H, on the other hand, is only found in the brain and testis

(Wang et al. 1999).

A second possible reason for the aforementioned diversity may be that is allows for expression of NOS1 at different stages of development. Evidence to support this hypothesis comes from a study performed by Lee et al. They found that, in the rat, expression of one of the alternate first exons is limited to embryonic tissue

(E18) (Lee et al. 1997). There is also evidence of developmental regulation of different transcripts in human tissue. Greg Hartt working in our lab found that exon

5’3(1D) was excluded from embryonic tissue but was present in adult brain tissue.

Another potential reason for the large number of promoters that drive expression of the NOS1 gene may be to provide a mechanism for selective increases in NOS expression due to various stimuli. Although originally believed to be constitutively expressed, NOS1 has been shown to be inducible in response to certain stimuli (Zhang et al. 1994; Sheehy et al. 1997; Rife et al. 2000; Sasaki et al. 2000). It is quite possible certain promoters are activated and thus certain transcripts are preferentially upregulated by certain stimuli. Some evidence exists to support this theory. Sasaki and colleagues, for example, showed that a promoter located in what is normally exon 2 is activated in mouse cortical cultures due to depolarization (Sasaki et al. 2000). Terrie Rife, working in our lab, showed that upregulation of NOS1 due to nerve growth factor treatment was mediated by the 5’2(1f) promoter (Rife et al. 2000).

As I will discuss in chapter 3, the promoter located in exon 2 is also responsible for the increases in NOS1 observed due to PACAP or forskolin treatment of PC12 cells.

140 There is also some evidence to support the notion that these alternate transcripts are translated with different efficiency. Wang and colleagues showed that the different 5’ ends effected translation in both cell free translation assays and in transfected cells (Wang et al. 1999). There are two other possible reasons for this transcript diversity for which there is no evidence at present. One of these is that these different transcripts have different half lives. A second is that these different transcripts are found in different regions of the cell.

To summarize, this chapter describes the cloning of two novel alternate first exons of human NOS1 and there associated upstream regions. These exons had previously been designated 5’3 and 5’4 (Hartt 2003). These expression of these alternate transcripts were subsequently shown to be driven by 2 separate promoters and the 5’3 promoter appeared much stronger in the Hela cell model utilized. At present, our lab and others have identified a total of 12 alternate first exons. The common second exon can be the first exon in some cases as well. All of these transcripts are translated into the same protein as the translation start site is located in exon 2. There are many possible reasons for the large number of promoter and transcript diversity found in this gene (Wang et al. 1999; Boissel et al. 2003; Hartt

2003). This diversity may allow for the differential temporal and spatial expression seen with NOS1. It also appears to effect translation as different transcripts appear to be translated with different efficiencies. Finally, these different transcripts may have different half lives or may be localized to different regions of the cell.

141 Final Thoughts:

The ability of PACAP38 to stimulate NOS1 expression may have significant biological implications. For example, both NOS1 derived NO and PACAP38 appear to be important in the regulation of bronchial relaxation (Linden et al. 1999; Belvisi et al. 2000). NO, produced by non-adrenergic non-cholingergic neurons, has been shown to be important in dilation of the bronchi in humans (and other species).

PACAP and the related peptide VIP are also potent bronchodilators and are present in nerves proximal to the airways (Linden et al. 1999). As NO cannot be made and stored like other neurotransmitters, the only way to alter the amount of NO released from nitrergic neurons is to modify the amount of NOS present. One could imagine a scheme where PACAP (or VIP), in addition to their bronchodilatory action, also increased NOS1 levels in nitrergic neurons. In addition to the PC12 studies described in this thesis, there is additional precedent for PACAP induction of NOS1. PACAP38 has been shown to stimulate NOS1 expression in rat pituitary gonadotrophs (Garrel et al. 2002). This increase in NOS1 would then augment the amount of NO produced upon stimulation of these nerves, enhancing bronchodilation. Finally, there is some evidence of non-adrenergic non-cholinergic dysfunction during allergic reactions and severe asthma (Lammers et al. 1992). Accordingly, one could speculate that defects in the ability of PACAP to stimulate the production of NOS1 could well play a role in the pathophysiology of asthma.

Both NO produced by NOS1 and PACAP appear to cause smooth muscle relaxation in the GI tract. Moreover, PACAP has been shown to directly stimulate NO

142 production (Zizzo et al. 2004). As with the tracheal innervation, both NOS1 and

PACAP containing neurons are present in the GI tract, thus the possibility that

PACAP stimulates NO production in nitrergic nerves exists in the GI tract as well.

In addition to actions on peripheral actions, NO and PACAP also appear to co- regulate many processes in the central nervous system. For example, both appear to be involved in learning and memory. As previously mentioned, both NO produced by

NOS1 and PACAP are both involved in release of LH and FSH from rat anterior pituitary gonadotrophs. In this model, NOS1 production is induced directly by

PACAP and this enhanced the release of the hormones (Garrel et al. 2002).

In conclusion, NOS1 and PACAP38 are both involved in the regulation of many of the same physiological functions. Included among these are learning and memory, hormone release and smooth muscle relaxation in both the GI tract and the airway. Further, PACAP is able to regulate NOS1 expression in some cells and tissue.

While little is presently known about the consequences of the interaction between

NOS1 and PACAP it is likely important in the regulation of these functions and may play a role in diseases such as asthma.

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