PROTEIN KINASE CJ311

AS AN EFFECTOR ENZYME OF THE

AQUAPORIN 3/PHOSPHOLIPASE 02/PHOSPHATIDYLGLYCEROL

SIGNALING MODULE

By

Lakiea J Bailey

Submitted to the Faculty of the College of Graduate Studies

of Georgia· Health Sciences University in partial fulfillment

of the Requirements of the Degree of

Doctor of Philosophy

March

2012 PROTEIN KINASE C~ll

AS AN EFFECTOR ENZYME OF THE

AQUAPORIN 3/PHOSPHOLIPASE D2/PHOSPHATIDYLGLYCEROL

SIGNALING MODULE

This thesis/dissertation is submitted by Lakiea J Bailey and has been examined and approved by an appointed committee of the faculty of the College of Graduate Studies of Georgia. Health Science University

The signature which appears below verifies the fact that all required changes have been incorporated and the thesis/dissertation has received final approval with reference to content, form and accuracy of presentation.

This thesis/dissertation is therefore in partial fulfillment of the requirement for the degree of Doctor of Philosophy

2~ Apnl Zot "Z­ Date

an, College of Gr?Iduate Studies ACKNOWLEDGEMENTS

This has been a long and arduous journey fraught with many difficulties along the way, yet by the grace of God, and with the assistance of many wonderful individuals, I have finally reached the finish line.

I would like to thank my advisor, Dr. Wendy Bollag, for her abundant patience and invaluable assistance throughout the many stages of this project.

Her contagious enthusiasm and genuine love of research has been an inspiration. Wendy taught me the necessity of flowing with, what I have come to think of as the rhythm of research, the value of being able to glean useful information from unexpected results, and, perhaps most importantly, the importance of being able· to think around seemingly insurmountable research obstacles.

I would also like to thank the members of my advisory committee: Drs.

Abdullah Kutlar and Dorathy Tuan for their patient assistance, and Drs. John

Johnson and Vidavel Ganapathy for their rigorous criticism and valuable insights.

I am also grateful for the time and effort of my readers Drs. Lynnette McCluskey and Somanath Shenoy.

I am thankful to all of the members of the Bollag laboratory for their technical assistance and advice, specifically Sara Chen, Mariya George, Purnima

Murai and Peter Parker. I am especially thankful to Dr. Brian Shapiro for his

iii assistance at the start of my project, for Lawrence Olala for his constant support and Mutsa Seremwe for her unyielding faith and encouragement. The Bollag laboratory has been a wonderful environment to learn in and I have enjoyed working with all of them.

I would also like to extend my thanks to Laura Hutchinson, Dr. Gretchen

Caughman, Dr. Edward Inscho, Dr. Patricia Cameron, Donna Wingrove and

Marvis Baynham. These wonderful individuals form an integral part of the

College of Graduate Studies and have each played an important role in helping me to accomplish this goal.

Finally, there is no one that I am more grateful to than the memb~rs of my family. Through troubleshooting and negative results, laboratory changes, surgeries and hospitalizations, they have never left me side. At various points, members of my family have served as everything from sounding boards and editors to research and administrative assistance. They are my greatest cheerleaders and most trusted critics. I would especially like to thank my mother,

Doris Bailey, who taught me the value of hard work and determination, and my sister, L'Bonne Bailey, whose belief in me has encouraged me to push through all obstacles to obtain my dreams. I would also like to thank my cousins: Ray,

Angie, Jason, Kristina, Ashley, Jasmine, Shayla and Alonzo, whose visits and phone calls kept me focused, and Pastors Anyta and Randy Hall, Mother Viola

Walker, Lauri Koth and Lane Holland (Ma), whose prayers kept me strong.

iv TABLE OF CONTENTS

ACKNOWLEDGEMENT ...... iii

LIST OF ABBREVIATIONS ...... viii

LIST OF FIGURES ...... x

CHAPTER ONE: INTRODUCTION ...... ; ...... 1 I. Skin overview ...... 1 II. Keratinocyte differentiation ...... 5 A. Gene expression ...... 5 Modifiers of keratinocyte differentiation ...... ·...... 6 1. Calcium ...... 6

2. 1 ,25(0H)2D3 ...... •.....•...... •.•...... •...... •.. ·.•...... 8 3. Cytokines and retinoids ...... 10 B. Regulation of expression ...... 11 C. PLD/AQP/PG signaling cascade ...... 13 1. Phospholipase D ...... 13 2. Aquaporins ...... ·...... 15 3. Glycerol and phosphatidylglycerol ...... 18 D. Protein kinase C isozymes (PKCs) ...... 22 1 . Structure and classification ...... 23 2. Expression, phosphorylation and translocation ...... 25 3. PKCs in keratinocyte differentiation ...... 30

4. Protein kinase C~ll ...... 32 E. Hypothesis ...... ·...... 37

v CHAPTER TWO: MATERIALS AND METHODS Materials ...... 38 Methods ...... 42 Animals ...... 42 Culture of primary mouse keratinocytes ...... 42 Preparation of PG liposomes ...... 43 Reverse transcriptase- polymerase chain reaction ...... 44 Primers ...... 44 Protein purification ...... 45 Western blot analysis ...... 45 3 [ H]Thymidine incorporation into DNA, a proliferation assay ...... 46 Immunocytochemistry ...... 47 Keratinocyte transfection ...... 48 Inhibitor experiments ...... 49

LY333531 PKC~II catalytic activity inhibitor ...... 49 V5 peptide fragment translocation inhibitor ...... 49 Statistics ...... 49

CHAPTER THREE: RESULTS

PKC~II is present in mouse keratinocytes and redistributed with stimulation of the AQP3/PLD2/PG signaling module ...... 51

Over-expression of PKC~II in primary mouse keratinocytes ...... 58

Over-expression of PKC~II had no effect on calcium-induced inhibition of proliferation ...... 59

vi PKCf311 over-expression induced an up-regulation of keratin 10 upon calcium-induced stimulation of differentiation ...... 60

Treatment of keratinocytes with a PKCf3 catalytic activity inhibitor had no effect on calcium-induced inhibition of proliferation ...... 64

Treatment of keratinocytes with a PKCf311 translocation inhibitor had no effect on calcium-induced inhibition of proliferation ...... 67

Inhibitors of PKCf3 catalytic activity or PKCf311 translocation had no effect on either autophosphorylation of PKCf311 or keratin 10 levels ...... 70

PG stimulated an increase in PKCf311 auto phosphorylation ...... 73

Over-expression of PKCf311 affected the pattern of keratin 10 distribution and resulted in an altered morphology in cells incubated in the presence of phosphatidylglycerol ...... 75

CHAPTER FOUR: DISCUSSION ...... 77

CHAPTER FIVE: REFERENCES OF LITERATURE CITED ..... , ...... 94

vii LIST OF ABBREVIATIONS

Abbreviation Definition [Ca2+]e extracellular calcium concentration [Ca2+]J intracellular calcium concentration 1a0Hase la-25 hydroxyvitamin D hydroxylase 1,25(0H)203 1 ,25-dihydroxyvitamin 0 3 PDK-1 3-phosphoinositide dependent protein kinase-1 7-DHC 7 -dehydrocholesterol AN OVA analysis of variance AP activator protein AQP aquaporin

C/EBP~ CCAAT/enhancer protein~ CaR calcium receptor CaMK calcium/calmodulin-dependent protein kinase DAG diacylglycerol EGF epidermal growth factor INV involucrin IP3 inositol 1 ,4,5-trisphosphate K1 keratin K5 keratin 5 K-10 keratin 10 K14 keratin 14 K-SFM keratinocyte-serum free medium

(Commercially available; 50~M CaCI 2) PG phosphatidylglycerol PIP2 pho.sphatidylinositol 4,5-bisphosphate PKC protein kinase C

viii PLC phospholipase C PLD phospholipase D RACKs receptors for activated C kinase SFKM serum-free keratinocyte medium

(Laboratory prepared; 251JM CaCI2) TG transglutaminase TGF transforming growth factor TPA 12-0-tetradecanoylphorbol-13-acetate VDR vitamin D receptor VDRE vitamin D response element

ix LIST OF FIGURES

Figure Page

1. Epidermal stratification, calcium gradient and markers of differentiation ...... 4

2. The domain structure of the classical PKCs ...... 24

3 .. Schematic of PKC{31 and PKC{311 splice variants ...... 34

4. A novel signaling module comprised of AQP3/PLD2/PG3 ...... 36

5. PKC{3 mRNA and PKC{31/ protein are present in primary mouse keratinocytes ...... 53

6. PKC{31/ is autophosphorylated and redistributed upon activation of the AQP3/PLD2/PG signaling module ...... 57

7. Over-expression of PKC{31/ and its autophosphorylation in mouse keratinocytes ...... 61

B. PKC{311 over-expression had no effect on calcium-induced inhibition of proliferation ...... 62

9. PKC{31/ over-expression increases up-regulation of calcium- induced levels of keratin 10 ...... 63

10. Treatment of keratinocytes with an inhibitor of PKC{3 catalytic activity had no effect on calcium-induced inhibition of proliferation ...... 66

11. A PKC{31/ translocation inhibitor had no effect on calcium- induced inhibition of keratinocyte proliferation ...... 69

12. Treatment of keratinocytes with the PKC{3-selective inhibitor had no effect on the auto-phosphorylation of PKC{311 or keratin 10 levels ...... 71

X 13. Treatment of keratinocytes with a PKC/311 translocation inhibitor had no effect on PKC/311 autophosphorylation or keratin 10 levels ...... 72

14. PG stimulated increases in PKC/311 autophosphorylation were not attenuated by L Y333531 or a PKC/311 translocation inhibitor ...... 74

15. Over-expression of PKC/311 affects the subcellular distribution of keratin 10 and resulted in an altered morphology of cells incubated in the presence of phosphatidylglycerol ...... 76

xi CHAPTER ONE: INTRODUCTION

Skin Overview

The body's largest organ, and arguably one of the most important, is the skin. It functions as the foremost barrier against ultraviolet radiation, trauma and infection and is essential for existence (8 Amirlak et al., 2011 ). Skin is composed of the epidermal, dermal and subcutaneous layers. The innermost layer, the subcutis, upon which the epidermis and dermis rest, is composed primarily of adipose and connective tissue. The middle layer, the dermis, is a complex structure comprised of the papillary dermis and the reticular dermis, the primary function of which is to support and maintain the epidermis (8 Amirlak et al.,

2011 ). The epidermis, a stratified squamous epithelium consisting primarily of keratinocytes, holds the distinction of performing the most important function of skin - providing the essential physical and water permeability barrier. This barrier is established and maintained by a careful balance between keratinocyte proliferation and differentiation (Goldsmith, 1991 ), resulting in a multi-layered structure composed of the stratum germinativum (basal layer), stratum spinosum

(spinous layer), stratum granulosum (granular layer) and stratum corneum

(cornified layer) (Fig.1 ).

1 2

The deepest layer of epidermal keratinocytes is found within the basal layer adjacent to the basement membrane. This single layer of cells does not differentiate, but possesses the ability to proliferate and continuously replenish the keratinocytes of the epidermis. The early stages of keratinocyte differentiation occur in the layer immediately above this, the spinous layer. The cells in this layer are no longer proliferating; instead they continue to differentiate outwards through the granular layer of late stage differentiation and terminating in the outer cornified layer of the epidermis. Keratinocytes within this outermost cornified layer have terminally differentiated and enucleated (DO Bikle & Pillai,

1993). This distinctly defined multi-layered skin is present at birth and is in a constant state of dynamic flux as the keratinocytes proliferate and differentiate outward from the basement membrane to the most superficial layer of squames.

Several human skin diseases, including psoriasis, a common hyperproliferative disorder of the epidermis, and the non-melanoma skin cancers (basal and squamous cell carcinoma), are characterized by excessive growth and aberrant differentiation of epidermal keratino~ytes resulting from dysregulation of this carefully controlled pathway (Yuspa, 1998). The National Psoriasis Foundation and the American Academy of Dermatology report that approximately 7.5 million

Americans and as much as 3 percent of the total world population live with psoriasis (Dermatology, 2011; Foundation, 2011 ). The American Academy of

Dermatologists also lists basal and squamous cell carcinomas as the two most common cancers in the world with more than 2 million new diagnoses each year

(Dermatology, 2011 ). The ultimate goal to decrease human suffering 3 produced by these diseases must first be prefaced by a better understanding of the molecular processes regulating keratinocyte proliferation and differentiation. 4

Comlftcatlon Cornified Cell Envelope Nuclear Breakdown

Granular Late Dlft'erentlatlon T•·ansglutaminase, Lo.-icrin Interme diate Differentiation lnvolutTin

Spinous Early Dlft'erentlatlon Keratins 1 and 10

Basal P ·ohfer~tion Ke1·atins Sand 14

Figure 1: Epidermal Stratification, calcium gradient and markers of differentiation. The epidermis is maintained by a balance between proliferation and differentiation resulting in multi-layered structure of basal, spinous, granular and cornified layers. Throughout the layers of the epidermis a defined pattern of genes are expressed or repressed in a specific temporal and spatial manner.

These include keratins 5 and 14 (expressed in the basal layer), keratins 1 and 10

(representing markers of early differentiation), and involucrin, transglutaminases and loricrin (representing markers of intermediate and late differentiation).

Calcium, a key regulator of keratinocyte maturation, is present in low levels within the actively proliferating basal layer and gradually increases in concentration upward throughout the suprabasallayers, triggering early, intermediate and keratinocyte differentiation. 5

Keratinocyte Differentiation

Gene Expression

Throughout the layers of the epidermis a defined pattern of genes encoding transcription factors, structural and cytoskeletal proteins and proteins integral in forming the epithelial barrier are expressed and/or repressed in a specific temporal and spatial manner (C Byrne, M Tainsky & Fuchs, 1994 ). The keratins comprise a major family of proteins the genes for which demonstrate precise epidermal expression. Within the basal layer genes encoding keratins 5 and 14

(K5/14) are expressed (Fuchs, 1995). These keratins become sequentially repressed and then replaced by keratins 1 and 10 (K1/1 0) as the proliferative cells of the basal layer cease to proliferate and enter early stage differentiation in the spinous layer (J Reichelt et al., 2001 ). Growth arrest of primary keratinocytes is an early event in the process of keratinocyte differentiation. It occurs prior to the expression of K1 and K1 0 early markers of differentiation, the expression of which has been demonstrated to be required for the increased mechanical resistance of these cells (Fuchs, 1995). In the upper epidermis the granular layer keratinocytes begin expressing genes encoding substrates involved in the assembly of the cornified envelope, including loricrin, filaggrin, involucrin (I NV) and transglutaminase (TG) (S Sinha et al., 2000). At this stage keratin 1 and keratin 10 become covalently cross-linked to the cornified envelope proteins that also provide the scaffolding upon which specialized lipids are tethered (S Sinha et al., 2000). The expression of many of these proteins, including K1, K1 0, loricrin and filaggrin (as well as C/EPB~, discussed below), are greatly 6 diminished in squamous cell carcinomas and tight regulation of the expression of these genes is a crucial component of maintaining skin health (S Zhu et al.,

1999).

Modifiers of Keratinocyte Differentiation

Calcium

Calcium is a key regulator of keratinocyte maturation and is essential for normar epidermal differentiation both in vitro and in vivo (DD Bikle & Pillai, 1993; GK

Menon et al., 1992; SH Yuspa et al., 1989). An increase in extracellular calcium concentration ([Ca2+]e) is sufficient to initiate the complete aforementioned process of differentiation including induction of the formation of cornified envelopes and the expression of differentiation-associated genes (D Hohl et al.,

1991; DC Ng et al., 1995; DF Gibson, AV Ratnam & Bilke, 1996; SM Thacher &

Rice, 1985). Keratinocytes grown in very low calcium (less than 1OOJ,JM) continue to proliferate, but fail to enter differentiation (DD Bikle, Y Oda & Xie,

2004b). Elevating the extracellular calcium concentration to 1001-JM stimulates a rapid redistribution from the cytosol to the membrane of components involved in the formation of intercellular contacts, including desmoplakin, cadherins, integrins and actinin (DD Bikle et al., 2004b). Within hours of this calcium switch markers of keratinocyte proliferation (K5, K14) are down-regulated and are replaced with differentiative markers (K1, K1 0), followed by subsequent up-regulation of filaggrin, loricrin, involucrin and transglutaminase (DC Ng et al., 1995; DD Bikle et al., 2004b). Extracellular calcium increases the rate of transcription of the 7 involucrin gene (possibly through its AP-1 site) as well as involucrin mRNA, revealing a role for calcium-dependent regulation of the involucrin gene during epidermal differentiation (DC Ng et al., 1995). A calcium gradient has been observed in the epidermis, with the lowest concentration found in the basal layer and increasing upwards towards the granular layer, indicative of the principal role that calcium plays in epidermal homeostasis also in vivo (GK Menon, S Grayson

& Elias, 1985).

The mechanism through which extracellular calcium initiates these changes is a complex and multiphasic process beginning with activation of the calcium­ sensing G protein coupled receptor (CaR) by its calcium ion ligand (CL Tu, Y

Oda & Bilke, 1999; N Chattopadhyay, A Mithal & Brown, 1996; SC Herbert &

2 Brown, 1995). The full-length version of CaR is required to modulate [Ca +]9 - induced stimulation of differentiation, which in turn results in the production of an alternatively spliced CaR variant lacking exon 5 (Y Oda et al., 1998). The Gq alpha subunit of G-protein couples the CaR to PLC, leading to direct stimulation of PLC activity. In addition, [Ca2+]e, through the CaR, induces an influx of calcium through one or more of the many calcium channels in the keratinocyte membrane, and this calcium influx can also stimulate the activity of phospholipase C (PLC). PLC, a hydrolytic enzyme, cleaves phosphatidylinositol

4,5-bisphosphate (PIP2) forming diacylglycerol (DAG) and inositol 1,4,5- trisphophate (IP3) (DD Bikle et al., 2004b ). DAG, which activates phorbol ester­ responsive kinases such as protein kinases C and D (PKC and PKD), and IP3, 8

which increases intracellular calcium concentration ([Ca2+]i) by stimulating the

release of calcium from intracellular calcium stores in the endoplasmic reticulum,

are reported to increase within minutes after exposure to elevated extracellular 2 calcium concentrations (S Jaken & Yuspa, 1988). The increase in [Ca +] 1 is itself

then able to activate other calcium-responsive enzymes such as

Ca2+/calmodulin-dependent kinases.

Vitamin D

A second major regulator of keratinocyte differentiation is the hormonally active

form of vitamin 03, 1 ,25-dihydroxyvitamin 0 3 [1 ,25(0H)203]. Evidence suggests

that" the action of 1 ,25-(0H)203 is biphasic with low doses stimulating proliferation

and high doses having the opposite effect (DO Bikle, S Pillai & Gee, 1991; M

Sebag et al., 1992; PH ltin, MR Pittelkow & Kumar, 1994; WB Bollag, J Ducote &

Harmon, 1995). Although the mechanism through which 1 ,25(0H)2D3 asserts it

pro-proliferative influence remains largely unknown, research suggests that

1 ,25(0H)2D3 promotes differentiation in much the same way as calcium.

1,25(0H) 2D3 has been shown to interact with the vitamin D receptor (VDR),

forming a heterodimer with retinoic acid receptor (RAR) or retinoid X receptor

(RXR), then binding to gene promoters possessing vitamin D response elements

(VDRE), such as involucrin and transglutaminase, to alter their expression (C

Carlberg & Polly, 1998; DO Bikle et al., 2004a). The VDR has been identified in

many normal and transformed cell types including epidermal keratinocytes.

These cells also contain 7 -dehydrocholesterol (7 -DHC), which initiates the 9 process by which vitamin 03 is formed upon exposure to ultraviolet (UV) light (DO

Bikle, Y Oda & Xie, 2005). Keratinocytes also express the enzymes necessary for the production of endogenous 1 ,25(0H)203 from its 7-DHC precursor. In this manner the keratinocyte is unique in its ability to form 1 ,25(0H)2D3 by UV­ mediated conversion of 7-DHC to vitamin 03 followed by two sequential hydroxylations by 25-vitamin D hydroxylase and 1a-25 hydroxyvitamin D hydroxylase (1a0Hase) (DD Bikle et al., 2005).

Calcium and 1 ,25(0H)203 have been shown to interact synergistically to regulate epidermal homeostasis. 1 ,25-(0H)2D3 also increases the expression of the CaR, enhancing keratinocyte sensitivity to calcium and resulting in an increase in intracellular calcium concentration, presumably by its ability to increase the expression of phosphoinositide-specific (PI)-PLCy1 (AV Ratnam, DD Bikle &

Cho, 1999; DD Bikle et al., 2004b; Z Xie & Bikle, 1997). Markers for keratinocyte differentiation are down-regulated in the epidermis of 1aOHase knockout mice

(1a0Hase _,_)which lack the ability to produce 1 ,25(0H)2D3 from its 7-DHC precursor. These mice demonstrate an inability to maintain the critical calcium gradient necessary for normal keratinocyte maturation (DO Bikle et al., 2004a).

Calcium and 1 ,25(0H)2D3 were found to synergistically induce involucrin and transglutaminase-1 mRNA and protein expression in normal human keratinocytes as well (Su MJ et al., 1994). Of interest, 1 ,25(0H)2D3 was also found to stimulate an increase in the expression of phospholipase 01 (PLD1) 10

(WB Bollag & Bollag, 2001.). The role of PLD in keratinocyte differentiation will be discussed in further detail below.

Cytokines and Retinoids

In addition to calcium and 1,25(0H)203, multiple other agents have been implicated in regulating keratinocyte differentiation. Among these are cytokines and the retinoids (vitamin A and its analogs). Cytokines such as TNF-a and IFN­ y have been shown to induce keratinocyte differentiation in response to stress

(NS Tan et al., 2001 ). Induction of differentiation through this pathway occurs in response to up-regulation of the PPAR{3/o gene through targeting of the AP-1 site in the PPAR{3/o promoter (NS Tan et al., 2001 ). The retinoids have been linked to epithelial cell organization in the literature dating as far back as 1925 (SR

Wolbach & Howe, 1925) and have been used to treat both psoriasis and precancerous skin lesions (Saurat, 1999). The method of action of the retinoids is mediated through interaction with two families of nuclear receptors, ligand­ dependent transactivation factor retinoid acid receptors (RARa, RAR~ and

RARy) and the retinoid X receptors (RXRa, RXR~ and RXRy) (Boehm MF et al.,

1995; Cham bon, 1994 ). RARa, RARy, RXRa and RXR~ are all expressed in the epidermis and topical application of tazarotene, a RAR-specific synthetic retinoid, is correlated with down-regulation of transglutaminase-1 expression and up­ regulation of the expression of filaggrin, indicating a role for retinoids in keratinocyte differentiation (Boehm MF et al., 1995; S Nagpal, J Athanikar &

Chandraratna, 1995; T Esgleyes-Ribot et al., 1994). 11

Regulation of Gene Expression

A growing body of evidence suggests that the regulation of epidermal gene expression is governed primarily by transcriptional regulatory sequences and factors. Among these are the activator protein (AP-1 and AP-2) family of heterodimeric proteins, SP1 and Ets family members (RL Eckert, JF Crish &

Robinson, 1997; S Sinha et al., 2000). AP-1 and ets gene induction has been linked to the regulation of suprabasal and basal epidermal gene expression (AS

Takaoka et al., 1998; J Casatorres et al., 1994; JH Lee et al., 1996; MW Sark et al., 1998; P Oettgen et al., 1997; Sl Jang, PM Steinert & Markova, 1996). A large number of studies in the epidermis, as well as multiple other cell types, reveal that most epidermal promoters contain functional binding sites for the AP-

2 family of transcription factors indicating a potential role of this transcription factor, in gene induction (A Leask, C Byrne & Fuchs, 1991; AM Snape, RS

Winning & Sargent, 1991; C Byrne & Fuchs, 1993; C Byrne et al., 1994; D

Warshawsky & Miller, 1995; LA McPherson & Weigel, 1999; M Moser et al.,

1995). Functional Sp1 sites have also been identified in epidermal .promoters, although there is little evidence linking Sp1 directly to cell-type spedfic gene expression (Byrne, 1997; C Byrne & Fuchs, 1993; JH Lee et al., 1996). In line with these data a study by Sinha et al. identified an epidermis-specific enhancer containing AP-2, AP-1 and ets sites, which is both necessary and sufficient for keratinocyte-specific gene expression (S Sinha et al., 2000). 12

1 1 Additional regulators, such as the p21war tciP cyclin-dependent kinase inhibitor,

CCAAT/enhancer protein ~ (C/EBP~), POU domain proteins, including the Brn transcription factor, and NF-KB, have also been directly implicated in regulation of

1 1 keratinocyte differentiation. The induction of p21 war tCIP , the promoter activity of which is modulated by p300, was shown to be necessary for initial commitment of keratinocytes to differentiation as well as for normal differentiation of human keratinocytes (P Wong, A Pickard & McCance, 201 0). C/EBPa and C/EBP~ are expressed in normal human and primary mouse keratinocytes. C/EBP~ expression is up-regulated during calcium-induced growth arrest and squamous differentiation and appears to be involved in the regulation of genes expressed during growth arrest as well as in K1 and K1 0 expression (S Zhu et al., 1999).

There is evidence suggesting that the POU domain proteins (Skn-1 all and Tst-

1/0ct-6) are involved in the down-regulation of K5 and K14 outside of the basal layer (8 Andersen et al., 1997; I Faus, HJ Hsu & Fuchs, 1994 ), while NK-KB enhances the ability of basal keratinocytes to respond to proliferative cues (CS

Seitz et al., 1998). Brn2, a POU domain-containing transcription factor, has also been reported to be an important regulator of keratinocyte differentiation (G Shi et al., 201 0). 13

PLD2/AQP3/PG Signaling Cascade in Keratinocyte Differentiation

Phospholipase D

Although the precise mechanisms regulating the induction and regulation of

keratinocyte differentiation remains largely unknown, our laboratory has

proposed a potential pathway involving phospholipase 02 (PLD2), aquaporin3

(AQP3) and phosphatidylglycerol (PG). PLD belongs to a superfamily of

phospholipases defined by the motif HXK(X)4D. This lipolytic enzyme catalyzes

the hydrolysis of phospholipids, in particular phosphatidylcholine, to generate

lipid molecules that are reported to play a role in keratinocyte signaling. In the

presence of water, PLD hydrolyzes phosphatidylcholine (PC) to phosphatidic

acid (PA) and choline. Lipid phosphate phosphatases, located on the outer

surface of the plasma membrane .or the luminal surface of internal membranes

(DN Brindley & Pilquil, 2009), dephosphorylate PA to yield diacylglycerol (DAG)

(DW Waggoner et al., 1999). Thus, the enzymatic action of PLD provides a

second mechanism, in addition to PLC, to generate DAG, which can then induce

the activation of PKD as well as the translocation and activation of PKCs involved

in epidermal keratinocyte function. In several systems PLD underlies a portion of

· sustained DAG production, although DAG generated in this way may differ from

PLC-derived DAG due to its different origin (PC rather than PIP2) (Nishizuka,

1995). These separate mechanisms for DAG generation may, in fact, account for

the biphasic response to DAG observed in keratinocytes and activate different

PKC isoforms. In the presence of a primary alcohol PLD also functions to 14 catalyze a transphosphatidylation reaction to generate phosphatidylalcohols.

Our laboratory has demonstrated the ability of PLD to use the physiological alcohol glycerol to generate phosphatidylglycerol in vitro (X Zheng, S Ray &

Bollag, 2003).

To date, two mammalian isoforms of PLDhave been identified (PLD1 and

PLD2), both of which require phosphatidylcholine (PC) as a substrate and PIP2 as a cofactor. These isofoms share about 50o/o amino acid similarity with the main structural differences at the N- and C-termini, leading to differential expression and activation (WB Bollag & Zheng, 2005). PLD1 associates primarily with intercellular membranes and is thought to play a role in secretory pathways (N Vitale et al., 2001; W Colley et al., 1997; Z Freyberg et al., 2001).

PLD2, on the other hand, localizes at the plasma membrane and is involved with cytoskeletal rearrangements and membrane ruffling (N Vitale et al., 2001; W

Colley et al., 1997; Z Freyberg et al., 2001 ). Both PLD1 and PLD2 are expressed in keratinocytes (RD Griner et al., 1999; V Muller-Wieprecht et al.,

1998) and a correlation has been uncovered between sustained PLD activation and the induction of keratinocyte differentiation (E Jung et al., 1999). PLD1 is highly conserved across species (92o/o identity, 97% similarity) and is up­ regulated in response to 1 ,2S(OH)203 (RD Griner et al., 1999). This enhanced

PLD1 expression was found to occur prior to the 1 ,25(0H)2D3-mediated increase in transglutaminases activity, but not prior to the inhibition of DNA synthesis or stimulation of K1 expression, suggesting a role for PLD1 in late keratinocyte 15 differentiation (RD Griner et al., 1999). PLD2 has also been linked to keratinocyte differentiation. Zheng and colleagues found that PLD is activated, and generates PG, in response to stimulation of keratinocyte differentiation (X

Zheng et al., 2003). Subsequent data suggested that PLD2 plays a role in keratinocyte differentiation due to its ability to generate PG as a potential lipid signaling molecule, through its interaction with the glycerol channel, AQP3 (see below) (WB Bollag et al., 2007).

Aquaporins

The aquaporins are a family of small (-30kDa) proteins containing two tandem repeats, each consisting of three membrane-spanning domains and a pore forming loop with a Asn-Pro-Aia (NPA) motif. To date, three groups totaling thirteen members of the aquaporin family have been identified (AQPO-AQP12).

The first group, AQPO, AQP1, AQP2, AQP4, AQPS, AQP6 and AQPB, are structurally similar to the bacterial water channel and allow only the passage of water. The second group, termed aquaglyceroporins, transport water as well as other small molecules such as urea and glycerol. The most recently identified aquaporins, AQP11 and AQP12, comprise the third, and functionally distinct subgroup of aquaporins. This group contains a divergent NPA motif, the exact effect of which remains under debate (DA Gorelick et al., 2006; K Yakata et al.,

2007). The aquaporins have been shown to play critical physiological roles in multiple organ systems, including vasopressin-dependent renal collecting duct water permeability (TH Kwon et al., 2001 ), re-absorption of cerebrospinal fluid, 16

osmoregulation and regulation of brain edema (GT Manley et al., 2000) and the

pathophysiology of muscular dystrophy (RH Crosbie et al., 2002). Of particular

interest is the apparent role of AQP3 in regulating the structure and function of the epidermis.

AQP3 is abundantly expressed in the epidermal keratinocytes and is localized within the basal and spinous layers (R Sougrat et al., 2002). Inspection via electron microscopy revealed that AQP3 primarily localizes in the plasma

membrane in both mouse and human epidermal keratinocytes (R Sougrat et al.,

2002; T Ma et al., 2002). The epidermis of mice genetically modified to be

deficient in AQP3 contains a lower glycerol content (M Hara, T Ma & Verkman,

2002) and a decreased water holding capacity(T Ma et al., 2002). These mice demonstrated delayed barrier recovery after subjection to stratum corneum

removal as well as impaired skin elasticity and delayed wound healing (M Hara et al., 2002; T Ma et al., 2002), suggesting a requirement for AQP3 in normal

epidermal function. Bollag and colleagues have provided evidence

demonstrating that AQP3 expression and activity are both modulated by extracellular calcium (X Zheng &·Bollag, 2003). Both mRNA and protein levels were determined to be drastically reduced after exposure to 1 mM elevated

2 [Ca +]e for 24 hours, as well as to 1,25(0H)2D 3, suggesting that AQP3 expression

is down-regulated at the transcriptional level in the presence of keratinocyte differentiation-inducing agents. Subsequent investigation revealed a differential

AQP3 pattern of expression in the presence of this 1mM concentration of 17

calcium, which promotes late differentiation, and a more physiologically relevant

125JJM calcium concentration. Primary mouse keratinocytes treated with a

moderately elevated (125JJM) calcium concentration showed a decrease in the

protein levels of unglycosylated AQP3, but an increase in the protein levels of

glycosylated AQP3 (H Qin et al., 2011 ). The authors hypothesize that

glycosylation, consistent with the protein expression observed in human skin,

· may help to regulate the cellular localization of AQP3 and may in fact help to

explain some controversy regarding the exact role of AQP3 in keratinocyte

maturation (although there is little doubt that AQP3 is an important player in

epidermal homeostasis).

As discussed previously, PLD also functions to catalyze the

transphosphatidylation of primary alcohols to phosphatidylalcohols. Although

frequently used as a method to monitor PLD activity, the cell may have retained

this function in order to use physiological primary alcohols, such as glycerol, to

generate specific phosphatidylalcohols like phosphatidylglycerol (PG). Using

detergent-free sucrose gradient ultracentrifugation as an isolation technique,

AQP3 and PLD2 were determined to be co-localized in caveolin-rich membrane

microdomains, a subset of lipid raft microdomains which provide discrete

platforms to promote efficient signal transduction (X Zheng & Bollag, 2003).

Additional evidence suggested that this co-localization was the result of direct

protein-protein interaction (X Zheng & Bollag, 2003). PLD was verified to

metabolize PC, in the presence of glycerol, to yield PG both in vitro and in intact 18 cells, and the production of PG was found to be increased in the presence of elevated extracellular calcium in a biphasic, dose-dependent manner (X Zheng et al., 2003). Ethanol, also a substrate for the PLD-catalyzed transphosphatidylation reaction, was able to significantly inhibit calcium-induced

PG formation indicating mediation by PLD. The maximal stimulation of PLD­ mediated PG formation was observed at the calcium .concentration optimal for stimulation of early keratinocyte differentiation markers. Taken together, these results provide evidence suggesting that caveolin-rich membrane microdomains may provide a compartmentalized scaffold for the functional interaction of AQP3 and PLD2, enabling glycerol entry into the cell where it is used in the generation of PG and further that this novel signaling cascade is involved in keratinocyte differentiation.

Glycerol and Phosphatidylglycerol

An effect of glycerol on the skin has been observed for over 75 years. Glycerol is known to prevent water loss and improve skin barrier properties by preventing the phase transition of lipids in the stratum corneum from a liquid to solid crystalline structure (Froebe, 1990). Glycerol has also been shown to play a role in desquamation by enhancing desmosomal degradation, reducing light scattering in skin, reducing the effect of chemical and mechanical irritation and accelerating wound healing (reviewed in (JW Fluhr, R Darlenski & Surber,

2008)). Glycerol can also function as a substrate for the biosynthesis of some lipids and is a crucial intermediate of energy metabolism (D Brisson et al., 2001 ). 19

Several potential sources for glycerol in the epidermis have been proposed.

Glycerol has been used as an ingredient in skin cosmetics and balms for many decades, providing an exogenous source of glycerol through topical application.

Glycerol has been found to be present in both amniotic fluid and the serum in a micro molar range (DA Harold & Reed, 1988; TE Graham et al., 2000). Local generation .of glycerol in the epidermis has also been indicated by the detection of several enzymes directly involved in lipid metabolism, such as lipases and phospholipases, with glycerol produced as a result of lipid catabolism (G Menon

& Ghadially, 1997).

The ability of AQP3 to transport glycerol into the cell has proven to be critical for

.epidermal function. As mentioned previously, the epidermis of AQP3 null mice exhibits multiple anomalies including decreased glycerol content and water holding capacity. The permeability of both water and glycerol was also greatly reduced in the epidermis of these mice despite normal glycerol concentrations in the dermis and serum, indicating a role for AQP3 in the transport of glycerol from the blood into the epidermis (M Hara et al., 2002). Subsequent studies revealed that glycerol metabolism remained unaltered by the decreased glycerol transport in AQP3-deficient mice and that oral and topical addition of glycerol, but not its structural analogues, could correct the functional defects found in AQP3 null mice (M Hara & Verkman, 2003). Consistent with this result was the finding that topical application of glycerol was able to accelerate barrier recovery in human skin (JW Fluhr et al., 1999). Taken together these results indicate an important 20

role for the AQP3 transport of glycerol in epidermal function. This conclusion is further supported by the observation that mice lacking sebaceous glands, the

asebia mouse model, exhibit an aberrant skin phenotype along with reduced

epidermal glycerol content, which can be corrected by topical glycerol

application, similar to the AQP3 knockout mouse (JW Fluhr et al., 2003).

Combined with an earlier finding that the epidermis contains the enzymes

necessary for lipid metabolism (discussed previously), the authors then proposed

that the function of the sebaceous gland may be to provide sebum as a substrate for the production of glycerol.

With multiple potential sources for epidermal glycerol established, a model

emerges wherein AQP3 transports glycerol into the cell where PLD2 converts it to PG. Taking into account that this pathway is modified by differentiative

agents, the question arises regarding what role, if any, PG plays in keratinocyte

homeostasis. Phosphatidylglycerol is a ubiquitous lipid found in almost all

bacterial types as well as in the membrane of both plants and animals. In

addition to PLD-mediated generation, PG is also synthesized from phosphatidic

acid through a cytidine diphosphate-diacylglycerol intermediate by the traditional

addition of glycerol-3-phosphate and subsequent removal of a phosphate group

(Christie, 2011 ). This second pool of PG is synthesized only in the mitochondria

in non-photosynthetic eukaryotes where it plays an essential role in mitochondrial function as a precursor for cardiolipin (diphosphatidylglycerol), a necessary

component in oxidative phosphorylation (C Lange et al., 2001; M Schlame, D 21

Rua & Greenberg, 2000). These separate biosynthetic routes may lead to

variations in PG stereochemistry (or racemization). PG has also been reported

to play a potential role in the stabilization of lamellar phases as well as the

spreading of dipalmitoylphosphatidylcholine, the main functional component of

lung surfactant (AD Postle, EL Heeley & Wilton, 2001; Christie, 2011 ). In

addition, PG has been implicated as a possible lipid second messenger in

multiple cell systems. In human spleen the protein kinase PK-P (PG-dependent

protein kinase) was found to be activated by both PG and phosphatidylinositol

(DJ Klemm; AL Kazim & Elias, 1988; DJ Klemm & Elias, 1988a; DJ Klemm &

Elias, 1988b ). PG was also found to stimulate the activity of PKC-8 in activated

human leukocytes (SF Pietromonaco et al., 1998). This study provided evidence

indicating a role for PG in meosin phosphorylation. A second study found that in

·fibroblasts entry into mitosis is dependent upon activation of the·classical protein

kinase C~ll (PKC~II) by PG (NR Murray & Fields, 1998). Of particular interest

was the observation of differential stimulation of PKC~II activity by differing PG

species, discussed below.

With PK-P, PKC-8 and PKC~II all proposed as possible PG-responsive agents

the question remains as to what role PG is playing in keratinocytes specifically.

In primary epidermal mouse keratinocytes, treatment with elevated [Ca2+]e, but

not 1 ,25-(0H)2D3, was found to induce PLD-mediated PG synthesis, as

mentioned previously (X Zheng et al., 2003). This increased PG production was

found to be dose-dependent and biphasic with maximal PG synthesis at 1251JM 22

2 Ca +, the calcium concentration optimal for K1 and K1 0 expression (X Zheng et al., 2003). In a separate study, direct provision of PG liposomes to rapidly dividing keratinocytes induced an inhibition of DNA synthesis in a dose­ dependent manner in the presence of both low (251JM) and intermediate (1251JM) calcium concentrations (WB Bollag et al., 2007). An enhancement of a calcium­ induced increase in involucrin protein was also observed in cells treated directly with PG in the same study. These findings indicate a role for PG in keratinocyte maturation including inhibition of proliferation and stimulation of differentiation. In conjunction with the background data that have been discussed thus far, it follows that this AQP3/PLD2/glycerol signaling cascade may exert its effect on keratinocyte maturation through a PKC signaling effector.

Protein Kinase Cs (PKCs)

Structure and Classification

The protein kinase C (PKC) isoforms belong to a family of protein kinases comprised of protein kinase A (PKA), protein kinase G (PKG) and PKC that all posses a regulatory domain and a highly conserved COOH-terminal catalytic domain (Steinberg, 2008). Within the framework of this superfamily exists the serine/threonine-specific PKC isozymes, composed of four conserved domains

(C1-C4) and five divergent variable domains (V1-V5) (Fig.2). Originally identified in 1977 as a cytoplasmic, calcium-activated, phospholipid-dependent kinase (M

Inoue et al., 1977), the PKC isoenzymes are now understood to include conventional, novel and atypical PKCs, differing in the NH 2-terminal regulatory 23 domain. The regulatory domain, made up of an autoinhibitory pseudosubstrate domain and two C1 and C2 membrane targeting modules along with V1-V3 variable domains, is believed to interact with the catalytic domain to retain PKC in an inactive conformation. The catalytic kinase domain, comprised of C3 and C4 along with V3-V5, contains the ATP binding site as well as the domain that is believed to interact with the substrate. The conventional PKCs (cPKCs ), consisting of PKCa, PKCJ31, PKCJ311 and PKCy, require negatively charged phospholipids (such as phosphatidylserine; PS), diacylglycerol (or phorbol ester) and calcium for optimal activation (H Hug & Sarre, 1993; Jaken, 1996). The regulatory domains of cPKCs contain a C1 domain (divided into twin C1A and

C1 8 domains) that binds DAG/phorbol ester and a C2 domain that binds anionic phospholipids in a calcium-dependent manner. The novel PKCs (nPKCs), consisting of PKC5, PKCE, PKC9, and PKCJ"]/L (mouse/human), are similar to the cPKCs in that they require negatively charged phospholipids and diacylglycerol

(or phorbol ester); however, the nPKCs do not require calcium for activation (FJ

Johannes et al., 1994; H Hug & Sarre, 1993). The regulatory domain of nPKCs also contain twin C1 domains and a C2 domain, the latter lacking the critical calcium-responsive acidic residues (Steinberg, 2008). The atypical PKCs

(aPKCs), consisting of PKCJ\11 (mouse/human) and PKC~, require only negatively charged phospholipids (Jaken, 1996). These PKCs lack a calcium-sensitive C2 domain and contain an atypical C1 domain that binds PIP3 or ceramide, but not

DAG/phorbol ester (Steinberg, 2008). 24

DAG ATP ('2 C_,"' C4

Regulntot·y Dom~1in u Kinase Domain Hinge

Figure 2. The domain structure of the classical PKCs (PKCa, PKCfjl, PKC/311 and PKCy). The serine/threonine-specific PKC isozymes are composed of four conserved domains (C1-C4) and five divergent variable domains (V1-V5).

Specifically, the regulatory domain of the cPKCs contain twin C1A and C1B domains that bind DAG/phorbol ester and a C2 domain that binds anionic phospholipids in a calcium-dependent manner. The catalytic kinase domain contains the A TP-binding site as well as the domain that is believed to interact with the substrate. The PKC/31 and PKC/311 splice variants differ at the C-terminal

V5 region (the PG-binding domain of PKC/311). PS represents the pseudosubstrate domain. Interaction of DAG with the C1A domain leads to a conformational change that displaces the autoinhibitory domain, thereby activating the PKC. 25

Expression, phosphorylation and translocation

Despite many similarities between the various PKC isoforms, a major distinguishing factor is their distinct tissue and subcellular localization. Extensive investigation in rat utilizing isoform-specific antisera revealed the presence of

PKCa in all of the tissues examined (brain, lung, heart, spleen, liver, testes, ovary, kidney) (WC Wetsel et al., 1992). In this study, PKC~I was present in all of the organs except the liver and kidney; PKC~II was present everywhere except the liver. Both PKC~I and PKC~II were present in the greatest amounts in brain and spleen, whereas PKCy was detected only in brain. PKCo and PKC~ were detected in all of the organs tested and PKCE was found only in brain and kidney tissue (WC Wetsel et al., 1992). This study provided one of the earliest investigations of the localization of the PKC isoforms. Subsequent study, undertaken with more sensitive tools, later revealed more extensive expression of the PKC isoforms. Investigation of multiple different cell types revealed further differences in the subcellular localization of the PKC isoforms. The cell types investigated differ greatly from each other and include GH4C1 rat pituitary cells

(Kiley et al., 1990), primary cultured heart cells (MH Disatnik, G Buraggi &

Mochly-Rosen, 1994b; Mochly-Rosen et al., 1990), a breast cancer cell line (MH

Disatnik et al., 1994a) and HL60 human promyelocytic leukemia cells (Hocevar

& Fields, 1991 ). The PKC isoforms have been implicated in a wide variety of cellular responses due in part to the pleiotropic actions of diacylglycerol as well as the distinct subcellular localization of the various isoforms and the variety of regulatory actions that this enables. As a result, PKC plays a pivotal role in a 26 variety of signal transduction pathways in response to G protein-coupled receptor and growth factor-dependent ligands, resulting in a vast array of cellular processes including proliferation and differentiation as well as the release of hormones and neurotransmitters (Nishizuka, 1986; Nishizuka, 1988).

The general model of PKC activation is based on initial observations of the activation of PKCa. Under basal conditions PKCa is located in the cytosol and only weakly interacts with the membrane in the absence of calcium or DAG. In the presence of agonists that stimulate an increase in intracellular calcium, PKCa exhibits a greater affinity for cell membranes. Calcium binds to the C2 domain, increasing its affinity for the membrane and allowing PKCa to then diffuse within the plane of the lipid bilayer (Steinberg, 2008). PKCa then interacts with DAG through its C1A domain. Of note, tumor promoters, such as the phorbol esters, are able to substitute for endogenous DAG and lead to PKC activation.

Membrane phosphatidylserine (PS) plays a critical role in this interaction by disru.pting the C1A/C2 interdomain electrostatic forces, thus freeing the C1A domain and allowing it to penetrate into the lipid bilayer and interact with DAG

(RV Stahelin et al., 2005). This secondary binding leads to a conformational change that displaces the autoinhibitory pseudosubstrate domain enabling PKC activation (Steinberg, 2008). Most models of PKC activation generally focus on this intramolecular interaction between the catalytic pocket of PKCs and its pseudosubstrate domain. This basic model of activation can be manipulated to create constitutively active forms of PKC by generating mutations that disrupt the 27 intramolecular interactions on which activation is based. An alanine---+glutamate phosphomimetic substitution in the pseudosubstrate domain sequence or a pseudosubstrate domain deletion is thus sufficient to generate a constitutively active PKC variant.

Phosphorylation and translocation are both indirect indicators of PKC activation.

In general, PKC isoforms must first be processed by three distinct phosphorylation events before they are competent to respond to second messengers (Keranen, Dutil & Newton, 1995; SE Tsutakawa et al., 1995). The

PKC isoforms undergo a series of "priming" Ser/Thr phosphorylations that lock the enzyme in a closed, stabilized, catalytically-competent, phosphatase/protease-resistant conformation (AC Newton & Johnson, 1998; DB

Parekh, W Aiegler & Parker, 2000). The first PKC phosphorylation, generally attributed to phosphoinositide-dependent kinase-1 (PDK-1 ), occurs at a threonine residue within the highly conserved, 20-30 residue sequence "activation loop" of the kinase domain (Steinberg, 2008). Research suggests that the structure of newly synthesized PKCs is originally in an open conformation allowing PDK-1 access to the exposed unphosphorylated hydrophobic motif sequence in the

COOH-terminal V5 domain (ED Sonnenburg, T Gao & Newton, 2001; EM Dutil, A

Taker & Newton, 1998; T Gao, A Taker & Newton, 2001 ). The activation loop assumes a different orientation in the active and inactive states and forms part of the peptide substrate binding surface, stabilizing the active conformation of the enzyme by introducing a neg.ative charge that aligns residues in the catalytic 28 pocket (Steinberg, 2008). PDK-1 is then released from the COOH-terminus in order to allow the subsequent autophosphorylation step. Both cPKC and nPKC participate in two additional autophosphorylations at conserved motifs in the

COOH-terminal V-5 domain: one at a conserved, typically proline-flanked "turn motif' and the second at the Ser/Thr motif bracketed by hydrophobic residues.

Specifically, autophosphorylation at Ser657 of PKCa controls accumulation of phosphates at other sites on the kinase, as well as contributing to the maintenance of the phosphatase-resistant conformation (Bornancin & Parker,

1997), and autophosphorylation at Ser660 of PKC~II induces a 10-fold increase in the enzyme's affinity for PS and calcium, both required for full activation

(Edwards & Newton, 1997). Collectively, hydrophobic motif autophosphorylations play an integral step in generating fully phosphorylated, catalytically competent PKCs.

In the case of cPKCs, hydrophobic motif autophosphorylations have also been implicated as a mechanism that regulates binding to membranes. PKC isoforms in general translocate to the plasma membrane in response to activation. cPKCs

(PKCa, PKC~I and PKC~II) rapidly and transiently translocate to the plasma membrane via a mechanism that seems to involve PLC-derived DAG accumulation. PKCa and PKC~II (but not PKC~I) are then released from the plasma membrane by a method requiring PKC catalytic activity (attributed to hydrophobic motif autophosphorylation) in cells that exhibit a biphasic DAG response (such as keratinocytes) (X Feng & Hannun, 1998; X Feng et al., 2000). 29

PKCa and PKC~II then accumulate in the perinuclear area as a result of sustained DAG synthesis involving PLD, but not PLC (T Hu & Exton, 2004 ). In general, PKC isoforms which migrate to the nucleus are phosphorylated (Neri et al., 2002). PKC isoforms have also been observed to translocate to specialized membrane compartments such as lipid rafts or caveolae (Steinberg, 2008).

Often used as a measure of activation in cells, translocation may represent a necessary step allowing PKCs to exert their cellular effects.

In addition to the general model of PKC activation, phosphorylation and translocation discussed above, a second body of research focuses upon the interaction of PKC with receptors for activated C kinases (RACKs) as an essential step for PKC activation and localization in some cellular processes.

The RACKs are a family of membrane-associated anchoring proteins that participate in PKC signaling by acting as molecular scaffolds bringing into proximity individual PKCs with their activators and substrates (D Schechtman &

Mochly-Rosen, 2001 ). The Mochly-Rosen group has provided evidence suggesting that individual PKC isoforms have specific, uniquely localized RACKs and that interaction with a particular RACK is an essential component of PKC isoform-specific cellular responses (D Mochly-Rosen et al., 1995). Through identification of the RACK-PKC complex binding sites, this group was able to show that short peptides containing an amino acid sequence that mimics the

PKC isoform binding site serve as isoform-selective competitive inhibitors of PKC 30 translocation and function (D Schechtman & Mochly-Rosen, 2001 ). This model of intramolecular interaction is of specific interest to the research presented here.

PKCs in keratinocyte differentiation

Keratinocytes have been described to express many of the PKC isozymes, and differential translocation of these isoforms has been observed upon stimulation of keratinocytes with elevated calcium (MF Denning et al., 1995). Data suggest disparate roles for these isoforms in regulating various aspects of keratinocyte differentiation. PKC11, on the other hand, has been shown to increase the transcription of transglutaminase, a marker of keratinocyte differentiation, more effectively than other PKC isoforms (E Ueda et al., 1996). The 11 isoform also stimulates transcription of the differentiation gene involucrin (H Takahashi et al.,

1998; T Efimova & Eckert, 2000), as does PKCa (H Takahashi et al., 1998),

PKCE and PKCo {T Efimova & Eckert, 2000). Adenovirus-mediated introduction of PKCo or 11 into human keratinocytes triggers their differentiation (M Ohba et al., 1998). PKCa also seems necessary since antisense ablation of this isoenzyme inhibits the expression of multiple differentiation markers (LC Yang,

DC Ng & Bikle, 2000; YS Lee et al., 1997). Therefore, it appears that specific

PKC isoforms may be responsible for determining the patterned expression of genes during keratinocyte differentiation.

A variety of studies determined that activation of PKC induces the expression of genes associated with the granular layer (late differentiation), including loricrin 31 and filaggrin, as well as the desmosomal cadherin isoform desmoglein 1 (AA

Dlugosz & Yuspa, 1993; Dlugosz & Yuspa, 1994; MF Denning et al., 1998).

Specifically, PKC11 was determined to be localized to the granular layer and to induce growth arrest as well as the expression of transglutaminase (TG) and involucrin (I NV) (Kashiwagi et al., 2002). In addition to TG and I NV, PKC activation has been linked to the induction of many of the other proteins involved in the water-barrier function provided by the covalent cross-linking of lipids and proteins that form the cornified envelope, such as loricrin, filaggrin and SPRR-1

(Dlugosz & Yuspa, 1994 ). Early studies focusing on terminal differentiation of interfollicular keratinocytes into squamous cells revealed potential roles for multiple PKC isoforms at several points in this process as well. In one such study PKC activation was found to induce the irreversible cell cycle withdrawal that initiates the process of differentiation, and inhibition of PKC could prevent cell cycle withdrawal (Tibudan, Wang & Denning, 2002). This latter observation also suggests the involvement of PKC in the early commitment of basal keratinocytes to enter the differentiation program. These authors suggested that activation of PKCa may be involved in this process. Subsequent research in normal human keratinocytes revealed induction of differentiation-associated growth arrest and inhibition of DNA synthesis by PKCa, the down-regulation of which delays growth arrest (Jerome-Marais et al., 2009). PKCa is also thought to facilitate keratinocyte involvement in innate and adaptive immunity and has been implicated in the epidermal inflammatory response. In response to PKCa activation, transgenic mice that over-express epidermal PKCa were shown to 32 express elevated levels of cytokines, chemoattractants and the inflammatory mediators TN Fa, MIP2 and COX2 (Cataisson et al., 2003; Wang & Smart, 1999).

Of specific interest to the research presented here is the evidence indicating a role for PKC~II in keratinocyte differentiation. Both PKC~I and PKC~II are expressed in a tissue-specific and developmentally regulated manner (CE

Chalfant et al., 1995). Although an initial report failed to detect PKC~ in mouse keratinocytes by northern analysis (Dlugosz et al., 1992), two subsequent studies found PKC~ in both human skin (GJ Fisher et al., 1993) and mouse keratinocytes

(SM Fischer et al., 1993). PKC~II was found to be preferentially reduced in psoriatic lesions in comparison to normal skin (GJ Fisher et al., 1993), despite increased activity of PI-PLC in active psoriatic lesions (GJ Fisher et al., 1990), indicating additional regulation of PKC~II separate from PI-PLC-generated DAG accumulation. Alterations in PKC~II expression have also been linked to changes in the proliferative status of colonic epithelium (Murray et al., 1999;

Murray et al., 2002) a~d was correlated to the median survival of patients with

PKC~II-expressing colorectal epithelial tumors (KG Spindler et al., 2009).

Protein Kinase C/31/ (PKC/311)

Within the structure of the PKC isozymes exist five divergent variable regions, as discussed previously. Carboxy-terminal to the catalytic core (C3 and C4 domains) of PKC sits the VS domains (Fig.2). This domain is composed of 50- to

70-amino acids, containing highly conserved turn and hydrophobic phosphorylation motifs, as well as an additional 7-21 residues at the very end of 33 the C-terminus, beyond the hydrophobic motif. These additional residues are highly variable in both length and sequence and share little to no sequence homology. Although largely ignored in early PKC studies, aside from use as epitopes to raise PKC isoform-specific antibodies for Western blotting and immunolocalization studies, V5 domains are now understood to contain important determinants of isoform-specific targeting and function (Steinberg, 2008). Of specific interest was the discovery that the PKC~ splice variants (PKC~I and

PKC~II) differ only at their C-terminal V5 region (Gokmen-Polar & AP Fields,

1998). PKC~I and PKC~II are alternatively spliced products of one gene encoding a PKC~ pre-mRNA. The PKC~II mRNA splice variant is produced from the inclusion of the PKC~II exon in the 3'-region, resulting in PKC~I and PKC~II proteins that differ only in their 50-52 C-terminal amino acids, resp~ctively (Fig.

3) (Nishizuka, 1986; Patel et al., 2003). Additionally, the priming phosphorylations integral in generating fully phosphorylated, catalytically competent PKCs (discussed previously) reside within this domain. For PKC~II these phosphorylations occur at Thr500, Thr641 and Ser660. 34

..------\"!' ------,

PKC11Jn-•RMA

s..,c•••• C-4 C4

PKC'PlmRIIA PKCPIImRIIA

Figure 3. PKC{JI and PKC{J/1 are alternatively spliced products of one gene encoding a PKC{J pre-mRNA. The PKC{31 and PKC{311 splice variants are translated from alternatively spliced products of the same gene. The PKC{3/I mRNA splice variant is produced from the inclusion of the PKC{31/ exon in the 3'- region and the generation of a stop codon at the boundary between PKC{31 and

PKC{31/, resulting in PKC{31 and PKC{311 proteins that differ only in 50-52 C- terminal amino acids, respectively (Patel eta/., 2003). 35

Of particular interest to our research was the discovery that the catalytic domain

of PKC~II contains the molecular determinants necessary for selective nuclear targeting of the enzyme (Walker et al., 1995) through interaction with a nuclear

membrane interaction factor (NMAF) that stimulated PKC~ activity 3-6 fold

above the level achieved in the pre.sence of optimal concentrations of calcium,

DAG and PS in human promyeolocytic (HL60) leukemia cells (Murray, Burns &

Fields, 1994 ). A subsequent study identified phosphatidylglycerol (PG) as the

NMAF and found that PG binds the unique C-terminal region of PKC~II in a

potent and selective manner (NR Murray & Fields, 1998). Specifically, it is the 13

C-terminal amino acids of the PKC~II VS region that bind PG and contain the

molecular determinant necessary for nuclear translocation and activation of

PKC~II (Gokmen-Polar & AP Fields, 1998). A soluble peptide corresponding to the VS region of PKC~II was able to inhibit PG-mediated activation of the isoform

(Gokmen-Polar & AP Fields, 1998). These findings, combined with the

observation that activation of PKC~II by PG is required for cell cycle progression

in human erythroleukemia (K562) cells, provide strong evidence that PKC~II may exert its cellular effects, in part, by acting as a PG-responsive effector enzyme.

Thus, we propose that AQP3 and PLD2 functionally interact to regulate the production of PG. Modulated by elevated extracellular calcium, we hypothesize that this AQP3/PLD2/PG complex represents a novel signaling module that exerts a pro-differentiative and/or anti-proliferative effect on keratinocytes through the PG-responsive signaling enzyme PKC~II (Fig.4). 36

epidennallipid amniotic hydrolysis fluid serum \ J skin-care ' Gl yceroI / products

., ... Keratinocyte ! _. .... Differentiation PKCPII --·

Figure 4. A novel signaling module comprised of aquaporin3 (AQP3), phospholipase D2 (PLD2) and phosphatidylglycerol (PG). Multiple sources for epidermal glycerol have been identified, including micromolar amounts in the serum and amniotic fluid. We propose that AQP3 and PLD2 functionally interact to regulate the production of PG through AQP3-mediated transport of glycerol into the cell and conversion by PLD2 to PG. We further propose that this

AQP3/PLD2/PG signaling module mediates pro-differentiative and/or anti- proliferative effects on keratinocytes in response to stimulation by extracellular calcium through the PG-responsive signaling lipid PKCf31/. 37

Hypothesis

We hypothesize that PG-responsive PKC~II functions as a down-stream effector enzyme involved in regulating calcium-induced keratinocyte differentiation in response to the novel AQP3/PLD2/PG signaling module.

We base this hypothesis on findings in the literature, as well as observations from our laboratory, demonstrating:

1. Maximal stimulation of calcium-induced, PLD-mediated, PG formation

was observed at calcium concentrations optimal for stimulation of early

keratinocyte differentiation markers K1 and K1 0.

2. The C-terminal PKC~II VS region binds PG in a potent and selective

manner and contains the molecular determinant necessary for nuclear

translocation and activation of the enzyme (Gokmen-Polar & AP

Fields, 1998; NR Murray & Fields, 1998).

3. PG selectively stimulates PKC~II activity 3-6 fold above the level

achieved in the presence of optimal concentrations of calcium, DAG

and PS in HL-60 cells (Murray et al., 1994). CHAPTER TWO: MATERIALS AND METHODS

MATERIALS

Animals

Male and female ICR (CD-1®) outbred mice were obtained from Harlan

Laboratories (North America division).

Culture of primary mouse keratinocytes

Serum Free Keratinocyte Medium (SFKM)

Calcium-free minimum essential medium (MEM) was from Biologos, lnc.(Montgomery, IL) and ITS+ (6.251-Jg/mL insulin, 6.251-Jg/mL transferrin,

6.25ng/mL selenous acid, 1.25 mg/mL BSA, 5.351-Jg/mL linoleic acid) was from

Collaborative Biomedical Products (Bedford, MA). Dialyzed fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA). Epidermal growth factor was purchased from Life Technologies, lnc.(Grand·lsland, NY). Glutamine, penicillin, streptomycin and fungizone were all from Mediatech (Herndon, VA). BD Falcon six-well plates were from Fisher Scientific (Hampton, NH) and trypsin was purchased from Invitrogen. SFKM .contains a total of 251-JM CaCI2.

Keratinocyte Serum Free Medium (K-SFM)Keratinocyte-SFM, human recombinant EGF and bovine pituitary extract were all from GIBCO, a subsidiary of Invitrogen. K-SFM is supplemented with 501-JM CaCI2.

38 39

Experimental Treatments

Experimental treatment materials were acquired as follows: 12-0-tetradecanoyl­ phorbol-13-acetate, glycerol and calcium chloride were from Sigma-Aldrich (St

Louis, MO). Egg phosphatidylglycerol was from Avanti Polar Lipids, Inc.

(Alabaster, AL). All peptide inhibitors were a generous gift from Dr. Daria

Mochly-Rosen (Stanford University, Stanford, CA). LY333531 mesylate

(Ruboxistaurin) was from Axon MedChem (AT Groningen, The Netherlands).

Immunocytochemistry

Collagen type I Cellware 4-well Culture Slides were acquired from BD

Biosciences, a subsidiary of Fisher Scientific, Inc. (Hampton, NH). ProLong Gold antifade reagent with DAPI was from Invitrogen. Normal goat serum and paraformaldehyde were from Sigma-Aldrich. 1OX phosphate buffered saline were from Bio-Rad Laboratories, Inc. (Hercules, CA). Triton X-1 00 was from Fisher

Scientific (Hampton, NH) and cover glasses were from Surgipath Medical

Industries (Eagle.River, WI).

Western blot

DC protein assay reagents were from Bio-Rad (Hercules, CA). Bovine serum albumin (BSA) was from Pierce (Rockford, IL). lmmobilon-FL transfer membranes were from Millipore (Billerica, MA). Tris-HCI powder was from

Research Organics. SDS, EDTA, EGTA, AEBSF and leupeptin were all from

Sigma. Tween 20 and ammonium persulfate (APS) were from Fisher. Running 40 buffer, (composed of 1OXTris/glycine/SDS), Triton X-1 00 and beta­ mercaptoethanol were purchased from BioRad (Herculeas, CA). Odyssey blocking buffer was from LI-COR Biosciences (Lincoln, NE).

Antibodies

Phospho- and total protein kinase C~ll antibodies used for the immunocytochemical experiments were a generous gift from Dr. Denise Cooper

(University of South Florida, Tampa, FL). Total PKC~II was raised against residues 657 to 673 (C-terminus specific to PKC~II) and the pPKC~II antibody recognizes phosphoserine 661, an autophosphorylation site. The keratin 10 mouse monoclonal antibody used in the immunocytochemistry experiments was from Abeam (Cambridge, MA). For use in western blotting experiments, protein kinase C~ll rabbit polyclonal was from Abeam, phospho-protein kinase C~ll

(pS660) rabbit monoclonal primary antibody from Epitomics (Burlingame, CA) and keratin 10 rabbit polyclonal from Covance. Actin (1-19) goat polyclonal antibody was from Santa Cruz (Santa Cruz, CA) and actin mouse monoclonal was from Sigma. Secondary antibodies were acquired as follows: Alexa Fluor®

64 7 chicken anti-rabbit lgG from Invitrogen; Cy3-conjugated goat anti-rabbit lgG

(H+L) secondary antibody; IRDye 800 conjugated goat anti-rabbit lgG secondary antibody and IRDye 680-conjugated goat anti-rabbit lgG secondary antibody from LI-COR; Alexa Fluor 680 goat-anti mouse and Alexa Fluor 800 donkey anti­ rabbit were from Molecular Probes. 41

RNA Isolation and RT-PCR

Trizol RNA isolation reagent as well as the forward and reverse primers for

mouse PKCa PKC~, and PKCy were acquired from Invitrogen. Both

Thermoscript RT -PCR System and JumpStart RedTaq reaction mix were

·purchased from Sigma-Aldrich (St. Louis, MO).

Transient Transfection of PKC{j/1

Empty vector or PKC~II plasm ids in a pcDNA3 vector backbone under the control

of CMV promoter were a generous donation from Dr. LanKa (Georgia Health

Sciences University, Augusta, GA). Amaxa Basic Nucleofector Kit for primary

endothelial cells was from Lanza Cologne AG (Basel, Switzerland).

Thymidine Assay

[Methyi-3H] Thymidine was from Moravek Biochemicals (Brea, CA). 42

METHODS

Animals

A breeding colony of ICR (CD-1®) mice was established in our laboratory.

Breeding pairs were maintained with a diet of standard chow and water ad libitum in the Georgia Health Sciences University Division of Laboratory Animal

Services. Treatment of mice conformed to policies set forth in the Guide for the

Care and Use of Laboratory Animals and monitored by the Institutional Animal

Care and Use Committee (IACUC) of Georgia Health Sciences University.

Culture of primary mouse keratinocytes

Primary murine epidermal keratinocytes were prepared from 1 to 3 day old neonatal ICR CD-1 outbred mice as described by Yuspa and Harris (Yuspa &

Harris, 197 4 ). The keratinocytes were obtained by first removing the skins as intact sheets followed by an overnight incubation in 0.25% trypsin at 4 oc. The epidermis and dermis were then mechanically separated using forceps, and keratinocytes were separated from the underside of the epidermis by gentle scraping. Keratinocytes were collected by centrifugation, plated at a density of

25,000 cells/cm2 in 6-well tissue culture plates, 60mm tissue culture plates or 4- well tissue culture slides and incubated overnight in an atmosphere of 95%> air I

5°/o carbon dioxide at 37°C in plating medium composed of calcium-free minimum essential medium alpha supplemented with 2%> dialyzed fetal bovine serum, 25J.JM CaCb, 5 ng/mL epidermal growth factor, 2 mM glutamine, ITS+, 43

100 U/ml penicillin, 100 JJg/mL streptomycin and 0.25 JJg/mL fungizone. After approximately 24 hours, plating media was replaced with either serum-free keratinocyte medium (SFKM) prepared in-house or commercially available keratinocyte-serum free medium (K-SFM). Initial experiments used SFKM containing 25 JJM CaCI2, 90 JJg/mL bovine pituitary extract, ITS+, 5 ng/ml epidermal growth factor, 2mM glutamine, 0.05°/o BSA, 100 U/ml penicillin, 100 ug/ml streptomycin and 0.25 JJg/mL fungizone as described by Griner et al. (RD

Griner et al., 1999). Our laboratory subsequently switched to Gibco K-SFM prepared by supplementing pre-prepared K-SFM with 2.5J..lg recombinant human

EGF and 25mg bovine pituitary extract. Medium was replaced every 1-2 days.

Preparation of phosphatidylglycerol (PG) liposomes

Egg PG was obtained commercially from Avanti Polar Lipids and aliquots were prepared as follows: briefly, 1mg of PG in 1OOJJI chloroform/methanol was aliquoted into amber glass vials. Evaporation of the chloroform was performed under a gentle flow of nitrogen to yield a lipid film, and the vials were sealed trapping nitrogen inside. All steps were performed on ice using only glass vials and pipets. Aliquots were stored at -20°C. To prepare liposomes from aliquots

0.5ml of serum-free keratinocyte medium was added to the vial in order to hydrate the lipid film and lipid vesicles were formed by sonication using a bath sonicator at 4 oc immediately prior to use. 44

RT-PCR Analysis

Near-confluent cultures of primary keratinocytes were incubated in SFKM (251JM

CaCb) or SFKM containing 1251JM CaCI2 at 37°C. After the desired incubation time cells were collected in 5001JL Trizol reagent, and RNA was extracted according to the manufacturer's protocol. Tissue was also collected and RNA extracted from mouse brain as a positive control. First-strand eDNA synthesis was performed using the Thermoscript RT -PCR System and oligo( dT) nucleotides according to the manufacturer's protocol. RT -PCR was performed using JumpStart RedTaq reaction mix and the primers listed below, also according to the manufacturer's protocol. The reaction parameters consisted of heat activation at 94°C for 2 minutes followed by 35 cycles of denaturation at

94°C for 15 seconds, annealing at 50°C for 30 seconds and elongation at 72°C for 30 seconds. The amplified product was resolved on a 1 o/o TAE agarose gel.

Primers

mPKCa 5' -ACCGGCGACTGTCCGAGGAA-3' (forward)

5'-TTTGTTGCCGGCAGGGCCAA-3' (reverse)

mPKC~ 5'-GCTGACAAGGGCCCAGCCTC-3' (forward)

5'-GTGTGGTTCCGTGCCGCAGAG-3' (reverse)

mPKCy 5' -CGGCTCAGCGTGGAGGTGTG-3' (forward)

5' -CCCGTCCGCACCCTCTCGTA-3' (reverse)

mGAPDH 5' -GCGGCACGTCAGATCCA-3' (forward)

5' -CATGGCCTTCCGTGTTCCCTA-3' (reverse) 45

Protein Purification

Near confluent (60-80%) cultures of keratinocytes were incubated in SFKM

(25J.JM CaCI2) or K-SFM (50J.JM CaCb) alone or with SFKM containing the desired treatments: elevated calcium (125J.JM or 1mM CaCI2), 1OOnM TPA, 0.2o/o glycerol or 1OOJ.Jg/ml PG. Cells were harvested at the desired time points by first washing once with phosphate-buffered saline lacking divalent cations (PBS-) and . . then collecting the cells in a lysis buffer to obtain total protein as described below.

For total protein 30J.JI/cm2 of heated buffer [containing 0.1875M Tris-HCI (pH 8.5),

3o/o SDS and 1.5mM EDTA] was added to each well. Protein concentrations were determined using a BioRad protein assay with BSA as the standard. After protein concentrations were determined, 3X sample buffer (containing 30o/o glycerol, 15o/o beta-mercaptoethanol and 1% bromophenol blue) was added to each sample to constitute Laemmli buffer (Laemmli, 1979). Total protein was also extracted from mouse brain, homogenized epide.rmis and freshly isolated keratinocytes. Tissue was sheared and lysed using an 18 gauge needle and cells were washed once in PBS-. Cells were further lysed and proteins collected in heated lysis buffer as above. Samples were stored at -20°C until western blot analysis was performed.

Western Blot Analysis

Protein samples were heated to near boiling (85-90°C) and equal amounts of protein were loaded onto 8o/o SDS polyacrylamide (SDS-PAGE) gels, separated 46

by electrophoresis and then transferred onto lmmobilon-FL transfer membranes.

Membranes were then washed with PBS- and blocked in Odyssey blocking

buffer for 1 hour at room temperature. The membranes were washed in PBS­

containing Tween-20 (PBS-T) and then incubated overnight in primary antibody

(pPKC~II, 1:1 0,000; K1 0, 1 :15,000; actin, 1 :15,000) diluted in Odyssey blocking

buffer containing Tween-20. Following overnight incubation in primary antibody

the membranes were washed three times in PBS-T and then incubated with

secondary Alexa Fluor fluorescent secondary antibodies (1: 10,000) diluted in

Odyssey blocking buffer containing Tween-20 for 1 hour at room temperature.

The membranes were then washed extensively in PBS-T followed by one wash

in PBS. Immunoreactive bands corresponding to the proteins of interest were

visualized via the Odyssey®SA infrared imaging system from LI-COR.

Fluorescent bands were analyzed using the Odyssey®SA internal software

according to the manufacturer's instructions, and the data were reported

normalized to actin levels.

fHJ-Thymidine Incorporation into DNA, a Proliferation Assay

DNA proliferation assays were performed on primary mouse keratinocytes

cultured in SFKM (251JM calcium) and over-expressing PKC~II or empty vector with or without an elevated extracellular calcium concentration. Proliferation assays were also performed on mouse keratinocytes cultured in K-SFM (501JM

calcium) in the presence and absence of either the ~II V5 fragment peptide translocation inhibitor (EG Stebbins & Mochly-Rosen, 2001) of PKC~II or the 47

LY333531 activity inhibitor of PKC~ (Wheeler, 2003). Briefly, cells were transfected and/or cultured as previously described. After the desired time of inhibition and/or exposure to experimental treatments, the cells were incubated with 1 ~Ci/ml [3H]thymidine for 1 hour at 37°C. Cold TCA was added to culture wells, following a wash with cold PBS, and maintained on ice for 10 minutes.

This step was repeated with fresh TCA for an additional 10 minutes and cells were solubilized in 0.3 M sodium hydroxide. [3H]Thymidine incorporation was measured using Ecoscint scintillation fluid in a Beckman Coulter LS 6500 multi­ purpose scintillation counter (Brea, CA). Experimental measures were repeated in duplicate and values presented as the means of multiple experiments.

lmmunocytochemisty

Primary murine keratinocytes were plated on glass BD BioCoat fibronectin­ coated slides and at near-confluence were incubated in SFKM (25~M CaCI2) or

SFKM containing either 125~M CaCI2, 1 mM CaCI2, 1OOnM TPA, 1 OO~g/ml PG or

0.2% glycerol at 37°C. After the desired incubation time, cells were washed with

PBS- and then fixed in 4% paraformaldehyde for 10 minutes at room temperature. The slides were then washed again in PBS-, and cells permeabilized with 0.2o/o Triton X-1 00 for 5 minutes. The slides were washed again and then blocked in buffer containing 10°/o goat serum and 1 o/o BSA in

PBS- for 1 hour at room temperature. After blocking, the slides were washed again and then incubated in primary antibody buffer containing either anti-PKC~II

(1 :1 000), anti-phospho PKC~II (1 :500) or anti-keratin-1 0 (1 :250). Following 48 overnight incubation the slides were washed and then incubated in Cy3- conjugated secondary goat anti-rabbit lgG antibody (1 :150) in 10% goat serum for 40 minutes at room temperature. The slides were mounted with Prolong

Antifade with DAPI and then visualized by multiphoton microscopy using a Zeiss

LSM 510 confocal laser scanning microscope with Meta System equipped with a

Coherent Mira 900 tunable Ti:Sapphire laser for multi-photon excitation at

488nm, 543nm and 760nm wavelengths (Carl Zeiss Microscopy, Germany).

Keratinocyte Transfection

Wild-type PKC~II, under the control of the CMV promoter, in a pcDNA3 vector backbone was used to transform JM1 09 chemically competent bacterial cells according to the manufacturer's instructions. Cells were also transformed with plasmid containing an empty vector (EV) for use as a control. Plasmid DNA was isolated and purified (with a Qiagen mini prep) and positive clones were verified by restriction digestion followed by agarose gel analysis of the plasmid DNA.

Primary mouse keratinocytes were then transfected with purified wild-type

PKC~II plasmid (or empty vector plasmid) via AMAXA nucleofection, according to the manufacturer's instructions. Transfected cells were then incubated in medium consisting of RPMI-1640, glutamine, HEPES and fetal bovine serum to stabilize the cells for 20 minutes, then plated in plating medium (described previously) and allowed to attach overnight. After 24 hours the plating medium was replaced with K-SFM (containing 501JM CaCI 2), which was replaced every 1-

2 days until desired confluence was reached. 49

Inhibitor Experiments

L Y333531 PKC/3 catalytic activity inhibitor

Primary mouse keratinocytes were cultured in K-SFM (containing 501JM CaCI2).

At near confluence cells were incubated with either basal (501JM) calcium, elevated extracellular (1251JM) calcium or PG liposomes (1 OO!Jg/ml) in combination with 11JM LY333531 (L Y), 300nM LY or no inhibitor (control) for 24 hours. The cells were then either harvested for western blot analysis or exposed to [3H]thymidine for the DNA synthesis assay, as described above.

PKC/31 and PKC/311 peptide translocation inhibitors

Primary mouse keratinocytes were cultured in K-SFM (containing 501JM CaCI2).

At near confluence cells were incubated with either basal (501JM) calcium, elevated extracellular (1251JM) calcium or PG liposomes (1 OO!Jg/ml) in combinatiofl with PI VS fragment PKCPI translocation inhibitor, PII VS-5 fragment

PKCPII translocation inhibitor (both conjugated to the 11 amino acid protein transduction sequence of TAT) or the unconjugated TAT peptide alone (as control). After 24 hours the cells were then either harvested for western 'blot analysis or exposed to [3H]thymidine for the DNA synthesis assay, as described above.

Statistics

All experiments were performed independently a minimum of three times as indicated. Values were analyzed for statistical significance by analysis of so variance or repeated measures analysis of variance with a Student-Newmann­

Keuls or Dunn's post-hoc test using lnStat or Prism (Graph Pad Software, San

Diego, CA) with statistical significance assigned at p<0.05. All quantitative data were expressed in the form of bar graphs, with the bars representing mean ± standard error (S.E.). CHAPTER THREE: RESULTS

PKC/311 is present in mouse keratinocytes and undergoes ·subcellular redistribution with stimulation of the AQP3/PLD2/PG signaling module

Although multiple PKC isozymes have been identified in keratinocytes, there has been some debate regarding the presence of PKC~ in these cells (Dlugosz et al.,

1992; GJ Fisher et al., 1993; SM Fischer et al., 1993). An initial report failed to detect PKC~ in mouse keratinocytes by northern analysis (Dlugosz et al., 1992).

Two subsequent studies found PKC~ in both human skin (GJ Fisher et al., 1993) and mouse keratinocytes (SM Fischer et al., 1993). To resolve this issue, we first sought to determine if any of the classical PKCs could be detected by a more sensitive technique, amplification from mRNA of primary mouse keratinocytes, cultured under basal calcium (25J,JM), intermediate calcium (125J,JM) or high

(1 mM) calcium conditions, by RT -PCR. Intact RNA was extracted following the manufacturer's instructions, as originally described by Chomczynski and Sacchi

(Chomczynski & Sacchi, 1987). Mouse brain was used as a positive control.

RT -PCR was performed as described in the Materials and Methods section using primers for PKCa, PKC~, PKCy and GAPDH. The genes for all three classical

PKCs were found to be transcribed in primary mouse keratinocytes (Fig. SA).

Total cellular lysates from identically treated primary mouse keratinocytes were

51 52 then analyzed by western blotting using an antibody recognizing PKC~II. This analysis revealed the expression of PKC~II in these cells (Fig. 58}. 53

A.

PKCa PKC~ PKCy GAPDH

B. Basal Int.

PKCPII

Actin

Figure 5. PKCJ3 mRNA and PKCJ311 protein are present in primary mouse keratinocytes. mRNA or protein was extracted from mouse brain and primary keratinocytes grown in the presence of basal (25JJM) or intermediate (125pM) calcium. (A) RT-PCR was performed using primers for the classical PKCs

(PKCa, PKC{3 and PKCy) as well as the housekeeping gene GAPDH. (B)

Western blots were performed using antibodies recognizing PKC/311 and actin as a loading control. 54

We have proposed that a signaling module, in which AQP3 mediates the

transport of glycerol into basal keratinocytes followed by the PLD2-catalyzed

transphosphatidylation of glycerol to PG, represents a novel lipid signaling

pathway involved in keratinocyte differentiation. With this in mind we next sought

to determine what effect, if any, the stimulation of this signaling cascade would

have on PKC~II. As discussed previously, autophosphorylation and translocation

are both considered indirect assays of PKC activation. High calcium

concentrations and phorbol esters, such as TPA, have been reported to activate

the classical PKCs (WB Bollag & Bollag, 2001; X Zheng et al., 2003). Upon

activation the cPKCs rapidly and transiently translocate to the plasma membrane

by a mechanism that is thought to involve phospholipase C (PLC)-derived

diacylglycerol (DAG) accumulation. The catalytic activity of PKC is thought to

play a role in the subsequent release of PKCa and PKC~II (but not PKC~I) from

the plasma membrane in cells like keratinocytes that exhibit a biphasic DAG

response (X Feng & Hannun, 1998; X Feng et al., 2000). PKCa and PKC~II

·have both been observed to then accumulate in the perinuclear area. This

accumulation appears to be independent of PLC and is, instead, the result of

sustained PLD-generated DAG (T Hu & Exton, 2004). These observations

indicate that translocation may be an integral step in the coordinated action of the

cPKCs. We utilized immunocytochemical techniques to visualize the cellular

localization of total and autophosphorylated PKC~II in the presence of a known

PKC activator, 12-0-tetradecanoylphorbol-13-acetate (TPA), a stimulator of 55 differentiation [elevated extracellular calcium ([Ca2+]e)] and components of the

AQP3/PLD2/PG signaling cascade (glycerol and PG).

We first established a positive control by verifying the reported ability of the phorbol ester TPA to activate and induce the translocation of PKC~II. Primary mouse keratinocytes were plated onto collagen-coated slides and cultured as described in the Materials and Methods section. Under basal conditions, autophosphorylated PKC~II (pPKC~II) was found diffusely throughout the entire cell with slightly increased staining around the perinuclear area (Fig. 6A, Basal panel). Treatment with the phorbol ester TPA stimulated a rapid and transient increase in pPKC~II staining at the perinuclear area (1 0 min), in the nucleus (1 hr) and a partial return to the perinuclear area (24hr) confirming the role of TPA as an activator of PKG~II (Fig.6A). Next we looked at both total- and phospho­

PKC~II in mouse keratinocytes stimulated with elevated extracellular calcium

(125~M) for one hour (Fig.68). In the unstimulated state, total (and phospho­

PKC~II) was located diffusely throughout the cytosol with a slight concentration around the perinuclear area (Fig. 68, Basal panel). In the presence of elevated extracellular calcium, PKC~II translocated primarily to the perinuclear area, and a substantial portion of the protein was in the autophosphorylated state (Fig. 68,

2 elevated Ca + panel). We then sought to determine the effect of a one hour treatment with PG, the proposed product of the AQP3/PLD2/PG signaling module, and glycerol, its proposed source (Fig.6C). Mouse keratinocytes, cultured as previously described, were exposed to either 0.2% glycerol or

1OO~g/ml egg-derived PG in the form of PG liposomes. Increased staining for 56 autophosphorylated PKC~II was observed at the perinuclear area upon addition of PG as well as with glycerol, although to a lesser extent. ·

These results confirm that PKC~II is present in mouse keratinocytes and is likely activated (i.e. autophosphorylates and trans locates) in response to stimulation by

TPA, a known activator of classical PKCs. These results further suggest that

PKC~II is activated in response to stimulation of differentiation by extracellular calcium as well as by direct stimulation of the AQP3/PLD2/PG signaling module by glycerol and phosphatidylglycerol. However, the site of translocation induced by TPA versus more physiological stimulators varies, with TPA inducing a nuclear localization of PKC~II, as compared to the perinuclear changes associated with the other agents. Figure 6. PKCPII is autophosphorylated and redistributed upon activation . of the AQP3/PLD2/PG signaling module. Mouse keratinocytes were cultured on collagen-coated slides in the presence of serum-free medium containing basal

(25J1M) calcium levels, elevated extracellular calcium (125J1M), phosphatidylglycerol (PG, 100J1g/mL), glycerol (0.2%) or the phorbol ester TPA

(100nM). The keratinocytes were then fixed and probed for total- or phospho­

PKC{31/. (A) Keratinocytes were treated with 12-0-tetradecanoylphorbol-13- acetate (TPA) for 10 min, 1 hour or 1 day and probed with phospho-PKC{31/

(Top). The image is merged with DAPI nuclear staining (Bottom). (B)

Keratinocytes were cultured in the presence of TPA, basal calcium or elevated calcium for 1 hour and probed for total PKC{31/ (Top) or phospho-PKC{31/

(Bottom). (C) Cells were subjected to direct addition of glycerol or PG for 1 hr and probed for phospho-PKC{311 (Top) or merged with DAPI nuclear staining

(Bottom). Shown are micrographs taken from a single experiment typical of multiple experiments conducted in separate keratinocyte cultures. 57

A. Basal TPA TPA TPA

I 0 .c a.= oullla1. .c~ c.. c.

B.

TPA Basal Elevated Ca 2+

I .c_0 o.­ oullla1. .s:.~ c.. c. c. Basal Glycerol PG

I 0 .s:. a.= oullla1. .c~ c.. c. 58

Over-expression of PKC/31/ in primary mouse keratinocytes

As previously discussed, calcium is a key regulator of keratinocyte maturation and is essential for normal epidermal differentiation. The observed ability of calcium to stimulate activation and translocation of PKC~II led us to suspect that

PKC~II may play a direct role in calcium-induced stimulation of differentiation. To explore this possibility we experimentally altered the expression of PKC~II and recorded the effect of this manipulation on keratinocyte proliferation and differentiation upon treatment with or without elevated extracellular calcium.

Primary mouse keratinocytes were transfected with either the empty vector or

PKC~II plasmid vector, as described in the Materials and Methods, and then cultured in the presence or absence of elevated extracellular calcium. Total

(Fig.7B) and autophosphorylated (Fig.7C) PKC~IIIevels were increased in mouse keratinocytes transfected with the PKC~II plasma vector (Fig.7). 59

Over-expression of PKCfJ/1 had no effect on calcium-induced inhibition of proliferation

Proliferation was assessed by measuring the incorporation of [3H]-thymidine into the DNA of dividing keratinocytes. In cells expressing basal, physiological levels of PKC~II, stimulation with elevated extracellular calcium resulted in a calcium­ induced inhibition of proliferation, as described elsewhere (C Tu, W Chang &

Bikle, 2001 ). Consistent with this, calcium-induced inhibition of proliferation was observed in keratinocytes transfected with an empty vector (Fig.S). Although there was a trend towards calcium-induced inhibition of proliferation in keratinocytes over-expressing PKC~II, this inhibition was not statistically significant (Fig. 8). Of interest, there was no statistical difference between the effect of transfection with empty vector or with PKC~II on calcium-induced inhibition of proliferation. Also note that these assays were analyzed only for cells that were shown to be clearly over-expressing PKC~II by western blot analysis (Fig. 7 A). 60

PKCPII over-expression induced an up-regulation of keratin 10 upon calcium-induced stimulation of differentiation

Protein levels of keratin 10, a marker of keratinocyte differentiation, were also examined in cells over-expressing PKC~II. There was no change in the keratin

10 levels of keratinocytes over-expressing PKC~II cultured under basal conditions. PKC~II over-expression in combination with elevated extracellular calcium, however, resulted in a substantial up-regulation in keratin 10 levels with p<0.01 versus all other conditions (Fig.9). These results suggest that PKC~II alone is not sufficient to induce an increase in keratin 10 expression, but instead works in concert with calcium to promote early differentiation, but not proliferation or growth arrest, in primary mouse keratinocytes. Figure 7. Over-expression of PKC/311 and its autophosphorylation in mouse keratinocytes. Mouse keratinocytes were transfected with PKC{311 ({311) or empty vector (EV) and then cultured in the presence of basal (25J1M) calcium or elevated (125J1M) extracellular calcium. (A) Totallysates were resolved on 8%

SDS PAGE gel, transferred to PVDF membranes and probed with antibQdies specific for total PKC{311, autophosphorylated PKC{311 (pPKC{311) and actin. Total

PKC{311 and pPKCf311 levels were quantified using LI-COR detection methods and normalized to actin. Data bars represent the mean ± SEM of (B) total PKC{311

[n=4; *p<0.05 {311 (elevated calcium) vs EV (basal) or EV(elevated)] or (C) autophosphorylated PKC{311 [n=7; #p<0.01 {311 (basal) vs. EV (basal); *p<0.01 {311

(elevated) vs EV (elevated); Ap<0.001 {311 (elevated) vs EV (basal); +p<0.05 EV

(elevated) vs {311 (basal)]. Values were calculated and presented relative to {311

(rei. to {311). 61

A Etevo1ted Ca+ EV

pPKCISfl

PKC~Sfl "- ~..;;

~-- ~- ~ --.:.... Actin

B c

,.,.. c.· U.vatedca· . 62

- ...... ' . . . .. ·· · .•Bas.~' ·ca· ·· · · Elevated c:a•

. ·· .. · .. · · .. · .. ·... · ... ·.· .....·.···· ... : .· ..... • ·:·.· ... : .... : . ·.· .. ·· :·· ..... ···.:·.· :.• · ... :·.·.·· ..... · ··. •:· .: · ....· .· ..... · . •Figure B.~: f!'KC/311· QV~r~expresslon had no effflct orr c.alciu.rri-induced .: .·

... : ·.:. ·. ' :·_: ·: ..: . ·. ·: . _: --. . . ·: _: ·: .: .. : .. -_ .. _· .. : .. _. ': . ·. ·: ...... :iflhibltioii o~ prolifera.tio.n .. •Mouse keratinocyte.s were.ttansfect~d with PKG{3/l .. • · .

·· {{311) oremptyv~cfcH· (EVj and then cultured in.the·presence·ofbasal (25J.1MJ or:

.. ·.. . . - ... ,'...... - ...... '.-...... '.'...... ·elevated (125J.1M) ca{ciurrt . Proliferatiqn assays were perforrnedby measuring . : ..

· ·lhe incorporation ofr~diolabeled thymidine into·DNA .[n=s;· *p

.·· ...... ' .. ··· ... · .... '·, ,·. · ... ·· ...· ·. .· · ...... ·: ..· . · ... · · .·vs~· {3/l(basal} or EV(b;:Jsal)]. :Values were. calculated at)d presented relative to l31I · . . .

' . . . (rei. to f311). ··· 63

·· ·a,asai ca+ · · Elevated ca+ EV. ·(ill . . EV . : (311 _· . ·.K~lo· -b:~:p~~. :~f[:~t::jl£)::. · A~tin _·. -~J: •-~~-~l-·

. ev ~~~.. . ~v-• pn _· . · . · ·aasai' ca•.. : ·Elevated. cat: ·· · ·

. . - ...... Figure 9.·PKC.J31i over~~xpre$Sion increases up~regtll_ation.•~t _calcium-._: ..

...... ·· ·. ·incl~ced level's ·of kera~iri :10. Mouse ~eratinocytes -w~re tran$fected with ·

...... ··. ·_·: ..... ··:: .. · ·.· ..· ... ··. · ... ·· .. ·:·.:_:_· ··.: :.·.: ·.... : .. _... ·~ PKC~II (~ll}'or enipttve_ctor {EV) ·and then cultur~d in the· pre~ence or absence ··

...... ·- '...... '. . . . . ' ' ...... ·. •of elevated extracell.ular.calcium (125J,JM). Totallysates were resolved ori·a% .

• SD~ PAGE gels, :tra11sferred •to PVDF membranes arid pro~~-d with an aQtibody.

. . - . . _specific ·far·keratin~to (K10)~. KtO-Ievels·were qu~ntifiedand•norl'!lalized·to actin· .. ·

...... _·· ... · ...... · ..... ·_ .. - ..... , .... _·.. . _-.- ·- ... - ...... · .. - .. . Data•barsrepresentthe :rnean·±-SEM [n=4;_.*p

. . . '. .. - ...... ' .... . ~-ll(basal) or' EV(.elevated.)]. •·. · ...... - ·- 64

Treatment of keratinocytes with a PKC/3 catalytic activity inhibitor had no effect on calcium-induced inhibition of proliferation

We next examined the effect of inhibition of PKC~II on calcium-induced inhibition of keratinocyte proliferation. Activation of PKCs relies heavily upon PDK-1 phosphorylation and autophosphorylation facilitated by the activation loop of the

ATP-binding site of the PKC kinase domain, as discussed in greater detail previously. PKC catalytic activity can thus be altered by point mutations within the ATP-binding site or deletions within the kinase domain. The kinase domain, however, is highly conserved among the PKC isoforms rendering this method of inhibition or down-regulation non-specific. In 2003 Eli Lilly announced the development of ruboxistaurin (L Y333531 ), an ATP-competitive PKC inhibitor reported to inhibit PKC~I (1Cso=4.7 nM) and PKC~II (1Cs0=5.9 nM) with a potency

76- and 61-fold greater, respectively in comparison to PKCa (H Ishii et al., 1996;

Jirousek et al., 1996; JL Burkey et al., 2002; Tang et al., 2008; Wheeler, 2003).

The ATP-dependent competitive inhibition of ruboxistaurin was reported to be selective for PKC in comparison to other protein kinases, such as protein kinase

A (PKA), calcium-calmodulin dependent protein kinase, casein kinase and src tyrosine kinase (Jirousek et al., 1996). Initially described as a potential treatment for diabetic retinopathy (Frank, 2002; H Ishii et al., 1996), ruboxistaurin was cited as one of the only drugs of its class that targets PKC~I and PKC~II in a highly selective manner, with minimal interaction with other PKC isozymes, including

PKCa and PKC~ {Tang et al., 2008). 65

Here ruboxistaurin (referred to hereafter as LY333531, or simply LY) was used to inhibit PKC~ catalytic activity in mouse keratinocytes. Primary mouse keratinocytes were grown in keratinocyte-serum free (K-SFM) medium containing either basal calcium (50J,JM) 1 or elevated extracellular calcium (125J,JM) in combination with no inhibitor (control) or 300nM LY or 1uM LY. After a 24-hour incubation period the cells were exposed to [3H]-thymidine for 1 hour and proliferation was determined by measuring radiolabeled thymidine incorporation into DNA. As expected, exposure to elevated extracellular calcium resulted in a substantial and significant decrease in proliferation (Fig. 10). Inhibition of the catalytic activity of PKC~, however, had no effect on calcium-induced inhibition of proliferation in the presence of either 300nM or 1 J,JM LY333531 (Fig. 10).

1 Due to changes in cell viability the laboratory protocol for preparing keratinocyte medium was altered. All keratinocyte experiments from this point forward were cultured in commercially available keratinocyte-serum free medium (Gibco) containing SO~M calcium. 66

...-.... i!!: -!·.· . .:en o· · ·.. ·.·.··!·.: ...... ~ .:S:: - . ~ •.. 'U) >· ·*. .· ... ~- :-..·-·· . .:...... z--· . .. _.. .•...... -

. . . .. · .. . : . ,. ' .... · .. : . . :~.. · . .... ' : .~ . c. . . (i ' (, ...... V· ·-~"··: -~" · L~ · ·· ·x ... · X· . . ,.'to ~ ,~ cP~ ~· ,. cP~ . . . 17 . .. . . ~(S . ll> .

. -- ... · ... : ...... · :. . . . - .: ... --. . . : ...... Figun~· 1·0. 'l~reatmenfof. :keratinocYte.s with an· inhibit()r·.of: P~CJ3 catalyti(;: ..

. . :·.·.' . _·. ' . _·_: .. : ·: __ ... _·_. ·_ _··_: ... ·:. _·:: _ .. _:: · __ . _·· . . -_. : . _-.' ·:_ .. . activity.· had n~ ·effect_:on calcl_um~iriduced i'~hibition·ol p.roliferation~ .

. . . . -_ ._·. : : _·. · __ -_.· .. -_· .. - .. _-_·: __ ._· ·_ ·__ .: _-_- .. · ...... -__ ,'' .. _._· ._- : Primary. mouse· keratinocYtes·:were treatedwith.SOf.JMcalcium· (Cont.), SOJ.JM. _·:

'.: ·_ .. : _--_-- _ ... · ___ ·: .: : ----:·--_: -·: _· .. ·_ . :. ·_ .... : .. _·_ .·-.. . ._- _· ·_·: . : _-_ ... ·:. _·: ·_ ·calcium- contaiiling. :300nM LY333531 (300nM LY) or 50f.JM cpnt~i-~ing .1 pM .. ·: ·.

·- ...... ·. . '._·: ..'...... _·: .. · .... _- '. ·: _· .... : .·· ... ' · ... '' .... ·_ · L Y333531 (1pM.LY) .. Mbl.JS~ .kere~tinocytes from the samecultur~ were also·

- . ' - ...... ' ...... ·. grown i·n· the presence :of: 12"5~-JM calcium (Cant+ C)_,: 1251JM calcium -coritairiing ·

...... :-. - . . : -- .~ . . . - :. . . : : . : . : . .. : ~ : . . . . . -. . . . . : .. .. : : . :. . -. '. ' . . : . : . . . ••· . 300nM LY333531 (300nM +·C), or t25~:M :calcium: containing 1pM LY333531 . . ·...... : . . . . . - ...... ' ' . ~ . ' ' . ' ' . . . : . . . ' . . .

...... -- -- .. · .. ' ... ·...... : '.. .. · .... '. . . . ' ''. "·. . :'' ... · (tJ.J.M +·c); Proliferation assays were: performecr by measuring. the incorporation : · ·

. ._-- .. ·. _ .. : '' ·. . ' . _· _· .. . . . ·... ·. .. . · ... :. ' .· .. : ...... ' ...·. ·; .· ·.· . . . .. · ·.· . . . + . of rad1olabeled thym1d1ne 1nto DNA (n=4; p<0.05-'(s·irCont or 30pnM)~: · 67

Treatment of keratinocytes with a PKCfJII translocation inhibitor had no

effect on calcium-induced inhibition of proliferation

Translocation of PKC isoforms to unique cell compartments upon activation has

been demonstrated here and in multiple other studies. The unique subcellular

localization of individual PKC isoforms likely plays a role in the varied cellular

responses to PKC activation. RACKs (receptors for activated C-kinase)

represent a family of anchoring proteins involved in the specificity of PKC

subcellular localization (D Schechtman & Mochly-Rosen, 2001 ). Individual

RACKs have been determined to bind active PKCs in an isoform-specific manner

and have been proposed to function, in part, by bringing the isoform into contact with a unique subset of substrates as well as by modulating the translocation of the signaling complex to a second location (D Ron et al., 1999b; D Schechtman

& Mochly-Rosen, 2001; Mochly-Rosen, 1995). Within the family of RACKs,

RACK1 has been shown to interact with PKC~ (D Mochly-Rosen et al., 1995; D

Ron et al., 1994) and was subsequently revealed to interact selectively with

PKC~II (D Ron et al., 1999a; EG Stebbins & Mochly-Rosen, 2001 ). Evidence

has been provided in other cell types revealing a co-localization of RACK1 and

PKC~II in the cell periphery. This RACK1-PKC~II complex translocates to the

perinuclear area upon a 10-minute stimulation with the phorbol ester TPA (D Ron et al., 1999a). Targeting of RACKs by peptide translocation inhibitors has been den;1onstrated to inhibit the translocation and function of PKC isoforms (Chen &

Mochly-Rosen, 2001; D Ron, J Luo & Mochly-Rosen, 1995; GC Mayne & Murray,

1998; KO Aley et al., 2000). Peptides with amino acid sequences that mimic the 68

C2 or V5 regions of PKC~II, the regions that contain the binding sites for PKC~II, inhibit PKC~II function by inhibiting the interaction between PKC~II and RACK1

(D Ron et al., 1999a; EG Stebbins & Mochly-Rosen, 2001 ).

Here we investigated the effect of small V5 region peptide inhibitors (the C2 regions of both PKC~I and PKC~II are identical) on keratinocyte proliferation.

Primary mouse keratinocytes were grown in commercially available keratinocyte­ serum free medium (K-SFM) containing either basal calcium (50J.JM) or elevated extracellular calcium (125J.JM) in combination with either TAT-conjugated ~I V5 peptide (PKC~I inhibitor), ~II V5 peptide (PKC~II inhibitor) or the TAT peptide control. After a 24 hour incubation period, the cells were exposed to [3H]­ thymidine for 1 hour and proliferation was again determined by measuring radiolabeled thymidine incorporation into DNA. Consistent with our previous observation, exposure to elevated extracellular calcium resulted in the expected decrease in proliferation (Fig. 11 ); however, treatment of keratinocytes with a

PKC~II translocation inhibitor showed no effect on this calcium-induced inhibition of proliferation (Fig. 11 ). These results were consistent with our previous observation of no effect of PKC~II over-expression on the calcium-induced inhibition of proliferation (Fig.S). 69

... #· ..

- .: : "•

' ..

10·. . ' •'i .·.' •• ,f ,.

0 ,;.I.·• """""'----l.__.l,;,;,··---- ...... ,...;;.;..;;;...: ...... ! ...... · '..-.,;;,.;;,;""'--.a;.;;.;.;...... -&._. """"-- .....·. ..,_; . . :TAT .. lSI' ...... ·- ...... - .: .Ba$al·ca·· .· E·levat.od·ca•:. ·

...... - ...... ·. '.. . .. · ... -. . . .·. . . ·· .. · Fig.ure ·11 ~ A_:PKCJ311. translocation:. inhibi.tor had_ no effect on calcium·~ ·

. . . . . indu.ced inhibltio.n of keratinocyte.pr()liferation ... Primary niouse·_ ..

. . . ' . . . . - ...... ' ' . ' ...... - ' . . . ' ' ' ...... k~rathiocytes.were:incubated'.in basal (501JM).or-ele\tated(t25pM) calCium· . : : .. · ... ·· :· · ... ::: .. ·· ...... ·.. ·· ..... · .. : : ·:_. ·: . : . : .··.. . .. · ·.· .. : ·:_:·: ·. : .. · .. : ·::' .... :containing: ~ither the .. protein: ·trarlsduction m()tif 'of TAT alone or V5 region·peptid~ .

.. . ' ...... - ...... · ...... ··.:translocation inhibitors:for·PKC-~.'(~1) or PKC~·I( (~ll)conjuga.tedto this TAT motif·: .·

· ::_ · . for2·4: hours_;· Proli~eratian· assays·were ·then perfor~ed:by meas,uring ·the··

·· · incorporation of radiol.abefed thymidine into DNA .(n=e~· #p<.0;05·_·vs:TA~(with ·· ·

· · basal calcium);_

I.· 70

Inhibitors of PKC/3 catalytic activity or PKC/311 translocation had no effect on either autophosphorylation of PKC/311 or keratin 10 levels

Next, we turned our attention to investigating the effect of these inhibitors on early keratinocyte differentiation, as measured by keratin-10 levels. Totallysates from primary mouse keratinocytes grown in culture containing basal calcium

(50JJM) or elevated extracellular calcium (125JJM) in combination with LY333531, a PKC~ catalytic activity inhibitor (Fig. 12) or VS derived PKC~ peptide translocation inhibitors (Fig. 13) were subjected to western blot analysis. The relative amounts of autophosphorylated PKC~II remained unchanged in the presence of either LY333531 (Fig. 128) or VS-derived translocation inhibitors

(Fig. 138). Although we would not necessarily expect inhibition of translocation to exert an effect on catalytic activity, we were surprised to observe that the purportedly selective PKC~II catalytic activity inhibitor, LY333531, did not inhibit

PKC~II autophosphorylation in our cells (Fig.12). Likewise, on average, there was no observable effect of either PKC~ catalytic inhibition (Fig. 12C) or translocation inhibition (Fig. 13C) on keratin-1 0 levels. These results were unexpected. At this point we began to suspect that although PKC~II was a strong candidate based on the literature as a PG-responsive effector mediating calcium-induced keratinocyte maturation and can trigger an increase in differentiation (keratin 10 levels) with overexpression, the enzyme may not be involved in the differentiative response to calcium under physiological conditions. 71

A Basal ElevatedCa

....~ ~~ .to ~ 'Oj ..~ pPKCIJII

K-10

Actin

B. c.

2.5

Elevated Ca

Figure 12. Treatment of keratinocytes with the PKCfJ-selective inhibitor had no effect on the autophosphorylation of PKCfJ/1 or keratin-10 levels.

Primary mouse keratinocytes were incubated in the presence of basal (50J,JM) or elevated (125pM) calcium alone or containing either 300nM or 1pM L Y333531 for

24hours. (A) Totallysates were resolved on 8% SDS PAGE gel, transferred to

PVDF membranes and probed with antibodies recognizing phospho-PKC/311, keratin-10 and actin. Immunoreactivity for (B) pPKCf3/l (n=B) and (C) keratin10

(n=B) were quantified and normalized to actin and expressed relative to the no inhibitor control. 72

A

8 c

Basal ElevatedCa Basal ElevatedCa

Figure 13. Treatment of keratinocytes with a PKC/311 translocation inhibitor had no effect on pPKC/311 autophosphorylation or keratin 10 levels. Primary mouse keratinocytes were incubated in the presence of basal (50JJM) or elevated

(125JJM) calcium containing either the protein transduction motif of TAT alone

(TAT) or V5 region peptide translocation inhibitors for PKC{31 ({31) or PKC{31/ ({311) conjugated to TAT for 24hours. {A) Totallysates were resolved on 8% SDS

PAGE gels, transferred to PVDF membranes and probed with antibodies specific for PKC{311 (pPKC{31/), keratin 10 or actin. Protein expression for (B) pPKC{31/

(n=B) and (C) keratin10 (n=B) were quantified and normalized to actin and expressed relative to TAT with basal calcium. 73

PG stimulated an increase in PKC/3/I autophosphorylation

At this point we began to assume that PKC~II was not the major PG-responsive agent activated by the keratinocyte AQP3/PLD2/PG signaling module in response to elevated extracellular calcium concentration. We did, however, observe a statistically significant increase in autophosphorylated PKC~II when cells were treated with phosphatidylglycerol (Fig. 14). However, this increase was not affected by inhibition of PKC~ catalytic activity with 300nM; however, a high concentration of LY (11-JM) appeared to inhibit PG-stimulated PKC~II activation, although this inhibition was not statistically significant (Fig. 14A).

Likewise in the presence of peptide translocation inhibitors we observed a trend towards PG-stimulated activation of PKC~II (Fig.14B). These results led us to think that PKC~II can be activated by PG in conjunction with signals that· stimulate keratinocyte differentiation, as observed by PKC~II's translocation in the presence of extracellular calcium (Fig. 6). 74 .

A . ·B.

Basal . ·· PG.. PG .

.. ' 1= :~ . ' • .:.-

. pPKCPII .. . pPK(:PII: ;.:~:-;c_.'~--~~W_':t····!e.•J. · .. Actin -...;..:~-----~~

PG Basal· PG ...

.. . . . ' . . . . ' .. :·. •. ·Figure .1:4~ .P~-stimuiat~d h1creases in P.KCPU a~toph~spho_rylati.on•were. ·· · .· .

. notatt~nuated:by•Ly33353t or~ PK:cJ3fltra~siQcati.on• in_hibitor.· Prirhary .

. . ' . . . ' . . . .. ' . .. ''' .. . mouse keratinqc}'tes: _were incubat~d ·in the p·resence _ofbasa_I.(S~pM) ·calcium or ·

.basal caiCiu.m• ~ith 1•6QJ,J_g/mL PG (in lipo~orne forrn) .• !he-ceils were' .incubated ....•. ·

.. . .· . '' .. · ...... · . . .· '...... with the (A) LY333531 PKC~ activity inhibitor(n=6;. *p

...... · ·vs ·r~gion·translocation :inhibitors· co'nJugat~d· tO: the ·TATprote-rn tran·sductio'n · ... · · · . ' ' ...... '

...... ~ ...... : . . . ' ...... : . . .. : . .. . .: . . . . : . ... '- . . : ...... ' .. . . --- . . . . . -: :peptide ·seqLience.•(n=.S).for 24hoUrs. Total' fysates were resqlved on _8°/o ~OS

. . . . . '. . . . . ' . . .· .·:PAGE ge.is; transferre.cf tO: PVDF .membranes aJKf proped :with anti.bodies· : ..

...... " recognizihg_:autophosphorylated PKCPH _(p.PKC~II}and· actin .• Leve.ls· of pPKC~II' .

.·: are displayed: normalized to actin. and ·expressed .·relative to no inhibitor (A) or .·. ·: .· .·: . : _· .. ·: -·: ... ·: ·. :. ·• .· TATcontrols {B).· 75

Over-expression of PKC/311 affected the pattern of keratin 10 distribution and resulted in an altered morphology in cells incubated in the presence of phosphatidylglycerol

Our previous findings made us curious about the' morphological effect of PG on keratinocytes over-expressing PKCf311. Mouse keratinocytes were again transfected with either PKCf311 or empty vector and were then cultured on collagen-coated slides in serum-free keratinocyte medium containing basal

(50J.JM) calcium, elevated extracellular calcium (125J.JM) or 1OOJ.Jg/ml egg PG (in the form of liposomes). The cells were then fixed, permeabilized and stained with an antibody specific for keratin-10 (Fig.15). Over-expression of PKCf311 resulted in an altered pattern of keratin-1 0 distribution. Interestingly, treatment with PG led to morphological changes consistent with entrance of the keratinocytes into a late stage of differentiation in cells transfected with PKCf311, but not in cells transfected with empty vector. This morphological change was not observed in keratinocytes that were expressing basal levels of PKCf311 and incubated with PG or in the absence of PG (Fig.6C).

Taken together these data provide evidence suggesting that stimulation of our proposed signaling cascade can activate PKCf311, but that the isoform alone is not sufficient to stimulate keratinocyte differentiation. Instead PKCf311 may function as only a minor PG effector enzyme to promote keratinocyte differentiation. This hypothesis is consistent with the observed ability of PKCf311 over-expression to increase keratin 10 levels in the presence of elevated calcium 76

Basal Elevated Ca+ PG

EV

Probed w/ K10

Figure 15. Over-expression of PKC/31/ affects the subcellular distribution of keratin 10 and resulted in an altered morphology of cells incubated in the presence of phosphatidylglycerol (PG). Mouse keratinocytes were transfected with PKCf311 ({311) or empty vector (EV) and then were cultured in keratinocyte-serum free medium containing either 50pM calcium (Basal), elevated extracellular calcium (125pM) or with phosphatidylglycerol (PG). The cells were stained with an antibody recognizing keratin 10. Shown are micrographs taken from a single experiment typical of multiple experiments conducted in separate keratinocyte cultures. CHAPTER FOUR: DISCUSSION

Disruption in the normal form and function of skin can result in a significant amount of human suffering. Several human skin diseases, such as psoriasis, a hyperproliferative disorder of the epidermis, and the non-melanoma skin cancers

(basal and squamous cell carcinoma) are the result of a break-down in the carefully controlled program regulating the proliferation and differentiation of keratinocytes, the primary cell type found in the epidermis of the skin. The

National Psoriasis Foundation and the American Academy of Dermatology report that approximately 7.5 million Americans and as much as 3 percent of the total world population suffers from the devastating· effects of psoriasis (Dermatology,

2011; Foundation, 2011 ). Although not a fatal condition, the impact of psoriasis on physical and emotional functioning has been reported to be comparable with that of other serious medical conditions, including heart and lung disease, depression and cancer (LH de Arruda & Moraes, 2001; Rapp et al., 1999). The

American Academy of Dermatologists also lists basal and squamous cell carcinomas as the two most common skin cancers in the world, with more than two million new diagnoses each year (Dermatology, 2011 ). This research seeks to contribute to the body of knowledge regarding the excessive growth and aberrant differentiation of epidermal keratinocytes that characterize these diseases.

77 78

AQP3/PLD2/PG Signaling Module

Although the precise mechanisms regulating the entrance and progression of keratinocytes through the multilayered structure of basal, spinous, granular and cornified layers of the epidermis remain unknown, our laboratory has provided evidence for a potential signaling module involving phospholipase 02 (PLD2), aquaporin 3 (AQP3) and phosphatidylglycerol (PG). Phospholipase D belongs to a superfamily of phospholipases that catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline in the presence of water. We have previously provided evidence indicating a functional interaction between PLD 2 and the glycerol channel AQP3 (X Zheng & Bollag,

2003). Our laboratory has further shown that PLD2 can utilize glycerol to generate PG and that elevated calcium increases PG and this increase is inhibited by ethanol (indicating mediation by PLD) (X Zheng et al., 2003). The maximal stimulation of PLD-mediated PG formation was observed at a calcium concentration optimal for stimulation of markers of early differentiation (K1, K1 0).

These findings, combined with the observation that the C-terminal PKC~II V5 region binds PG and contains the molecular determinant necessary for translocation and activation of the enzyme (Gokmen-Polar & AP Fields, 1998;

NR Murray & Fields, 1998) led us to suspect that PKC~II and PG may interact in a functional manner to modify keratinocyte maturation.

The research presented here was designed to investigate PKC~II as a possible

PG-responsive signaling enzyme involved in down-stream signal transduction in I 79

this pathway. PKC~II is a classical calcium-dependent PKC isozyme requiring

calcium, diacylglycerol (DAG) and phosphatidylserine (PS) for activity. PKC~II

differs from its altern?ttiye splice variant PKC~I by 13 amino acids in the C-

terminal VS region. This region of PKC~II is the location of the PG-binding ,/' domain apd contains the molecular determinant necessary for nuclear

translocation and enzyme activation (Gokmen-Polar & AP Fields, 1998; NR

Murray & Fields, 1998). In HL60 cells, PG, which resides in the nuclear

membrane, selectively stimulates PKC~II activity 3-6 fold above the level

achieved in the presence of optimal concentrations of calcium, DAG and PS

(Murray et al., 1994). In fibroblasts, entry into mitosis is dependent upon

activation of PKC~II by PG (DB Parekh et al., 2000). Thus, PKC~II is a likely

candidate as a PG-responsive signaling enzyme involved in promoting

keratinocyte differentiation. · .. 80·.

...... Here, we•preseht ~vidence that:-_-_

' ' .. -' ...... ' . . .. :. ' ...... •· PKCf311 is present in mouse keratinocytes ·C)nd redistributes with .· __ stimulation of the AQP3(PLD2/PG .signaling module . . . - . . . . ' .

...... -_ . . . . . : .. . -_. . .. : ._·.' :. - . ·_. • ··- PKCf311 over~expre·ssion i11duces an .up~re•gulat_ion -bf keratin 10 -__ -_upon :calciuril~induced _strmulaHon of: differentiation, but has_ no:­ .·effect on :calcium-induced :inhibition of proliferation·:. ·

_· :· - .. ··:·· ...... :. : .. ·.. . .. ·. : .. -. _··_. : .... ·_· ...... • - _Treatment ·afkeratinocytes with a PKCf3 ·catalytic activity inhibitor or .· ··a F>KCf3JI tra:nsl()catiqn .inhi_bitor _h~d no effect dJ1 either calciu,m~-: ___ _ .induce.d inhibition of proliferation_ or stimulation of. keratin fO :levels:_-.

' : . . . ~ . : - ' . . ' ' . ' . . . . . : ...... : ~ ...... : . . ' : ' . . - . . ~ . . . . . : . . . . : .. ' : . ; ...... •• ·.. Over~:expression orPJ

. ' . . . . . -· . . . .

' ...... · ...... ' . '...... 81

PKCPII is Present in Mouse Keratinocytes and Redistributes with

Stimulation of_ the AQP3/PLD2/PG Signaling Module

Multiple lines of evidence have been provided suggesting the ability of PG to activate PKCf311. There has, however, been some controversy in the literature regarding whether or not PKCf311 is even present in keratinocytes. An initial report was unable to detect PKCf3 in basal and differentiating mouse keratinocytes by northern analysis (Dlugosz et al., 1992). In contrast, two pther studies reported the presence of PKCf3 protein in normal and psoriatic adult human skin (GJ Fisher et al., 1993), as well as in mouse keratinocyte cultures

(SM Fischer et al., 1993). We used a sensitive method of RNA amplification and subsequent western blot analysis of protein to show that PKCf311 is both transcribed and translated in primary mouse keratinocytes (Fig. 5).

Calcium functions as a precise regulator of keratinocyte maturation and is essential for normal differentiation (DO Bikle & Pillai, 1993; GK Menon et al.,

1992; SH Yuspa et al., 1989). An increase in extracellular calcium concentration initiates the process of differentiation. Thus, keratinocytes grown in a low­ calcium medium proliferate and maintain a basal-like state in vitro, but will transition into a more differentiated state when exposed to raised extracellular calcium levels (DC Ng et al., 1995; DO Bikle et al., 2004b). Consistent with this, a calcium gradient has been observed in the epidermis in situ, with the lowest concentration found in the basal layer where keratinocytes are actively proliferating and gradually increasing outward towards the more differentiated granular layer (GK Menon et al., 1985). We have proposed that functional 82 interaction between PLD2 and AQP3 regulates the synthesis of PG, which may serve as a novel signaling lipid important in mediating keratinocyte differentiation in response to elevated extracellular calcium levels. With this in mind we next sought to determine the effect of stimulation by elevated extracellular calcium, as well components of our proposed signaling module, on PKC~II activation, as determined by PKC~II autophosphorylation and translocation (indirect measurments of PKC activation).

We utilized immunocytochemical techniques to visualize the cellular localization of total and autophosphorylated PKC~II in the presence of a known PKC activator, 12-0-tetradecanoylphorbol-13-acetate (TPA), a stimulator of differentiation (elevated extracellular calcium ([Ca+]e) and components of the

AQP3/PLD2/PG signaling modul.e (glycerol and PG). High calcium concentrations and phorbol esters, such as TPA, have been reported to activate the classical PKCs (WB Bollag & Bollag, 2001; X Zheng et al., 2003). Thus, we first verified the reported ability of TPA to activate and induce translocation of

PKC~II. Our findings confirmed the ability of TPA to stimulate an increase in autophosphorylation of PKC~II (Fig.6A). Incidentally, we also observed an apparent decrease in staining with prolonged (24 hours) exposure to TPA, consistent with reports of TPA-induced down-regulation of cPKCs (Bouche et al.,

1995; M Savart et al., 1992). We then examined the effect of an elevated extracellular calcium level (125tJM), glycerol or PG on autophosphorylated

PKC~II levels. In all three cases there was an increase in activation

(autophosphorylation) and translocation to the perinuclear area (Fig.6B,C). 83

These results confirm that PKC~II is present in mouse keratinocytes (Fig.SA,B), is activated in response to TPA (Fig.6) and responds to PG, the product of the proposed AQP3/PLD2/PG signaling module, as well as glycerol, its proposed source (Fig.6C). The pattern of autophosphorylated PKC~II staining appears to be the most distinct in cells treated with elevated calcium and PG, suggesting that stimulation of differentiation by calcium and direct addition of PG may induce

PKC~II activity above that achieved under basal conditions. 84

PKCPII Over-expression Induces an Up-regulation of Keratin-1 0 Upon Calcium-Induced Stimulation of Differentiation, but has no Effect on Calcium-induced Inhibition of Proliferation

The observation that extracellular calcium was able to stimulate the translocation of PKC~II led us to suspect that PKC~II may play a direct role in calcium-induced stimulation of differentiation. To address this, we experimentally altered the expression of PKC~II and recorded the effect of this manipulation on keratinocyte proliferation and differentiation. We began by verifying over-expression in primary mouse keratinocytes that were transfected with PKC~II (Fig. 7). Over- expression of PKC~IIIed to a clear up-regulation of PKC~II autophosphorylation,

under basal (251JM) and elevated (125J,JM) calcium conditions (Fig.7B). With the total PKC~II antibody, significant over-expression was observed only in calcium- stimulated conditions. We suspect that the lack of statistical significance observed with PKC~II over-expression under basal conditions may be related to observations we made regarding the affinity of the total PKC~II antibody. In our hands, total PKC~II was not as reliable an antibody when used in western analysis, despite multiple experiments with several different commercially available antibodies, as the antibody that recognized autophosphorylated

PKC~II. The lack of reliability of the total PKC~II antibody resulted in fewer analyzable western blots, with the smaller number of values included precluding an ability to attain statistical significance.

Proliferation was assessed by measuring the incorporation of [3H]-thymidine into

DNA in cells showing clear over-expression of PKC~II. In cells expressing basal, 85

physiological levels of PKC~II, stimulation with elevated extracellular calcium resulted in calcium-induced inhibition of proliferation (Fig.1 0, control), as described elsewhere (C Tu et al., 2001 ). Consistent with this, calcium-induced inhibition of proliferation was observed in keratinocytes transfected with the empty plasmid (Fig.8). Curiously, over-expression of PKC~II in the presence of elevated extracellular calcium resulted in slightly less of a reduction in [3H]­ thymidine incorporation, although this difference was not statistically significant

(Fig.8). Thus, calcium induced a 45% inhibition in cells transfected with empty vector, but only a 27% inhibition in PKC~II transfected cells. Although not statistically significant, this might suggest that the over-expression of PKC~II slightly blunts the effect of calcium on growth arrest. This would be counter to our hypothesis that PKC~II functions, in part, as a PG-responsive element to promote keratinocyte differentiation, but consistent with the reported pro­ proliferative, pro-mitotic role for PKC~II in Hela cells· (Ruvolo et al., 2011;

Thompson & Fields, 1996).

Although not statistically significant, the previous finding that PKC~II over­ expression may attenuate calcium-induced growth arrest suggested the possibility that PKC~II could potentially exert a pro-proliferative, instead of a pro­ differentiative, effect. Evidence has been provided in other cells types consistent with this idea. For example, transgenic mice that express elevated PKC~II in the colonic epithelium were found to exhibit hyperproliferation in these cells and were prone to carcinogen-induced colon cancer (Murray et al., 1999; Murray et al., 86

2002). In vascular smooth muscle cells expressing PKC~II, the effect could be pro-proliferative or pro-differentitative depending upon the glucose levels (Patel et al., 2003). Hence, PKC~II signaling, under normal conditions, was found to suppress vascular smooth muscle cell proliferation by attenuating the G1/S transition; whereas, in the presence of high glucose, a low molecular weight protein was found to bind to the region encoding the C-terminal region of PKC~II mRNA resulting in a destabilization of PKC~II mRNA and a down-regulation of protein (M Yamamoto et al., 2000; Patel et al., 2003; Yamamoto et al., 1998).

Thus, the precise role that PKC~II plays in a given system may be heavily dependent upon multiple additional factors.

In an effort to determine if PKC~II plays a role in keratinocyte differentiation, we first evaluated the effect of PKC~II over-expression on the levels of keratin 10, a marker of early differentiation, in the presence and absence of elevated extracellular calcium. Although we did not detect a statistically significant change in the levels of keratin-1 0 under basal conditions, PKC~II over-expression, in combination with elevated extracellular calcium, resulted in a substantial up­ regulation of keratin 10 expression, with p<0.01 versus all other conditions

(Fig.9). These results suggest that PKC~II alone is not sufficient to induce an increase in keratin 10 expression, but instead works in concert with calcium to promote early differentiation, but not proliferation or growth arrest, in primary mouse keratinocytes. 87

Treatment of Keratinocytes with an Inhibitor of PKCP Catalytic Activity or a

PKCPII Translocation Inhibitor Had No Effect on Either Calcium-induced

Inhibition of Proliferation or Stimulation of Differentiation

Next we examined the effect of inhibition of PKC~II on calcium-induced inhibition

of proliferation, as measured by thymidine incorporation, or stimulation of

differentiation, as measured by the early differentiation marker keratin 10. For

this purpose we obtained ruboxistaurin (L Y333531 ), an ATP-competitive PKC

inhibitor reported to selectively inhibit PKC~I and PKC~II,. with minimal inhibition

of other PKC isozymes, including PKCa and PKC~ (Jirousek et al., 1996; JL

Burkey et al., 2002; Tang et al., 2008; Wheeler, 2003). We found that treatment

of mouse keratinocytes with ruboxistaurin had no effect on the calcium-induced

inhibition of proliferation (Fig.1 0). Previously, we observed that over-expression

of PKC~II also had no effect on calcium-induced inhibition of proliferation (Fig.S),

suggesting that PKCJ311 does not play a role in growth inhibition in primary mouse

keratinocytes.

We then turned our-attention to the effect of PKC~ inhibition on early keratinocyte

differentiation. Stimulation with elevated extracellular calcium did not induce an

increase in PKCJ311 autophosphorylation. Furthermore, we found that

ruboxistaurin had no effect on autophosphorylation of PKCJ311, even at the high

11JM dose (Fig.12B). Although surprising, this finding is perhaps not entirely

unexpected. There is evidence suggesting that autophosphorylation may, in

some cases, not be a sufficiently sensitive parameter to detect inhibition by an

ATP-competitive inhibitor. In the case of protein kinase 01 (PKD1 ), 88 autophosphorylation of Ser916 was determined to be less sensitive to the AlP­ competitive inhibitor Go6976 than transphosphorylation of substrates (VO Rybin,

J Guo & Steinberg, 2008). Additionally, ruboxistaurin was originally described as an orally active inhibitor reported to ameliorate some of the vascular complications observed in diabetic rats (H Ishii et al., 1996). The specificity of ruboxistaurin was evaluated by measuring inhibition and translocation of PKCa and PKC~II in vascular smooth muscle cells over-expressing PKC~II upon stimulation with PMA (H Ishii et al., 1996). The specificity of ruboxistaurin inhibition was found to be increased only 10 fold for PKC~I (IC 50=4.7) and PKC~II

(1Csa=5.9) over PKC11 (ICsa=52nM), suggesting possible inhibition of a broader range of PKC isozymes in in vitro systems (H Ishii et al., 1996). Nevertheless, our findings suggest that the PKC~ inhibitor ruboxistaurin may not inhibit PKC~II in intact primary mouse keratinocytes (Fig.12B, n=8).

We also examined keratin 10 levels in cells treated with or without ruboxistaurin in both the presence and absence of elevated extracellular calcium (Fig.12C).

Although there was a trend towards calcium-induced up-regulation of keratin 10 in untreated cells (no inhibitor), consistent stimulation of calcium-induced keratin

10 expression was not achieved during these experiments. Likewise, ruboxistaurin had no observable effect on keratin 10 expression (Fig. 12C).

These experiments were performed in a commercially available keratinocyte­ serum free medium (K-SFM) containing a basal level of 501-JM calcium. Our previous medium was prepared internally and contained a basal calcium level of

251-JM. It is possible that this change in medium and the resulting increase in the 89 calcium concentration resulted in a higher basal level of keratin 10 expression.

Under these conditions we suspect that changes in keratin 10 expression might be harder to detect. Thus, although in our hands there was a trend for an increase in keratin 10 expression with elevated extracellular calcium (125J.JM) in cells cultured in the absence of ruboxistaurin, this increase failed to reach statistical significance.

We also examined the effect of inhibition of translocation by treating keratinocytes with a peptide inhibitor that blocks the ability of PKC~II to interact with RACK1. As previously discussed, PKCs interact selectively with their

RACKs at the C2 and V5 domains. This interaction has been demonstrated to initiate translocation of the RACK-PKC complex to a unique subcellular location.

Small peptides that mimic the C2 or V5 regions of the PKC block its interaction with RACK, presumably inhibiting translocation. Using peptides that mimic the

VS region of PKCJ31 and PKCJ311, we investigated the effect of inhibition of translocation on calcium-induced inhibition of proliferation or stimulation of differentiation. These VS region peptides were conjugated to the protein transduction domain of Tat (Trans-activator of transcription), allowing the peptide's entrance into the cell. We used the protein transduction domain of Tat alone as our control. We found that inhibition of PKCJ311 translocation had no effect on calcium-induced inhibition of proliferation (Fig.11 ). We also found that inhibition of translocation had no effect on PKCJ311 autophosphorylation or K1 0 levels (Fig.13). Although we would not necessarily expect inhibition of translocation to affect autophosphorylation, we were again surprised to learn that 90 blocking translocation had no effect on keratin 10 levels. These findings, combined with the observation that autophosphorylation of PKC~II did not increase in our control experiments (no LY inhibitor or Tat), led us to suspect that although PKC~II was the logical PG-responsive candidate involved in the

AQP3/PLD2/PG signaling module, it is not the main signaling effector in this pathway. 91

Over-expression of PKC{j/1 Affects the Pattern of Keratin 10 Distribution and Results in an Altered Morphology of Cells Grown in the Presence of

Phosphatidylglycerol

Although we saw no changes in PKC~II autophosphorylation in the presence of elevated extracellular calcium, we did observe a statistically significant increase in autophosphorylated PKC~II when cells were treated with phosphatidylglycerol

(Fig. 14A). This increase was not affected by LY333531 inhibition of catalytic activity at a concentration of 300nM; however, in the presence of 11JM of inhibitor this increase was no longer statistically significant (Fig. 14A). Likewise, in the presence of PKC~ peptide translocation inhibitors we observed a trend towards

PG-stimulated activation of PKC~II (Fig.14B). It is important to note that RACKs may not be necessary for translocation of PKC~II due to its ability to bind with

PG, a membrane lipid; thus, the direct interaction with PG of PKC~II may also function to mediate translocation. These results suggest that PKC~II can be activated by PG, along with signals that stimulate keratinocyte differentiation, as observed by the translocation in the presence of extracellular calcium (Fig. 6); however, under physiological conditions, PKC~II does not appear to contribute significantly to calcium-induced growth arrest or early differentiation. This made us curious about the morphological effect of PG on keratinocytes over­ expressing PKC~II. Interestingly, treatment with PG led to morphological changes consistent with entrance into late stage differentiation in cells transfected with PKC~II, but not in cells transfected with empty vector (Fig.15). . 92

. .. . ' .. . . . ' .

-- . - . . . -- ...... : . . . ' . . . . ·This morphological charige:was not detected in keratinocytes that were ...

...... ' ' .

expressing basal·leVels of PKC~II (Fig.6C).

......

. . . . . :' . . '.. . ·...... ·. : .·' .·· . ·.:: : ·.. · · . In summary, .. we· s~ow. that ·PKC~11· is presehf .in· mouse· keratinocytes .and ·i.s : ·.

' .. . . . '' ...... ·.redistributed upon· induction of the AQP3/PLD2/PG·.sigr1aling ·l'!l·a.dule. We· ..

...... · : · ...... ·. . . .. · ...· .· ...... · .· ·.··. ·: ·. .· .. · .· .· .... ·. ·. provide {urt~er evidence suggestrng that stimulation .of AQP3/PLD2/PG can· ..... : ·.· ... · .. ··.· .· .. ·...... ·.· ... : .. ·: . • ..... '.: . · .. ·...... ~ctivate.·PKC~•u•, .. but that the isoform alone is •not sufficient to• stimulate· ·

. ' .. . . -.- . ·' - ...... -- . -. . . - .. . keratinocyte.·differenti.ation .ctnd rnay be only·a rninor.plaY~r in the keratino~yte ·• .. . . . - .... · .... "' . . . . .· . . . . .·. : response to elevated· extracellular calcium leve.ls~

- .. - . .. . . - . . ' . . - ... -. . . · · · .·Frorilthis· we· co.nc.lude: ··

.. ·. . . ' ' ...... '. • . Alth.ough PKC~.i I is ·activated:by and responds to ..

...... ' ' .. . . · phosphatidyiglycerol, it is: not the primary PG~res.po'rtsive ·elenient. ·· ·

----:·· _-.·. ·: ·.: _· __ ... ·.· ·:.' . . -. ·. ·. _-· ..... :·: ... -_ .... ' ... · .. ·: .. ' _·: ·_ .. -_ :·:' .involved· fn. the AQP3/PL[)2/PG signafi.ng :mod~le upon acti\latio·n. :by :

.· '' . ' - ,, -- ' ' . . ' . . ' ...... - . . - . . . . . elevated extracell.ular calcium levels~

. . -...... · · • ·:!he Elf Lilly inhibitor•,: R.uboxistaurin. (L Y33353.1.) ·does not app~·ar .~·o .· .

' . . ... -_ ...... · ..... ·.· . ·. . ... · .. · .. _ ...... ·.- -.- ...... · : · : be ·an ~·ff~ctive. inhibit()r.ofautoph,o~phorylati.on· of 'PKC~IJ in, i~tact..

. . . . - ...... primary mot1se keratinocytesund:er the: conditions tested .. ·· 93

Future Directions

Multiple lines of evidence in the literature pointed to PKC~II as the most logical choice for a PG-responsive effector mediating the effects of the AQP3/PLD2/PG signaling cascade. Here we performed a series of experiments that eliminated this enzyme as the major effector. We conclude that PKC~II plays only a minor role in this pathway. PG has also been implicated as a possible lipid second messenger activating PG effector enzymes in several other cell types.

Specifically, protein kinase PK-P (PG-dependent protein kinase) was found to be activated by both PG and phosphatidylinositol in human spleen (DJ Klemm et al.,

1988; DJ Klemm & Elias, 1988a; DJ Klemm & Elias, 1988b). PG was also found to stimulate the activity of PKC-8 in activated human leukocytes (SF

Pietromonaco et al., 1998). We propose that future studies focus on investigating PK-P, PKC8 or other PG-responsive signaling effectors...... · . . . . .

...... : ...... - ...... CHAPTER FIVE: REFERENCES OF LITERATURE CITED ·

. . . . A:LEASK, C. BYRN.E &_ -FUCH.S, E. (1 991 ). Tra~scription: factor AP2. and its r()le· in . .

.. ·. . .. . ·.. . :...... : .· .. epidermai..;specific gene expression. Proc. Nat/. A· cad. Sci USA 88, 7948- . 7952.

...... _·:.. . :.: .· ...... ·. : ..·· _·. : .. . . . · .... ' ... · AA DL.uGosz & Yu~PA;. s-. (1 ~93); Coor~ioate:chariges in gene expressiorrwhich · . · mark the spinous to .gr~riulat cell trC)nsition ·ih epidermis. are regulated by .· · · . : . . .. prqteinkina~e.c;·J Ceii:B!o/120, 2-17~25.- : · .· · . . . . : . · · . . . .. ·. ·AC NEWTON &:JOHNSc)N,:J. {1998) .. :"Protein kina~e c: aparadign:forregulation of.. ·· protein_· function by twa: membrane-targeting modules. Biochim .Biophys. ·: · . . . Acta 137_6~.1:55~172~: .· ...... · .. · . . . . AD POSTLE, -EL HEELEY :&.W.ILTON, D .. {2001).·A·_cbmparison. of the molecular.·. . .. species composition of mammalian lung. surfactant phospholipids . .Camp .. · . . . Bfochem. Physio/129,-. 65~73,·. --: ·.. · .. · . . · . ·. : · · . . . . ···.AM SNAPE, R.S WINNING~ S~RGENT,. T.~ (1:991 ). Transcription factor AP-2 is .. · .. ·. · tissue~specific in· Xer1opus: arid Closely related e>r identical·to .ker~tin. .· : · ·. · . transcription factor ·1 .{KTF""1 ). Development 113,. 283~293~-. · ·· · · As TAKAo·KA, r YA[VlADA;. ·M GoroH, v KANAI; ·K: :IMA(& HIRo~AsHI; ·s; (1.99~).· · · . . . . CJoning .and. characterizatior1 of the humc:1n beta :4-:-integrin·:gene .prqmoter' : · · · · : and enha.ncers. J. Bioi ·chem 273. 33848..;33855.- ·: · : : :: : : · · · : :· . · ·: ·· . . . . . ' ...... ' ...... ·... ·Av RATNAM,· DD BIKLE &_.CHo,·J~:_(19.99). 1 ,2~~Dihydro>_:F~ &.PARKER,· P.· J: (1997).. Phosphoryration ofprotein.kinase: C-a.lpha · · o~ serin·e 65_7 .. co'ntro.ls t~e accumulation of active enzyme and contributes: ...... to. itspho.spha_tase_-resistarit state:. J Bioi- Chern :2.72, 3544-9. . · .• ...... · B()UCHE, M:, ZAPP_~LLI, .. F_ .. ;.Pc)UrytENI~.M.~, ADArv10.,· S~, W_~jS~L~ W~ C.; SEN.NtM.t & . . .MOLINAR(),_ M: (1.995). Rapid activa~ibn ar1d d~w~~regulation. of protein .: kinase C alpha in 12~o~tetradecarioylphorbol~13-induced :differentiation of: .: · human rhabdomyosarcoma ·cells.: Cell Growth Differ 6, 845-8S,2~ ·· · ·

. . . . .·. <•··· ......

...... 94 .. . . 95

BYRNE, C. (1997). Regulation of gene expression in developing epidermal epithelia. Bioessays 19, 691-698. C BYRNE & FUCHS, E. (1993). Probing keratinocyte and differentiation specificity of the KS promoter in vitro and in transgenic mice. Mol Cell Bio/13, 3176- 3190. C BYRNE, M TAINSKY & FUCHS, E. {1994 ). Programming gene expression in developing epidermis. Development 120, 2369-2383. C CARLBERG & POLLY, P. {1998). Gene regulation by vitamin D3. Grit Rev Eukaryot Gene Expr 8, 19-4 C LANGE, JH NETT, BL TRUMPOWER & HUNTE, C. {2001 ). Specific roles of protein­ phospholipid interactions in the yeast chytochome bc1 complex structure. EMBO J 20, 6591-6600. C Tu, W CHANG & BIKLE, D. (2001 ). The extracellular calcium-sensing receptor is required for calcium-induced differentiation in human keratinocytes. J Bioi Chern 276, 41 079-41085. CATAISSON, C., JOSELOFF, E., MURILLAS, R., WANG, A., ATWELL, C., TORGERSON, S., GERDES, M., SUBLESKI, J., GAO, J. L., MURPHY, P._M., WILTROUT, R. H., VINSON, C. & YUSPA, S. H. (2003). Activation of cutaneous protein kinase C alpha induces keratinocyte apoptosis and intraepidermal inflammation by independent signaling pathways. J /mmuno/171, 2703-13. CE CHALFANT, H MISCHAK, JE WATSON, BC WINKLER, J GOODNIGHT, RV FARESE & CooPER, D. (1995). Regulation of alternative splicing of protein kinase C-~ by insulin. J Bioi Chern 270, 13326-13332. CHAMBON, P. (1994). The retinoid signaling pathways: molecular and genetic analysis. Semin. Cell Bio/5, 115-125. CHEN, C. & MOCHL Y-ROSEN, D. {2001 ). Opposing effects of delta and xi PKC in ethanol-induced cardioprotection. J Mol Cell Cardio/33, 581-5. CHOMCZYNSKI, P. & SACCHI, N. {1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156-9. CHRISTIE, W. (2011 ). Phosphatidylglycerol and Related Lipids: Structure, Occurrence, Composition and Analysis In The Lipid Library (ed. A. 0. C. Society). Scottish Crop Research Institute, lnvergowrie, Dundee, Scotland. CL Tu, Y 0DA & BILKE, D. (1999). Effects of a calciium receptor activator on the cellular response to calcium in human keratinocytes. J Invest Dermatol 1113, 340-345. CS SEITZ, Q LIN, H DENG & KHAVARI, P. {1998). Alterations in NF-kappaB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF­ kappaB. Proc. Nat/. Acad. Sci USA 95, 2307-2312. D BRISSON, MC VOHL, J ST-PIERRE, T J HUDSON & GAUDET, D. {2001 ). Glycerol: a neglected variable in metabolic processes? Bioessays 23, 534-542. D HOHL, U LICHTI, D BREITKREUTZ, PM STEINERT & ROOP, D. {1991 ). Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid. J Invest Dermato/96, 414-418. 96

D MOCHLY-ROSEN, BL SMITH, CH CHEN, MH DISATNIK.& RON, D. (1995). Interaction of protein kinase C with RACK1, a receptor for activated C­ kinase: a role in beta protein kinase C mediated signal transduction. Biochern Soc Trans 23, 596-600. D RON, CH CHEN, J CALDWELL, L JAMIESON, E ORR & MOCHLY-ROSEN, D. (1994). Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins. Proc Nat/ A cad Sci US A 91, 839-43. D RON, J Luo & MOCHL Y-ROSEN, D. {1995). C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo. J Bioi Chern 270, 24180-7. D RON, Z JIANG, L YAO, A VAGTS & DIAMOND, I. (1999a). Coordinated Movement of RACK1 with activated ~IIPKC. J Bioi Chern 274, 27039. D RON, Z JIANG, L.YAO, A VAGTS, I DIAMOND & GORDON, A. (1999b). Coordinated movement of RACK1 with activated betaiiPKC. J Bioi Chern 274, 27039- 46. D SCHECHTMAN & MOCHLY-ROSEN, D. {2001 ). Adaptor protein in protein kinase C­ mediated signal transduction. Oncogene 20, 6339-6347. D WARSHAWSKY & MILLER, L. {1995). Tissue-specific in vivo protein-DNA interactions at the promoter region of the Xemopus 63 KDa keratin gene during metamorphosis. Nucleic Acids Res 23, 4502-4509. DA GORELICK, J PRAETORIUS, T TSUMEMARI, S NIELSEN & AGRE, P. {2006). Aquaporin-11: a channel protein lacking apparent transport function expressed in brain. BMC Biochern 7, 14. DA HAROLD & REED, A. (1988). Interference by endogenous glycerol in an enzymatic assay of phosphatidylglycerol in amniotic fluid. Clin Chern· 34, 560-563. DB PAREKH, W AIEGLER & PARKER, P. {2000). Multiple pathways control protein kinase C phosphorylation. EMBO J 19, 496-503. DC NG, M Su, R KIM & BIKLE, D. (1995). Regulation of lnvolucrin Gene Expression By Calcium in Normal Human Keratinocytes. Front Biosci 1, a16-24. DD BIKLE & PILLAI, S. (1993). Vitamin D, calcium, and epidermal differentiation. Endocr Rev 14, 3-19. DD BIKLE, S CHANG, D CRUMRINE, J ELALIEH, MQ MAN, EH CHOI, 0 DARDENNE, Z XIE, RS ARNAUD, K FEINGOLD & ELIAS, P. {2004a). Hydroxyvitamin D1 alpha-hydroxylase is required for optimal epidermal differentiation and permeability barrier homeostasis. J Invest Derrnato/ 122, 984-992. DD BIKLE, S PILLAI & GEE, E. (1991 ). Squamous carcinoma cell lines produce 1 ,25 dihydroxyvitamin D, but fail to respond to its prodifferentiating effect. J Invest Derrnatol 97' 435-441 . DD BIKLE, Y ODA & XIE, Z. (2004b ). Calcium and 1 ,25(0H)2 D: interacting drivers of epidermal differentiation J Steroid Biochern Mol Bioi 89-90, 355-360. DD BIKLE, Y ODA & XIE, Z. (2005). Vitamin D and skin cancer: A problem in gene regulation. J of Steroid Biochern Mol Bio/97, 83-91. 97

DERMATOLOGY, A. A. 0. (2011 ). Dermatology A to Z: Stats and Facts. DF GIBSON, AV RATNAM & BILKE, D. (1996). Evidence for separate control mechanisms at the message, protein, and enzyme activation levels for transglutaminase during calcium-induced differentiation of normal and transformed human keratinocytes. J Invest Derrnatol 106, 154-161. DJ KLEMM, AL KAZIM & ELIAS, L. (1988). Phosphatidylglycerol-modulated protein kinase activity from human spleen: I. Enzyme purification and proterties. ·Arch Biochein Biophys 265, 496-505. DJ_ KLEMM & ELIAS, L. (1988a). Phosphatidylglycerol-modulated protein kinase activity from human spleen: II. Interaction with phospholipid vesicles. Arch Biochern Biophys 265, 506-513. DJ KLEMM & ELIAS, L. (1988b). Purification and assay of a phosphatidylglycerol­ stimulated protein kinase from murine leukemic cells and its perturbation in response to IL-3 and PMA treatment. Exp. Hernatol16, 855-860. DLUGOSZ, A. A., MISCHAK, H., MUSHINSKI, J. F. & YUSPA, S. H. (1992). Transcripts encoding protein kinase C-alpha, -delta, -epsilon, -zeta, and -eta are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells. Mol Carcinog 5, 286-92. DLUGOSZ, A. A. & YUSPA, S. H. (1994). Protein kinase C regulates keratinocyte transglutaminase (TGK) gene expression in cultured primary mouse epidermal keratinocytes induced to terminally differentiate by calcium. J Invest Derrnatol102, 409-14. DN BRINDLEY & PILQUIL, C. (2009). Lipid phosphate phosphatases and signaling. J Lipid Res 50, S225-S230. DWWAGGONER, J Xu, I SINGH, R JASINSKA, Q-XZHANG & BRINDLEY, D. (1999). Structural organization of mammalian lipid phosphate phosphatases: Implications for signal transduction. Biochirn Biophys Acta 1439, 299-316. E JUNG, S BETANCOURT-CALLE, R MANN-BLAKENEY, RD. GRINER & BOLLAG, W. (1999). Sustained phospholipase D activation is associated with keratinocyte differentiation. Carcinogenesis 20, 569-576. E UEDA, S OHNO, T KUROKI, E LIVNEH, KYAMADA, KYAMANISHI & YASUNO, H. (1996). The fl isoform of protein kinase C mediates transcriptional activation of the human transglutaminase 1 gene. J Bioi Chern 271, 9790- 9794. ED SONNENBURG, T GAO & NEWTON, A. (2001 ). The phosphoinositide-dependent kinase, PDK-1, phosphorylates conventional protein kinase C isozymes by a mechanism that is independent of phosphoinositide 3-kinase. J Bioi Chern 276. EDWARDS, A. S. & NEWTON, A. C. (1997). Phosphorylation at conserved carboxyl­ terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C. J Bioi Chern 272, 18382-90. EG STEBBINS & MOCHL Y-ROSEN, D. (2001 ). Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C. J Bioi Chern 276, 29644-50. 98

EM DUTIL, A TOKER & NEWTON, A. (1998). Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1. Curr Biol8, 1366-1375. FJ JOHANNES, J PRESTLE, S EIS, P 0BERHAGEMANN & PFIZENMAIER, K. (1994 ). PKCS is a novel atypical member of the protein kinase C family. J Bioi Chern 269, 6140-6148. FOUNDATION, N. P. (2011 ). About Psoriasis- Statistics. FRANK, R.N. (2002). Potential new medical therapies for diabetic retinopathy: protein kinase C inhibitors. Am J Ophthalmol133, 693-8. FROESE, C. (1990). Prevention of stratum corneum lipid phase transition in vitro by glycerol - an alternative mechanism for skin moisturization. J Soc Cosmet Chern 41, 51-65. FucHs, E. (1995). Keratins and the skin. Annu Rev Cell Dev Bio111, 123-153. G MENON & GHADIALLY, R. (1997). Morphology of lipid alterations in the epidermis: a review. Microsc. Res. Tech 37, 180-192. G SHI, K-C SOHN, D-K CHOI, Y-J KIN, S-J KIM, B-S Ou, Y-J PIAO, YH LEE, T-J YOON, Y LEE, Y-J SEO, CD KIM & LEE, J.-H. (2010). Brn2 is a transcription factor regulating keratinocyte differentiation with a possible role in the pathogenesis of lichen planus. PLoS ONE 5, e13216. GC MAYNE & MURRAY, A. (1998). Evidence that protein kinase Cepsilon mediates phorbol ester inhibition of calphostin C- and tumor necrosis factor-alpha­ induced apoptosis in U937 histiocytic lymphoma cells. J Bioi Chern 273, 24115-21. GJ FISHER, A TAVAKKOL, K LEACH, D BURNS, P BASTA, C LOOMIS, CEM GRIFFITHS, KD COOPER, NJ REYNOLDS, JT ELDER, E LIVNECH & VOORHEES, J. (1993). Differential expression of protein kinase C isoenzymes in normal and psoriatic adult human skin: reduced expression of protein kinase C-beta II in psoriasis. J Invest Dermatol1 01, 553-9. GJ FISHER, HS TALWAR, JJ BALDASSARE, PA HENDERSON & JJ VOORHEES. (1990). Increased phospholipase C-catalyzed hydrolysis of phosphatidylinositol- 4,5-bishosphate and 1 ,2-sn-diacylglycerol content in psoriatic involved compared to uninvolved and normal epidermis. J Invest Dermatol88, 220- 222. GK MENON, PM ELIAS, SH LEE & FEINGOLD, K. (1992). Localization of calcium in marine epidermis following disruption and repair of the permeability barrier. J Invest Dermatol 84, 508-512. GK MENON, S GRAYSON & ELIAS, P. (1985). Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry. J Invest Dermatol 84,508. GOKMEN-POLAR, Y.· & AP FIELDS. (1998). Mapping of a molecular determinant for protein kinase C betall isozyme function. J Bioi Chern 273, 20261-6. GOLDSMITH, L. (1991 ). Physiology, biochemistry and molecular biology of the skin. Oxford University Press, New York. 99

GT MANLEY, M FUJIMARA, T MA, N NOSHITA, F FILIZ, AW BOLLEN, P CHAN & VERKMAN, A. (2000). Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med 6, 159-163. H HuG & SARRE, T. (1993). Protein kinase C isoenzymes: diverfence in signal transduction? Biochern. J. 291, 329-343. H ISHII, MR JIROUSEK, D KOYA, C TAKAGI, P XIA, A CLERMONT, SE BURSELL, TS KERN, LM BALLAS, WF HEALTH, LE STRAMM, EP FEENER & KING, G. (1996). Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science 272, 728-731. H QIN, X ZHENG, X ZHONG, AK SHETTY, PM ELIAS & BOLLAG, W. (2011 ). Aquaporin-3 in keratinocytes and skin: Its role and interaction with phospholipase D2. Arch Biochern Biophys 508, 138-143. H TAKAHASHI, KASANO, A MANABE, M KINOUCHI, A ISHIDA-YAMAMOTO & IIZUKA, H. (1998). The a and 11 isoforms of protein kinase C stimulate transcription of human involucrin gene. J Invest Derrnatol110, 218-223. HocEVAR, B. A. & FIELDS, A. P. (1991 ). Selective translocation of p 11-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells. J Bioi Chern 266, 28-33. I FAus, HJ Hsu & FucHs, E. (1994 ). Oct-6: a regulator of keratinocyte gene expression in stratified squamous epithelia. Mol Cell Biol14, 3263-3275. J CASATORRES, JM NAVARRO, M BLESSING & JORCANO, J. (1994 ). Analysis of the control of expression and tissue specificity of the keratin 5 gene, characteristic of basal keratinocytes. Fundamental role of an AP-1 element. J Bioi Chern 269, 20489-204996. J REICHELT, H BUSSOW, C GRUND & MAGIN, T. (2001). Formation of Normal Epidermis Supported by Increased Stability of Keratins 5 and 14 in Keratin 10 Null Mice. Mol Bioi Cell12, 1557-1568. JAKEN, S. (1996). Protein kinase C isozymes and substrates. Curr Opin Cell Bioi 8, 168-173. JEROME-MORAIS, A., RAHN, H. R., TIBUDAN, S. S. & DENNING, M. F. (2009). Role for protein kinase C-alpha in keratinocyte growth arrest. J Invest Derrnatol 129, 2365-75. JH LEE, Sl JANG, JM YANG, NG MARKOVA & STEINERT, P. (1996). The proximal promoter of the human transglutaminase 3 gene. Stratified squamous epithelial-specific expression in cultured cells is mediated by binding of Sp1 and ets transcription factors to a proximal promoter element. J Bioi Chern 271, 4561-4568. JIROUSEK, M. R., GILLIG, J. R., GONZALEZ, C. M., HEATH, W. F., MCDONALD, J. H., 3RD, NEEL, D. A., RITO, C. J., SINGH, U., STRAMM, L. E., MELIKIAN-BADALIAN, A., BAEVSKY, M., BALLAS, L. M., HALL, S. E., WINNEROSKI, L. L. & FAUL, M. M. (1996). (S)-13-[(dimethylamino)methyl]-1 0,11, 14, 15-tetrahydro-4,9:16, 21-dimetheno-1 H, 13H-dibenzo[e,k]pyrrolo[3,4- h][1 ,4, 13]oxadiaiacyclohexadecene-1 ,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta. J Med Chern 39, 2664-71. 100

JL BURKEY, KM CAMPANALE, DO O'BANNON, JW CRAMER & FARID, N. {2002). Disposition of L Y333531, a selective protein kinase C beta inhibitor, in the Fischer 344 rat and beagle dog. Xenobiotica 32, 1045-52. JW FLUHR, M GLOOR, L LEHMANN, S LAZZERININ, F DISTANTE & BERARDESCA, E. (1999). Glycerol accelerates recovery of barrier function in vivo. Acta Derrn Venereal 79, 418-421 . JW FLUHR, M MAO-QIANG, BE BROWN, PW WERTZ, 0 CRUMRINE, JP SUNDBERG, KR FEINGOLD & ELIAS, P. (2003). Glycerol regulates stratum corneum hydration in sebaceous gland deficient (asebia) mice. J Invest Derrnatol 120, 728-737. JW FLUHR, R DARLENSKI & SURBER, C. {2008). Glycerol and the skin: holistic approach to its origin and functions. Br J Derrnatol159, 23-24. K YAKATA, Y HIROAKI, K ISHIBASHI, E SCHARA, S SASAKI, K MITSUOKA & FUJIYOSHI, Y. (2007). Aquaporin-11 containing a divergent NPA motis has normal water channel activity. Biochirn Biophys Acta 1768, 688-693. KASHIWAGI, M., 0HBA, M., CHIDA, K. & KUROKI, T. {2002). Protein kinase C eta (PKC eta): its involvement in keratinocyte differentiation. J Biochern 132, 853-7. KERANEN, L. M., DUTIL, E. M. & NEWTON, A. C. {1995). Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr Bioi 5, 1394-1403. KG SPINDLER, J LINDEBJERG, M LAHN, KJAER-FRIFELDT, S. & JAKOBSEN, A. {2009). Protein kinase C-f3 II (PKC-f311) expression in patients with colorectal cancer. lnt J Colorectal Dis 24, 641-645. KILEY, S., SCHAAP, 0., PARKER, P., HSIEH, L. L. & JAKEN, S. {1990). Protein kinase C heterogeneity in GH4C1 rat pituitary cells. Characterization of a Ca2(+)­ independent phorbol ester receptor. J Bioi Chern 265, 15704-12. KO ALEY, RO MESSING, 0 MOCHL Y-ROSEN & LEVINE, J. {2000). Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C. J Neurosci 20, 4680-5. LA McPHERSON & WEIGEL, R. {1999). AP2 alpha and AP2 gamma: a comparison of binding site specificity and trans-activation of the estrogen receptor promoter and single site promoter constructs. Nucleic Acids Res 27, 4040- 4049. LAEMMLI, U. (1979). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. LC YANG, DC NG & BIKLE, D. (2000). Protein kinase C alpha expression is required for calcium dependent keratinocyte differentiation. J Invest Derrnatol 114,811a. LH DE ARRUDA & MORAES, A. D. {2001 ). The impact of psoriasis on quality of life. Br J Derrnatol144 Suppl 58, 33-6. M HARA, T MA & VERKMAN, A. (2002). Selectively reduced glycerol in skin of aquaporin-3 deficient mice may account for impaired skin hydration, elasticity and barrier recovery. J Bioi Chern 277,46616-46621. 101

M HARA & VERKMAN, A. (2003). Glycerol replacement corrects defective skin hydration, elasticity, and barrier function in aquaporin-3-deficient mice. Proc Nat/ Acad Sci USA 100, 7360-7365. M INOUE, A KISHIMOTO, Y TAKAI & NISHIZUKA, Y. (1977). Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues II. J Bioi Chern 252, 7610-7616. M MOSER, A IMHOF', A PSCHERER, R BAUER, W AMSELGRUBER, F SINOWATZ, F HOFSTADTER, R SCHULE & BUETTNER, R. (1995). 'Cloning and characterization of a second AP-2 transcription factor:· AP-2 beta. Development 121, 2779-2788. M 0HBA, K ISHINO, M KASHIWAGI, S KAWABE, K CHIDA, N HUH & KUROKI, T. (1998). Induction of differentiation in normal human keratinocytes by adenovirus­ mediated introduction of the f1 and o isoforms of protein kinase C. Mol Bioi Cell18, 5199-5207. M SAVART, P LETARD, S BUL TEL & DUCASTAING, A. (1992). Induction of protein kinase C down-regulation by the phorbol ester TPA in a cal pain/protein kinase C complex. lnt J Cancer 52 399-403. M SCHLAME, D RUA & GREENBERG, M. (2000). The biosynthesis and functional role of cardiolipin. Prog Lipid Res 39, 257-288. M SEBAG, J HENDERSON, J RHIM & KREMER, R. (1992). Relative resistance to 1,25- dihydroxyvitamin D3 in a keratinocyte model of tumor progression. J Bioi Chern 267, 12162-12167. M YAMAMOTO, M ACEVEDO-DUNCAN, CE CHALFANT, NA PATEL, JE WATSON, J. & CooPER, D. (2000). Acute glucose-induced downregulation of PKC-betall accelerates cultured VSMC proliferation. Am J Physiol Cell Physio/279, C587-95. MF DENNING, AA DLUGOSZ, EK WILLIAMS, EK WILLIAMS, Z SZALLASI, PM BLUMBERG & YUSPA, S. (1995). Specific protein kinase C isozymes mediate the induction of keratinocyte differentiation markers by calcium. Cell Growth Differ6, 149-157. MF DENNING, SG GUY, SM ELLERBROEK, SM NORVELL, AP KOWALCZYK & KJ GREEN. (1998). The expression of desmoglein isoforms in cultured human keratinocytes is regulated by calcium, serum, and protein kinase C. Exp Cell Res 239, 50-9. MH DISATNIK, AR WINNIER, D MOCHL Y-ROSEN & ARTEAGA, C. (1994a). Distinct responses of protein kinase C isozymes to c-erbB-2 activation in SKBR-3 human breast carcinoma cells. Cell Growth Differ 5, 873-80. MH DISATNIK, G BURAGGI & MOCHLY-ROSEN, D. (1994b). Localization of protein kinase C isozymes in cardiac myocytes. Exp Cell Res 210, 287-97. MocHL Y-RosEN, D. (1995). Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 268, 24 7-51. MOCHLY-ROSEN, D., HENRICH, C. J., CHEEVER, L., KHANER, H. & SIMPSON, P. C. (1990). A protein kinase C isozyme is translocated to cytoskeletal elements on activation. Cell Regu/1, 693-706. 102

MURRAY, N. R., BURNS, D. J. & FIELDS, A. P. (1994). Presence of a ~II protein kinase C-selective nuclear membrane activation factor in human leukemia cells. J Bioi Chern 269, 21385-90. MURRAY, N. R., DAVIDSON, L.A., CHAPKIN, R. S., CLAY GUSTAFSON, W., SCHATTENBERG, D. G. & FIELDS, A. P. (1999). Overexpression of protein kinase C betall induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J Cell Biol145, 699-711. MURRAY, N. R., WEEMS, C., CHEN, L., LEON, J., Yu, W., DAVIDSON, L.A., JAMIESON, L., CHAPKIN, R. S., THOMPSON, E. A. & FIELDS, A. P. (2002). Protein kinase C betall and TGFbetaRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis. J Cell Biol157, 915-20. MW SARK, OF FISHER, E DE MEIJER, P VAN DE PUTTE & BACKENDORF, C. (1998). AP-1 and ets transcription factors regulate the expression of the human SPRR1A keratinocyte terminal differentiation marker. J Bioi Chern 273, 24683-24692. N CHATTOPADHYAY, A MITHAL & BROWN, E. (1996). The calcium-sensing receptor: a window into the physiology and pathophysiology of mineral ion metabolism. Endocr Rev 17, 289-307. N VITALE, AS CAUMONT, S CHASSEROT-GOLA, G Du, S. W., VA SCIORRA, AJ MORRIS, MA FROHMAN & BADER, M. (2001 ). Phospholipase 01: a key factor for the exocytotic machinery in neuroendocrine cells. EMBO J 20, 2424-2434. NERI, l. M., BORTUL, R., BORGATTI, P., TABELLINI, G., BALDINI, G., CAPITAN!, S. & MARTELLI, A.M. (2002). Proliferating or differentiating stimuli act on different lipid-dependent signaling pathways in nuclei of human leukemia cells. Mol Bioi Cell13, 947-64. NISHIZUKA, Y. (1986). Studies and perspectives of protein kinase C. Science 233, 305-312. NISHIZUKA, Y. (1988). The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334, 661-665. NtSHIZUKA, Y. (1995). Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9, 484-496. NR MURRAY & FIELDS, A. (1998). Phosphatidylglycerol is a physiologic Activator of Nuclear Protein Kinase C. J Bioi Chern 273, 11514-11520. NS TAN, L MICHALIK, N Nov, R YASMIN, C PACOT, M HElM, B FLUHMANN, B DESVERGNE & WAHLI, W. (2001 ). Critical roles of PPAR~/0 in keratinocyte response to inflammation. Genes Dev 15, 3263-3277. P 0ETTGEN, RM ALANI, MA BARCINSKI, l BROWN, Y AKBARALI, J BOL TAX, C KUNSCH, K MUNGER & LIBERMANN, T. (1997). Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family. Mol Cell Biol17, 4419-4433. P WONG, A PICKARD & McCANCE, D. (201 0). p300 alters keratinocyte cell growth and differentiation through regulation of p21Waf'1/CIP1 PLoS ONE 5, e8369. PATEL, N. A., EICHLER, D. C., CHAPPELL, D. S., ILLINGWORTH, P. A., CHALFANT, C. E., YAMAMOTO, M., DEAN, N. M., WYATT, J. R., MEBERT, K., WATSON, J. E. & 103

CoOPER, D. R. (2003). The protein kinase C beta II exon confers mRNA instability in the presence of high glucose concentrations. J Bioi Chem 278, 1149-57. PH ITIN, MR PITTELKOW & KUMAR, R. (1994). Effects of vitamin D metabolites on proliferation and differentiation of cultured human epidermal keratinocytes grown in serum-free or defined culture medium. Endocrinology 135, 1793- 1798. R SOUGRAT, M MORAND, C GONDRAN, P BARRE, R GOBIN, F BONTE, M DUMAS & VERBAVATZ, J. (2002). Functional expression of AQP3 in human skin epidermis and reconstructed epidermis. J Invest Dermatol 118, 678-685. RAPP, S. R., FELDMAN, S. R., EXUM, M. L., FLEISCHER, A. B., JR. & REBOUSSIN, D. M. (1999). Psoriasis causes as much disability as other major medical diseases. JAM Acad Dermatol41, 401-7. RD GRINER, F QIN, E JUNG, CK SUE-LING, KB CRAWFORD, R MANN-BLAKENEY, RJ BoLLAG & BOLLAG, W. (1999). 1 ,25-dihydroxyvitamin D3 induces phospholipase D-1 expression in primary mouse epidermal keratinocytes. J Bioi Chem 274, 4663-4670. RH CROSBIE, SA DOVICO, JD FLANAGAN, JS CHAMBERLAIN, CL OWNBY & CAMPBELL, K. (2002). Characterization of aquaporin-4 in muscle and muscular dystrophy. FASEB J 16, 943-949. RL ECKERT, JF CRISH & ROBINSON, N. (1997). The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation. Physiol Rev 77, 397-424. RUVOLO, P. P., ZHOU, L., WATT, J. C., RUVOLO, V. R., BURKS, J. K., JIFFAR, T., KORNBLAU, S., KONOPLEVA, M. & ANDREEFF, M. (2011 ). Targeting PKC­ mediated signal transduction pathways using enzastaurin to promote apoptosis in acute myeloid leukemia-derived cell lines and blast cells. J Cell Biochem 112, 1696-707. RV STAHELIN, J WANG, NR BLATNER, JD RAFTER, D MURRAY & CHO, W. (2005). The origin of C1A-C2 interdomain interactions in protein kinase Ca. J Bioi Chem 280, 36452-36463. S JAKEN & YusPA, S. (1988). Early signals for keratinocyte differentiation: role of 2 Ca + -mediated inositol lipid metabolism in normal and neoplastic epidermal cells. Carcinogenesis 9, 1033. S NAGPAL, J ATHANIKAR & CHANDRARATNA, R. (1995). Separation of transactivation and AP1 antagonism functions of retinoic acid receptor alpha. J Bioi Chem 270, 923-927. S SINHA, l DEGENSTEIN, C COPENHAVER & FUCHS, E. (2000). Defining the regulatory factors required for epidermal gene expression. Mol Cell Bioi 20, 2543-2555. S ZHU, H OH, M SHIM, E STERNECK, PF JOHNSON & SMART, R. (1999). C/EBP~ Modulates the Early Events of Keratinocyte Differentiation Involving Growth Arrest and Keratin 1 and Keratin 10 Expression. Mol Cell Biol19, 7181-7190. 104

SAURAT, J. (1999). Retinoids and psoriasis: novel issues in retinoid pharmacology and implications for psoriasis treatment. JAM A cad Dermatol41, S2-6. SC HERBERT & BROWN, E. (1995). The extracellular calcium receptor. Curr Opin Cell Biol7, 289-307. SE TSUTAKAWA, KF MEDZIHRADSZKY, AJ FLINT, AL BURLINGAME & DE KOSHLAND, J. (1995). Determination of in vivo phosphorylation sites in protein kinase C. J Bioi Chern 270, 26807-12. SF PIETROMONACO, PC SIMONS, A ALTMAN & ELIAS, L. (1998). Protein kinase C-9 phosphorylation of moesin in the actin-binding sequence. J Bioi Chern 273, 7594-7603. SH YUSPA, AE KILKENNY, PM STEINERT & RooP, D. (1989). Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol109, 1207-1217. Sl JANG, PM STEINERT & MARKOVA, N. (1996). Activator protein 1 activity is involved in the regulation of the cell type-specific expression from the proximal promoter of the human profilaggrin gene. J Bioi Chern 271, 24105-24114. SM FISCHER, ML LEE, RE MALDVE, RJ MORRIS, D TRONO, DL BUROW, AP BUTLER, A PAVONE & WARREN, B. (1993). Association of protein kinase C activation with induction of ornithine decarboxylase in murine but not human keratinocyte cultures. Mol Carcinog 7, 228-37. SM THACHER & RICE, R. (1985). Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell40, 685-695. SR WOLBACH & HowE, P. (1925). Tissue changes following deprivation of fat­ soluble A vitamin. J Exp Med 43, 753-777. STEINBERG, S. (2008). Structural Basis of Protein Kinase C lsoform Function. Physiol Rev88, 1341-1378. Su MJ, BIKLE DD, MANCIANTI ML & S., P. (1994). 1 ,25-Dihydroxyvitamin D3 potentiates the keratinocyte response to calcium. J Bioi Chern 269, 14723- 9. T EFIMOVA & EcKERT, R. (2000). Regulation of human involucrin promoter activity by novel protein kinase C isoforms. J Bioi Chern 275, 1601-1607. T ESGLEYES-RIBOT, RA CHANDRARATNA, DA LEW-KAVA, J SEFTON & DUVIC, M. (1994 ). Response of psoriasis to a new topical retinoid, AGN 190168. J. Am. Acad. Dermatol30, 581-590. T GAo, A ToKER & NEWTON, A. (2001 ). The carboxyl terminus of protein kinase C provides a switch to regulate its interaction with the phosphoinositide­ dependent kinase, PDK-1. J Bioi Chern 276, 19588-19596. T Hu & ExTON, J. (2004 ). Protein kinase Ca translocates to the perinuclear region to activate phospholipase D1. J Bioi Chern 279, 35702-35708. T MA, M HARA, R SOUGRAT, JM VERBAVATZ & VERKMAN, A. (2002). Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3. J Bioi Chern 277, 17147-17153. 105

TANG, S., XIAO, V., WEI, L., WHITESIDE, C. I. & KOTRA, L. P. {2008). Protein kinase C isozymes and their selectivity towards ruboxistaurin. Proteins 72, 447- 60. TE GRAHAM, JW HELGE, DA MACLEAN, B KIENS & RICHTER, E. {2000). Caffeine ingestion does not alter carbohydrate or fat metabolism in human skeletal muscle during exercise. J Physio/529, 837-84 7. TH KWON, H HAGER, LN NEJSUM, ML ANDERSEN, J FROKAIER & NIELSEN, S. {2001 }. Physiology and pathophysiology of renal aquaporins. Sernin Nephro/21, 231-238. THOMPSON, L. & FIELDS, A. {1996). ~II Protein Kinase C is Required for the G2/M Phase Transition of Cell Cycle. J Bioi Chern 271, 15045-15053. TIBUDAN, S. S., WANG,'(. & DENNING, M. F. (2002). Activation of protein kinase C triggers irreversible cell cycle withdrawal in human keratinocytes. J Invest Derrnato/119, 1482-9. V MULLER-WIEPRECHT, C RIEBELING, C ALEXANDER, F-R SCHOLZ, A HOER, T WIEDER, CE 0RFANOS & GEILEN, C. {1998). Expression and regulation of phospholipase D in the human keratinocyte cell line HaCaT. FEBS Letters 425, 199-203. VO RYBIN, J Guo & STE,INBERG, S. (2008). Protein kinase D1 autophosphorylation via distinct mechanisms at Ser744/Ser7 48 and Ser916. J Bioi Chern 284, 2332-2343. W COLLEY, TC SUNG, R :RoLL, J JENCO, SM HAMMOND, YM AL TSHULLER, D BAR­ SAG!, AJ MORRIS & FROHMAN, M. {1997). Phospholipase D2, a PLD1- related isoform with novel regulatory properties and discrete subcellular localization that provokes cytoskeletal reorgnization. Curr Opin Cell Bio/7, 191-201. WALKER, S.D., MURRAY, N. R., BURNS, D. J. & FIELDS, A. P. {1995). Protein kinase C chimeras: catalytic domains of alpha and beta II protein kinase C contain determinants for isotype-specific function. Proc Nat/ Acad Sci US A 92, 9156-9160. WANG, H. Q. & SMART, R. C. (1999). Overexpression of protein kinase C-alpha in the epidermis of ~ransgenic mice results in striking alterations in phorbol ester-induced infl.ammation and COX-2, MIP-2 and TNF-alpha expression but not tumor promotion. J Cell Sci 112 ( Pt 20), 3497-506. WB BOLLAG & BOLLAG, ~· (2001 }. 1 ,25-Dihydroxyvitamin D3, phospholipase D and protein kinase C in keratinocyte differentiation. Mol Cell Endocrine/ 177, 173-182. WB BOLLAG, D XIE, X ZH:ENG & ZHONG, X. {2007). A potential role for the phospholipase D2-Aquaporin-3 signaling module in early keratinocyte differentiation: Production of a phosphatidylglycerol signaling lipid. J Invest Derrnatol 127' 2823-2831 . WB BOLLAG, J DUCOTE & HARMON, C. {1995). Biphasic effect of 1,25- dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation. J Cell Physio/163, 248-256.