1

Pdx-1 activates islet α- and β-cell proliferation via a TRPC3/6- and ERK 1/2-regulated 1

mechanism 2

Running title: Pdx-1 induces islet cell replication via TRPCs and ERK 3

Heather L. Hayes1,2, Larry G. Moss1,3, Jonathan C. Schisler1,4, Jonathan M. Haldeman1,2, 4

Zhushan Zhang3, Paul B. Rosenberg1,3, Christopher B. Newgard1,2,3 and Hans E. Hohmeier1,3 5

1Sarah W. Stedman Nutrition and Metabolism Center and Duke Institute of Molecular 6

Physiology 7

Departments of Pharmacology and Cancer Biology2 and Medicine3 8

Duke University Medical Center, Durham, NC 27704, USA 9

4Present address: McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 10

27599, USA 11

Corresponding Author: 12

Hans E. Hohmeier, MD, PhD 13

Mailing address: Sarah W. Stedman Nutrition and Metabolism Center 14

Duke Independence Park Facility 15

4321 Medical Park Drive, Suite 200 16

Durham, NC 27704 17

Phone: (919) 479-2342 18

Fax: (919) 477-0632 19

Email: hans.hohmeier@duke.edu 20

21

Copyright © 2013, American Society for Microbiology. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.00469-13 MCB Accepts, published online ahead of print on 12 August 2013

2

Abstract 22

The homeodomain transcription factor, Pdx-1, has important roles in pancreatic development and 23

β-cell function and survival. In the present study, we demonstrate that adenovirus-mediated 24

overexpression of Pdx-1 in rat or human islets also stimulates cell replication. Moreover, co-25

overexpression of Pdx-1 with another homeodomain transcription factor, Nkx6.1, has an additive 26

effect on proliferation compared to either factor alone, implying discrete activating mechanisms. 27

Consistent with this, Nkx6.1 stimulates mainly β-cell proliferation, whereas Pdx-1 stimulates 28

both α- and β-cell proliferation. Furthermore, cyclins D1/D2 are upregulated by Pdx-1 but not by 29

Nkx6.1, and inhibition of cdk4 blocks Pdx-1- but not Nkx6.1-stimulated islet cell proliferation. 30

Genes regulated by Pdx-1 and not Nkx6.1 were identified by microarray analysis. Two members 31

of the transient receptor potential cation (TRPC) channel family, TRPC3 and TRPC6, are 32

upregulated by Pdx-1 overexpression, and siRNA-mediated knockdown of TRPC3/6 or TRPC6 33

alone inhibits Pdx-1-induced but not Nkx6.1-induced islet cell proliferation. Pdx-1 also 34

stimulates ERK1/2 phosphorylation, an effect partially blocked by knockdown of TRPC3/6, and 35

blockade of ERK1/2 activation with a MEK1/2 inhibitor partially impairs Pdx-1-stimulated 36

proliferation. These studies define a pathway by which overexpression of Pdx-1 activates islet 37

cell proliferation that is distinct from and additive to a pathway activated by Nkx6.1. 38

39

40

41

42

3

Introduction 43

Type 1 diabetes mellitus is caused by autoimmune destruction of pancreatic islet β-cells, whereas 44

type 2 diabetes involves the combined loss of glucose-stimulated insulin secretion (GSIS) and 45

functional β-cell mass by non-autoimmune mechanisms (1-3). Because both forms of diabetes 46

are characterized by insulinopenia, transplantation of functional β-cells or delivery of agents that 47

induce β-cells to replicate in a controlled manner have been considered as therapeutic strategies. 48

These potential interventions require identification of pathways that maintain or augment islet 49

proliferation with retention of function, but such strategies have remained elusive, especially 50

when dealing with human islets (4). 51

In most cases, factors that induce β-cell replication also cause loss of desired phenotypes, such as 52

insulin content and GSIS (5, 6). Rare exceptions to this include cyclin D or cdk6 overexpression, 53

which are sufficient to promote human β-cell proliferation with no discernible loss of function 54

(7), although recent studies suggest that these factors may also promote DNA damage and 55

eventual cell cycle arrest (8). In addition, our laboratory has shown that Nkx6.1 overexpression 56

is sufficient to promote proliferation while potentiating GSIS in isolated rat islets (9). It should 57

be noted that in another study with inducible Nkx6.1 transgenic mice, an increase in islet cell 58

proliferation was not observed (10), which may be attributed to the level of Nkx6.1 59

overexpression or difference in species. It is also important to devise methods to protect islet 60

cells against cytotoxic agents encountered in diabetes, including cytokines, elevated lipids and 61

toxins produced by immune responses (11, 12). Thus, factors that maintain functionality, provide 62

protection and stimulate proliferation are of great interest. Pdx-1 is known to regulate pancreatic 63

islet function and protect against cell death (13-16). Therefore, the current investigation was 64

focused on determining if Pdx-1 could be used as a tool for inducing islet cell proliferation. 65

4

Many years of research have led to an understanding of a temporal sequence of expression of a 66

family of transcription factors that coordinate the development of α-, β- and δ-cells in pancreatic 67

islets. Brn4, Pax4, Pax6, Mafa, Mafb, Nkx2.2, Nkx6.1 and Pdx-1 are among the factors that are 68

important for late-stage differentiation of mature α-, β- and δ-cells (17). These factors are also 69

important for maintaining differentiated functions of adult islet cells. Pdx-1 is essential for 70

pancreatic development, as demonstrated by complete pancreatic agenesis in Pdx-1-/- mice (18, 71

19). Reduced expression of Pdx-1 leads to impaired GSIS (13), but importantly, Pdx-1 72

overexpression does not impair function (20). A potential concern is raised by a recent report 73

linking Pdx-1 to malignant phenotypes in pancreatic cancers (21). In contrast, no evidence of an 74

oncogenic phenotype was reported in pancreas of Pdx-1 transgenic mice (22). Pdx-1 is also 75

necessary for maintenance of β-cell mass as demonstrated by studies in β-cell specific Pdx-1+/- 76

mice (23). Moreover, Pdx-1 deficiency leads to increased apoptosis, autophagy and susceptibility 77

to ER stress (14-16), suggesting that Pdx-1 is essential for β-cell survival. Pdx-1 expression has 78

been associated with proliferation or increased β-cell mass in remnant islets (24) and in 79

pancreatic ductal cells after partial (90%) pancreatectomy (25). While Pdx-1 transgenic mice 80

have a two-fold increase of 5-bromo-2-deoxyuridine (BrdU) labeling in β-cells as compared to 81

wild-type mice (22), the impact of acute expression of Pdx-1 on proliferation in isolated islets 82

has not been studied, and the mechanisms by which Pdx-1 might induce proliferation are 83

unknown. 84

In the present study, we show that Pdx-1 overexpression stimulates rat islet cell proliferation as 85

measured by [3H]-thymidine incorporation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation and 86

phospho-histone H3 (pHH3) staining. We also show that Pdx-1 overexpression stimulates [3H]-87

thymidine incorporation in human islets. Moreover, we demonstrate that the co-overexpression 88

5

of Pdx-1 and Nkx6.1 results in an additive proliferative effect and that the two factors activate 89

islet proliferation via two separate pathways. We show that unlike Nkx6.1, which stimulates 90

mainly β-cell proliferation, Pdx-1 stimulates both α- and β-cell proliferation. We also 91

demonstrate that the cyclin D/cdk4 complex is essential for Pdx-1- but not Nkx6.1-stimulated 92

proliferation. Finally, we show that the transient receptor potential cation (TRPC) channels, 93

TRPC3 and TRPC6, as well as activated ERK1/2 are required to support the proliferative effect 94

of Pdx-1. Our findings map out a new pathway for stimulation of β-cell replication that may 95

contain targets for expansion of functional β-cell mass in diabetes. 96

Materials and Methods 97

Cell culture, reagents and use of recombinant adenoviruses 98

INS-1-derived 832/13 rat insulinoma cells were cultured as previously described (26). Pancreatic 99

islets were isolated from male Wistar rats and cultured as previously described (9, 27, 28) under 100

a protocol approved by the Duke University Institutional Animal Care and Use Committee. 101

Human islets were obtained from the Integrated Islet Distribution Program (http://iidp.coh.org). 102

The Cdk4 inhibitor, PD0332991, was a kind gift from Dr. Ned Sharpless at UNC-Chapel Hill. 103

Cyclosporin A and the MEK1/2 inhibitor, U0126, were purchased from Calbiochem. 104

For gene overexpression studies, CMV promoter-driven recombinant adenoviruses containing 105

hamster Nkx6.1, mouse Pdx-1, bacterial β-galactosidase (βgal), green fluorescent protein (GFP) 106

and constitutively active calcineurin (CnA) cDNA were used as previously described (20, 29, 107

30). We constructed CMV promoter-driven human Flag-tagged TRPC3 and human Myc-tagged 108

TRPC6 adenoviruses by cloning cDNA constructs into the pAdTrack shuttle vector and using the 109

Ad-Easy system to generate the recombinant adenoviruses as previously described (31). 110

6

For gene suppression studies in isolated rat islets, adenoviruses containing siRNAs specific to rat 111

TRPC3/TRPC6 (siT3/T6), rat TRPC6 (siT6) or with no known gene homology (siScr) were 112

constructed and used as described previously (32). 113

All recombinant adenoviruses were shown to be E1A deficient using a RT-PCR screen as 114

previously described (33). Pools of 200 islets were cultured in 2 ml of RPMI medium (10% FCS 115

and 8 mM glucose), treated with viruses at a concentration of ~2x109 particles/ml medium for 18 116

h and then cultured in virus-free medium until the islets were collected for assays 78 h post-117

transduction unless otherwise noted in the figure legends. For gene overexpression studies in 118

832/13 cells, cells were treated with viruses at a concentration of ~0.2x109 particles/ml medium 119

for 18 h and then cultured in virus-free medium until the cells were collected for assays 48 h 120

post-transduction. 121

For gene suppression studies in 832/13 cells, siRNA duplexes targeting TRPC3 and TRPC6 were 122

purchased and used according to the manufacturer’s protocol at a final concentration of 50 nM 123

(Dharmacon). A duplex with no known target (siScr) was used as a control (34). Before the 124

transfection of the siRNA duplexes, 832/13 cells were first treated with an adenovirus 125

overexpressing Pdx-1 for 4 h. Media was then changed, and the transfection was performed 2 h 126

later. Cells were harvested after an additional 72 h of culture. 127

[3H]-thymidine incorporation 128

DNA synthesis rates were measured as described previously (9, 35) with some modifications. 129

Briefly, [3H]-thymidine was added at a final concentration of 1 µCi/ml to pools of approximately 130

200 islets during the last 18 h of cell culture. Three groups of 20 islets were picked, washed once 131

in media and washed once in 1xPBS. Islets were collected by centrifugation at 1,000 rpm for 5 132

7

min at RT. DNA was precipitated by adding 500 µl of cold 10% trichloroacetic acid (TCA) for 133

20 min on ice followed by centrifugation at 13,000 rpm at 4°C for 20 min. The pellet was 134

solubilized with 80 µl of 0.3 N NaOH. The amount of [3H]-thymidine incorporated into DNA 135

was measured by liquid scintillation counting and normalized to total cellular protein (36). 136

EdU incorporation 137

For EdU labeling, a 1:1000 dilution of EdU labeling reagent (Invitrogen) was added to islet 138

culture medium during the last 18 h of cell culture. For immunohistochemistry, islets were fixed 139

in 4% paraformaldehyde for 2 h at room temperature. Islets were prepared for 140

immunhistochemistry as previously described (35) with some modifications. After mixing islets 141

with Affi-Gel blue beads (Bio Rad), warm histogel (55°C) was added to the slurry. After 142

cooling, the histogel containing the islet/bead mixture was embedded in paraffin and sectioned. 143

Five-micrometer sections were deparaffinized and subjected to antigen retrieval as previously 144

described (9). EdU was detected using the Click-iT kit (Invitrogen) following the manufacturer’s 145

protocol. For insulin and glucagon staining, slides were incubated overnight with goat anti-146

guinea pig insulin (Dako cat#A0564) and goat anti-rabbit glucagon (Dako cat#A0565) antibodies 147

followed by detection with an AlexaFluor488-conjugated goat anti-guinea pig secondary 148

antibody and AlexaFluor647-conjugated goat anti-rabbit secondary antibody, respectively 149

(Invitrogen cat#A11073 and cat#A21245, respectively). Slides were counterstained with DAPI. 150

Images were captured and analyzed using OpenLab software, and cells were quantitated using 151

ImageJ software. 152

For immunofluorescence, islets were dispersed using trypsin/EDTA, plated on poly-D-lysine 153

coated coverslips (BD Biosciences) and fixed using neutral buffered formalin. EdU detection, 154

8

insulin staining, pHH3 (Cell Signaling cat#2577) staining and phospho-gamma H2AX (Cell 155

Signaling cat#3377) staining were performed as described above for IHC. Cells were 156

counterstained with DAPI. Images were captured and analyzed using OpenLab software, and 157

cells were quantitated using ImageJ software. 158

Microarray analysis 159

Rat islets were left untreated (NV) or treated for 18 h with recombinant adenovirus (βgal or Pdx-160

1) and cultured for an additional 30 h. Islets were harvested 48 h post-transduction, and total 161

RNA was isolated using the RNeasy kit (Qiagen). cDNA microarray analysis was performed on 162

the Rat Genome 230 2.0 array (Affymetrix) with 31,042 probe sets corresponding to over 28,000 163

annotated genes in the Duke University Microarray Core Facility. Replicate (n=5) microarray 164

studies were performed for each treatment. Analysis of gene expression data was conducted with 165

modules from GenePattern (http://genepattern.broadinstitute.org). An expression matrix was 166

generated from the raw Affymetrix data using the Robust Multi-array Average (RMA) algorithm 167

in the ExpressionFileCreator module. The raw intensity values were background corrected, log2 168

transformed and then quantile normalized. A linear model was next fit to the normalized data to 169

obtain an expression measure for each probe set on each array. The data set was filtered using the 170

PreprocessDataset module to set thresholds and eliminate genes exhibiting minimal changes. 171

Differential expression between the experimental and control conditions was determined using 172

the ComparativeMarkerSelection module. Testing was done by a two-sided t-test with cutoffs 173

determined by False Discovery Rate (FDR) by the Benjamini and Hochberg procedure (<0.05) 174

with p-values less than 0.05. GO analysis was performed using DAVID v6.7. Data files have 175

been deposited in the NCBI GEO database. 176

9

Electrophysiology 177

Currents were recorded in the whole-cell voltage clamp mode using a MultiClamp-700A 178

amplifier with the Digidata 1322A interface and analyzed with pCLAMP software (Axon 179

Instruments) as described previously (37). The patch pipettes had a resistance of 2-3 MΩ when 180

filled with a pipette solution containing 140 mM Cs aspartate, 5 mM NaCl, 1 mM Mg-ATP, 10 181

mM HEPES and 10 mM BAPTA (pH 7.3). The external solution contained 140 mM NaCl, 2.8 182

mM KCl, 2 mM BaCl2, 1 mM MgCl2, 10 mM HEPES and 5 mM glucose (pH 7.4). Currents 183

were induced by a 200 ms voltage ramp protocol (1 mV/ms; from 100 to -100 mV) every 3 s 184

from a holding potential of 0 mV (K+ channel blocked by Cs in the internal solution; L-type Ca2+ 185

channel blocked by 10 µM verapamil in external solution; voltage-dependent Na+ channel 186

inactivated by the stimulation protocol; and chloride current inhibited by reduced and equal Cl- 187

concentration in the external and internal solutions or by 1 mM anthracene-9-carboxylic acid (9-188

AC) in the external solution. Experiments were performed at room temperature (20-22°C) with a 189

sample rate of 4 KHz (filtered at 2 kHz). Currents were measured at -80 and +80 mV, and the 190

currents were normalized by membrane capacitance. 191

Measurement of RNA levels 192

RNA was isolated using the RNeasy kit (Qiagen), and cDNA was made using iScript (Bio Rad). 193

Real-time PCR assays were performed using the ViiA7 detection system and software (Applied 194

Biosystems). The primers used for rat cyclins E1, E2, D1, D2 and D3 as well as rat cdk4 were 195

TaqMan-based Assay on Demand (Applied Biosystems). All other primers were designed and 196

used with SYBR green (Bio Rad). Primer sequences are available upon request. 197

Immunoblot analysis 198

10

Cells or islets were harvested and lysed in ice-cold RIPA buffer (Sigma) containing protease (BD 199

biosciences) and phosphatase inhibitors (Sigma). Lysates were precleared by centrifugation 200

(13,000 rpm at 4°C for 10 min), resolved on 4-12% NuPAGE gels (Invitrogen) and transferred to 201

polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% milk in TBS-T 202

for 30 min followed by overnight incubation at 4°C with the following diluted primary 203

antibodies: Nkx6.1 (Iowa Development Hybridoma Bank cat#F55A10), Pdx-1 (Abcam 204

cat#47267), γ-Tubulin (Sigma cat#T5326), TRPC6 (Genetex cat#GTX113858), Myc (Abcam 205

cat#34773-100), Flag M2 Peroxidase (Sigma cat#A8592), Phospho-p42/44 MAPK (Cell 206

Signaling cat#4370 and Millipore cat#05-797R clone AW39R) and p42/44 MAPK (Cell 207

Signaling cat#4695). Sheep anti-mouse (1:10,000) and goat anti-rabbit (1:10,000) antibodies (GE 208

Healthcare cat#NXA931 and cat#NA934V, respectively) coupled to horseradish peroxidase were 209

used to detect primary antibodies followed by detection with SuperSignal West Femto 210

Chemiluminescent Substrate (Thermo Scientific). Goat anti-mouse IRDye 800CW (1:10,000) 211

and AlexaFluor 680 goat anti-rabbit IgG (1:10,000) antibodies were also used to detect primary 212

antibodies followed by detection using the Odyssey CLx system (LI-COR). Quantitation of 213

immunoblots was performed by measuring pixel density using Adobe Photoshop CS4. 214

Statistical analysis 215

Data are presented as mean ± SEM. For statistical significance determinations, data were 216

analyzed by the two-tailed t-test. For multiple group comparisons, ANOVA with Bonferroni post 217

test or Tukey’s test was used. P-values less than 0.05 were considered significant. 218

219

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11

Results 221

Pdx-1 overexpression stimulates rat and human islet cell proliferation and is additive to 222

Nkx6.1 223

Pdx-1 is essential for pancreatic development, β-cell function and β-cell survival (14, 18, 23), but 224

its effects on islet cell proliferation are not well defined. Here, we tested if adenovirus-mediated 225

overexpression of Pdx-1 is sufficient to increase proliferation either alone or combined with 226

Nkx6.1 overexpression, a transcription factor that we have previously reported to stimulate 227

proliferation in isolated rat islets while enhancing GSIS (9, 20). Pdx-1 overexpression stimulated 228

[3H]-thymidine incorporation in rat islets by approximately 7- and 6-fold compared to control 229

islets treated with no virus (NV) or with a β-galactosidase (βgal)-expressing adenovirus, 230

respectively (Figure 1A). Importantly, our previous work has shown that overexpression of Pdx-231

1 does not interfere with GSIS in rat islets (20). When Pdx-1 and Nkx6.1 were co-overexpressed, 232

an additive proliferative effect was observed (15-fold increase) as compared to the effect of 233

either transcription factor alone (8- and 10-fold increase with Pdx-1 or Nkx6.1 expression, 234

respectively) (Figure 1A). Pdx-1 and Nkx6.1 protein levels for these experiments are shown in 235

Figure 1B. These data demonstrate that Pdx-1 and Nkx6.1 can independently stimulate rat islet 236

proliferation and that the combination of both factors has an additive proliferative effect. 237

We also studied the effect of Pdx-1 overexpression in human islets and found a significant 238

enhancement (approximately 50%) in [3H]-thymidine incorporation compared to the βgal control 239

in experiments involving 10 separate islet donors (Figure 1C). The modest proliferative effect in 240

human islets relative to that observed in rat islets is similar to what we have reported for Nkx6.1 241

(8) and is consistent with the generally refractory nature of human islets to proliferative stimuli. 242

12

Pdx-1 induces proliferation of both α- and β-cells whereas Nkx6.1 induces mainly β-cell 243

proliferation 244

In both our prior study (9) and the current study, the adenoviruses utilized to overexpress Nkx6.1 245

or Pdx-1 use the CMV promoter to drive gene expression, which is active in all mammalian 246

cells. To identify the islet cell types that proliferate in response to overexpression of each 247

transcription factor, islets were transduced with AdCMV-Pdx-1, AdCMV-Nkx6.1 or both 248

adenoviruses (Pdx-1+Nkx6.1). Sections of paraffin-embedded rat islets from four independent 249

experiments were treated with antibodies and were imaged by fluorescence microscopy (Figure 250

2A). Among the four experiments, a total of 62,833 cells were counted with at least 7,900 cells 251

counted for each treatment. The percentage of EdU positive cells among all islet cells was 6.3, 252

8.3 and 13.6% with overexpression of Pdx-1, Nkx6.1 or Pdx-1+Nkx6.1, respectively, relative to 253

NV and βgal controls, in which <1% of islet cells were EdU positive (Figure 2B). Importantly, 254

the additive effect of Nkx6.1+Pdx-1 on EdU incorporation (Figure 2B) is in full agreement with 255

the [3H]-thymidine incorporation data (Figure 1A). When considering cell-type specificity, a 256

significant increase in β-cell proliferation was observed when the two factors were co-expressed 257

(12.1±1.2% INS+EdU+ cells) relative to either transcription factor alone (Figure 2C). 258

Interestingly, Pdx-1 caused a much larger increase in EdU incorporation into non-β-cells (mainly 259

α-cells) relative to Nkx6.1 (9.5 vs. 3.6% INS-EdU+ cells, respectively; p<0.05) (Figure 2D). 260

Conversely, Nkx6.1 caused a significant increase in EdU incorporation in insulin-positive cells 261

(β-cells) relative to Pdx-1 (p<0.05) (Figure 2C). In agreement with our previous work (9), 262

Nkx6.1 stimulated mainly β-cell proliferation (approximately 93% of EdU+ cells were insulin 263

positive), whereas Pdx-1 stimulated replication of both α- and β-cells (approximately 68 and 264

31% of EdU+ cells were insulin or glucagon positive, respectively) (Figure 2E). Co-265

13

overexpression of Pdx-1 and Nkx6.1 created a pattern of EdU incorporation resembling that 266

observed with Pdx-1 overexpression. Taken together, these data show that the impact of 267

combined overexpression of Nkx6.1+Pdx-1 on [3H]-thymidine incorporation (Figure 1A) is due 268

to increased β-cell proliferation induced by both factors as well as additional α-cell proliferation 269

that is mainly driven by Pdx-1. 270

Pdx-1 and Nkx6.1 increase phospho-histone H3 (pHH3), and Pdx-1 does not significantly 271

increase the DNA damage marker phospho-γH2AX in rat islets 272

EdU incorporation is a marker for DNA replication, an early step in cell division. To determine 273

the extent to which cells also completed DNA synthesis and moved past the G2 checkpoint to 274

undergo mitosis, we used phospho-histone H3 (pHH3) as a marker. The total number of cells 275

positive for pHH3 was less than the number of EdU positive cells for all conditions (Figures 3A 276

and 3B), which can be attributed to the transient phosphorylation of HH3 during the cell cycle. 277

Nevertheless, there were approximately 7-, 9- and 21-fold more double positive (EdU+pHH3+) 278

cells in the AdCMV-Pdx-1-, AdCMV-Nkx6.1- and ACMV-Pdx-1+AdCMV-Nkx6.1-treated 279

islets, respectively, as compared to AdCMV-βgal-treated islets. Moreover, co-expression of Pdx-280

1 and Nkx6.1 increased the number of double positive cells relative to either factor alone (Figure 281

3B). These data demonstrate that Pdx-1 and Nkx6.1 are inducing both early and late events in the 282

cell cycle. 283

Recent studies involving overexpression of other proliferative factors, such as HNF4α, cdk6 and 284

cyclin D3, in human islets have demonstrated clear increases in early proliferative markers but 285

have also reported that the same factors induce markers of DNA damage and eventual cell cycle 286

arrest (8). To investigate this important issue, we used phospho-histone variant γH2AX (γH2AX) 287

14

as a marker for DNA damage response (Figure 3C). Control cells (NV or βgal) had <1% EdU 288

incorporation and also were <1% positive for γH2AX staining with only rare EdU+γH2AX+ 289

cells (Figure 3D). Although the percentage of γH2AX+ cells in Pdx-1-treated islets was slightly 290

increased (1.97±0.77%) as compared to βgal-treated islets (0.99±0.38%; p>0.05), the percentage 291

of Edu+ cells was far greater than the percentage of cells that were γH2AX+ or EdU+γH2AX+ 292

(p<0.01 and p<0.001, respectively) (Figure 3D). Importantly, when considering only the EdU+ 293

cells in islets treated with the Pdx-1 adenovirus, approximately 75% of the EdU+ cells were 294

negative for γH2AX staining, suggesting that the majority of cells induced to proliferate by Pdx-295

1 lack a DNA damage response (Figure 3E). 296

The cyclin D/cdk4 complex is necessary for Pdx-1- but not Nkx6.1-stimulated proliferation 297

We next measured the mRNA levels of cyclins E1, E2, D1, D2 and D3 to understand their 298

regulation by Pdx-1 and Nkx6.1. Pdx-1, Nkx6.1 and Pdx-1+Nkx6.1 increased the mRNA levels 299

of both cyclins E1 and E2 compared to controls (Figure 4A). In contrast, increases in cyclins D1 300

and D2 mRNA only occurred in response to Pdx-1 (alone or in combination with Nkx6.1; Figure 301

4B). Thus, these data suggest that Pdx-1 and Nkx6.1 exert their effects on proliferation via 302

different components of the core cell cycle machinery. 303

To further test if cyclins D1 and D2 are necessary for Pdx-1- or Nkx6.1-stimulated rat islet 304

proliferation, we tested the effect of disrupting the cyclin D/cdk4 complex with the cdk4-specific 305

inhibitor, PD0332991. The inhibitor completely blocked Pdx-1-stimulated rat islet proliferation 306

but did not significantly affect Nkx6.1-stimulated rat islet proliferation (Figure 4C). Importantly, 307

the cdk4 inhibitor did not affect the mRNA levels of Pdx-1, Nkx6.1, cyclin D1, cyclin D2 or 308

15

cdk4 (Figure 4D). Together, these data show that Pdx-1, but not Nkx6.1, stimulates islet cell 309

proliferation via activation of the cyclin D/cdk4 complex. 310

Pdx-1 stimulates rat islet proliferation and upregulates TRPC3/6 expression 48 h after 311

adenoviral transduction 312

We next studied the time course of Pdx-1-mediated islet cell proliferation. Pdx-1 stimulates rat 313

islet proliferation as early as 48 h and maintains proliferation through 96 h post-transduction 314

(Figure 5A) while maintaining constant Pdx-1 protein levels (Figure 5B). This experiment 315

demonstrates another important difference between Pdx-1- and Nkx6.1-stimulated proliferation, 316

as Nkx6.1-stimulated proliferation has a slower time course, with the first detectable effects on 317

[3H]-thymidine incorporation occurring at 72 h (9). 318

To further investigate the pathway by which Pdx-1 stimulates islet cell replication, we performed 319

microarray analyses on untreated islets and islets treated with either βgal or Pdx-1 adenoviruses 320

at the 48 h time point. RNA from five independent islet samples for each of the three conditions 321

was hybridized to the Rat Genome 230 2.0 Affymetrix microarray. As compared to the βgal 322

control and considering only those genes that exhibited a 2-fold change in expression level with 323

a p-value less than 0.05, 264 genes were upregulated by Pdx-1, and 44 genes were 324

downregulated by Pdx-1 (Supplemental Table 1). To elucidate the Pdx-1-specific effect on rat 325

islet proliferation, we investigated only those genes induced by Pdx-1 and not by Nkx6.1 by 326

comparing the present microarray data to data previously collected from rat islets transduced 327

with AdCMV-Nkx6.1 for 48 h (Tessem et al., in revision). To further classify Pdx-1-regulated 328

genes, the microarray data was subjected to gene ontology and pathway analysis. GO pathway 329

analysis revealed a cluster of genes related to calcium homeostasis and signaling that was 330

16

strongly induced by Pdx-1 but not Nkx6.1, including Calbindin 1, Calbindin 2, 331

Calcium/calmodulin-dependent protein kinase type 1D (Camk1d), Cannabinoid receptor 1 (CB1) 332

and Protein kinase C β1 (PKCβ1). Treatment of islets with overexpression adenoviruses for each 333

of these genes or expression of constitutively active forms of Camk1d and PKCβ1 was not 334

sufficient to increase islet cell proliferation (data not shown). Moreover, treatment of Pdx-1-335

transduced islets with Camk1d siRNA, CB1 siRNA, Camk1d dominant negative (DN) or 336

PKCβ1DN adenoviruses did not affect Pdx-1-induced islet cell replication (data not shown). 337

In addition to the abovementioned genes, two members of the TRPC channel family, TRPC3 and 338

TRPC6, were significantly upregulated in rat islets overexpressing Pdx-1 but not Nkx6.1. 339

Importantly, TRPC3 and TRPC6 have been implicated in cellular proliferation in other cellular 340

systems (38-40). To verify the microarray data, we measured mRNA levels via real-time PCR 341

and found that TRPC3 and TRPC6 mRNA levels were approximately 150- and 3-fold higher, 342

respectively, in AdCMV-Pdx-1-treated islets compared to control islets (Figure 5C). Although 343

the Pdx-1-induced increase in TRPC6 mRNA at 48 h was not statistically significant compared 344

to control islets, the increase in TRPC6 mRNA levels induced by Pdx-1 at 96 h was significant 345

(Figure 7E). Pdx-1 overexpression also induced TRPC6 protein levels at 48 h post-transduction 346

in 832/13 cells (Figure 5D). These data demonstrate that Pdx-1 stimulates rat islet proliferation 347

as early as 48 h and suggest that TRPC3/6 may be involved in the mechanism of Pdx-1-348

stimulated proliferation. 349

Pdx-1, TRPC3 or TRPC6 overexpression increases channel activity in 832/13 cells 350

To test if Pdx-1, TRPC3 and TRPC6 regulate channel activity in β-cells as suggested by the GO 351

analysis, we measured membrane currents (INSC) by a whole-cell voltage clamp method in 352

17

832/13 rat insulinoma cells following adenovirus-mediated overexpression of Pdx-1, Flag-353

TRPC3 or Myc-TRPC6 (Figures 6A-6D). Currents were significantly higher in cells 354

overexpressing Pdx-1, Flag-TRPC3 and Myc-TRPC6 compared to control cells at -80 mV 355

(p<0.05) (Figure 6E). As expected for TRPC channels, the current was increased after perfusion 356

with 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable analog of diacylglycerol (DAG), and 357

was inhibited by gadolinium (Gd) (Figures 6C-6D). OAG increased INSC by 4- to 5-fold in cells 358

overexpressing Pdx-1, Flag-TRPC3 or Myc-TRPC6 but caused only a slight increase 359

(approximately 50%) in GFP-treated control cells (Figure 6F). These data demonstrate that 360

overexpressed TRPC3 and TRPC6 proteins are fully functional and that Pdx-1 induces a current 361

consistent with increased expression of these same channels. 362

TRPC3/6 is necessary but not sufficient for Pdx-1-stimulated rat islet proliferation 363

We next tested if overexpression of TRPC3 and TRPC6 was sufficient to induce rat islet 364

proliferation. Neither Flag-TRPC3 nor Myc-TRPC6 expression stimulated rat islet proliferation 365

compared to control islets (Figure 7A) even with robust protein overexpression (Figure 7B), and 366

co-overexpression of Flag-TRPC3 and Myc-TRPC6 also had no effect (data not shown). These 367

data demonstrate that increased expression of TRPC3 and TRPC6 is not sufficient to promote rat 368

islet proliferation. 369

To test if TRPC3/6 were necessary for Pdx-1-stimulated proliferation, we treated rat islets with 370

adenoviruses expressing a siRNA targeting a sequence common to TRPC3 and TRPC6 371

(siT3/T6), a separate siRNA specific for TRPC6 (siT6) or a non-targeting scrambled siRNA 372

sequence (siScr). We then treated the same islets with adenoviruses overexpressing βgal, Pdx-1 373

or Nkx6.1. Treatment with the siT3/T6 virus resulted in a 75 and 70% knockdown of TRPC3 374

18

and TRPC6 mRNA levels, respectively, whereas an approximate 50% knockdown of TRPC6 375

mRNA levels was obtained in response to treatment with the siT6 adenovirus. Pdx-1-stimulated 376

rat islet proliferation was inhibited by 38 and 50% in response to knockdown of TRPC3/6 and 377

TRPC6, respectively (Figures 7C-7E). The similar impact of the two knockdown adenoviruses is 378

likely due to the fact that endogenous expression of TRPC6 is higher than that of TRPC3, 379

making TRPC6 the more physiologically relevant channel. In contrast, Nkx6.1-stimulated rat 380

islet proliferation was unaffected by knockdown of both TRPC3 and TRPC6 or TRPC6 alone 381

(Figure 7C). Importantly, downregulation of TRPC3/6 and TRPC6 caused a significant decrease 382

in cyclin D2 mRNA levels (Figure 7F). Together, these data demonstrate that TRPC3 and 383

TRPC6 are not sufficient but are necessary for maximal Pdx-1-mediated proliferation. These 384

results further define distinct mechanisms by which Pdx-1 and Nkx6.1 regulate rat islet 385

proliferation. 386

Activated ERK1/2 is downstream of TRPC3/6 and is required for maximal Pdx-1-387

stimulated rat islet proliferation 388

Because TRPC3/6 have been shown to activate calcineurin/NFAT signaling (41), which has been 389

implicated in β-cell proliferation, function and survival (42, 43), we next tested if this pathway 390

was necessary for Pdx-1-stimulated rat islet cell proliferation by blocking calcineurin activity 391

with cyclosporin A (CsA). While CsA completely blocked rat islet proliferation induced by CnA 392

overexpression, it was unable to block Pdx-1-stimulated rat islet proliferation (Figure 8A). 393

We next measured the phosphorylation levels of several signaling molecules and found that Pdx-394

1 clearly increased ERK1/2 phosphorylation levels in rat islets compared to controls (Figure 8B). 395

Pdx-1 overexpression and simultaneous knockdown of TRPC3/6 (approximately 60% 396

19

knockdown of TRPC3/6 as verified by real-time PCR; data not shown) in 832/13 cells inhibited 397

Pdx-1-induced phosphorylation of ERK1/2 by approximately 40% relative to siScr control cells 398

(Figures 8C-8D). Furthermore, inhibition of ERK1/2 with the specific MEK1/2 inhibitor, U0126, 399

caused a 35% decrease in Pdx-1-stimulated rat islet proliferation (Figure 8E). Overall, these data 400

define a novel pathway in which Pdx-1 overexpression induces expression of the calcium 401

channel proteins, TRPC3/6, which in turn activate ERK1/2. This pathway is required for full 402

activation of rat islet proliferation and the cyclin D/cdk4 complex in response to Pdx-1 403

overexpression (see Model in Figure 8F). 404

405

20

Discussion 406

Loss of functional β-cell mass is central to the development of both type 1 and type 2 diabetes. 407

Much effort is therefore being directed towards developing strategies for inducing the remaining 408

β-cells to proliferate in a controlled manner or to grow functional β-cells ex vivo for 409

transplantation into diabetic patients. To achieve these goals, it is crucial to understand 410

mechanisms for inducing β-cell proliferation with retention of key functions. In the present 411

study, we demonstrate that Pdx-1 induces rat islet cells to proliferate and that co-overexpression 412

of Pdx-1 and Nkx6.1 causes an additive proliferative effect. Subsequent experiments show that 413

the two transcription factors mediate proliferation by completely separate mechanisms, including 414

differences in cell type specificity, timing of proliferation, target genes, signaling pathways, and 415

activation of distinct components of the core cell cycle machinery. 416

Whereas Nkx6.1 induces mainly β-cell proliferation, Pdx-1 stimulates both α- and β-cell 417

proliferation, even though both transcription factors are being expressed in all islet cells from the 418

constitutive CMV promoter. While the importance of increasing β-cell proliferation is well 419

known (44), the significance of increasing α-cell proliferation has not been fully recognized until 420

recently. Recent studies have suggested that α-cells can act as a pool of precursors for α- to β-cell 421

conversion leading to β-cell regeneration (45, 46). Several models involving β-cell depletion 422

have been used to demonstrate transdifferentiation of mature α-cells into β-cells. In one study 423

involving β-cell ablation with diphtheria toxin and lineage tracing, several months were required 424

for complete α- to β-cell conversion, but impressively, mice eventually became normoglycemic, 425

suggesting that α-cells might be a physiologically relevant depot of β-cell progenitors (45). In 426

another model using pancreatic duct ligation coupled with destruction of β-cells with alloxan, α- 427

to β-cell conversion was demonstrated within two weeks (46). More recently, forced expression 428

21

of Pdx-1 in early endocrine progenitors was shown to cause α- to β-cell conversion (47). Thus, if 429

Pdx-1 overexpression is sufficient to promote proliferation of mature α-cells, as demonstrated in 430

the present study, perhaps Pdx-1 can be used to enhance the efficiency and extent of α-cell to β-431

cell transdifferentiation by increasing the pool of α-cells. Further lineage tracing studies will be 432

required to determine if α-cells that are proliferating as a result of Pdx-1 expression undergo cell 433

type conversion. 434

Pdx-1 and Nkx6.1 also differ with regard to timing of their proliferative effects such that Pdx-1 435

stimulates proliferation as early as 48 h post-transduction (the present study) and Nkx6.1 436

activates proliferation 72 h post-transduction (9). We have recently found that Nkx6.1-mediated 437

proliferation requires upregulation of the orphan nuclear receptors, Nr4a1 and Nr4a3. These 438

genes are induced by Nkx6.1 in the first 24-48 h of Nkx6.1 overexpression and then require an 439

additional 48 h to exert their effects, thereby explaining the slow induction of proliferation by 440

Nkx6.1 (Tessem et al., in revision). In contrast, Pdx-1 induces TRPC3/6 expression, ERK 441

activation and cyclin D upregulation within 48 h of its expression. 442

With regard to downstream target genes, both Pdx-1 and Nkx6.1 affect the expression of a 443

diverse array of genes. When comparing microarray datasets obtained in primary rat islets 48 h 444

after Pdx-1 or Nkx6.1 overexpression, only 20 genes were found to be commonly induced, 445

whereas approximately 260 and 430 genes were induced by Pdx-1 and Nkx6.1 alone, 446

respectively. In addition to the TRPC channels, other calcium-related genes found to be highly 447

upregulated by Pdx-1 in the microarray analysis included Calbindin 1, Calbindin 2, Camk1d, 448

CB1 and PKCβ1. These other genes have been previously implicated in proliferation, although 449

mostly in non-islet studies. However, overexpression or inhibition of these genes via knockdown 450

or use of dominant-negative constructs did not affect islet cell proliferation or interfere with Pdx-451

22

1-stimulated proliferation. In contrast, knockdown of TRPC3 and/or TRPC6 significantly 452

inhibited Pdx-1-stimulated rat islet proliferation, underscoring the specific and selective role of 453

these channels in mediating the Pdx-1 proliferative effect. TRP channels are divided into seven 454

subfamilies, and three subfamilies (TRPC, TRPM and TRPV) have been detected in pancreatic 455

β-cells (48). TRP channels are involved in sensing intracellular calcium levels in β-cells to 456

mediate different aspects of insulin secretion (49, 50) and may be involved in β-cell stress and 457

islet inflammation via regulation of neuropeptide release from surrounding neuronal cells (51). 458

While TRP channels have been implicated in cellular proliferation in other cell types (38-40), 459

only TRPV2 has been linked to β-cell proliferation in studies with the mouse insulinoma cell 460

line, Min6 (52). Thus, to our knowledge, the present study is the first to implicate TRPC3/6 in 461

the regulation of proliferation in adult islet cells. 462

A pathway downstream of TRP channels that is known to lead to hypertrophy (41) and β-cell 463

proliferation (42, 43) is the calcineurin/NFAT pathway. However, our studies demonstrate that 464

the calcineurin/NFAT pathway is not required for Pdx-1-induced proliferation. Thus, we 465

investigated other potential downstream signaling pathways and found that Pdx-1 increases the 466

phosphorylation of ERK1/2, a known cell cycle regulator (53) that is activated in concert with β-467

cell expansion and induction of HNF4α (54). Furthermore, it has been shown that calcium influx 468

via TRPC3 can sustain PKCβ and ERK1/2 activation in B-cells (55) consistent with our finding 469

that TRPC3/6 expression is necessary for the full activation of ERK1/2 by Pdx-1. The fact that 470

TRPC3/6 knockdown or ERK1/2 inhibition only partially impairs Pdx-1-mediated islet cell 471

proliferation suggests that Pdx-1 may activate additional pathways upstream of the cyclin D/cdk4 472

complex. Further studies will be required to identify such pathways. 473

23

We attempted to define a mechanism by which Pdx-1 regulates TRPC channels by transfecting 832/13 474

insulinoma cells with an 1800 bp fragment of the human TRPC3 promoter driving a luciferase reporter 475

gene (30) and then overexpressing Pdx-1 via our adenoviral vector. In these assays, Pdx-1 caused only a 476

trend for increased luciferase reporter activity that was not statistically significant. Moreover, we 477

analyzed a recent ChIP-Seq analysis of Pdx-1 binding sites (56) and found that it did not reveal any sites 478

in the vicinity of the TRPC3 or TRPC6 genes, consistent with the lack of signal in the reporter gene 479

analysis. Possible explanations for these findings could include the need for other regulatory sequences 480

at some distance from the TRPC genes or that Pdx-1 is exerting its effects via an indirect mechanism. 481

The final difference between Pdx-1- and Nkx6.1-stimulated proliferation uncovered in this study is the 482

involvement of different core cell cycle factors. Previous studies have shown that cdk4, cyclin D2 or 483

cyclin D1/D2 knockout mice have decreased β-cell mass resulting in diabetes (57, 58). The present 484

study shows that the cyclin D/cdk4 complex is required for Pdx-1-stimulated proliferation but also 485

demonstrates that these core cell cycle molecules are not required in all instances of adult β-cell 486

proliferation because blocking the cyclin D/cdk4 complex does not inhibit Nkx6.1-stimulated 487

proliferation. This is consistent with our prior studies showing that Nkx6.1 induces the expression of 488

cyclins E, A and B but not D, and that overexpression of cyclin E is sufficient to activate islet cell 489

replication (9). Interestingly, a previous study has shown that cyclin D2/cdk4/GLP1 overexpression 490

preferentially induces β-cell proliferation, whereas cyclin D2/cdk4 overexpression preferentially induces 491

α-cell proliferation (59). This suggests the possibility that additional factors acting upstream of cyclin 492

D/cdk4 may exist that focus the proliferative effects of Pdx-1 in β-cells. Further studies are required to 493

elucidate the potential cell-type specific mechanisms of Pdx-1-stimulated proliferation. 494

24

In conclusion, our findings map out a novel pathway by which Pdx-1 stimulates islet cell 495

replication. Knowledge of this pathway and the separate one by which Nkx6.1 induces 496

proliferation may lead to identification of small molecules that target key components of these 497

pathways for expansion of functional β-cell mass in diabetes. 498

25

Acknowledgements 499

This work was supported by a grant from the National Institutes of Health β-cell biology 500

consortium (BCBC) (U01 DK-089538) to HEH and CBN, Juvenile Diabetes Research 501

Foundation (JDRF) grants 17-2011-15 (to CBN) and 17-2011-614 (to HEH) and a JDRF 502

postdoctoral fellowship to HLH (3-2009-561). The authors would like to thank Drs. Samuel 503

Stephens and Jeffery Tessem for helpful advice and discussion as well as Danhong Lu, Helena 504

Winfield, Lisa Poppe, and Paul Anderson for expert technical assistance. We would like to thank 505

the Duke Microarray Core facility (a Duke National Cancer Institute and a Duke Institute for 506

Genome Sciences and Policy shared resource facility) for their assistance in generating the 507

microarray data reported in this manuscript. 508

509

26

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Figure Legends

Figure 1. Effects of overexpressed Pdx-1 and Nkx6.1 on rat islet cell proliferation and effect 687

of Pdx-1 on human islet cell proliferation. Primary rat islets were left untreated or treated with 688

recombinant adenoviruses (AdCMV) expressing βgal, Pdx-1+βgal, Nkx6.1+βgal or Pdx-689

1+Nkx6.1 as indicated for 18 h and cultured for an additional 78 h. (A) [3H]-thymidine 690

incorporation was measured. Data represent the mean ± SEM of five independent experiments. * 691

p<0.01, ** p<0.001, ns=not significant; # p<0.01 vs. Pdx-1; $ p<0.001 vs. Nkx6.1 and Pdx-692

1+Nkx6.1 (n=5). (B) Pdx-1 and Nkx6.1 protein expression levels as measured by immunoblot 693

analysis. Immunoblot is representative of five independent experiments. (C) Human islets from 694

10 separate donors were treated with adenoviruses expressing either βgal or Pdx-1 for 18 h and 695

cultured for an additional 78 h. [3H]-thymidine incorporation was measured. Data represent the 696

mean ± SEM of ten independent experiments. * p<0.01 according to a paired t-test. 697

Figure 2. Pdx-1 stimulates proliferation of both α- and β-cells whereas Nkx6.1 stimulates 698

mainly β-cell proliferation. Primary rat islets were left untreated or treated with recombinant 699

adenoviruses (AdCMV) expressing βgal, Pdx-1+βgal, Nkx6.1+βgal or Pdx-1+Nkx6.1 as 700

indicated for 18 h and cultured for an additional 78 h. Islets were collected, embedded in 701

paraffin, sectioned and used for histochemical analysis. (A) EdU was detected using the Click-iT 702

kit (top panels), and antibodies that detect insulin and glucagon were used to stain the sections 703

(overlay of EdU, insulin and glucagon; bottom panels). Yellow arrows indicate glucagon-704

positive cells (α-cells) with EdU incorporation, and white arrow indicates insulin- and glucagon-705

negative cells with EdU incorporation. All other Edu+ cells shown are insulin-positive cells (β-706

cells). Percentage of (B) total cells expressing EdU, (C) insulin-positive cells (β-cells) expressing 707

EdU and (D) insulin-negative cells (mainly α-cells) expressing EdU. * p<0.05, ** p<0.01, *** 708

35

p<0.001 and ns=not significant (n=4). (E) Comparison of insulin-positive and insulin-negative 709

EdU+ cells (%). # p<0.001 vs. Pdx-1 and Pdx-1+Nkx6.1 for INS+. $ p<0.001 vs. Pdx-1 and Pdx-710

1+Nkx6.1 for INS-. Data represent the mean ± SEM of four independent experiments. 711

Figure 3. Both Pdx-1 and Nkx6.1 promote increased pHH3 staining but Pdx-1 does not 712

significantly increase γH2AX staining. (A and B) Primary rat islets were left untreated or 713

treated with recombinant adenoviruses (AdCMV) expressing βgal, Pdx-1+βgal, Nkx6.1+βgal or 714

Pdx-1+Nkx6.1 as indicated for 18 h and cultured for an additional 78 h. (A) EdU was detected 715

using the Click-iT kit (top panels), and antibodies that detect insulin and pHH3 were used for 716

staining (overlay of EdU, pHH3 and insulin; right panel). Yellow arrows indicate double positive 717

cells (EdU+pHH3+). (B) Percentages of total EdU+, pHH3+ and EdU+pHH3+ cells were 718

calculated. Data represent the mean ± SEM of three independent experiments. * p<0.001 vs. NV 719

and βgal; ** p<0.001 vs. Pdx-1 and Nkx6.1; $ p<0.001 vs. Nkx6.1; and # p<0.01 vs. Pdx-1 720

(n=3). (C-E) Primary rat islets were left untreated or treated with recombinant adenoviruses 721

expressing βgal or Pdx-1 as indicated for 18 h and cultured for an additional 78 h. (C) EdU was 722

detected using the Click-iT kit (top panels), and antibodies that detect insulin and γH2AX were 723

used for staining (overlay of EdU, γH2AX and insulin; right panel). Yellow arrow indicates 724

double positive cells (EdU+γH2AX+). (D) Percentages of total EdU+, γH2AX+ and 725

EdU+γH2AX+ cells were calculated. * p<0.001 and ** p<0.001. (E) Percentage of γH2AX 726

staining of EdU+ cells in Pdx-1-treated islets. Data represent the mean ± SEM of three 727

independent experiments. 728

Figure 4. Pdx-1 but not Nkx6.1 increases cyclin D1/D2 levels and requires the cyclin D/cdk4 729

complex to stimulate rat islet proliferation. Primary rat islets were left untreated or treated 730

with recombinant adenoviruses (AdCMV) expressing βgal, Pdx-1+βgal, Nkx6.1+ βgal or Pdx-731

36

1+Nkx6.1 as indicated for 18 h and cultured for an additional 78 h. (A and B) Quantitative RT-732

PCR (qRT-PCR) was used to measure mRNA levels of cyclins E1, E2, D1, D2 and D3. Data 733

represent the mean ± SEM of five independent experiments. # p<0.001 and * p<0.01 as 734

compared to NV or βgal; @ p<0.05 as compared to NV; & p<0.01 as compared to βgal; $ 735

p<0.01; ** p<0.001; and ns = not significant (n=5). (C and D) Primary rat islets were treated 736

with recombinant adenoviruses (AdCMV) expressing βgal, Pdx-1 or Nkx6.1 as indicated for 18 737

h and cultured for an additional 78 h. During the last 48 h of culture, vehicle or 300 nM 738

PD0332991 (specific cdk4 inhibitor) was added to the culture media. (C) [3H]-thymidine 739

incorporation was measured. (D) qRT-PCR was used to measure mRNA levels of Pdx-1, 740

Nkx6.1, cyclin D1, cyclin D2 and cdk4. Data represent the mean ± SEM of three independent 741

experiments. * p<0.01 vs. control (n=3). 742

Figure 5. Pdx-1 stimulates rat islet proliferation and upregulates TRPC3/6 expression as 743

early as 48 h post-transduction. (A) Time course of [3H]-thymidine incorporation into rat islets 744

using AdCMV-Pdx-1, AdCMV-GFP or NV control. Data represent the mean ± SEM of three 745

independent experiments. ** p<0.001 as compared to NV and GFP (n=3). (B) Time course of 746

Pdx-1 protein expression levels as measured by immunoblot analysis. Immunoblot is 747

representative of three independent experiments. (C) qRT-PCR was used to measure mRNA 748

levels of Pdx-1, TRPC3 and TRPC6 at 48 h post-transduction of AdCMV-Pdx-1, AdCMV-βgal 749

or NV control. Data represent the mean ± SEM of three independent experiments. * p<0.01, ** 750

p<0.001 and ns=not significant as compared to NV or βgal controls (n=3). (D) 832/13 cells were 751

left untreated or treated with AdCMV- βgal or AdCMV-Pdx-1. Cells were harvested 48 h post-752

transduction. TRPC6 and Pdx-1 protein levels were measured via immunoblot analysis. 753

Immunoblot is representative of three independent experiments. 754

37

Figure 6. Pdx-1, TRPC3 and TRPC6 increase TRP channel activity in 832/13 cells. 755

Membrane currents (INSC) were recorded by a whole-cell voltage clamp method in 832/13 cells 756

with adenovirus-mediated overexpression of Pdx-1, TRPC3, or TRPC6 for 48 h. The current was 757

induced by a 200 ms voltage ramp protocol (1 mV/ms, from 100 mV to –100 mV and holding 758

potential of 0 mV; see inset), and it was normalized by membrane capacitance. Examples of I-V 759

relation of INSC and OAG responses recorded from individual 832/13 cells expressing (A) GFP, 760

(B) Pdx-1, (C) TRPC3 and (D) TRPC6. To verify TRP channel activity, 20 µM Gd was added, 761

which blocks TRP channel activity. (E) Group mean values of baseline INSC at -80 mV and +80 762

mV in control GFP-expressing cells (n=20), Pdx-1-expressing cells (n=39), TRPC3-expressing 763

cells (n=20) and TRPC6-expressing cells (n=14). * p<0.05. (F) Group mean changes (%) of INSC 764

at -80mV caused by perfusion of 50 µM OAG in cells expressing GFP, Pdx-1, TRPC3 and 765

TRPC6. * p<0.05. 766

Figure 7. TRPC3/6 are necessary but not sufficient for Pdx-1-stimulated rat islet 767

proliferation. Primary rat islets were treated with recombinant adenoviruses (AdCMV) 768

expressing βgal, Pdx-1, Flag-TRPC3 or Myc-TRPC6 as indicated for 18 h and cultured for an 769

additional 78 h. (A) [3H]-thymidine incorporation was measured. Data represent the mean ± 770

SEM of three independent experiments. * p<0.01 (n=3). (B) Protein expression levels as 771

measured by immunoblot analysis. Immunoblot is representative of three independent 772

experiments. (C-F) Primary rat islets were treated with recombinant adenoviruses containing 773

siScr, siT3/T6 or siT6 as indicated for 18 h. Media was then changed, and islets were treated 774

with adenoviruses (AdCMV) overexpressing βgal, Pdx-1 or Nkx6.1 as indicated for 18 h and 775

cultured for an additional 84 h. (C) [3H]-thymidine incorporation was measured. Data represent 776

the mean ± SEM of three independent experiments. Solid bars indicate three independent 777

38

experiments using siSCr and siT3/T6 adenoviruses, and lined bars indicate three independent 778

experiments using siScr and siT6 adenoviruses. * p<0.01 and ** p<0.001 as compared to siScr 779

(n=3). (D-F) qRT-PCR was used to measure mRNA levels of (D) TRPC3, (E) TRPC6 and (F) 780

cyclin D2. Data represent the mean ± SEM of three independent experiments. * p<0.01 and ** 781

p<0.001 as compared to siScr (n=3). 782

Figure 8. Pdx-1 requires ERK1/2 activation for maximal proliferative effect. Primary rat 783

islets were treated with recombinant adenoviruses (AdCMV) expressing βgal, Pdx-1 or 784

constitutively active calcineurin (CnA) as indicated for 18 h and cultured for an additional 78 h. 785

During the last 48 h, 1 µM cyclosporin A (CsA) was added to the culture media. (A) [3H]-786

thymidine incorporation was measured. ** p<0.001 as compared to control (n=3). (B) Primary 787

rat islets were left untreated or treated with recombinant adenoviruses (AdCMV) expressing βgal 788

or Pdx-1 as indicated for 18 h and cultured for an additional 78 h. Phosphorylation and protein 789

levels were measured by immunoblot analysis. Immunoblot is representative of three 790

independent experiments. (C and D) 832/13 cells were left untreated or treated with an 791

adenovirus overexpressing Pdx-1 for 4 h. Media was then changed, and transfection of 50 nM 792

siRNAs was performed 2 h later. Cells were harvested after an additional 72 h of culture. 793

Immunoblot is representative of three independent experiments. (D) Quantification of protein 794

levels. Fold change is the pixel density ratio of phosphorylated ERK1/2 protein levels to total 795

ERK1/2 protein levels normalized to siScramble control. * p<0.05 (n=3). (E) Primary rat islets 796

were left untreated or treated with recombinant adenoviruses (AdCMV) expressing βgal or Pdx-1 797

as indicated for 18 h and cultured for an additional 78 h. During the last 48 h, 10 µM U0126 was 798

added to the culture media. [3H]-thymidine incorporation was measured. ** p<0.001 as 799

compared to control (n=3). Data represent the mean ± SEM of three independent experiments. 800

39

(F) Model of Pdx-1-stimulated rat islet proliferation. Dashed line and grey text indicate partial 801

inhibition. 802

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