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Originally published In Press as doi:10.1074/jbc.M706105200 on January 23, 2008

J. Biol. Chem., Vol. 283, Issue 13, 8723-8735, March 28, 2008
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Glucagon-like Peptide-1 Activation of TCF7L2-dependent Wnt Signaling Enhances Pancreatic Beta Cell Proliferation*Formula

Zhengyu Liu and Joel F. Habener1

From the Laboratory of Molecular Endocrinology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Received for publication, July 25, 2007 , and in revised form, January 18, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The insulinotropic hormone GLP-1 (glucagon-like peptide-1) is a new therapeutic agent that preserves or restores pancreatic beta cell mass. We report that GLP-1 and its agonist, exendin-4 (Exd4), induce Wnt signaling in pancreatic beta cells, both isolated islets, and in INS-1 cells. Basal and GLP-1 agonist-induced proliferation of beta cells requires active Wnt signaling. Cyclin D1 and c-Myc, determinants of cell proliferation, are up-regulated by Exd4. Basal endogenous Wnt signaling activity depends on Wnt frizzled receptors and the protein kinases Akt and GSK3β but not cAMP-dependent protein kinase. In contrast, GLP-1 agonists enhance Wnt signaling via GLP-1 receptor-mediated activation of Akt and beta cell independent of GSK3β. Inhibition of Wnt signaling by small interfering RNAs to β-catenin or a dominant-negative TCF7L2 decreases both basal and Exd4-induced beta cell proliferation. Wnt signaling appears to mediate GLP-1-induced beta cell proliferation raising possibilities for novel treatments of diabetes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The gut-derived insulinotropic hormone GLP-1 (glucagon-like peptide-1) and its long acting agonist exendin-4 (Exd4) are new agents for the treatment of diabetic patients (1). GLP-1 is a peptide hormone arising by its alternative enzymatic cleavage from proglucagon, the prohormonal precursor of GLP-1 (2, 3). GLP-1 is released from the enteroendocrine cells of the gut in response to meals, stimulates glucose-dependent insulin secretion, and lowers blood glucose levels in type 2 diabetic subjects. Initial observations established that GLP-1 is a potent insulin secretagogue (4). Subsequently, multiple anti-diabetogenic actions of GLP-1 were discovered, including the stimulation of the proliferation and the inhibition of the apoptosis of insulin-producing pancreatic beta cells (5-8).

GLP-1 induces multiple signaling pathways intrinsic to beta cell function. Activation of the GLP-1 receptor (GLP-1R) by GLP-1 or Exd4 leads to the accumulation of cAMP and the activation of cAMP-dependent protein kinase A (PKA),2 mediated through the activation of adenylyl cyclase and the stimulatory G protein G{alpha}S. Recent studies determined that GLP-1 receptors activate several other second messengers, including mitogen-activated protein kinase (9), phospholipase C (10), intracellular Ca2+ (11), and phosphatidylinositol 3-kinase (12). The ability of the GLP-1 receptor to regulate such diverse responses appears to result from its promiscuous G protein coupling and its actions to mediate intracellular receptor crosstalk. For example, GLP-1 is reported to enhance beta cell proliferation via transactivation of the EGF receptor (EGFR) and its downstream effector PI3K (13).

The canonical β-catenin-dependent Wnt signaling pathway is important in the modulation of cell proliferation, survival, migration and differentiation, and in organ development (14-17). It is also a regulator of stem cell fate determination and cancer (18, 19). In the absence of Wnt ligand, cytoplasmic β-catenin is phosphorylated by GSK3β (glycogen synthase kinase 3β), within a protein complex containing axin and adenomatous polyposis coli (APC) protein. The phosphorylation of β-catenin by GSK3β results in its ubiquination and degradation (inactivation) (15, 20). The binding of Wnt ligands to the frizzled receptors activates the intracellular protein, Dishevelled (Dvl), which inhibits APC-GSK3β-axin activity, leading to the accumulation of free cytosolic β-catenin. Subsequently, β-catenin translocates to the nucleus and forms a transcriptionally productive complex with members of the lymphocyte enhancer factor (LEF)/T cell factor (TCF) family of transcription factors, such as TCF7L2, to activate the expression of Wnt signaling target genes (19).

The role of Wnt signaling in pancreas development and functions is unclear. The expression of several Wnt and frizzled receptor genes has been detected in the developing pancreatic mesenchyme and epithelium. Misexpression of Wnt1 and Wnt5a in the early foregut results in agenesis or hypoplasia of the pancreas, respectively (21). Deletion of β-catenin within the pancreatic epithelium results in either a loss (22) or no change (23) in beta cell mass. Ectopic stabilization of β-catenin deregulates the normal mechanisms that control embryonic pancreas formation and postnatal organ growth (24).

Based on a preliminary study of pancreatic islet gene expression profiling on microarrays, we recognized that GLP-1 and Exd4 induced the expression of genes in both the Wnt signaling pathway per se and target genes of the Wnt signaling pathway (Table S1). These genes are activated by the transcriptional complex of β-catenin and TCF/LEF. These microarray findings prompted us to examine in greater detail the regulation of Wnt signaling in pancreatic beta cells by GLP-1 agonists and the role of Wnt signaling in the GLP-1 agonist stimulation of beta cell proliferation. In this study, we examined the Exd4 activation of Wnt signaling in isolated islets and the INS-1 beta cell line using a β-catenin/TCF-activated reporter gene assay and found that GLP-1 and Exd4 enhance Wnt signaling. By using specific protein kinase inhibitors, dominant-negative isoforms of the kinases and of TCF7L2, and siRNA knockdown of β-catenin, we find that the basal Wnt signaling is dependent on endogenous Wnt ligands and requires active Akt and the inactivation of GSK3β. In marked contrast, GLP-1 and Exd4 activate Wnt signaling through the GLP-1 receptor coupled to the activation of cAMP-dependent protein kinase A (PKA), and the prosurvival kinase Akt/PKB, independent of GSK3β. Exd4-mediated activation of PKA phosphorylates β-catenin on Ser-675, a mechanism that appears to stabilize β-catenin and to enhance TCF7-L2 activation of gene expression in beta cells. We also provide evidence that active Wnt signaling stimulates the proliferation of beta cells as a dominant-negative TCF7L2 inhibits both basal and Exd4-stimulated INS-1 cell proliferation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transient Transfection—INS-1 cells (insulin-producing strain obtained from C. Wolheim, Geneva, Switzerland) were maintained in RPMI supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 µg/ml), and streptomycin (0.25 µg/ml) at 37 °C under 5% CO2 and at 95% humidity. Transfections were done with Lipofectamine 2000 (Invitrogen) according to the manufacturer's manual. For stable transfections, INS-1 cells were cotransfected with pcDNA3-myc-dnTCF7L2, encoding the dominant-negative mutant of TCF7L2 (dnTCF7L2), or a control empty vector and puromycin vector, containing a puromycin-selectable marker. Two days after their transfection, cells were selected for resistance to puromycin for 2 weeks. Puromycin-resistant clones were screened for dnTCF7L2 expression with a Myc monoclonal antibody against the Myc epitope after 2 weeks of selection, and positive clones were pooled.

Plasmids—The TOPflash and FOPflash luciferase vectors were gifts from R. Moon, University of Washington. Dominant-negative form of the regulatory subunit of PKA was from G. S. McKnight, University of Washington. Dominant-negative form of the PI3K was from J. Du, Baylor College of Medicine. Dominant-negative forms of CREB were from Clontech.

Isolated Mouse Pancreatic Islets—Mouse islets were isolated from the pancreata of TOPGAL mice transgenic for the LEF-lacZ Wnt signaling reporter (25). Freshly isolated islets were treated for 4 h with Exd4 with and without the addition of the PKA inhibitor H89 or the GLP-1R antagonist Exd-(9-39). β-Galactosidase activity was determined by incubation of the islets with X-gal for 6 h. For BrdUrd proliferation assay, islets were treated with 0.01% trypsin for 5 min while lightly disrupting them with a pipette. Islets were then resuspended in a mixture of media and/or retrovirus and then spun down at 2000 rpm for 60 min.

Wnt Signaling Luciferase Reporter Assay (TOPflash)—INS-1 cells were plated into 24-well dishes 24 h before transfection with TOPflash (1 µg/well) or FOPflash (1 µg/well) using Lipofectamine 2000 (Invitrogen). Various concentrations of GLP-1 or Exd4 were then added to the culture medium 24 h following transfection for the indicated times. In studies in which inhibitors were used, LY294002 (10 µM), AG1478 (10 µM), H89 (10 µM), U0126 (10 µM), PD98059 (10 µM), SB203580 (1 µM), or Akt inhibitor IV (10 nM) were added concomitantly with Exd4. In studies in which wild-type, dominant-negative (dn), or constitutively active (ca) forms of kinase were used, dnPI3K, dnPKA, caPKA, dnGSK3β, caGSK3β, wild-type CREB, dnCREB, dnAkt, or caAkt (0.5 µg/well) was co-transfected with TOPflash. Luciferase activity in transfected cells was determined with a luciferase assay kit (Promega).

Cyclic AMP-response Element Luciferase Reporter Assay—INS-1 cells were plated into 24-well dishes 24 h before transfection with cAMP-response element (CRE) luciferase (1 µg/well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Various concentrations of GLP-1 or Exd4 were then added to the culture medium 24 h following transfection for the indicated time.

dnTCF7L2 Retroviral Plasmids and Retroviral Infection of INS-1 Cells and Isolated Islets—A dnTCF7L2 fragment was subcloned into the BamHI and XhoI sites of retroviral vector pBMN (Orbigen). Retrovirus was prepared by transfecting phoenix cells with control (pBMN) or dnTCF7L2 retroviral vector (pBMN-dnTCF7L2). Virus-containing medium was collected 36 h after transfection and passed through a 0.45-µm syringe filter. Polybrene (hexadimethrine bromide; Sigma) was added to a final concentration of 8 µg/ml. This medium was then applied to subconfluent (50%) INS-1 cells (10-cm plates) or isolated islets for 36 h, and the infected cells were used in the subsequent cell proliferation assay.

Wnt3A Conditioned Media—Wnt3A conditioned media and blank conditioned media were obtained from the ATCC. Wnt3A conditioned medium was prepared from TH L-M(TK-) cells transfected with a Wnt3A expression vector and selected in medium containing G418. Selected stable clones encode a secreted biologically active Wnt3A protein. Blank conditioned medium was collected from a parental cell line.

Mfz8CRD-IgG Conditioned Media—Mfz8CRD-IgG was produced in 293 cells that were transiently transfected by the mfz8CRD-IgG cDNA (a kind gift from Dr. J. Nathans, The Johns Hopkins University) using Lipofectamine 2000. One day after transfection, cells were transferred to serum-free Dulbecco's modified Eagle's medium, and the conditioned medium was harvested after an additional 24 h. Control conditioned medium was obtained from untransfected 293 cells.

Real Time RT-PCR—Real time RT-PCR was carried out by using the SYBR® Green QPCR kit (Stratagene). Briefly, INS-1 cells were treated with 10 nM Exd4 or PBS vehicle control for 4 h. Total RNA was reverse-transcribed to cDNA using Super-Script II reverse transcriptase (Invitrogen). PCR was performed to amplify cyclin D1 by using the following primers, forward 5'-TGTTTGAGACCTTCAACACC-3' and reverse 5'-CCAGACAGCACTGTGTTGGC-3', and to amplify Myc by using the following primers, forward 5'-CTGCTCTCCGTCCTATGTTG-3' and reverse 5'-CCTGGATGATGATGTTCTTGATG-3'. For measurements of β-galactosidase and mRNA levels, real time quantitative PCR was used. Isolated islets prepared from TOPGAL mice were treated with 10 nM Exd4 or PBS vehicle control for 4 h. Total RNA was prepared from isolated islets of TOPGAL mice and reverse-transcribed to cDNA. Real time RT-PCR was performed to amplify the β-galactosidase encoding lacZ transcript by using the following primers, forward 5'-GTACGGCAGTTATCTGGAAG-3' and reverse 5'-CATAACCACCACGCTCATCG-3'.

Western Immunoblots—Membrane immunoblots were prepared from extracts of INS-1 cells and were interrogated with antisera to β-catenin as follows: total protein, the destabilizing GSK-3 phosphorylation sites (UpState catalog number 46-626), and the stabilizing PKA-mediated phosphorylation site, Ser-675, that stabilizes β-catenin (AnaSpec catalog number 29619).

siRNA-mediated Knockdown of β-Catenin Expression—siRNAs against rat β-catenin (GenBankTM accession number NM_053357 [GenBank] ) were from Dharmacon (siRNA1 catalog number J100628-05, siRNA2 catalog number J-100628-06). 50 nM siRNAs were transfected into INS-1 cells using the Dharma reagent. siRNA-transfected cells were grown for 48 or 72 h, and aliquots of cells were harvested for Western immunoblot analysis and used for the TOPflash/FOPflash Wnt signaling reporter assay.

Cell Proliferation Assay—The proliferation of dispersed isolated islets and INS-1 cells, stably transfected or transiently infected, was determined by measuring the incorporation of BrdUrd into newly synthesized DNA of proliferating cells. Cells were plated at 10,000/well or 20 islets/well in 96-well plates and treated with Exd4 or PBS overnight. Cells were pulse-labeled with BrdUrd 4 h before measurement. BrdUrd staining was measured by the DELFIA cell proliferation kit (PerkinElmer Life Sciences).

Chromatin Immunoprecipitation (ChIP) Assays—For ChIP assays, INS-1 cells were treated with formaldehyde (1% final concentration) for 10 min at 37 °C. Cells were then pelleted for 4 min at 2000 rpm and resuspended in 200 µl of SDS lysis buffer. After sonication, cell supernatant was diluted 10-fold in ChIP dilution buffer. Anti-TCF7L2 (Santa Cruz Biotechnology) or β-catenin (Sigma) antibodies were added and incubated overnight at 4 °C. Afterward 60 µl of salmon sperm DNA/protein A-agarose slurry was added to the supernatant. After 1 h of incubation at 4 °C, the antibody-histone complex was collected by centrifugation. After elution and reverse cross-linking, DNA was recovered by phenol/chloroform extraction. DNAs were resuspended in 100 µl of TE buffer and analyzed by PCR using the following primers directed to cyclin D1 promoter, forward 5'-TGGAACTGCTTCTGGTGAAC-3' and reverse 5'-CAGGAGAGGAAGTTGTTGGG-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GLP-1 and Exd4 Activate Wnt Signaling in INS-1 Cells and in Islets ex Vivo via the GLP-1 Receptor—Several of the following observations suggested to us that GLP-1 agonists may be involved in the activation of Wnt signaling in pancreatic beta cells. 1) GLP-1 agonists are known to enhance the proliferation (9) and the neogenesis (8) of beta cells as well as the differentiation of both somatic (26-28) and embryonic (29-33) stem cells into insulin-producing beta cells. 2) Components of the Wnt signaling pathway are known to be expressed in the pancreas during development, and experimentally induced genetic disruption of Wnt signaling impairs pancreas growth and functions (21, 22, 24, 34). Wnt signaling is an important pathway in the maintenance of the proliferation and the differentiation of embryonic and somatic stem cells (23, 35-39). Collectively, these observations prompted us to examine the expression of genes expressed in Wnt signaling in beta cells using a focused Wnt signaling gene microarray (SuperArray, Invitrogen) and the clonal beta cell line INS-1 cells. Of the 118 probes represented on the Wnt signaling gene array, 37 were expressed above background in cultured INS-1 cells. Furthermore, we found that a 4-h exposure of the cells to Exd4 enhanced the expression of 14 of the genes, strongly suggesting that GLP-1 agonists activate components and target genes of the Wnt signaling pathway in INS-1 cells (supplemental Table S1).

To investigate in more detail whether GLP-1 and Exd4 induce Wnt signaling in INS-1 cells, we used a Wnt signaling reporter assay (TOPflash/FOPflash) to measure Wnt signaling in INS-1 cells stimulated by Exd4. The TOPflash and FOPflash constructs contain the luciferase reporter either under the control of consensus TCF7L2-binding sites or mutated TCF7L2-binding sites, respectively. The ratio of TOPflash activity to FOPflash activity indicates the intensity of Wnt signaling. INS-1 cells were transfected with TOPflash or FOPflash, and 24 h later GLP-1 or derivatives thereof were added to the cell culture at the indicated doses. Luciferase activity was measured after 4 h of incubation (Fig. 1A) or the indicated times (Fig. 1B). The intensity of active Wnt signaling was determined by the TOPflash/FOPflash ratio. GLP-1-(7-36) and Exd4 activated Wnt signaling dose-dependently with maximum responses achieved at 5 and 1 nM, respectively (Fig. 1A). Rapidly after its secretion, intact GLP-1-(7-36) is proteolytically cleaved by the enzyme dipeptidyl peptidase IV, yielding the metabolite GLP-1-(9-36). Exendin-(9-39), a derivative of Exendin-4 (Exd4), is a specific and competitive antagonist of the GLP-1 receptor. These two inactive GLP-1R ligands did not activate Wnt signaling (TOPflash/FOPflash activity) at all concentrations tested (Fig. 1A). The activation of Wnt signaling by Exd4 occurred as early as 1 h and reached maximum levels after 4 h of exposure to the hormone (Fig. 1B). Furthermore, the Exd4 activation was antagonized by co-incubation with increasing amounts of the Exd4-(9-39) antagonist (Fig. 1A), indicating that the activation of Wnt signaling by GLP-1 or Exd4 occurs via the GLP-1 receptor.

To further determine whether Exd4 induces Wnt signaling, mouse islets were isolated from a commonly used Wnt signaling reporter mouse model (TOPGAL mice) to measure the Wnt signaling stimulated by Exd4. TOPGAL mice are transgenic mice harboring a reporter gene TOPGAL, a β-galactosidase-encoding gene (lacZ) under the control of a regulatory sequence consisting of three consensus LEF/TCF-binding motifs upstream of a minimal c-fos promoter. This mouse provides an effective model for studying the Wnt signaling pathway (40). Islets from TOPGAL mice were incubated with Exd4 or vehicle for 4 h and then treated with β-galactosidase substrate (X-gal) for an additional 4 h. The reaction products were examined under a phase contrast microscope. Islets treated with Exd4 exhibited a distinct blue color, representative of active Wnt signaling (Fig. 1C, upper panels). β-Galactosidase expression was inhibited by co-incubation with the GLP-1R antagonist Exd-(9-39) (Fig. 1C, lower panels). To quantitate the activation of lacZ by Exd4, we used real time PCR to measure levels of lacZ mRNA in islets treated with Exd4 or vehicle. lacZ mRNA increased by 2.6-fold in Exd4-treated TOPGAL islets, providing ex vivo evidence of Exd4-induced Wnt signaling activation in pancreatic islets (Fig. 1D).


Figure 1
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  		FIGURE 1.
Exd4 and GLP-1-(7-36) but not Exd4-(9-39) and GLP-1-(9-36) activate Wnt signaling in INS-1 cells and isolated mouse islets. Wnt signaling is indicated by the value of TOPflash luciferase activity divided by FOPflash activity. A, ratio of TOPflash to FOPflash activity in INS-1 cells treated with increasing doses of GLP-1-(7-36), GLP-1-(9-36), Exd4, and Exd-(9-39). Increasing doses of Exd-(9-39) antagonize Exd4 activation of Wnt signaling. The x axis scale for the Exd4 and Exd-(9-39) experiment refers to the peptide concentration of Exd-(9-39) antagonist added at increasing doses to a fixed concentration (2 nM) of the Exd4 agonist. All values are relative to the zero peptide concentration. Data are normalized for transfection efficiency by co-transfected β-galactosidase and represent means ± S.D. of three experiments. B, a time course of the TOPflash:FOPflash ratio of INS-1 cell treated with 2 nM Exd4. C, phase contrast microscopic images of islets isolated from TOPGAL mice treated with Exd4 (top) or vehicle (middle) or Exd4 and Exd-(9-39) (bottom). D, real time RT-PCR results of lacZ transcription in islets of TOPGAL mice treated with Exd4 or vehicle or Exd4 and Exd-(9-39). Expression values are displayed relative to vehicle-treated condition (control). Data are normalized by using the housekeeping gene GAPDH as internal control. Statistical significance is depicted as * (p < 0.05) when compared with control values.

 
GLP-1 Interactions with the GLP-1R in INS-1 Cells and in Isolated Mouse Islets Are Coupled to the cAMP/PKA/CREB Signaling Pathway—Additional evidence that GLP-1 and Exd4 are acting on the known GLP-1R in INS-1 cells, and activate the cAMP/PKA/CREB signal transduction axis, was obtained by showing that both GLP-1-(7-36) and Exd4 agonists, and not the GLP-1-(9-36) and Exd-(9-39) antagonists, activated transcription from a luciferase reporter driven by a cAMP-response element (CRE) (Fig. 2A). The CRE-mediated transcription in response to GLP-1-(7-36) was attenuated by the PKA inhibitor H89 indicating that cAMP and active PKA are generated by GLP-1 activation of the GLP-1R in INS-1 cells (Fig. 2B). In addition, H89 abrogated Exd4-stimulated activation of β-galactosidase enzymatic activity and mRNA expression in islets isolated ex vivo from the TOPGAL Wnt signaling reporter mouse (Fig. 2, C and D).

Insulin Does Not Activate Wnt Signaling in INS-1 Cells—Because GLP-1 agonists are insulin secretagogues, the effects of insulin on Wnt signaling were examined in INS-1 cells. Insulin (100 nM) had no effects on TOPFlash activities in conditions in which insulin readily stimulated the phosphorylation of insulin receptor substrate-1 (serine 1101), GSK3{alpha} (serine 21), and GSK3β (serine 9) (supplemental Fig. 2). We also found that there is no effect of insulin on GLP-1 stimulation of Wnt signaling in the INS-1 cells, as determined by the activation of the TopFlash reporter (supplemental Fig. 3).

Wnt Ligands and Frizzled Receptors Mediate Basal Endogenous Wnt Signaling in INS-1 Cells—We observed that the TOPflash activity in INS-1 cells is 20-fold higher than FOPflash activity, indicating that these cells have substantial basal levels of Wnt signaling. To investigate whether the basal Wnt signaling in INS-1 cells is mediated by the canonical Wnt signaling pathway, we observed that the basal endogenous Wnt signaling is stimulated by the Wnt ligand Wnt3A in a dose-dependent manner (Fig. 3A) and is inhibited by the Wnt receptor antagonist mFz8CRD-IgG (41) (Fig. 3B). Wnt3A conditioned media is a widely used generic Wnt ligand, and Fz8CRD-IgG is a generalized antagonist of frizzled (Fz) receptors for Wnt ligands by virtue of containing an isolated cysteine-rich domain (CRD) (41).

Basal Endogenous Wnt Signaling in INS-1 Cells Requires Both Active Akt Kinase, Active TCF7L2 Transcription Factor, and the Inactivation of GSK3β—The binding of GLP-1 or Exd4 to the GLP-1R couples to the G{alpha}S protein, which stimulates adenylate cyclase, leading to an increase in intracellular cAMP levels and the activation of PKA. To determine whether PKA is required for maintaining the basal level of Wnt signaling activity, we used the PKA inhibitor H89 to inhibit endogenous PKA activity and measured basal levels of TOPflash activity. H89 did not inhibit basal levels of TOPflash activity, indicating that PKA is not required to maintain basal levels of Wnt signaling (Fig. 3C).


Figure 2
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  		FIGURE 2.
The GLP-1 receptor is coupled to the cAMP/PKA/CREB signaling pathway in INS-1 cells and isolated mouse islets. GLP-1 and Exd4, but not GLP-1-(9-36) and Exd-(9-39), activate a CRE transcriptional reporter in INS-1 cells. INS-1 cells expressing the pCRE-LUC reporter plasmid were treated for 4 h at 37. A, dose-response of CRE luciferase reporter gene expression in response to increasing concentrations of GLP-1, Exd4, GLP-1-(9-36), and Exd-(9-39). B, stimulation of CRE luciferase gene expression with GLP-1 (10 nM) was abolished by co-incubation of H-89 (10 µM). All values are relative to the 1st (leftmost) bar. Data are normalized for transfection efficiency by co-transfected β-galactosidase and represent means ± S.D. of three experiments. Statistical significance is depicted as * (p < 0.05) when compared with control values. C, mouse islets isolated ex vivo from TOPGAL Wnt signaling reporter mice were incubated for 4 h with Exd4 (2 nM) in the presence and absence of the cAMP/PKA inhibitor H89 (10 µM) (see legend to Fig. 1C). D, real time RT-PCR of lacZ transcription in islets of TOPGAL mice treated with vehicle or Exd4 or Exd4 and H89. Expression values are displayed relative to vehicle-treated control. Data are normalized by using the housekeeping gene GAPDH as the internal control. Statistical significance is depicted as * (p < 0.05) when compared with control values.

 
Furthemore, a dominant-negative PKA (dnPKA) antagonized PKA activity in INS-1 cells and did not inhibit basal Wnt signaling (Fig. 3D). This observation, combined with the results that the GLP-1R antagonist Exd-(9-39) and the inactive metabolite GLP-1-(9-36) did not inhibit basal levels of Wnt signaling (Fig. 1, B and D), indicates that the endogenous GLP-1/GLP-1R/PKA axis does not mediate the basal Wnt signaling in INS-1 cells.

The CRE-binding protein (CREB) has been linked to GLP-1-mediated cell proliferation and survival (42). CREB is a major substrate for phosphorylation, is activated by both PKA and Akt, and belongs to the ATF/CREB transcription factor family that interacts with CREs in the promoters of cAMP-responsive genes. To evaluate the role of CREB in maintaining basal level Wnt signaling, we transfected INS-1 cells with dominant-negative CREB (dnCREB) and found no effects on basal TOPflash activity, indicating that CREB does not regulate Wnt signaling in INS-1 cells (Fig. 3D).

GLP-1 has been reported to enhance beta cell proliferation via transactivation of the EGF receptor (EGFR), and such activation is sensitive both to the EGFR kinase inhibitor AG1478 and to the PI3K inhibitor LY294002 (43). We therefore investigated whether EGFR and PI3K activations are required for basal Wnt signaling in INS-1 cells. The kinase inhibitors AG1475 and LY294002 had no effects on basal levels of TOPflash activity, indicating that EGF receptor activation and PI3K are not required for maintaining basal levels of Wnt signaling activity (Fig. 3, C and D). These results were further confirmed by the finding that a dominant-negative form of PI3K did not inhibit basal TOPflash activity (Fig. 3D).

The prosurvival kinase Akt is known to be activated by the PI3K, and yet PI3K appeared not to be involved in the basal signaling of INS-1 cells. We therefore investigated the role of Akt. Akt is implicated in the GLP-1-mediated proliferation and survival of beta cells (6, 44). GLP-1 increases Akt levels in beta cells, both in vivo in db/db mice and in vitro in INS-1 cells (45, 46). The ablation of Akt abrogates the effect of GLP-1 in the prevention of staurosporine-induced apoptosis in INS-1 cells (45). To determine whether Akt is required for maintaining basal Wnt signaling, we used the Akt inhibitor IV to inhibit the endogenous Akt activity and measured basal levels of TOPflash activity (Fig. 3C). We found that the TOPflash activity was enhanced by caAkt (see Fig. 4C) and suppressed by either the Akt inhibitor IV (Fig. 3C) or dnAkt (Fig. 3D). Therefore, Akt appears to be required for basal Wnt signaling. Akt is known to be a kinase that phosphorylates and inactivates GSK3β.We investigated whether inactivation of GSK3β is required for basal Wnt signaling in INS-1 cell. A constitutively active GSK3β (caGSK3β) inhibited basal Wnt signaling, findings consistent with canonical Wnt signaling in INS-1 cells (Fig. 3D).

TCF7L2 is a member of the TCF family that has been implicated in the regulation of the proglucagon gene in intestinal enteroendocrine cells (47). Although reported not to be expressed in pancreatic beta cells or islets (47), we observed the expression of TCF7L2 both in islets (data not shown) and in INS-1 cells (Fig. 5B). This circumstance prompted us to investigate whether active TCF7L2 is necessary for basal Wnt signaling in INS-1 cells. INS-1 cells were transfected with a dominant-negative TCF7L2 construct (dnTCF7L2), which lacks the domain that interacts with β-catenin and thereby inhibits the canonical Wnt signaling pathway downstream of β-catenin. The expression of dnTCF7L2 substantially reduced TOPflash activity when compared with cells transfected with empty control vector (Fig. 3D).

The Activation of Wnt Signaling by Exd4 Requires the Participation of Akt and PKA and p42/44 MAPK but Not EGF Receptor Signaling, Active PI3K, and Inactivation of GSK3β—To explore whether PKA, EGF receptor, activation of PI3K, Akt, and inactivation of GSK3β are required for Exd4-enhanced TOPflash activity, INS-1 cells were transfected with several different activating and inhibitory forms of kinases and signaling molecules. TOPflash luciferase reporter assays were performed under the condition in which INS-1 cells were treated with Exd4 (2 nM) or vehicle for 4 h. caPKA had no effect on Exd4-activated TOPflash activity, although it elevated Wnt signaling in the absence of Exd4 (Fig. 4A). dnPKA partially attenuated Exd4 actions (Fig. 4A). These results indicate that although PKA does not participate in maintaining basal Wnt signaling, it is required for Exd4-stimulated Wnt signaling. This conclusion is further confirmed by the finding that the PKA inhibitor H89 inhibited Exd4-stimulated Wnt signaling (Fig. 4B).


Figure 3
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  		FIGURE 3.
Endogenous Wnt signaling mediated via Wnt ligands and Frizzled receptors in INS-1 cells. A, increasing concentrations of Wnt3a conditioned media stimulate TOPflash reporter activity. Wnt signaling is indicated by the value of TOPflash luciferase activity divided by FOPflash activity. Results show ratio of TOPflash to FOPflash activity in INS-1 cells treated with increasing concentrations of normal medium (INS-1 cell culture medium), Wnt3a conditioned medium, or control medium (L cell culture medium) overnight. B, Frizzled receptor antagonist, m8FzCRD-IgG, inhibits basal TOPflash reporter activity in INS-1 cells. Results show ratio of TOPflash to FOPflash activity in INS-1 cells treated with increasing concentrations of normal medium (INS-1 cell culture medium), m8FzCRD-IgG conditioned medium, or control medium (L cell culture medium) overnight. Statistical significance is depicted as * (p < 0.05) when compared with control values (cells treated with normal medium). C and D, roles of PKA, PI3K, Akt, EGF receptor, CREB, GSK3β, and TCF4 in the basal level of Wnt signaling in INS-1 cells. The Akt inhibitor (Akt inhibitor IV), dnAkt, dominant-negative TCF7L2, and constitutively active GSK3β inhibit basal level TOPflash activity. The PI3K inhibitor (LY294002) and dnPI3K, PKA inhibitor (H89), dnPKA, dnCREB, and EGF receptor inhibitor (AG1478) do not inhibit basal level TOPflash activity in INS-1 cells. All values are relative to the 1st (leftmost) bar. Statistical significance is depicted as * (p < 0.05) when compared with control values (leftmost bar). All values are relative to the 1st (leftmost) bar. Data are normalized for transfection efficiency by co-transfected β-galactosidase and represent means ± S.D. of three experiments.

 
The activation of EGFR and PI3K was not involved in Exd4-induced Wnt signaling, as indicated by the observations that the EGFR inhibitor AG1478, the PI3K inhibitor LY294002 (Fig. 4B), and the dnPI3K (Fig. 4D) had no effect on Exd4-induced TOPflash activity.

Notably, we found that active Akt is also required for the effects of Exd4 on Wnt signaling. Exd4-stimulated TOPflash activity is inhibited by either the Akt inhibitor IV (Fig. 4B) or dnAkt (Fig. 4C). caAkt did not augment Exd4-induced activity indicating that Exd4 had achieved maximum Akt-mediated effects (Fig. 4C). The inhibition of either Akt alone (Fig. 4C) or PKA alone (Fig. 4A) does not suppress TOPflash activity to that of basal levels (without Exd4), suggesting that both Akt and PKA contribute to Wnt signaling. Although the activation of Wnt signaling is dependent on active PKA (Fig. 4A), CREB does not regulate TOPflash activity as two dominant-negative isoforms of CREB, KCREB and CREB S133A, had no significant effect (Fig. 4E).

Surprisingly, both caGSK3β and dnGSK3β had no effect on Exd4-induced TOPflash activity (Fig. 4F). Therefore, the conventional mechanism of Akt activation that leads to GSK3β phosphorylation and inactivation and eventual stabilization of β-catenin seems not to be in play in the Exd4 stimulation of Wnt signaling in INS-1 cells. Nevertheless, the participation of β-catenin and TCF7L2 is required for the Wnt signaling as shown by the inhibitory actions of dnTCF7L2 (Fig. 4G).

GLP-1 is reported to stimulate ERK1/2 (p42/44 MAPK) via cAMP and PKA (48) and to activate beta cell replication in an ERK1/2-dependent manner (49). Involvement of p38 MAPK and an atypical protein kinase C isoform, PKC{zeta}, was demonstrated in GLP-1-induced replication of INS-1 cells (9). We therefore investigated whether p42/44 MAPK or p38 MAPK is required for basal and Exd4-induced Wnt signaling. We found that the activation of p42/44 MAPK but not p38 MAPK contributes to basal and Exd4-induced Wnt signaling, as indicated by the observations that basal and Exd4-induced TOPflash activity were inhibited by both the p42/44 MAPK inhibitor PD98059 and the inhibitor of its upstream kinase MEK1/2 (U1026), but not by the p38 MAPK inhibitor SB203580 (Fig. 4H).

Requirement for Active β-Catenin for Exd4-mediated Wnt Signaling—The activation of downstream Wnt signaling requires the association of TCF7L2 with active β-catenin (16, 19, 20), TCF7L2 is a DNA-binding protein that in the absence of β-catenin serves as a transcriptional repressor. β-Catenin contributes the activation domain to the transcriptional complex via its association with TCF7L2. Therefore, in addition to examining the effects of the inhibition of TCF7L2 by dnTCF7L2 on Exd4-mediated transcriptional activation by Wnt signaling (see above), we examined the requirement for active β-catenin in this signaling. To investigate whether Exd4 induces the stabilization of β-catenin, the level of active β-catenin (unphosphorylated on Ser-33 and Ser-37) was examined in INS-1 cells. An immunoblot of cell lysates treated with Exd4 for different periods of time was probed with an antibody specific for unphosphorylated β-catenin. In response to Exd4, the level of active β-catenin dramatically increased as early as 5 min after the treatment of the INS-1 cells with Exd4 (Fig. 5A). To further investigate the role of β-catenin in regulating Wnt signaling in INS-1 cells, INS-1 cells were transfected with siRNA against β-catenin. Immunoblotting analysis revealed that 72 h after transfection more than 70% of endogenous β-catenin protein was knocked down by expressing either type of siRNA or expressing both siRNAs, whereas an siRNA with scrambled sequence had no effect on the expression of β-catenin (Fig. 5B). siRNAs to β-catenin markedly inhibited both basal and Exd4-mediated TopFlash activity (Fig. 5C). Thus, canonical Wnt signaling drives TOPflash activity in the INS-1 cells. These findings substantiate the validity of the TOPflash reporter, and further indicate that β-catenin and TCF7L2 is required for the transcriptional activity reported by TOPflash.


Figure 4
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  		FIGURE 4.
Roles of PKA, PI3K, Akt, EGF receptor, CREB, GSK3β, and TCF4 in the basal levels and Exd4-induced Wnt signaling in INS-1 cells. A, caPKA activates basal level TOPflash activity while having no effect on Exd4 activated TOPflash activity. dnPKA inhibits Exd4 activated Wnt signaling without effect on basal signaling. B, the PKA inhibitor (H89) and the AKT inhibitor (AKT inhibitor IV), but not the PI3K inhibitor (LY294002) and the EGF receptor inhibitor (AG1478), inhibit Exd4-activated TOPflash. The Akt inhibitor, but not the other inhibitors, attenuates TOPflash activity. C, caAkt stimulates basal endogenous Wnt signaling (TOPflash activity) whereas dnAkt inhibits both basal and Exd4-induced TOPflash activity. D, dnPI3K has no effect on either basal or Exd4-induced TOPflash activity. E, wild-type CREB (wtCREB) and two dominant-negative forms of CREB (KCREB and CREB133) have no effect on Exd4-induced TOPflash activity. F, dominant-negative forms and constitutively active forms of GSK3β have no effect on Exd4 induced TOPflash activity. G, dominant-negative TCF7L2 inhibits both basal level and Exd4-induced TOPflash activity. Data are expressed as luciferase activity relative to the value of the leftmost bar. H, MEK inhibitor (U0126) and ERK inhibitor (PD98059), but not the p38MAPK inhibitor (PB203580), inhibit basal and Exd4-activated TOPflash. All values are relative to the 1st (leftmost) bar. Data are normalized for transfection efficiency by co-transfected galactosidase and represent means ± S.D. of three independent experiments. Statistical significance is depicted as * (p < 0.05) when compared with control values (leftmost blue bar) and # (p < 0.05) when compared with Exd4 values (leftmost red bar).

 
Phosphorylation on Serine 675 by PKA Stabilizes β-Catenin—Based on these observations, we hypothesized that the activation of the GLP-1 receptor by GLP-1 agonists, and the subsequent activation of PKA, triggers a direct response that induces the stabilization and thereby the activation of β-catenin. It has been shown that β-catenin is phosphorylated by PKA in vitro and in intact cells at two novel sites, Ser-552 and Ser-675 (50). The phosphorylation by PKA promotes the stability and the transcriptional activity of β-catenin without affecting GSK3β-dependent phosphorylation of β-catenin. It has also been reported that PKA phosphorylates Ser-675 and prevents its ubiquination and thereby stabilizes β-catenin (51). Therefore, we determined whether the treatment of INS-1 cells would result in the phosphorylation of Ser-675 in β-catenin. Within 5 min after the addition of Exd4 (2 mM) to the cells, phosphorylation of Ser-675 was detected by Western immunoblotting of cell extracts (Fig. 5D). Moreover, the Exd4-stimulated phosphorylation of Ser-675 was inhibited by both the GLP-1R antagonist, Exd-(9-39), and the PKA antagonist, H89 (data not shown). These findings further indicate that PKA directly activates β-catenin and stimulates Wnt signaling in INS-1 cells.

Exd4 Induces Cyclin D1 Transcription via TCF7L2 and β-Catenin—Cyclin D1 and c-Myc are well studied pro-proliferation target genes of Wnt signaling. The TCF7L2/β-catenin-binding site in their promoters allows transactivation by β-catenin-dependent Wnt signaling (52-54). In light of our finding that Exd4 activated Wnt signaling in INS-1 cells, we determined whether Exd4 activated cyclin D1 and c-Myc transcription through a β-catenin-TCF7L2 activator complex. By Western immunoblot TCF7L2 is expressed in INS-1 cells (Fig. 6A). Myc-tagged TCF7L2 was used as a control marker for the immunoblot (Fig. 6A). Exd4 treatment induced cyclin D1 mRNA expression by 14-fold in INS-1 cells and induced c-Myc mRNA expression by 1.8-fold (Fig. 6B). Exd4 also enhanced the interaction of TCF7L2 and β-catenin with the cyclin D1 promoter as shown by using chromatin immunoprecipitation (ChIP) assays. In the presence of Exd4, incubation of cross-linked protein-DNA complexes with either TCF7L2 or β-catenin antibodies efficiently precipitated a cyclin D1 promoter fragment containing the TCF7L2-binding sites of cyclin D1 (Fig. 6C). Without Exd4, the cyclin D1 fragment co-immunoprecipitated poorly with TCF7L2 and did not co-immunoprecipitate with β-catenin (Fig. 6C). These results demonstrate that Exd4 increases the interaction of TCF7L2 and β-catenin with the cyclin D1 promoter.


Figure 5
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  		FIGURE 5.
Exd4 treatment stabilizes β-catenin, siRNA-mediated knockdown of β-catenin abrogates Exd4-stimulated Wnt signaling, and Exd4 enhances phosphorylation of β-catenin on the stabilizing PKA site, Ser-675. A, Exd4 stabilizes cytosolic β-catenin in INS-1 cells. INS-1 cell cultures were stimulated with Exd4 for indicated times. The cells were collected and sonicated on ice. The soluble cytosolic fractions were obtained and used (2 mg of protein/lane) for immunoblot analysis with anti-β-catenin (top) or anti-unphosphorylated β-catenin (bottom). Time course study shows Exd4 (2 nM) significantly increased the accumulation of cytosolic β-catenin within 5 min of stimulation. B, siRNAs to β-catenin decrease levels of β-catenin in INS-1 cells determined by Western immunoblot. Cells were incubated for 72 h with 50 nM siRNAs 1 or 2, or their combination (1 + 2). Scrambled RNA served as a control for the siRNA experiments and actin for the protein loading of the immunoblot. C, siRNAs to β-catenin inhibit both basal (Control) and Exd4-stimulated Wnt signaling. Cells were transfected and pre-incubated for 48 h with the combination TOPflash or FOPflash and a combination of either siRNAs 1 and 2, or scrambled siRNA as a control, and then treated for 4 h with either Exd4 or control vehicle. D, Exd4 stimulates the phosphorylation of Ser-675 on β-catenin. Exd4 (2 mM) was added to INS-1 cells at time 0. Aliquots of cells were extracted at the times indicated after the addition of Exd4 and were analyzed by immunoblotting using an antiserum specific for phospho-Ser-675 of rat β-catenin.

 


Figure 6
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  		FIGURE 6.
Exd4 treatment enhances cyclin D1 and c-Myc transcription through a β-catenin/TCF4-binding site. A, immunoblot shows endogenous expression of TCF7L2 in INS-1 cells. Left lane shows transient ectopic expression of Myc-tagged TCF7L2 in INS-1 cells. Right lane shows endogenous expression of TCF7L2 in INS-1 cells. B, real time RT-PCR results show that Exd4 enhances cyclin D1 mRNA expression by 14-fold and Myc mRNA expression by 1.8-fold. Expression values are displayed relative to vehicle-treated condition (control). Data are normalized by using the housekeeping gene GAPDH as internal control and represent means ± S.D. of three independent experiments. Statistical significance is depicted as * (p < 0.05) when compared with control values. C, ChIP analysis monitoring the interaction of TCF7L2 and β-catenin with the promoter of cyclin D1 in INS-1 treated for 4 h with either PBS (Cont) or 2 nM Exd4 (+Exd4). Cyclin D1 promoter sequence in the immunoprecipitate was measured by semi-quantitative RT-PCR.

 
Active Wnt Signaling Is Required for Both Basal and Exd4-stimulated Proliferation of INS-1—To investigate whether active Wnt signaling is required for beta cell proliferation, we established an INS-1 cell line (INS-1-dnTCF7L2) stably transfected with a dominant-negative TCF7L2. The BrdUrd incorporation assay showed that INS-1-dnTCF7L2 inhibits the proliferation rate of INS-1 cells by 50% that of control cells expressing an empty vector (INS-1-pcDNA3) (Fig. 7A). These findings indicate that active TCF7L2 and active Wnt signaling upstream of TCF7L2 are required for maintaining the basal levels of INS-1 cell proliferation. Furthermore, an overnight treatment of INS-1 cells with Exd4 (2 nM) accelerates the proliferation of INS-1-pcDNA3 cells by 70%, but it has no effect on the proliferation of INS-1-dnTCF7L2 cells deficient in active TCF7L2. Because of concerns about possible clonal selection in the stable dnTCF7L2 cell line, these findings were further confirmed by similar experiments using transient infection, rather than stable transfection, of INS-1 cells and dispersed islets by dnTCF7L2 retrovirus (Fig. 7, B and D). In addition we observed that the siRNA to β-catenin inhibits the proliferation of the INS-1 cells (Fig. 7C). Therefore, both active TCF7L2 and active β-catenin are required for maintaining both basal and Exd4-stimulated proliferation of beta cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We find that GLP-1 agonists activate TCF7L2-dependent Wnt signaling in isolated mouse pancreatic islets and in pancreas-derived INS-1 beta cells and that Wnt signaling is involved in the proliferation of INS-1 cells. We provide evidence that INS-1 cells maintain high basal levels of Wnt signaling via Wnt ligands and Frizzled receptors. In contrast to basal signaling, we show that GLP-1 agonists enhance Wnt signaling through their binding to the GLP-1 receptor (GLP-1R), a G-protein-coupled receptor coupled to G{alpha}S and the activation of PKA. Although the GLP-1/GLP-1R/PKA axis is not involved in maintaining basal levels of Wnt signaling, it is essential for the enhancement of Wnt signaling by Exd4, a long acting agonist of GLP-1. Furthermore, we show that the prosurvival protein kinase, Akt, along with active MEK/ERK signaling, is required for maintaining both basal and Exd4-induced Wnt signaling. Both β-catenin and TCF7L2 are required for Exd4-mediated transcriptional responses and cell proliferation. A model depicting a summary of our tentative findings of the Wnt signaling pathways utilized in INS-1 cells under basal resting conditions (endogenous signaling) and in response to Exd4 is shown in Fig. 8.


Figure 7
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  		FIGURE 7.
Wnt signaling is required for the proliferation of INS-1 cells; BrdUrd incorporation cell proliferation assay. A, INS-1 cells that were stably transfected with expression vectors encoding dnTCF7L2 (INS-1-dnTCF7L2) or control empty vector (INS-1-pcDNA3) were treated with either PBS (Control) or Exd4 (2 nM) (+Exd4) overnight, and their proliferation rate was examined by BrdUrd incorporation assays the next day. INS-1-dnTCF7L2 cells exhibit 50% of the proliferation rate of that of INS-1-pcDNA3 under basal condition (left panel). After overnight Exd4 treatment, INS-1-pcDNA3 cells exhibited an 80% increase of proliferation rates, whereas INS-1-dnTCF7L2 cells stayed unchanged. B, INS-1 cells were infected with dnTCF7L2 retrovirus (dnTCF7L2) or vector retrovirus (Control) and were subsequently treated with either PBS (Control) or Exd4 (2 nM) (+Exd4) overnight, and their proliferation rates were examined by BrdUrd incorporation assays the next day. dnTCF7L2-infected INS-1 cells exhibit 67% of the proliferation rate of that of vector-infected INS-1 cells under basal condition (left panel). After overnight Exd4 treatment, vector-infected INS-1 cells exhibited a 62% increase in proliferation rate, whereas dnTCF7L2-infected INS-1 cells remained unchanged. C, INS-1 cells were treated with siRNAs (1 + 2) to β-catenin for 48 h followed by treatment with Exd4 (2 mM) for 20 h. The siRNAs significantly inhibited BrdUrd incorporation (proliferation) by 27%. Control scrambled siRNA had no significant effect on proliferation. D, dispersed islets were infected with dnTCF7L2 retrovirus (dnTCF7L2) or vector retrovirus (Control) and were subsequently treated with either PBS (Control) or Exd4 (2 nM) (+Exd4) overnight, and their proliferation rates were examined by BrdUrd incorporation assays the next day. After overnight Exd4 treatment, vector-infected INS-1 cells exhibited a 94% increase in proliferation rate, whereas dnTCF7L2-infected INS-1 cells showed no significant effect on proliferation. All values are relative to the 1st (leftmost) bar. A and B, data represent means ± S.D. of three experiments. C, data are the average of two experiments. Statistical significance is depicted as * (p < 0.05) when compared with control values (leftmost blue bar) and # (<0.05) when compared with vector transfected cells treated with Exd4 (rightmost blue bar). C and D, data are represents means ± S.D. of two experiments. Statistical significance is depicted as * (p < 0.05) when compared with control values (leftmost blue bar) and & (p < 0.1) when compared with scramble siRNA transfected cells (C) or vector-infected islets (D) treated with Exd4 (rightmost blue bar).

 
Endogenous Basal Wnt Signaling—Endogenous Wnt signaling in INS-1 cells appears to be mediated by canonical Wnt signaling components, including endogenous Wnt ligands, Frizzled receptors, GSK3β, β-catenin, and TCF7L2. A dominant-negative TCF (dnTCF7L2), defective in its β-catenin interaction domain, inhibited basal and Exd4-induced β-catenin/TCF-mediated transcriptional reporter activity and the proliferation of INS-1 cells. In addition, we found that caGSK3β inhibited Wnt signaling. dnAkt inhibited and caAkt stimulated Wnt signaling. Taken together, these results suggest that the inactivation of GSK3β by an active Akt is important for the signaling pathway that maintains the basal, endogenous Wnt signaling in INS-1 cells.

Although Akt is not usually considered to be a component of the canonical Wnt signaling pathway, Akt and its upstream activating kinase PI3K are well known to modulate Wnt signaling by the phosphorylation and inactivation of GSK3β (55, 56). Notably, it appears from our studies that Akt modulates the canonical Wnt signaling in INS-1 cells through a PI3K-independent mechanism. Some controversy exists regarding the requirement for PI3K in the GLP-1-mediated activation of Akt and the proliferation of beta cells. The activation of Akt in INS-1 cells is reported to be inhibited by wortmannin thereby implicating PI3K as an essential requirement for the activation of Akt (45). However, another report indicates that PI3K is questionably required for the activation of Akt (57). Furthermore, in mice deficient in PI3K{gamma}, no growth impairment of beta cells was observed (58). Although in most cell systems the actions of Akt in the activation of Wnt signaling are downstream of PI3K (56, 59), there are exceptions; Akt may be activated by PI3K-independent mechanisms. For example, agents that raise intracellular cAMP in 293 EBNA cells activate Akt (60, 61). But this activation is not sensitive to wortmannin, indicating that it is independent of PI3K. Interestingly, Wnt signaling in PC12 and Int5 cells directly activates Akt, and activated Akt, in association with Dvl, enhances the phosphorylation and inactivation of GSK3β in the APC-axin complex resulting in the stabilization of β-catenin (55).


Figure 8
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  		FIGURE 8.
Schematic model of Wnt signaling pathways in INS-1 cells. A, basal endogenous signaling in the absence of GLP-1 or other exogenous agonists. B, signaling in response to GLP-1 agonists such as Exd4. The thicker arrow indicates a major pathway of GLP-1/GLP-1R to cAMP/PKA phosphorylating Ser-675 on β-catenin and thereby stabilizing against degradation. The thin arrows with Xs indicate that the pathway designated by the arrow is not involved in GLP-1-activated Wnt signaling at the level of activating gene transcription via β-catenin/TCF7L2. The cross-bar indicates the inhibition of GSK3 activity by Gao and disheveled (DVL), an upstream pathway of canonical Wnt signaling.

 
GLP-1 Agonist-stimulated Wnt Signaling—In marked contrast to the pathways involved in endogenous basal Wnt signaling, Exd4-enhanced Wnt signaling occurs entirely independently of GSK3β, but requires the GLP-1R, PKA, and Akt, whereas cross-activation of the EGF receptor and PI3K by GLP-1 (43) is unlikely to be involved. However, active MEK/ERK signaling contributes to both basal and Exd4-induced Wnt signaling. The role of MEK/ERK in GLP-1-mediated signaling and the mechanism by which GLP-1 activates MEK/ERK remain unclear. In human islet cells, glucose and glucagon-like peptide-1 activate ERK through the Rap and B-Raf signaling module (62). Inhibiting PKA by H89 prevented GLP-1-induced Erk1/2 phosphorylation (48). The ERK inhibitor PD98059 inhibits the proliferation of INS-1 cells in response to GLP-1 (49).

Recent evidence suggested cross-talk between MEK/ERK signaling and Wnt signaling. For example, stimulation of the nonreceptor tyrosine kinase v-Src activates β-catenin/TCF-mediated transcription partially through the ERK pathway in tumor cells (63), and stimulation of rat neural progenitors by SDF1 resulted in cytoplasmic accumulation of β-catenin through the activation of ERK by the G protein-coupled CXCR4 receptor (64). In addition, insulin and IGF-1 stimulate the β-catenin pathway in liver cells via the activation of Ras-MEK-ERK (65).

The activation of Wnt signaling by GLP-1 agonists is likely a direct result of G-protein-coupled receptor-mediated activation of signaling. There are several reports indicating the existence of cross-talk between Wnt signaling and G-protein signaling (66). The Fz receptors are structurally related to G-protein-coupled receptors. Signaling by G{alpha}O and G{alpha}Q contributes to Wnt-mediated disruption of GSK3β-axin complexes and the stabilization of β-catenin (67, 68). For example, the direct activation of G proteins by GTP{gamma}S disrupts GSK3β-axin2 complexes and stabilizes β-catenin (69). Constitutively active G{alpha}S stimulates TOPflash activity in HEK293T and colon cancer cells. Parathyroid hormone activates Wnt signaling via cAMP-activated PKA (70).

Our findings strongly suggest that Exd4 activates the GLP-1R and its downstream effector PKA. PKA is known to stabilize and thereby activate β-catenin either directly (51) or indirectly (49) or through the activation of the intermediate signaling kinase Akt, which in turn stabilizes β-catenin (71, 72). In one study, PKA phosphorylates β-catenin at Ser-675 and enhances its transcriptional activity by enhancement of its interactions with CREB-binding protein (50). In another study, phosphorylation of Ser-675 on β-catenin by PKA inhibits its ubiquitination and degradation (51). We found that in INS-1 cells Exd4 increased the level of unphosphorylated and active β-catenin and resulted in the phosphorylation of Ser-675 on β-catenin within 10 min after the addition of Exd4 (Fig. 5C).

Notably, the Wnt signaling in INS-1 cells is not dependent upon CREB. Although the CREB kinases, Akt and PKA, are involved in the Wnt signaling in INS-1 cells, both endogenous and Exd4-induced Wnt signaling, as reported by transcription mediated by β-catenin/TCF (TOPflash activity), were not affected by dominant-negative inhibitors of CREB. Several reports implicate CREB as a downstream target of Wnt signaling in other cell systems. In mouse pre-somatic mesoderm Wnt ligands activate CREB and a CRE-reporter gene through adenylyl cyclase (73). Transcriptional activation of the cyclin D promoter by gastrin and β-catenin in gastric cancer (AGSE) cells (74) and by Wnt3a and integrin-linked kinase in mammary epithelial cells (MCF7) (52) is attenuated by dominant-negative CREB (74) and dominant-negative TCF (74). Studies of the promoter of WISP1 (Wnt-inducible secreted protein) show that the activation of CREB by a stabilized β-catenin is required for the activation of the promoter as it is inhibited by dnCREB, whereas the activation of a β-catenin/TCF reporter (TOPflash) is unaffected (75).

Wnt Signaling and Pancreas Development—The involvement of Wnt signaling and β-catenin in pancreas development remains unclear. The expression of the frizzled receptor antagonist, mFz8CRD-IgG, in pancreatic progenitor cells of transgenic mice results in a 75% reduction in overall pancreatic mass and 50% reduction in absolute beta cell numbers (34). In agreement with this reported finding, we found that dominant-negative TCF7L2 and siRNA to β-catenin-inhibited Exd4 induce INS-1 cell proliferation, implying that active Wnt signaling is crucial for beta cell proliferation. Several other studies in mice show that by using the Pdx-1 promoter to target the expression of either Wnt5a (76) or a stabilized β-catenin (24) results in perturbations in the mass of pancreatic islets. Targeted disruptions of β-catenin in mice are reported to reduce (22) or have no effect (77) on beta cell mass. Since the initial submission of this manuscript, three reports have appeared supporting a role for Wnt signaling in beta cell proliferation (78-80).

TCF7L2 and Diabetes—Although TCF7L2 is reported either not to be expressed in mouse pancreatic beta cells (47) or is expressed in human beta cells (81), we found robust expression of TCF7L2 in rat INS-1 cells. We show that TCF7L2 plays a central role in mediating both basal and Exd4-induced Wnt signaling in INS-1 cells. Of particular note, we found that dominant-negative TCF7L2 inhibited the proliferation of INS-1 cells, suggesting that impairments of TCF7L2 actions could result in a reduction in beta cell mass. Recent genetic studies in humans from several laboratories have identified a close association of polymorphisms in the TCF7L2 (previously known as TCF4) gene and susceptibility toward type 2 diabetes (81-87). These studies suggest that alterations in the expression of TCF7L2 confer risk for genotypes associated with impaired beta cell function but not with insulin resistance (83). Therefore, our results provide physiological evidence supporting these genetic epidemiological studies that suggest an important role of TCF7L2 in maintaining beta cell mass and/or function.

Our finding that Exd4 up-regulates pro-proliferative genes, including cyclin D1 and c-myc via the Wnt signaling pathway, provides a new insight into GLP-1 action in regulating beta cell growth. GLP-1 and Exd4 have been shown to induce cyclin D1 transcription (49, 88) through CREB (88). Our results provide Wnt signaling as a novel mechanism by which GLP-1 regulates cyclin D1 transcription. These two mechanisms are not necessarily mutually exclusive.

Our results further suggest the possibility that GLP-1 agonists might trigger a self-sustaining paracrine auto-regulatory cycle within islets. In human islets the alpha cells and beta cells are extensively intermingled such that 90% of alpha cells are in contact with beta cells (89). The proglucagon gene (Gcg), which encodes glucagon and GLP-1, is regulated by TCF7L2 (TCF7L2) and is among the known targets of the Wnt signaling pathway (47). Gcg is known to be expressed in islet alpha cells, which are known to express GLP-1Rs (90). Therefore, it is tempting to speculate that Exd4, or other GLP-1 agonists, may activate Wnt signaling and thereby induce Gcg expression in alpha cells, resulting in enhanced secretion of GLP-1 that then acts on adjacent beta cells to stimulate Wnt signaling and cellular proliferation. Further studies are warranted to test the hypothesis that GLP-1 agonists induce Wnt signaling in beta cells and subsequently turn on the synthesis of endogenous GLP-1 in the islets and further enhance beta cell growth and functions via paracrine mechanisms.

GLP-1 and Wnt Signaling in Stem Cells—It is further tempting to speculate that Wnt signaling in INS-1 cells may be involved in maintaining the cells in an undifferentiated state. Active Wnt signaling has been demonstrated to be required for conserving the pluripotency and proliferation of embryonic stem cells (35, 38) as well as in stem cell fate determination (91, 92). Wnt signaling is also involved in maintaining the undifferentiated state of somatic stem cells, including preadipocytes (93), neural stem cells (89), hematopoietic stem cells (94), mes-enchymal stem cells (36), and neural crest stem cells (37). We have shown previously that clonal beta cell lines, including INS-1 cells, contain mixtures of undifferentiated and differentiated cells (95). The stimulation of Wnt signaling by GLP-1 agonists demonstrated here in INS-1 cells may be a clue to the findings of the expression of the GLP-1 receptor in embryonic stem cells (29) and the efficacy of GLP-1 agonists in differentiating embryonic stem cells into insulin-producing cells (30-33) and in differentiating somatic tissue-derived cells into insulin-producing cells (27, 96).

Our findings of active Wnt signaling in insulin-producing beta cells, and its stimulation by GLP-1 agonists, may have potential implications for the treatment of individuals with diabetes. They link cAMP/PKA signaling via the GLP-1R, a widely utilized signaling pathway in cell biology, to Wnt signaling, a key pathway in the maintenance and differentiation of stem/progenitor cells. It seems possible that the treatment of diabetes that is caused by a reduction in beta cell mass with GLP-1-based therapies may stimulate the generation of new beta cells.


   FOOTNOTES
 
* This work was supported in part by United States Public Health Service Grants DK55365 and DK30834 (to J. F. H.) and National Institutes of Health Pilot and Feasibility Award P30 DK57521 (to Z. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. 1-3. Back

1 To whom correspondence should be addressed: Laboratory of Molecular Endocrinology, 55 Fruit St., Thier 306, Massachusetts General Hospital, Boston, MA 02114. E-mail: jhabener{at}partners.org.

2 The abbreviations used are: PKA, cAMP-dependent protein kinase; siRNA, small interfering RNA; CRE, cyclic AMP-response element; CREB, CRE-binding protein; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI3K, phosphatidylinositol 3-kinase; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; APC, adenomatous polyposis coli; dn, dominant-negative; ca, constitutively active; ChIP, chromatin immunoprecipitation; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuridine; EGF, epidermal growth factor; EGFR, EGF receptor; LEF, lymphocyte enhancer factor; TCF, T cell factor; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; Fz, frizzled. Back


   ACKNOWLEDGMENTS
 
We thank Tom Boodry and Katherine Chau for technical assistance and Michael Rukstalis, Violeta Stanojevic, and Melissa Thomas for helpful suggestions and guidance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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