Stromal cell-derived factor-1 promotes survival of pancreatic beta cells by the stabilisation of beta-catenin and activation of transcription factor 7-like 2 (TCF7L2)

Abstract

Aims/hypothesis: Stromal cell-derived factor-1 (SDF-1) is a chemokine produced in stromal tissues in multiple organs. Earlier we reported on levels of SDF-1 and SDF-1 receptor (CXCR4) in the insulin-producing beta cells of the mouse pancreas and determined that the SDF-1/CXCR4 axis is important for beta cell survival through activation of the prosurvival kinase, protein kinase B (AKT). Since AKT is known to modulate the wingless-type MMTV integration site family (WNT) signalling cascade, we examined the effects of SDF-1/CXCR4 on WNT signalling in beta cells and whether this signalling is important for cell survival.

Methods: Activation of downstream WNT signalling (beta-catenin and transcription factor 7-like 2, [TCF7L2]) in response to SDF-1 was examined in the islets of WNT signalling reporter (Tcf-optimal promoter beta-galactosidase) mice and in INS-1 and MIN6 beta cells. Cytoprotection of beta cells by SDF-1 in response to the induction of apoptosis was assessed by caspase 3 and TUNEL assays.

Results: SDF-1 induced WNT signalling in beta cells of isolated islets and in INS-1 cells via CXCR4-mediated activation of Galphai/o-coupled signalling and the phosphatidylinositol 3-kinase/AKT signalling cascade resulting in the inhibition of glycogen synthase kinase 3-beta. The key WNT signalling regulators, beta-catenin and AKT, were activated by SDF-1 at the transcriptional and post-translational levels. Specific inhibition of beta-catenin in the WNT signalling cascade reversed the anti-apoptotic effects of SDF-1.

Conclusions/interpretation: SDF-1 promotes pancreatic beta cell survival via activation of AKT and downstream WNT signalling via the stabilisation and activation of beta-catenin/TCF7L2 transcriptional activators. These findings suggest a mechanism for SDF-1 based glucose-lowering therapies by enhancing beta cell mass through increasing cell survival.

Fig. 1

SDF-1 (1 nmol/l) activates WNT-signalling in isolated islets from the TOPGAL WNT signalling reporter mouse. In comparison to control (a), isolated islets expressed reporter-driven beta-galactosidase (blue colour) (b), an indicator of activation of the WNT signalling reporter LacZ transgene. Specificity of SDF-1 for its receptor CXCR4 is shown by the inhibition of beta-galactosidase expression by AMD3100 (c), a specific antagonist of CXCR4. d Coupling of the SDF-1/CXCR4 axis i to the G-protein, Galphai/o, is shown by inhibition of beta-galactosidase expression with pertussis toxin (PTX). e Polymerase chain reaction showing changes in beta-galactosidase mRNA levels in islets shown above. *p<0.05 compared with control

Fig. 2

SDF-1 activates WNT signalling, as indicated by TOPflash luciferase activity:FOPflash activity, in INS-1 cells. a Time course of the TOPflash:FOPflash ratio of INS-1 cells treated with 1 nmol/l SDF-1. b Ratio of TOPflash:FOPflash activity in INS-1 cells treated with increasing doses of SDF-1. c Increasing doses of AMD3100 (AMD) antagonised 1 nmol/l SDF-1 activation of WNT signalling. The x-axes for b and c are logarithmic scales. All values are relative to the value of untreated INS-1 cells. Data were normalised for transfection efficiency by co-transfected beta-galactosidase and are presented as means±SD of three experiments

Fig. 3

Roles of PI3K, AKT, epidermal growth factor (EGF receptor) and GSK3β in basal levels of and SDF-1-induced WNT signalling in INS-1 cells. a Constitutively active GSK3β (caGSK3b) inhibited TOPflash activity whereas dominant-negative GSK3β (dnGSK3b) had no effect. b The inhibitors pertussis toxin (PTX) (Galphai), LY294002 (PI3K) and PD98059 (ERK) did not affect basal level TOPflash activity. In contrast, pertussis toxin and LY294002, but not PD98059, did inhibit SDF-1 induced TOPflash activity. The AKT inhibitor SH-5 inhibited basal and SDF-1-induced TOPflash activity. c Constitutively active AKT (caAKT) stimulated basal endogenous WNT signalling (TOPflash activity), whereas dominant-negative AKT (dnAKT) inhibited basal and SDF-1-induced TOPflash activity. All values are relative to the first (leftmost) bar. Data were normalised for transfection efficiency by co-transfected beta-galactosidase and are presented as means±SD of three experiments. White bars, vehicle treatment (control); black bars, 1 nmol/l SDF-1 treatment. *p<0.05 compared with vehicle treated vector only

Fig. 4

Inhibition of TCF7L2 or beta-catenin suppresses SDF-1-induced TOPflash activity. a Dominant-negative (dn)TCF7L2 inhibits basal (white bars) and SDF-1-induced (black bars) TOPflash activity. b Scrambled siRNAs to beta-catenin siRNAs inhibit SDF-1-stimulated WNT signalling in INS-1 cells. INS-1 cells were transfected and preincubated with TOPflash and the combination of either siRNAs 1 and 2, or scrambled siRNA, and then treated for 4 h with 1 nmol/l SDF-1 or control vehicle. Data were normalised for transfection efficiency by co-transfected beta-galactosidase and are presented as means±SD of three experiments. All values are relative to the first (leftmost) bar. *p<0.05 compared with control values (first bar). c Immunoblot analysis with anti-beta-catenin or anti-unphosphorylated beta-catenin (active beta-catenin) showing that SDF-1 stabilised beta-catenin through GPCR-coupled Galphai/o. INS-1 cell cultures were stimulated with SDF-1 for the indicated times. A time-course study showed that SDF-1 (1 nmol/l) significantly increased the accumulation of cellular beta-catenin within 30 min of stimulation. d Co-incubation of Galphai inhibitor pertussis toxin (PTX) inhibited the accumulation of beta-catenin

Fig. 5

Inhibition of apoptosis by SDF-1 in INS-1 and MIN-6 cells is reversed by knock-down of beta-catenin. a Western blot analysis of extracts from MIN6 cells that were mock-treated, thapsigargin-treated (1μmol/l) and/or siRNA-transfected (0.25 mg/ml) by using anti-sera against cleaved caspase-3 and cleaved PARP, and actin antibody as a loading control. b Caspase-3 activity (fluorescence [mg protein]⁻¹ × 10⁻⁵) of INS-1 or (c) MIN6 cells treated as above (a). Cell treatments from left to right: white bars, control; light grey, beta-catenin siRNA; dark grey, scramble siRNA; black, SDF-1; mottled, SDF-1 and beta-catenin siRNA; striped, SDF-1 and scramble siRNA. Results are presented as mean±SD for n=3 independent experiments. All values are relative to the first (leftmost) bar. *p<0.05 compared with untreated control values (leftmost bar); †p<0.05 compared with 1 mmol/l thapsigargin treated control values. d Representative images of TUNEL staining of INS-1 cells of n=3 independent experiments. e Quantification of apoptotic cells expressed as per cent apoptotic cells relative to total cells counted. f The proliferation rates of INS-1 cells after SDF-1 (grey bars) or exendin-4 (black bars), presented as relative BrdU and (g) MTT incorporation values compared with control-treated cells. *p<0.05 when compared with control values (leftmost bar)

Fig. 6

Schematic models of signalling pathways used by SDF-1/CXCR4 and GLP-1/GLP-1R in the activation of beta catenin/TCF7L2-mediated transcriptional expression of genes involved in beta cell survival and proliferation a SDF-1 activation of WNT signalling. Upstream SDF-1/CXCR4 actions are transmitted to beta-catenin/TCF7L2 predominantly via action of PI3K/AKT, the inhibition of GSK3 by AKT and the resultant stabilisation of beta catenin. Beta-catenin/TCF7L2 activated by SDF-1 results in the transcription of genes involved in cell survival. A direct action of AKT on the stabilisation of beta-catenin has been suggested [34, 35], but remains conjectural. b GLP-1 activation of WNT signalling. Upstream GLP-1/GLP-1R actions are transmitted to beta-catenin/TCF7L2 via cAMP/PKA and the direct stabilisation of beta-catenin by PKA. PI3K/AKT and GSK3 are minimally or not at all involved in the GLP-1-mediated pathway. The actions of beta-catenin/TCF7L2 mediated by GLP-1 are primarily to activate the transcription of genes responsible for cell proliferation. APC, adenomatous polyposis coli; AXIN, axis inhibition protein; B-CAT, beta-catenin; E-CAD, epithelial cahedrin; DVL, dishevelled, dsh homologue 1; G_βγ, G protein beta subunits; G_αo, G protein alpha unit o; Gαs, G protein alpha unit s; Gαi, G protein alpha unit i; FZ, frizzled; LRP, low-density lipoprotein receptor-related protein; MEK, mitogen activated protein kinase; P, phosphorylation; Raf, v-raf-1 murine leukaemia viral oncogene homologue 1; Ras, rat sarcoma viral oncogene homologue