STAT3 modulates β-cell cycling in injured mouse pancreas and protects against DNA damage

Abstract

Partial pancreatic duct ligation (PDL) of mouse pancreas induces a doubling of the β-cell mass mainly through proliferation of pre-existing and newly formed β-cells. The molecular mechanism governing this process is still largely unknown. Given the inflammatory nature of PDL and inflammation-induced signaling via the signal transducer and activator of transcription 3 (STAT3), the activation and the role of STAT3 in PDL-induced β-cell proliferation were investigated. Duct ligation stimulates the expression of several cytokines that can act as ligands inducing STAT3 signaling and phosphorylation in β-cells. β-Cell cycling increased by conditional β-cell-specific Stat3 knockout and decreased by STAT3 activation through administration of interleukin-6. In addition, the level of DNA damage in β-cells of PDL pancreas increased after deletion of Stat3. These data indicate a role for STAT3 in maintaining a steady state in the β-cell, by modulating its cell cycle and protection from DNA damage.

Figure 1

STAT3 expression and activity are stimulated in β-cells of PDL pancreas. (a) Immunostaining for phospho-STAT3 (Y705) (P-STAT3) and insulin in Sham tail, PDL head and PDL tail. (b) Quantification of data in a: percentage of P-STAT3⁺ INS⁺ cells at day 7 and 14 (D7 and D14) postsurgery (mean±S.E.M., n=3, ***P<0.001, ns: P>0.05, two-way ANOVA). (c) Stat3 mRNA in β-cells isolated at D7 from the tail of Sham- and PDL pancreas of MIP-RFP mice. Data are expressed as fold change versus Sham (=1) (n=3, ^#P<0.0001, Sham tail versus PDL tail by unpaired two-tailed t-test). (d) STAT3-activating factors whose expression increased 10-fold or more in PDL pancreas. Data represent the fold change in expression of each factor in PDL tail as compared with its expression in Sham tail (1) (D3: n=4, D7: n=4, D14: n=3, *P<0.05, **P<0.005, ***P<0.001, ^#P<0.0001, ns: P>0.05, Sham tail versus PDL tail by unpaired two-tailed t-test)

Figure 2

Ki67⁺ β-cells differ from P-STAT3⁺ β-cells in PDL pancreas. (a) β-Cells with active cell cycle (Ki67⁺) at D7 and D14 post surgery (mean±S.E.M., D7: n=3, D14: n=4-6, ***P<0.001, ns: P>0.05, PDL tail versus Sham tail or PDL head by two-way ANOVA). (b) Proliferating β-cells (Ki67⁺ INS⁺) and (c) β-cells with activated STAT3 (P-STAT3⁺ INS⁺) cells in small (≤20 INS⁺ cells) and large (>20 INS⁺ cells) islets, at D7 and D14 post surgery (mean±S.E.M., D7: n=3, D14: n=4-6, **P<0.005, ***P<0.001, ns: P>0.05, small versus large islets in PDL and Sham by unpaired two-tailed t-test). (d) Ki67 and P-STAT3 in β-cells in PDL pancreas. Ki67⁺ β-cells are P-STAT3⁻ (white arrows)

Figure 3

β-Cell-specific Stat3 knockout stimulates β-cell cycling after PDL. (a) Schematic illustration of the inducible transgenic mouse model, RIP (rat insulin gene promotor), CreERT (causes recombination fused to TAM-inducible estrogen receptor), R26 (Rosa26 promotor), loxP (locus of crossing over P1), STOP (transcriptional STOP sequence), YFP, TAM. (b) Recombination efficiency as shown by immunostaining for YFP in INS+ cells in PDL tail of RipCre^ERTR26^YFP mouse. (c) Immunostaining for STAT3 in PDL tail from WT versus Stat3^−/− mice, quantified in (d) as the percentage of STAT3⁺ INS⁺ cells in PDL tail from WT and Stat3^−/− mice. (e) Cycling β-cells (Ki67⁺ INS⁺) were observed in the islets of Stat3^−/− mice that appeared normal based on the distribution of β- and α-cells (GCG⁺). (f) β-Cell cycling (Ki67⁺ INS⁺) in PDL head and tail pancreas from WT and Stat3^−/− mice at D14 post surgery. (g) Percentage INS⁺ cells that express Ki67⁺, in small (≤20 INS⁺ cells) and large (>20 INS⁺ cells) islets of PDL head and tail pancreas from WT and Stat3^−/− mice at D14 post surgery. (h) Total insulin content (μg insulin per mg tissue) in PDL head and tail pancreas from WT and Stat3^−/− mice. (i) β-Cell volume (μl) in PDL tail pancreas from WT and Stat3^−/− mice. (d and f–i) mean±S.E.M., in (d and f–h) n=4, in (i) n=3–4, *P<0.05, **P<0.005, ***P<0.001, ^#P<0.0001, ns: P>0.05, WT versus Stat3^−/− by unpaired two-tailed t-test

Figure 4

Recombinant IL6 stimulates STAT3 activation and blunts β-cell cycling in PDL pancreas. (a) Left: short-term effect of recombinant (r) IL6, injected at D7 post surgery into PDL pancreas that was collected 1 h post injection. Right: long-term effect of recombinant (r) IL6 injected at D6 post surgery into PDL pancreas that was collected 24 h post injection. (b) Immunostaining for P-STAT3 and insulin (INS) in PDL tail pancreas 1 h after (r)IL6 or vehicle injection at D7 post PDL. (c) Percentage of P-STAT3⁺ β-cells in PDL tail 1 h after vehicle or (r)IL6 injection (mean±S.E.M., n=4, ***P<0.001, vehicle versus (r)IL6-injected PDL tail pancreas, by unpaired two-tailed t-test). (d) Percentage P-STAT3⁺ β-cells 24 h after vehicle, (r)IL6, isotype or anti-IL6 injection in PDL tail at D7 post PDL (n=4, **P<0.005, ns: P>0.05, vehicle versus (r)IL6-injected PDL tail pancreas and isotype versus anti-IL6-injected PDL pancreas, two-way ANOVA). (e) Cycling β-cells (Ki67⁺ INS⁺) in PDL tail injected with vehicle, (r)IL6, isotope or anti-IL6 at D7 post surgery (mean±S.E.M., n=4, *P<0.05, ns: P>0.05, vehicle versus (r)IL6-injected PDL tail pancreas and isotype versus anti-IL6-injected PDL pancreas, two-way ANOVA)

Figure 5

STAT3 protects β-cells from DNA damage in PDL pancreas. (a) Abundance of mRNA encoding inflammatory cytokines (Ifng, Tnfa and Il1b) in PDL tail. Data are expressed as fold change compared with transcript level in Sham tail (1) (D3: n=4, D7: n=4, D14: n=3, *P<0.05, **P<0.005, ***P<0.001, ^#P<0.0001, ns: P>0.05, Sham tail versus PDL tail by unpaired two-tailed t-test). (b) Percentage β-cell apoptosis (cCASP3⁺ INS⁺) in PDL head and tail pancreas from WT and Stat3^−/− mice at D14 post surgery (n=4, *P<0.05, ns: P>0.05, WT versus Stat3^−/−, two way ANOVA). (c) Abundance of miR375 in plasma from WT and Stat3^−/− mice at D1, D3, D5, D7, D10 and D14 post PDL surgery and in plasma of positive control for β-cell death 24 h after Alloxan injection (ALX). Data are expressed as Ct values. WT, n=5; Stat3^−/−, n=4; *P<0.05, ns: P>0.05, WT versus Stat3^−/− by unpaired two-tailed t-test) (d) Immunostaining for Ki67 and gH2AX in PDL tail pancreas from Stat3^−/− mice showing two forms of gH2AX nuclear staining: homogenous nuclear labeling in proliferating β-cells (white arrows) and nuclear foci (yellow arrows). (e) gH2AX in PDL tail from WT and Stat3^−/− mice at D14 post surgery. (f) Percentage non-cycling β-cells with DNA damage (gH2AX⁺ Ki67⁻ INS⁺) in PDL tail from WT and Stat3^−/− mice at D14 post surgery (n=4, *P<0.05, ns: P>0.05, WT versus Stat3^−/−, by unpaired two-tailed t-test). (g) Percentage non-cycling β-cells with DNA damage (gH2AX⁺ Ki67⁻ INS⁺) in PDL tail pancreas injected with vehicle, (r)IL6, isotype or anti-IL6 at D7 post surgery (n=4, *P<0.05, ns: P>0.05, two-way ANOVA)