PDGF signalling controls age-dependent proliferation in pancreatic β-cells

Abstract

Determining the signalling pathways that direct tissue expansion is a principal goal of regenerative biology. Vigorous pancreatic β-cell replication in juvenile mice and humans declines with age, and elucidating the basis for this decay may reveal strategies for inducing β-cell expansion, a long-sought goal for diabetes therapy. Here we show that platelet-derived growth factor receptor (Pdgfr) signalling controls age-dependent β-cell proliferation in mouse and human pancreatic islets. With age, declining β-cell Pdgfr levels were accompanied by reductions in β-cell enhancer of zeste homologue 2 (Ezh2) levels and β-cell replication. Conditional inactivation of the Pdgfra gene in β-cells accelerated these changes, preventing mouse neonatal β-cell expansion and adult β-cell regeneration. Targeted human PDGFR-α activation in mouse β-cells stimulated Erk1/2 phosphorylation, leading to Ezh2-dependent expansion of adult β-cells. Adult human islets lack PDGF signalling competence, but exposure of juvenile human islets to PDGF-AA stimulated β-cell proliferation. The discovery of a conserved pathway controlling age-dependent β-cell proliferation indicates new strategies for β-cell expansion.

Figure 1. Age-dependent attenuation of Pdgfr-α limits β-cell Ezh2 expression and proliferation in neonatal and juvenile mice

a, Pdgfr-α and insulin immunostaining on wild-type mouse pancreas sections at indicated ages. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 25 µm. b, c, mRNA levels of indicated genes in FACS-purified β-cells (b) or wild-type islets (c) at specified ages. n = 3–5 experiments per group or time point. *P < 0.05, **P < 0.01 compared to samples from 2 weeks (b) or 15 days (c). Insr. refers to mRNA encoding insulin receptor. d–g, Relative mRNA levels of Pdgfra (d) and Ezh2 (e) in isolated islets, and percentage β-cell BrdU incorporation (f) and pancreatic β-cell mass (g) of βPdgfαKO and control mice at specified ages. n = 3–5 independent islet preparations or mice per group. *P < 0.05, **P < 0.01 for the comparison as indicated. Error bars denote s.e.m.

Figure 2. Pdgfra loss impairs β-cell regeneration in STZ-induced diabetes

a, b, Immunostaining of Pdgfr-α and insulin on pancreas sections (a), and relative mRNA levels of Pdgfra and Pdgfrb in islets (b) from 8-week-old wild-type mice 1 week after vehicle or STZ (100 mg kg ⁻¹ body weight) treatment. n = 3–5 mice per group. a, Yellow arrows mark Pdgfr-α induction in a subset of insulin⁺ cells. c, Representative images showing immunostaining for insulin, Ezh2 and BrdU on pancreatic sections from indicated 6–7-week-old mice 3 weeks after STZ (150 mg kg⁻¹ body weight) treatment. d, Percentage of BrdU⁺ insulin⁺ cells (β-cells) or BrdU⁺ acinar cells 3 weeks after STZ challenge. n = 3 or 6 mice per group. e, f, Pancreatic β-cell mass (e) and blood glucose (f) levels during ad libitum feeding after STZ treatment. n = 3–8 mice (e) or 18–21 mice (f) per time point per group. *P < 0.05, **P < 0.01 for the comparison as indicated or versus control. Scale bars, 25 µm. Error bars denote s.e.m.

Figure 3. Activated PDGFR-α delays age-dependent Ezh2 loss and replication failure in pancreatic β-cells

a, Relative mRNA levels of human PDGFRA and mouse Pdgfra in islets from littermate control and βPDGFRαTg mice at 3 months of age. n = 3–7 mice per group. b–d, Pancreatic β-cell mass (b), and β-cell proliferation, assessed by Ki67 expression (c) or BrdU incorporation (d) in βPdgfαKO and control mice at indicated ages. n = 3–5 mice per group. e, f, Relative mRNA levels of Ezh2 (e) in islets, and immunostaining (f) for Ezh2, BrdU and insulin on pancreatic sections from control and βPDGFRαTg mice at indicated ages. n = 3–7 mice per group. Scale bars, 25 µm. g, Postprandial blood glucose levels in mice at indicated ages n = 14–24 mice per group per time point. h, Glucose tolerance assessed in 14-month-old control and βPDGFRαTg mice. n = 6 or 10 mice per group. *P < 0.05, **P < 0.01 in comparisons indicated. Error bars denote s.e.m.

Figure 4. PDGFR-α promotes β-cell expansion through Ezh2

a, b, mRNA levels for human PDGFRA (a) and mouse Ezh2 (b) in islets from mice with indicated genotypes at 3–4 months of age. n = 3–5 mice per group. c–f, β-cell BrdU incorporation (c), β-cell mass (d), postprandial plasma insulin (e) and blood glucose (f) levels in 3–4-month-old mice with indicated genotypes. Each dot in f represents a measurement from an individual mouse. n = 4–5 mice for c and d, and n = 5–18 mice for e and f per genotype. *P < 0.05, **P < 0.01. NS, not significant in comparisons indicated. Error bars denote s.e.m.

Figure 5. Pdgfr signalling governs Erk and Rb/E2f regulation of Ezh2 in islet β-cells

a, Western blot analysis of total and phosphorylated Erk1/2, Akt and PLCγ in islet proteins from 3–4-month-old βPDGFRαTg and littermate control mice. Similar results were obtained from multiple independent experiments. b, Relative phosphorylated protein level compared to total protein was quantified by densitometry. c, Immunostaining for phospho-Erk1/2 (pErk1/2), phospho-Rb-Ser 780 (p-Rb(Ser780)) and insulin in 3-month-old littermate control and βPDGFRαTg pancreatic sections. Scale bar, 25 µm. d, Percentage of phospho-Rb-Ser 780⁺ insulin⁺ cells from the indicated mice quantified by morphometry. n = 4 mice per group. e, Cyclin D1 (Ccnd1) or cyclin D2 (Ccnd2) mRNA levels in 3-month-old control and βPDGFRαTg islets. n = 4 or 6 mice per group. f, g, ChIP analyses of the Ezh2 and β-actin (Actb) loci with antibodies to E2f1 (f) and E2f4 (g) using the indicated amplicons (see Supplementary Information) in 3–4-month-old littermate control and βPDGFRαTg islets. n = 3–4 independent experiments per antibody with independent islet samples. *P < 0.05, **P < 0.01 for control versus βPDGFRαTg. Error bars denote s.e.m.

Figure 6. PDGFR-α regulates human β-cell EZH2 expression and proliferation

a, b, Representative images showing immunostaining for PDGFR-α (a), phospho-ERK1/2 (pERK1/2), phospho-RB-Ser 780 (pRB(Ser780)), EZH2 (b) and insulin (a and b) on pancreatic sections from juvenile and adult human subjects. PDGFR-α was detected in juvenile β-cells (arrows) but not in adult β-cells (arrowheads). Scale bars, 25 µm. c–f, Assessment of effects on human juvenile or adult islets after exposure to PDGF-AA (50 ng ml⁻¹) for 2 days, with or without Sunitinib (2 µM) or U0126 (10 µM)co-treatment. c, Islet EZH2 mRNA levels after the indicated treatments. n = 3–5 independent experiments. d, EZH2 locus ChIP analysis with anti-E2F1 antibody or IgG in human juvenile islets. n = 3–4 for E2F1, n = 2 for IgG. e, f, Human islet β-cell proliferation changes after the indicated treatments. Average percentage of BrdU⁺ insulin⁺ cells (e) was quantified by morphometry from sectioned islets immunostained (f) for insulin (green), glucagon (white) and BrdU (red). n = 3–6 independent experiments. Scale bar, 25 µm. g, Illustration summarizing how Pdgfr-α signalling regulates β-cell Ezh2 and proliferation by activating Erk/Rb/E2f pathways sensitive to the indicated inhibitors. *P < 0.05, **P < 0.01 as indicated. Error bars denote s.e.m.