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. 2016 Oct 21;291(43):22630-22637.
doi: 10.1074/jbc.M116.747840. Epub 2016 Sep 1.

Epidermal Growth Factor Receptor Signaling Regulates β Cell Proliferation in Adult Mice

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Free PMC article

Epidermal Growth Factor Receptor Signaling Regulates β Cell Proliferation in Adult Mice

Zewen Song et al. J Biol Chem. .
Free PMC article

Abstract

A thorough understanding of the signaling pathways involved in the regulation of β cell proliferation is an important initial step in restoring β cell mass in the diabetic patient. Here, we show that epidermal growth factor receptor 1 (EGFR) was significantly up-regulated in the islets of C57BL/6 mice after 50% partial pancreatectomy (PPx), a model for workload-induced β cell proliferation. Specific deletion of EGFR in the β cells of adult mice impaired β cell proliferation at baseline and after 50% PPx, suggesting that the EGFR signaling pathway plays an essential role in adult β cell proliferation. Further analyses showed that β cell-specific depletion of EGFR resulted in impaired expression of cyclin D1 and impaired suppression of p27 after PPx, both of which enhance β cell proliferation. These data highlight the importance of EGFR signaling and its downstream signaling cascade in postnatal β cell growth.

Keywords: beta cell (β cell); cell proliferation; cyclin D1; diabetes; epidermal growth factor receptor (EGFR).

Figures

FIGURE 1.
FIGURE 1.
EGFR is up-regulated in islets after 50% partial pancreatectomy. A, β cell proliferation increased 7 days after PPx, compared with sham-operated C57BL/6 mice, shown by representative immunostaining images for BrdU, insulin, and Hoechst (HO). B and C, EGFR levels in islets by RT-qPCR (B), and by Western blotting analysis (C). D, RT-qPCR analyses of mRNA of major cell-cycle regulators. E and F, Western blotting analyses of major cell-cycle regulators, shown by quantification (E), and by representative blots (F). n = 5. All results were mean ± S.E. *, p < 0.05. NS, no significance. Scale bars are 100 μm.
FIGURE 2.
FIGURE 2.
Generation of Pdx1-CreERT; EGFRfx/fx mice. A, EGFR was specifically targeted and depleted in pancreatic β cells in Pdx1-CreERT; EGFRfx/fx transgenic mice. In these mice, the expression of CreERT is driven by the Pdx1 promoter, but the recombinase stays in the cytoplasm. In the presence of tamoxifen, CreERT translocates into the nucleus and deletes exon 3 of the EGFR gene, leading to early termination of translation. B and C, EGFR levels in mouse islets were determined 2 weeks after tamoxifen injection in Pdx1-CreERT; EGFRfx/fx mice, and control Pdx1-CreERT and control EGFRfx/fx mice, by RT-qPCR (B, p < 0.05), and by Western blotting analysis (C, p < 0.05). D, mouse body weight. n = 5. All results were mean ± S.E. *, p < 0.05. NS, no significance.
FIGURE 3.
FIGURE 3.
EGFR is required for baseline and workload-induced β cell proliferation. A, male Pdx1-CreERT; EGFRfx/fx, control Pdx1-CreERT mice and control EGFRfx/fx mice received PPx or sham operated at 8 weeks of age, 2 weeks after tamoxifen treatment. These mice were fed with BrdU drinking water for 1 week to aid in immunohistochemical analysis of β cell replication. Ki-67 expression in β cells from these mice was also analyzed at the same time. B and C, blood glucose (B) and glucose tolerance (C) were measured in these mice at 1 week after PPx/Sham treatment. D and E, BrdU+/insulin+ cells were quantified, shown by quantification (D), and by representative images (E). n = 5. All results were mean ± S.E. *, p < 0.05. NS, no significance. Scale bars are 50 μm.
FIGURE 4.
FIGURE 4.
Ki-67+/insulin+ cell quantification and β cell mass. A and B, Ki-67+/insulin+ cells were quantified, shown by representative images (A), and by quantification (B). C, β cell mass in the head portion of the pancreas was analyzed at 1 week after PPx/sham treatment. n = 5. All results were mean ± S.E. *, p < 0.05. NS, no significance. Scale bars are 50 μm.
FIGURE 5.
FIGURE 5.
Cyclin D1 and p27 may be involved in the regulation of baseline β cell proliferation by EGFR signaling. A, mRNA of cyclin D1, cyclin D2, Cdk4, and p27 was analyzed in purified islets by RT-qPCR. B and C, protein of cyclin D1, cyclin D2, Cdk4, and p27 was analyzed in purified islets by Western blotting analysis, shown by representative blots (B), and by quantification (C). n = 5. All results were mean ± S.E. *, p < 0.05. NS, no significance.
FIGURE 6.
FIGURE 6.
Cyclin D1 and p27 regulation in β cells may be impaired in Pdx1-CreERT; EGFRfx/fx mice after PPx. Islets were isolated from both Pdx1-CreERT; EGFRfx/fx mice and control EGFRfx/fx mice after either PPx or sham surgery for analysis of the expression of cell cycle regulators. A, representative Western blots for EGFR and cell cycle regulators. B–F, protein quantification of EGFR (B), cyclin D1 (C), cyclin D2 (D), Cdk4 (E), and p27 (F). n = 5. All results were mean ± S.E. *, p < 0.05. NS, no significance.

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