Cyclin D2 protein stability is regulated in pancreatic beta-cells

Abstract

The molecular determinants of beta-cell mass expansion remain poorly understood. Cyclin D2 is the major D-type cyclin expressed in beta-cells, essential for adult beta-cell growth. We hypothesized that cyclin D2 could be actively regulated in beta-cells, which could allow mitogenic stimuli to influence beta-cell expansion. Cyclin D2 protein was sharply increased after partial pancreatectomy, but cyclin D2 mRNA was unchanged, suggesting posttranscriptional regulatory mechanisms influence cyclin D2 expression in beta-cells. Consistent with this hypothesis, cyclin D2 protein stability is powerfully regulated in fibroblasts. Threonine 280 of cyclin D2 is phosphorylated, and this residue critically limits D2 stability. We derived transgenic (tg) mice with threonine 280 of cyclin D2 mutated to alanine (T280A) or wild-type cyclin D2 under the control of the insulin promoter. Cyclin D2 T280A protein was expressed at much higher levels than wild-type cyclin D2 protein in beta-cells, despite equivalent expression of tg mRNAs. Cyclin D2 T280A tg mice exhibited a constitutively nuclear cyclin D2 localization in beta-cells, and increased cyclin D2 stability in islets. Interestingly, threonine 280-mutant cyclin D2 tg mice had greatly reduced beta-cell apoptosis, with suppressed expression of proapoptotic genes. Suppressed beta-cell apoptosis in threonine 280-mutant cyclin D2 tg mice resulted in greatly increased beta-cell area in aged mice. Taken together, these data indicate that cyclin D2 is regulated by protein stability in pancreatic beta-cells, that signals that act upon threonine 280 limit cyclin D2 stability in beta-cells, and that threonine 280-mutant cyclin D2 overexpression prolongs beta-cell survival and augments beta-cell mass expansion.

Figure 1

Cyclin D2 expression is stimulated by 50% partial pancreatectomy (a model of adaptive β-cell proliferation, chiefly by posttranscriptional mechanisms. A, Representative Western blot of islet extracts from individual mice 5 d after partial pancreatectomy. B, Representative RT-PCR analysis of pooled islets from multiple mice (n = 4) from each treatment group. Results are expressed as mean ± sd for four replicates. C, Representative pancreatic immunohistochemistry of 6-wk-old male mice 5 d after partial pancreatectomy, continuously treated with BrdU for 5 d before being killed, stained for cyclin D2 (top images), stained with cyclin D2 (red), BrdU (green), and DAPI (blue) (bottom images). Scale bars, 100 μm.

Figure 2

Threonine 280 limits cyclin D2 stability in β-cells. A, Tg expression strategy; B and C, representative RT-PCR of tg islets with primers that are transgene specific (B) or directed against total cyclin D2 (C), with results expressed as mean ± sd for four replicates; D, Western blot of tg islets; E and F, cycloheximide time course. Primary islets from tg mice were cultured with cycloheximide to stop translation of cyclin D2 mRNA. Western blot was performed with lysates using cyclin D2 antisera (top) or tubulin antisera (bottom).

Figure 3

Threonine 280 limits cyclin D2 nuclear localization. Representative pancreatic immunofluorescent images, stained for cyclin D2. Top, Long exposure cyclin D2 images (1 sec); middle, short exposure cyclin D2 images (240 msec); bottom, merge of short exposure cyclin D2 images (red), insulin (green), and DAPI (blue). Scale bar, 100 μm.

Figure 4

Cyclin D2 T280A tg expression promotes β-cell growth in aged mice. Representative immunofluorescent images of mice at various ages are shown. Mice were infused with BrdU in the drinking water for 2 wk before being killed. Merged images contain insulin (red), BrdU (green), and DAPI (blue). Scale bars, 100 μm.

Figure 5

Pancreatic immunofluorescent image of a large tumor within a cyclin D2 T280A tg at 18 months. Mouse was infused with CldU and then IdU for 2 wk each in the drinking water before being killed. Top, ×5 objective image; bottom, ×40 objective images of insets from above. Merged images contain CldU (red), IdU (green), and insulin (blue). Scale bars, 100 μm.

Figure 6

Representative RT-PCR of pooled tg islets from multiple (four to six) mice at 8 wk of age. Results are expressed as mean ± sd for four replicates.