TGF-β Signaling Regulates Pancreatic β-Cell Proliferation through Control of Cell Cycle Regulator p27 Expression

Abstract

Proliferation of pancreatic β-cells is an important mechanism underlying β-cell mass adaptation to metabolic demands. Increasing β-cell mass by regeneration may ameliorate or correct both type 1 and type 2 diabetes, which both result from inadequate production of insulin by β-cells of the pancreatic islet. Transforming growth factor β (TGF-β) signaling is essential for fetal development and growth of pancreatic islets. In this study, we exposed HIT-T15, a clonal pancreatic β-cell line, to TGF-β signaling. We found that inhibition of TGF-β signaling promotes proliferation of the cells significantly, while TGF-β signaling stimulation inhibits proliferation of the cells remarkably. We confirmed that this proliferative regulation by TGF-β signaling is due to the changed expression of the cell cycle regulator p27. Furthermore, we demonstrated that there is no observed effect on transcriptional activity of p27 by TGF-β signaling. Our data show that TGF-β signaling mediates the cell-cycle progression of pancreatic β-cells by regulating the nuclear localization of CDK inhibitor, p27. Inhibition of TGF-β signaling reduces the nuclear accumulation of p27, and as a result this inhibition promotes proliferation of β-cells.

Keywords: HIT-T15 cells; TGF-β signaling; inhibition; p27; pancreatic β-cell proliferation.

Fig. 1

Effects of TGF-β signaling on cell proliferation of HIT-T15 cells. (A) HIT-T15 cells were cultured in 10% FBS RPMI 1640 medium with or without 5 ng/ml TGF-β1 for 1 day, 2 days, 3 days and 4 days (6 independent repeats of experiments). (B) HIT-T15 cells were cultured in 10% FBS RPMI 1640 medium containing DMSO or 2 µM SB-431542 for 1 day, 2 days, 3 days and 4 days. Six repeats of the experiment were done independently. The data are mean±SD (error bars) of a representative experiment. *p<0.01 (n=8). (C, D) Cell cycle distribution of HIT-T15 cells was analyzed by BrdU incorporation assay after treatment with or without 5 ng/ml TGF-β1, and/or with DMSO or 2 µM SB-431542, for 48 hr. (C) Upper row: BrdU staining (green). Lower row: Merged with nuclear staining (PI; red). (D) Results are displayed as the percentage of cells in S phase of the cell cycle. Six repeats of the experiment were done independently. The data are mean±SD (error bars) of a representative experiment. *p<0.01 (n=5).

Fig. 2

Expression of CKIs in HIT-T115 cells. Cells were incubated for 2 days and fixed, and then stained with different CKI antibodies. Upper row: Expression of different indicated CKIs (green). Lower row: Merged with nuclear staining (PI; red). (A) p15; (B) p16; (C) p18; (D) p19; (E) p21; (F) p27; (G) p57. (H) Western blotting of the whole cell extracts for p15, p16, p19 and p27.

Fig. 3

Effects of TGF-β signaling on nuclear localization of p27 (green). To stimulate TGF-β signaling, HIT-T15 cells were transfected with pcDNA.3.1.mRFP1 (red), which served as a transfectant indicator, and either empty vector, pcDNA3.1 (A) or a constitutively active form of TRβ-I, ALK5* (B). (C) Nuclear p27 integrated density (A. U., arbitrary unit) in (A) and (B). To inhibit TGF-β signaling, HIT-T15 cells were treated with DMSO (D) or 2 µM SB-431542 (E). (F) Nuclear p27 integrated density (A. U.) in (D) and (E). Nuclei were detected with TO-PRO-3 iodide (blue) (A, B) or PI (red) (D, E). The data are mean±SD (error bars) of a representative experiment. *p<0.01.

Fig. 4

Effects of TGF-β signaling on expression of p27. (A) HIT-T15 cells were treated with or without 5 ng/ml TGF-β1, and/or with DMSO or 2 µM SB-431542, for 2 days. Western blotting of the whole cell extracts for p27, and GAPDH, which was used as a control. (B) Results are displayed as the relative protein level of p27. Six repeats of the Western blotting were done independently. The data are mean±SD (error bars) of a representative experiment. *p<0.001. (C, D) Luciferase reporter assays in HIT-T15 cells. Cells were transfected with either p27 luciferase reporter p27PF or empty luciferase reporter pGVB2. In addition, transfection was performed, to stimulate TGF-β signaling, together with or without ALK5* in (C), or to inhibit TGF-β signaling, together with treatment with either DMSO or 2 µM SB-431542 in (D). All data shown are mean±SD (error bars) of a representative experiment performed in at least triplicate. n.s.: not significant.