The negative cell cycle regulators, p27^Kip1, p18^Ink4c, and GSK-3, play critical role in maintaining quiescence of adult human pancreaticβ-cells and restrict their ability to proliferate

Abstract

Adult human pancreatic β-cells are primarily quiescent (G0) yet the mechanisms controlling their quiescence are poorly understood. Here, we demonstrate, by immunofluorescence and confocal microscopy, abundant levels of the critical negative cell cycle regulators, p27(Kip1) and p18(Ink4c), 2 key members of cyclin-dependent kinase (CDK) inhibitor family, and glycogen synthase kinase-3 (GSK-3), a serine-threonine protein kinase, in islet β-cells of adult human pancreatic tissue. Our data show that p27(Kip1) localizes primarily in β-cell nuclei, whereas, p18(Ink4c) is mostly present in β-cell cytosol. Additionally, p-p27(S10), a phosphorylated form of p27(Kip1), which was shown to interact with and to sequester cyclinD-CDK4/6 in the cytoplasm, is present in substantial amounts in β-cell cytosol. Our immunofluorescence analysis displays similar distribution pattern of p27(Kip1), p-p27(S10), p18(Ink4c) and GSK-3 in islet β-cells of adult mouse pancreatic tissue. We demonstrate marked interaction of p27(Kip1) with cyclin D3, an abundant D-type cyclin in adult human islets, and vice versa as well as with its cognate kinase partners, CDK4 and CDK6. Likewise, we show marked interaction of p18(Ink4c) with CDK4. The data collectively suggest that inhibition of CDK function by p27(Kip1) and p18(Ink4c) contributes to human β-cell quiescence. Consistent with this, we have found by BrdU incorporation assay that combined treatments of small molecule GSK-3 inhibitor and mitogen/s lead to elevated proliferation of human β-cells, which is caused partly due to p27(Kip1) downregulation. The results altogether suggest that ex vivo expansion of human β-cells is achievable via increased proliferation for β-cell replacement therapy in diabetes.

Dual Effect of Raptor on Neonatal β-Cell Proliferation and Identity Maintenance

“…Cell cycle inhibitors were reported to be equally important in regulating b-cell proliferation (36)(37)(38). In our study, most factors that promote cell cycle progression were downregulated: only four cell cycle inhibitors were significantly upregulated (p57Kip2, Tgfb2, Tgfb3, Gadd45b) (Fig.…”

MicroRNAs 106b and 222 Improve Hyperglycemia in a Mouse Model of Insulin-Deficient Diabetes via Pancreatic β-Cell Proliferation

“…β-cells have post-injury regenerative capacity, but the processes are slow and highly restricted (Bouwens and Rooman, 2005). Particularly, in human adult pancreatic β-cells, Cip/Kip family members, such as p27 kip1 (Stein et al, 2014, Stein et al, 2013) and p57 kip2 (Avrahami et al, 2014), reportedly play key roles in maintaining quiescence, leading to very limited proliferation in response to the numerous mitogens, growth factors, and nutrients that have been proposed to induce β-cell expansion (Kulkarni et al, 2012). Therefore, the identification of these miRNAs, which suppress Cip/Kip family members in β-cells, might overcome this important obstacle to therapeutic application of β-cell regeneration.…”

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation

“…TAg inhibits pRb and p53 activity and thus short circuits entry into the G 1 and S stages of the cell cycle (39). p18 inhibits cyclin-CDK4/CDK6 complexes that are required for G 0 /G 1 progression (40), and p21 prevents G 1 progression and entry into S phase by blocking cyclin-CDK2/CDK4 activity (41,42). Taken together, our data indicate that mature beta cells reside in G 0 /G 1 and that silencing p18 and p21 lowers the threshold for G 1 /S phase entry.…”