Targeting the cell cycle inhibitor p57Kip2 promotes adult human β cell replication
Dana Avrahami, … , Benjamin Glaser, Klaus H. Kaestner
Dana Avrahami, … , Benjamin Glaser, Klaus H. Kaestner
Published January 16, 2014
Citation Information: J Clin Invest. 2014;124(2):670-674. https://doi.org/10.1172/JCI69519.
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Brief Report Metabolism

Targeting the cell cycle inhibitor p57Kip2 promotes adult human β cell replication

Abstract

Children with focal hyperinsulinism of infancy display a dramatic, non-neoplastic clonal expansion of β cells that have undergone mitotic recombination, resulting in paternal disomy of part of chromosome 11. This disomic region contains imprinted genes, including the gene encoding the cell cycle inhibitor p57Kip2 (CDKN1C), which is silenced as a consequence of the recombination event. We hypothesized that targeting p57Kip2 could stimulate adult human β cell replication. Indeed, when we suppressed CDKN1C expression in human islets obtained from deceased adult organ donors and transplanted them into hyperglycemic, immunodeficient mice, β cell replication increased more than 3-fold. The newly replicated cells retained properties of mature β cells, including the expression of β cell markers such as insulin, PDX1, and NKX6.1. Importantly, these newly replicated cells demonstrated normal glucose-induced calcium influx, further indicating β cell functionality. These findings provide a molecular explanation for the massive β cell replication that occurs in children with focal hyperinsulinism. These data also provided evidence that β cells from older humans, in which baseline replication is negligible, can be coaxed to re-enter and complete the cell cycle while maintaining mature β cell properties. Thus, controlled manipulation of this pathway holds promise for the expansion of β cells in patients with type 2 diabetes.

Authors

Dana Avrahami, Changhong Li, Ming Yu, Yang Jiao, Jia Zhang, Ali Naji, Seyed Ziaie, Benjamin Glaser, Klaus H. Kaestner

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Figure 1

Suppression of p57Kip2 in human islets.

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Suppression of p57Kip2 in human islets. (A) Molecular pathology of foc...
(A) Molecular pathology of focal hyperinsulinism. Left: Maternal and paternal copies of chromosome 11 inherited by the fetus. The paternal allele carries a recessive mutation in either ABCC8 or KCNJ11, the two subunits of the ATP-sensitive potassium channel. During fetal life, a break in the maternal chromosome followed by DNA repair using the paternal chromosome as a template occurs in a single β cell. Uniparental disomy of the short arm of chromosome 11 results, as shown in the right panel, and expression of the imprinted p57Kip2 (CDKN1C) gene is silenced. (B) Suppression of p57Kip2 using shRNA lentiviral particles. Quantitative RT-PCR (qPCR) showing mRNA levels of p57Kip2 in HEK293 cells transfected with p57Kip2-specific pGIPZ shRNA constructs. Transfected cells were FACS sorted for GFP-positive and GFP-negative fractions. Expression levels were normalized to an NT shRNA control (n = 3). (C) qRT-PCR of CDKN1C mRNA levels in cultured human islets transduced with lentiviral particles containing pGIPZ shRNA (clone p57-c) against p57Kip2. Five days after transduction, islet were dispersed and FACS sorted for GFP-positive (transduced) and GFP-negative fractions. Expression levels were normalized to NT shRNA control (n = 3 human islet donors). (D) Immunostaining of human islets transduced with pGIPZ shRNA lentivirus against p57Kip2. Turbo-GFP (green), insulin (white), and DNA (blue). Arrows point to costaining of insulin and turbo-GFP. (E) Immunostaining of recovered human islet grafts for turbo-GFP, which allows for tracking of shRNA expression. Turbo-GFP (green), PDX1 (white), and DNA (blue). Arrows point to turbo-GFP/PDX1–positive cells. Scale bars: 20 μm.