OBJECTIVEMost knowledge on human β-cell cycle control derives from immunoblots of whole human islets, mixtures of β-cells and non-β-cells. We explored the presence, subcellular localization, and function of five early G1/S phase molecules—cyclins D1–3 and cdk 4 and 6—in the adult human β-cell.RESEARCH DESIGN AND METHODSImmunocytochemistry for the five molecules and their relative abilities to drive human β-cell replication were examined. Human β-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse.RESULTSHuman β-cells contain easily detectable cdks 4 and 6 and cyclin D3 but variable cyclin D1. Cyclin D2 was only marginally detectable. All five were principally cytoplasmic, not nuclear. Overexpression of the five, alone or in combination, led to variable increases in human β-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control β-cells). A single molecule, cdk6, proved to be capable of driving human β-cell replication in vitro and enhancing human islet engraftment/proliferation in vivo, superior to normal islets and as effectively as the combination of cdk6 plus a D-cyclin.CONCLUSIONSHuman β-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent β-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal β-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human β-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human β-cell proliferation and function in vivo.

Expansion of pancreatic β-cells is a key goal of diabetes research, yet induction of adult human β-cell replication has proven frustratingly difficult. In part, this reflects a lack of understanding of cell cycle control in the human β-cell. Here, we provide a comprehensive immunocytochemical “atlas” of G1/S control molecules in the human β-cell. This atlas reveals that the majority of these molecules, previously known to be present in islets, are actually present in the β-cell. More importantly, and in contrast to anticipated results, the human β-cell G1/S atlas reveals that almost all of the critical G1/S cell cycle control molecules are located in the cytoplasm of the quiescent human β-cell. Indeed, the only nuclear G1/S molecules are the cell cycle inhibitors, pRb, p57, and variably, p21: none of the cyclins or cdks necessary to drive human β-cell proliferation are present in the nuclear compartment. This observation may provide an explanation for the refractoriness of human β-cells to proliferation. Thus, in addition to known obstacles to human β-cell proliferation, restriction of G1/S molecules to the cytoplasm of the human β-cell represents an unanticipated obstacle to therapeutic human β-cell expansion.

Harnessing control of human β-cell proliferation has proven frustratingly difficult. Most G1/S control molecules, generally presumed to be nuclear proteins in the human β-cell, are in fact constrained to the cytoplasm. Here, we asked whether G1/S molecules might traffic into and out of the cytoplasmic compartment in association with activation of cell cycle progression. Cdk6 and cyclin D3 were used to drive human β-cell proliferation and promptly translocated into the nucleus in association with proliferation. In contrast, the cell cycle inhibitors p15, p18, and p19 did not alter their location, remaining cytoplasmic. Conversely, p16, p21, and p27 increased their nuclear frequency. In contrast once again, p57 decreased its nuclear frequency. Whereas proliferating β-cells contained nuclear cyclin D3 and cdk6, proliferation generally did not occur in β-cells that contained nuclear cell cycle inhibitors, except p21. Dynamic cytoplasmic-nuclear trafficking of cdk6 was confirmed using green fluorescent protein–tagged cdk6 and live cell imaging. Thus, we provide novel working models describing the control of cell cycle progression in the human β-cell. In addition to known obstacles to β-cell proliferation, cytoplasmic-to-nuclear trafficking of G1/S molecules may represent an obstacle as well as a therapeutic opportunity for human β-cell expansion.

Parathyroid hormone-related protein (PTHrP) contains a classical bipartite nuclear localization signal. Nuclear PTHrP induces proliferation of arterial vascular smooth muscle cells (VSMC). In the arterial wall, PTHrP is markedly up-regulated in response to angioplasty and promotes arterial restenosis. PTHrP overexpression exacerbates arterial restenosis, and knockout of the PTHrP gene results in decreased VSMC proliferation in vivo. In arterial VSMC, expression of the cell cycle inhibitor, p27, rapidly decreases after angioplasty, and replacement of p27 markedly reduces neointima development. We have shown that PTHrP overexpression in VSMC leads to p27 down-regulation, mostly through increased proteosomal degradation. Here, we determined the molecular mechanisms through which PTHrP targets p27 for degradation. S-phase kinase-associated protein 2 (skp2) and c-myc, two critical regulators of p27 expression and stability, and neointima formation were up-regulated in PTHrP overexpression in VSMC. Normalization of skp2 or c-myc using small interfering RNA restores normal cell cycle and p27 expression in PTHrP overexpression in VSMC. These data indicate that skp2 and c-myc mediate p27 loss and proliferation induced by PTHrP. c-myc promoter activity was increased, and c-myc target genes involved in p27 stability were up-regulated in PTHrP overexpression in VSMC. In primary VSMC, PTHrP overexpression led to increased c-myc and decreased p27. Conversely, knockdown of PTHrP in primary VSMC from PTHrP(flox/flox) mice led to cell cycle arrest, p27 up-regulation, with c-myc and skp2 down-regulation. Collectively, these data describe for the first time the role of PTHrP in the regulation of skp2 and c-myc in VSMC. This novel PTHrP-c-myc-skp2 pathway is a potential target for therapeutic manipulation of the arterial response to injury.