Irene Cozar-Castellano

Induction of Human β-Cell Proliferation and Engraftment Using a Single G1/S Regulatory Molecule, cdk6

OBJECTIVE Most 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 METHODS Immunocytochemistry 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. RESULTS Human 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. CONCLUSIONS Human 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.

Survey of the Human Pancreatic β-Cell G1/S Proteome Reveals a Potential Therapeutic Role for Cdk-6 and Cyclin D1 in Enhancing Human β-Cell Replication and Function In Vivo

OBJECTIVES To comprehensively inventory the proteins that control the G1/S cell cycle checkpoint in the human islet and compare them with those in the murine islet, to determine whether these might therapeutically enhance human β-cell replication, to determine whether human β-cell replication can be demonstrated in an in vivo model, and to enhance human β-cell function in vivo. RESEARCH DESIGN AND METHODS Thirty-four G1/S regulatory proteins were examined in human islets. Effects of adenoviruses expressing cdk-6, cdk-4, and cyclin D1 on proliferation in human β-cells were studied in both invitro and in vivo models. RESULTS Multiple differences between murine and human islets occur, most strikingly the presence of cdk-6 in human β-cells versus its low abundance in the murine islet. Cdk-6 and cyclin D1 in vitro led to marked activation of retinoblastoma protein phosphorylation and cell cycle progression with no induction of cell death. Human islets transduced with cdk-6 and cyclin D1 were transplanted into diabetic NOD-SCID mice and markedly outperformed native human islets in vivo, maintaining glucose control for the entire 6 weeks of the study. CONCLUSIONS The human G1/S proteome is described for the first time. Human islets are unlike their rodent counterparts in that they contain easily measurable cdk-6. Cdk-6 overexpression, alone or in combination with cyclin D1, strikingly stimulates human β-cell replication, both in vitro as well as in vivo, without inducing cell death or loss of function. Using this model, human β-cell replication can be induced and studied in vivo.

Lessons from the first comprehensive molecular characterization of cell cycle control in rodent insulinoma cell lines.

OBJECTIVE—Rodent insulinoma cell lines may serve as a model for designing continuously replicating human β-cell lines and provide clues as to the central cell cycle regulatory molecules in the β-cell. RESEARCH DESIGN AND METHODS—We performed a comprehensive G1/S proteome analysis on the four most widely studied rodent insulinoma cell lines and defined their flow cytometric profiles and growth characteristics. RESULTS—1) Despite their common T-antigen–derived origins, MIN6 and BTC3 cells display markedly different G1/S expression profiles; 2) despite their common radiation origins, RINm5F and INS1 cells display striking differences in cell cycle protein profiles; 3) phosphorylation of pRb is absent in INS1 and RINm5F cells; 4) cyclin D2 is absent in RINm5F and BTC3 cells and therefore apparently dispensable for their proliferation; 5) every cell cycle inhibitor is upregulated, presumably in a futile attempt to halt proliferation; 6) among the G1/S proteome members, seven are pro-proliferation molecules: cyclin-dependent kinase-1, -2, -4, and -6 and cyclins A, E, and D3; and 7) overexpression of the combination of these seven converts arrested proliferation rates in primary rat β-cells to those in insulinoma cells. Unfortunately, this therapeutic overexpression appears to mildly attenuate β-cell differentiation and function. CONCLUSIONS—These studies underscore the importance of characterizing the cell cycle at the protein level in rodent insulinoma cell lines. They also emphasize the hazards of interpreting data from rodent insulinoma cell lines as modeling normal cell cycle progression. Most importantly, they provide seven candidate targets for inducing proliferation in human β-cells.

The Cell Cycle Inhibitory Protein p21cip Is Not Essential for Maintaining β-Cell Cycle Arrest or β-Cell Function In Vivo

p21cip1, a regulatory molecule upstream of the G1/0 checkpoint, is increased in β-cells in response to mitogenic stimulation. Whereas p21cip1 can variably stimulate or inhibit cell cycle progression, in vitro studies suggest that p21cip1 acts as an inhibitor in the pancreatic β-cell. To determine the functional role of p21cip1 in vivo, we studied p21-null mice. Surprisingly, islet mass, β-cell replication rates, and function were normal in p21-null mice. We next attempted to drive β-cell replication in p21-null mice by crossing them with rat insulin II promoter–murine PL-1 (islet-targeted placental lactogen transgenic) mice. Even with this added replicative stimulus of PL, p21-null islets showed no additional stimulation. A G1/S proteome scan demonstrated that p21cip1 loss was not associated with compensatory increases in other cell cycle inhibitors (pRb, p107, p130, p16, p19, and p27), although mild increases in p57 were apparent. Surprisingly, p18, which had been anticipated to increase, was markedly decreased. In summary, isolated p21cip1 loss, as for pRb, p53, p18, and p27 and other inhibitors, results in normal β-cell development and function, either because it is not essential or because its function is subserved or complimented by another protein. These studies underscore marked inhibitory pressure and the complexity and plasticity of inhibitory pathways that restrain β-cell replication.

Cellular Mechanism Through Which Parathyroid Hormone-Related Protein Induces Proliferation in Arterial Smooth Muscle Cells. Definition of an Arterial Smooth Muscle PTHrP/p27kip1 Pathway

Parathyroid hormone–related protein (PTHrP) is present in vascular smooth muscle (VSM), is markedly upregulated in response to arterial injury, is essential for normal VSM proliferation, and also markedly accentuates neointima formation following rat carotid angioplasty. PTHrP contains a nuclear localization signal (NLS) through which it enters the nucleus and leads to marked increases in retinoblastoma protein (pRb) phosphorylation and cell cycle progression. Our goal was to define key cell cycle molecules upstream of pRb that mediate cell cycle acceleration induced by PTHrP. The cyclin D/cdk-4,-6 system and its upstream regulators, the inhibitory kinases (INKs), are not appreciably influenced by PTHrP. In striking contrast, cyclin E/cdk-2 kinase activity is markedly increased by PTHrP, and this is a result of a specific, marked, PTHrP-induced proteasomal degradation of p27kip1. Adenoviral restoration of p27kip1 fully reverses PTHrP-induced cell cycle progression, indicating that PTHrP mediates its cell cycle acceleration in VSM via p27kip1. In confirmation, adenoviral delivery of PTHrP to murine primary vascular smooth muscle cells (VSMCs) significantly decreases p27kip1 expression and accelerates cell cycle progression. p27kip1 is well known to be a central cell cycle regulatory molecule involved in both normal and pathological VSM proliferation and is a target of widely used drug-eluting stents. The current observations define a novel “PTHrP/p27kip1 pathway” in the arterial wall and suggest that this pathway is important in normal arterial biology and a potential target for therapeutic manipulation of the arterial response to injury.

Tissue-Specific Deletion of the Retinoblastoma Protein in the Pancreatic β-Cell Has Limited Effects on β-Cell Replication, Mass, and Function

Animal studies show that G1/S regulatory molecules (D-cyclins, cdk-4, p18, p21, p27) are critical for normal regulation of β-cell proliferation, mass, and function. The retinoblastoma protein, pRb, is positioned at the very end of a cascade of these regulatory proteins and is considered the final checkpoint molecule that maintains β-cell cycle arrest. Logically, removal of pRb from the β-cell should result in unrestrained β-cell replication, increased β-cell mass, and insulin-mediated hypoglycemia. Because global loss of both pRb alleles is embryonic lethal, this hypothesis has not been tested in β-cells. We developed two types of conditional knockout (CKO) mice in which both alleles of the pRb gene were inactivated specifically in β-cells. Surprisingly, although the pRb gene was efficiently recombined in β-cells of both CKO models, changes in β-cell mass, β-cell replication rates, insulin concentrations, and blood glucose levels were limited or absent. Other pRb family members, p107 and p130, were not substantially upregulated. In contrast to dogma, the pRb protein is not essential to maintain cell cycle arrest in the pancreatic β-cell. This may reflect fundamental inaccuracies in models of β-cell cycle control or complementation for pRb by undefined proteins.

Mutant Parathyroid Hormone-Related Protein, Devoid of the Nuclear Localization Signal, Markedly Inhibits Arterial Smooth Muscle Cell Cycle and Neointima Formation by Coordinate Up-Regulation of p15Ink4b and p27kip1

Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (DeltaNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro. This is of clinical relevance because adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which DeltaNLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, DeltaNLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G(0)/G(1) arrest. This pRb underphosphorylation was associated with stable levels of cdks 2, 4, and 6, the D and E cyclins, p16, p18, p19, and p21, but was associated with a dramatic decrease in cdk-2 and cdk4 kinase activities. Cyclin A was reduced, but restoring cyclin A adenovirally to normal did not promote cell cycle progression in DeltaNLS-PTH-related protein VSMC. More importantly, p15(INK4) and p27(kip1), two critical inhibitors of the G(1/S) progression, were markedly increased. Normalization of both p15(INK4b) and p27(kip1) by small interfering RNA knockdown normalized cell cycle progression. These data indicate that the changes in p15(INK4b) and p27(kip1) fully account for the marked cell cycle slowing induced by DeltaNLS-PTH-related protein in VSMCs. Finally, DeltaNLS-PTH-related protein is able to induce p15(INK4) and p27(kip1) expression when delivered adenovirally to primary murine VSMCs. These studies provide a mechanistic understanding of DeltaNLS-PTH-related protein actions, and suggest that DeltaNLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis.

Hepatocyte growth factor gene therapy for islet transplantation.

Recent clinical studies have documented that human islet transplantation has the potential to replace pancreatic endocrine function in patients with type 1 diabetes. These studies have also highlighted an enormous shortage of human islets that impedes the use of islet transplantation in clinical practice on a larger scale. To address this problem, one potential approach is to use islet growth factors to increase beta cell replication, to improve beta cell function and to enhance beta cell survival. In that context, transgenic mice overexpressing hepatocyte growth factor (HGF) in the pancreatic beta cell display increased beta cell proliferation, function and survival. More importantly, HGF-overexpressing transgenic mouse islets markedly improve transplant performance in severe combined immunodeficiency (SCID) mice and reduce the number of islets required for successful islet transplantation. Recently, adenoviral-mediated gene transfer of HGF into normal rodent islets has confirmed the beneficial effects of HGF in improving islet transplant outcomes in two marginal mass islet transplant models in rodents: islet transplant under the kidney capsule in SCID mice; and portal islet allograft transplantation in rats treated with the Edmonton immunosuppressive regimen. These studies suggest that ex vivo HGF gene therapy has the potential to reduce the number of human islets required for successful islet transplantation.

Molecular engineering human hepatocytes into pancreatic beta cells for diabetes therapy

Type 1 (formerly “juvenile”) diabetes is believed to be an autoimmune disease in which host immune cells fail to recognize the insulin-producing beta cells contained within pancreatic islets of Langerhans as being “self” and inappropriately elect to destroy these critical insulin-producing cells. The traditional methods of treating type 1 diabetes are good, but not perfect. The studies reported by Sapir et al. (1) in this issue of PNAS suggest that new, more acceptable approaches to beta cell replacement may be feasible. In the current treatment of type 1 diabetes, insulin is given by injection or insulin pump. Patients must frequently check their blood glucose by finger stick to calculate and adjust their insulin doses. Undertreatment (hyperglycemia) and overtreatment (hypoglycemia) are common. The ideal therapy for diabetes would mimic the two essential features of normal beta cells: the ability to sense glucose continually coupled with intelligent and appropriate release of insulin in response to changes in blood glucose. One solution could be an implantable, glucose-sensing insulin delivery system. Attempts to develop such a computer-driven, “closed-loop” insulin delivery system have been aided substantially by the miniaturization of computer chips and insulin pumps. Important hurdles relate to development of reliable, long-term glucose sensors, miniaturization, replacement of batteries, and refillable reservoirs for the necessary insulin pumps. Whole pancreas transplant is another option. Long-term graft function is excellent, but the procedure requires major surgery, is limited by the small numbers of pancreata available for transplant, and is accompanied by morbidity from immunosuppressive drugs used to prevent organ rejection (2). In 2000, a group in Edmonton, Canada, demonstrated the feasibility of pancreatic islet transplantation (3), which requires only trivial outpatient surgery. The Edmonton demonstration that long-term islet graft survival and function are possible has reinvigorated attempts at beta cell replacement therapy. In broad terms, there are …