Cell Replication in Mice Transgenic for Hepatocyte Growth Factor and Placental Lactogen
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Original Article Evaluation of -Cell Replication in Mice Transgenic for Hepatocyte Growth Factor and Placental Lactogen Comprehensive Characterization of the G1/S Regulatory Proteins Reveals Unique Involvement of p21cip Irene Cozar-Castellano, Matthew Weinstock, Marcia Haught, Silvia Vela´zquez-Garcia, Darinka Sipula, and Andrew F. Stewart We hypothesized that combined transgenic overexpression ing belief that -cells cannot replicate and that, if induced of hepatocyte growth factor (HGF) and placental lactogen to replicate, they inevitably would de-differentiate. In in islets would lead to even greater increases in -cell mass contrast, we (3–5) and others (6–10) have demonstrated and replication than either growth factor alone. This did that human and rodent -cells can replicate and retain not occur, suggesting that -cell replication is saturable or their differentiated phenotype in vivo. A recent study has subject to molecular restraint. We therefore performed the suggested that replication of -cells is the principal mech- first comprehensive G1/S cell cycle survey in islets, cata-  loguing the broad range of kinases, cyclins, and kinase anism through which new -cells are generated (6). Thus, a clear and comprehensive understanding of -cell repli- inhibitors that control the G1/S transition in islets from normal, HGF, placental lactogen, and doubly transgenic cation will be important for developing novel approaches mice. Many of the G1/S checkpoint regulators (E2Fs; pRb; to treating and preventing diabetes. p107; p130; cyclins D1,2,3, A, and E; cdk-2; cdk-4; p15; p16; Important lessons regarding -cell replication have been p18; p19; p21; p27; MDM2; p53; c-Myc; and Egr-1) are revealed from mouse genetic models. For example, com- present in the murine islet. Most of these proteins were bined knockout of the p53 and pRb tumor suppressor unaltered by overexpression of HGF or placental lactogen, genes leads to pancreatic islet tumors (11,12). Similarly, either alone or in combination. In contrast, p21cip was uniquely, dramatically, and reproducibly upregulated in overexpression of simian virus 40 viral large T-antigen also placental lactogen and HGF islets. p21cip was also present leads to islet hyperplasia, and ultimately to islet tumori- in, and upregulated in, proliferating human islets, localiz- genesis (13). Cyclin D1 is the most abundant oncogene in ing specifically in -cells and translocating to the nucleus human insulinomas (14), and cyclin D1 overexpression on mitogenic stimulation. Homozygous p21cip loss releases leads to -cell hyperplasia in transgenic mice (15). Cyclin islets from growth inhibition, markedly enhancing prolifer- D2 has been shown to be essential for achieving and ation in response to HGF and placental lactogen. Diabetes maintaining -cell mass (16,17) and deletion of E2F1 and 55:70–77, 2006 E2F2 transcription factors results in -cell failure (18,19). Knockout of cdk-4 results in -cell failure and diabetic ketoacidosis (20,21), and overexpression of constitutively active cdk-4 results in -cell hyperplasia and ultimately rogress in pancreatic islet transplantation (1) and -cell tumorigenesis (20). Adenoviral overexpression of in immunosuppression directed against the auto- ϳ cyclin D1 and cdk-4 in human islets leads to an 10-fold immunity of type 1 diabetes (2) has focused  attention on a need to understand the cellular increase in the rate of -cell replication, without any P  apparent loss of function (5). These observations are mechanisms that control -cell replication. Surprisingly, significant in aggregate, for they demonstrate that the cell little is known regarding the molecular control of cell  cycle events in the -cell, in part, because of a longstand- cycle in the -cell is accessible to study and manipulation. Importantly, because all of the molecules described above are involved in the progression or arrest of the cell cycle at From the Division of Endocrinology, The University of Pittsburgh School of the G1/S checkpoint, they suggest that this is a particularly Medicine, Pittsburgh, Pennsylvania. important site of control in the -cell. Address correspondence and reprint requests to Irene Cozar-Castellano, PhD, Division of Endocrinology, BST E-1140, University of Pittsburgh School Because most or all of the molecules described above of Medicine, 200 Lothrop St., Pittsburgh, PA 15213. E-mail: cozari@dom. are present in all cells, it is not surprising that disturbing pitt.edu. these molecules might disrupt cell cycle control in the Received for publication 18 May 2005 and accepted in revised form 30  September 2005. -cell. In contrast, the large group of cell cycle control Additional information for this article can be found in an online appendix at molecules that lie upstream of these molecules, exempli- http://diabetes.diabetesjournals.org. fied by the inhibitory kinase (INK), cyclin inhibitory pro- CIP, cyclin inhibitory protein; HGF, hepatocyte growth factor; INK, inhibi- teins or kinase inhibitory proteins (CIP/KIP), p53, c-Myc, tory kinase; KIP, kinase inhibitory protein; PTHrP, parathyroid hormone– related protein; RIP, rat insulin promoter; RIP-PL, RIP-placental lactogen. and others (rev. in 22–24), are less ubiquitous and often © 2006 by the American Diabetes Association. tissue-specific in their expression and/or function. Surpris- The costs of publication of this article were defrayed in part by the payment of page ingly, very little is known about their presence or function charges. This article must therefore be hereby marked “advertisement” in accordance kip with 18 U.S.C. Section 1734 solely to indicate this fact. within the -cell. Only one of these, p27 , has been 70 DIABETES, VOL. 55, JANUARY 2006 I. COZAR-CASTELLANO AND ASSOCIATES FIG. 1. A: Low-power photomicrographs of whole pancreatic insulin-stained sections from the four genotypes. Islet mass and islet size are increased in all three transgenic mouse types, but the double-transgenic mice are not different from their single-transgenic littermates. B: Quantitative pancreatic histomorphometry on pancreata of the four genotypes. As reported previously in the RIP-HGF and RIP-PL mice (3,4,26,27), islet mass is increased as shown. There is no further increase in either islet mass or number in the double-transgenic mice. *P < 0.05; **P < 0.01. n, the number of animals studied in each group. implicated in control of the -cell cycle, and this observa- dine incorporation as described in detail previously (5). Briefly, p21Ϫ/Ϫ and tion is very recent (25). normal mouse islets were freshly isolated and placed in RPMI medium containing 5 mmol/l glucose and 10% fetal bovine serum in 24-well plates, with We have previously described three types of transgenic 50 islets per well. HGF or prolactin was added 24 h later in the concentrations mice in which parathyroid hormone–related protein shown in the legend of Fig. 8 (for HGF, for both p21ϩ/ϩ and p21Ϫ/Ϫ,11 (PTHrP) (26), hepatocyte growth factor (HGF) (3,27), or mmol/l glucose was used), and the islets were incubated for an additional 48 h. placental lactogen (4,28) has been overexpressed in the Tritiated thymidine (0.5 Ci/well) was added 24 h later in RPMI medium -cell under the control of the rat insulin promoter (RIP). containing the growth factors but without fetal bovine serum, and the islets Whereas many growth factors have been demonstrated to were allowed to incubate for an additional 24 h (for a total of 48 h with HGF  or prolactin and 24 h with tritiated thymidine). The remainder of the activate -cell mitogenesis in vitro, these three RIP-PTHrP, procedure was as described previously (5). RIP-placental lactogen (RIP-PL), and RIP-HGF mouse Cyclin D1 and cdk-4 adenoviruses. The adenoviral cyclin D1 and cdk-4 lines represent the only examples of growth factor–driven studies were performed using adenoviruses, conditions, and reagents de- -cell hyperplasia and insulin-mediated hypoglycemia. Be- scribed in detail previously (5). Briefly, murine and human islets were cause the RIP-PL and RIP-HGF mice have a marked transduced for 1 h with 500 multiplicity of infection of adenoviruses express- ing cyclin D and cdk-4 under the control of the cytomegalovirus promoter. increase in -cell replication rates, and because HGF and 1 Islet isolation and semiquantitative RT-PCR. Mouse islets were isolated placental lactogen signal through different pathways, we as described previously (3–5,26,27). Human islets were provided by the hypothesized that mice doubly transgenic for placental University of Pittsburgh Human Islet Isolation Facility as described in detail lactogen and HGF would display even greater increases in previously (5). Semiquantitative PCR was performed as described in detail -cell proliferation rates, -cell mass, and overall -cell previously (3–5,26,27) using the primer pairs shown in Supplemental Table function. 1, which is detailed in the online appendix (available at http://diabetes. Here, we demonstrate that this hypothesis is incorrect, diabetesjournals.org) (see table legend for full details). suggesting that intrinsic, rate-limiting controls exist that Cell cycle protein immunoblots. Islet extracts were prepared using nuclear  extraction buffer as described previously (5). Antisera and dilutions used are prevent unrestricted growth factor–driven -cell expan- shown in Supplemental Table 2, which is detailed in the online appendix sion. To define these molecular controls, we have compre- (available at http://diabetes.diabetesjournals.org). Each immunoblot