Development of a reliable automated screening system to identify small molecules and biologics that promote human ß-cell regeneration.

Numerous compounds stimulate rodent ß-cell proliferation; however, translating these findings to human ß-cells remains a challenge. To examine human ß-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human ß-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled ß-cells with high specificity using both nuclear and cytoplasmic markers. ß-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1ß signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ ß-cells, whereas treatment with other compounds had limited to no effect on human ß-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human ß-cell proliferation, thus allowing for increased testing of candidate human ß-cell mitogens.

Calcium-Sensing Receptor Promotes Breast Cancer by Stimulating Intracrine Actions of Parathyroid Hormone-Related Protein.

Parathyroid hormone-related protein (PTHrP) contributes to the development and metastatic progression of breast cancer by promoting hypercalcemia, tumor growth, and osteolytic bone metastases, but it is not known how PTHrP is upregulated in breast tumors. Here we report a central role in this process for the calcium-sensing receptor, CaSR, which enables cellular responses to changes in extracellular calcium, through studies of CaSR-PTHrP interactions in the MMTV-PymT transgenic mouse model of breast cancer and in human breast cancer cells. CaSR activation stimulated PTHrP production by breast cancer cells in vitro and in vivo Tissue-specific disruption of the casr gene in mammary epithelial cells in MMTV-PymT mice reduced tumor PTHrP expression and inhibited tumor cell proliferation and tumor outgrowth. CaSR signaling promoted the proliferation of human breast cancer cell lines and tumor cells cultured from MMTV-PyMT mice. Further, CaSR activation inhibited cell death triggered by high extracellular concentrations of calcium. The actions of the CaSR appeared to be mediated by nuclear actions of PTHrP that decreased p27(kip1) levels and prevented nuclear accumulation of the proapoptotic factor apoptosis inducing factor. Taken together, our findings suggest that CaSR-PTHrP interactions might be a promising target for the development of therapeutic agents to limit tumor cell growth in bone metastases and in other microenvironments in which elevated calcium and/or PTHrP levels contribute to breast cancer progression. Cancer Res; 76(18); 5348-60. ©2016 AACR.

Definition of a Skp2-c-Myc Pathway to Expand Human Beta-cells.

Type 2 diabetes (T2D) is characterized by insulin resistance and reduced functional ß-cell mass. Developmental differences, failure of adaptive expansion and loss of ß-cells via ß-cell death or de-differentiation have emerged as the possible causes of this reduced ß-cell mass. We hypothesized that the proliferative response to mitogens of human ß-cells from T2D donors is reduced, and that this might contribute to the development and progression of T2D. Here, we demonstrate that the proliferative response of human ß-cells from T2D donors in response to cdk6 and cyclin D3 is indeed dramatically impaired. We show that this is accompanied by increased nuclear abundance of the cell cycle inhibitor, p27(kip1). Increasing nuclear abundance of p27(kip1) by adenoviral delivery decreases the proliferative response of ß-cells from non-diabetic donors, mimicking T2D ß-cells. However, while both p27(kip1) gene silencing and downregulation by Skp2 overexpression increased similarly the proliferative response of human ß-cells, only Skp2 was capable of inducing a significant human ß-cell expansion. Skp2 was also able to double the proliferative response of T2D ß-cells. These studies define c-Myc as a central Skp2 target for the induction of cell cycle entry, expansion and regeneration of human T2D ß-cells.

Augmented Stat5 Signaling Bypasses Multiple Impediments to Lactogen-Mediated Proliferation in Human ß-Cells.

Pregnancy in rodents is associated with a two- to threefold increase in ß-cell mass, which is attributable to large increases in ß-cell proliferation, complimented by increases in ß-cell size, survival, and function and mediated mainly by the lactogenic hormones prolactin (PRL) and placental lactogens. In humans, however, ß-cell mass does not increase as dramatically during pregnancy, and PRL fails to activate proliferation in human islets in vitro. To determine why, we explored the human PRL-prolactin receptor (hPRLR)-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5)-cyclin-cdk signaling cascade in human ß-cells. Surprisingly, adult human ß-cells express little or no PRLR. As expected, restoration of the hPRLR in human ß-cells rescued JAK2-STAT5 signaling in response to PRL. However, rescuing hPRLR-STAT5 signaling nevertheless failed to confer proliferative ability on adult human ß-cells in response to PRL. Surprisingly, mouse (but not human) Stat5a overexpression led to upregulation of cyclins D1-3 and cdk4, as well as their nuclear translocation, all of which are associated with ß-cell cycle entry. Collectively, the findings show that human ß-cells fail to proliferate in response to PRL for multiple reasons, one of which is a paucity of functional PRL receptors, and that murine Stat5 overexpression is able to bypass these impediments.

Early and Late G1/S Cyclins and Cdks Act Complementarily to Enhance Authentic Human ß-Cell Proliferation and Expansion.

ß-Cell regeneration is a key goal of diabetes research. Progression through the cell cycle is associated with retinoblastoma protein (pRb) inactivation via sequential phosphorylation by the "early" cyclins and cyclin-dependent kinases (cdks) (d-cyclins cdk4/6) and the "late" cyclins and cdks (cyclin A/E and cdk1/2). In ß-cells, activation of either early or late G1/S cyclins and/or cdks is an efficient approach to induce cycle entry, but it is unknown whether the combined expression of early and late cyclins and cdks might have synergistic or additive effects. Thus, we explored whether a combination of both early and late cyclins and cdks might more effectively drive human ß-cell cell cycle entry than either group alone. We also sought to determine whether authentic replication with the expansion of adult human ß-cells could be demonstrated. Late cyclins and cdks do not traffic in response to the induction of replication by early cyclins and cdks in human ß-cells but are capable of nuclear translocation when overexpressed. Early plus late cyclins and cdks, acting via pRb phosphorylation on distinct residues, complementarily induce greater proliferation in human ß-cells than either group alone. Importantly, the combination of early and late cyclins and cdks clearly increased human ß-cell numbers in vitro. These findings provide additional insight into human ß-cell expansion. They also provide a novel tool for assessing ß-cell expansion in vitro.

Diabetes mellitus--advances and challenges in human ß-cell proliferation.

The treatment of diabetes mellitus represents one of the greatest medical challenges of our era. Diabetes results from a deficiency or functional impairment of insulin-producing ß cells, alone or in combination with insulin resistance. It logically follows that the replacement or regeneration of ß cells should reverse the progression of diabetes and, indeed, this seems to be the case in humans and rodents. This concept has prompted attempts in many laboratories to create new human ß cells using stem-cell strategies to transdifferentiate or reprogramme non-ß cells into ß cells or to discover small molecules or other compounds that can induce proliferation of human ß cells. This latter approach has shown promise, but has also proven particularly challenging to implement. In this Review, we discuss the physiology of normal human ß-cell replication, the molecular mechanisms that regulate the cell cycle in human ß cells, the upstream intracellular signalling pathways that connect them to cell surface receptors on ß cells, the epigenetic mechanisms that control human ß-cell proliferation and unbiased approaches for discovering novel molecules that can drive human ß-cell proliferation. Finally, we discuss the potential and challenges of implementing strategies that replace or regenerate ß cells.

Human pancreatic ß-cell G1/S molecule cell cycle atlas.

Expansion of pancreatic ß-cells is a key goal of diabetes research, yet induction of adult human ß-cell replication has proven frustratingly difficult. In part, this reflects a lack of understanding of cell cycle control in the human ß-cell. Here, we provide a comprehensive immunocytochemical "atlas" of G1/S control molecules in the human ß-cell. This atlas reveals that the majority of these molecules, previously known to be present in islets, are actually present in the ß-cell. More importantly, and in contrast to anticipated results, the human ß-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 ß-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 ß-cell proliferation are present in the nuclear compartment. This observation may provide an explanation for the refractoriness of human ß-cells to proliferation. Thus, in addition to known obstacles to human ß-cell proliferation, restriction of G1/S molecules to the cytoplasm of the human ß-cell represents an unanticipated obstacle to therapeutic human ß-cell expansion.

Cytoplasmic-nuclear trafficking of G1/S cell cycle molecules and adult human ß-cell replication: a revised model of human ß-cell G1/S control.

Harnessing control of human ß-cell proliferation has proven frustratingly difficult. Most G1/S control molecules, generally presumed to be nuclear proteins in the human ß-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 ß-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 ß-cells contained nuclear cyclin D3 and cdk6, proliferation generally did not occur in ß-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 ß-cell. In addition to known obstacles to ß-cell proliferation, cytoplasmic-to-nuclear trafficking of G1/S molecules may represent an obstacle as well as a therapeutic opportunity for human ß-cell expansion.

Overexpression of hepatocyte nuclear factor-4α initiates cell cycle entry, but is not sufficient to promote ß-cell expansion in human islets.

The transcription factor HNF4α (hepatocyte nuclear factor-4α) is required for increased ß-cell proliferation during metabolic stress in vivo. We hypothesized that HNF4α could induce proliferation of human ß-cells. We employed adenoviral-mediated overexpression of an isoform of HNF4α (HNF4α8) alone, or in combination with cyclin-dependent kinase (Cdk)6 and Cyclin D3, in human islets. Heightened HNF4α8 expression led to a 300-fold increase in the number of ß-cells in early S-phase. When we overexpressed HNF4α8 together with Cdk6 and Cyclin D3, ß-cell cycle entry was increased even further. However, the punctate manner of bromodeoxyuridine incorporation into HNF4α(High) ß-cells indicated an uncoupling of the mechanisms that control the concise timing and execution of each cell cycle phase. Indeed, in HNF4α8-induced bromodeoxyuridine(+,punctate) ß-cells we observed signs of dysregulated DNA synthesis, cell cycle arrest, and activation of a double stranded DNA damage-associated cell cycle checkpoint mechanism, leading to the initiation of loss of ß-cell lineage fidelity. However, a substantial proportion of ß-cells stimulated to enter the cell cycle by Cdk6 and Cyclin D3 alone also exhibited a DNA damage response. HNF4α8 is a mitogenic signal in the human ß-cell but is not sufficient for completion of the cell cycle. The DNA damage response is a barrier to efficient ß-cell proliferation in vitro, and we suggest its evaluation in all attempts to stimulate ß-cell replication as an approach to diabetes treatment.

Regulated and reversible induction of adult human ß-cell replication.

Induction of proliferation in adult human ß-cells is challenging. It can be accomplished by introduction of cell cycle molecules such as cyclin-dependent kinase 6 (cdk6) and cyclin D1, but their continuous overexpression raises oncogenic concerns. We attempted to mimic normal, transient, perinatal human ß-cell proliferation by delivering these molecules in a regulated and reversible manner. Adult cadaveric islets were transduced with doxycycline (Dox)-inducible adenoviruses expressing cdk6 or cyclin D1. End points were cdk6/cyclin D1 expression and human ß-cell proliferation, survival, and function. Increasing doses of Dox led to marked dose- and time-related increases in cdk6 and cyclin D1, accompanied by a 20-fold increase in ß-cell proliferation. Notably, Dox withdrawal resulted in a reversal of both cdk6 and cyclin D1 expression as well as ß-cell proliferation. Re-exposure to Dox reinduced both cdk/cyclin expression and proliferation. ß-Cell function and survival were not adversely affected. The adenoviral tetracycline (tet)-on system has not been used previously to drive human ß-cell proliferation. Human ß-cells can be induced to proliferate or arrest in a regulated, reversible manner, temporally and quantitatively mimicking the transient perinatal physiological proliferation that occurs in human ß-cells.

c-myc and skp2 coordinate p27 degradation, vascular smooth muscle proliferation, and neointima formation induced by the parathyroid hormone-related protein.

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.

A human islet cell culture system for high-throughput screening.

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.

The scaffolding protein EBP50 promotes vascular smooth muscle cell proliferation and neointima formation by regulating Skp2 and p21(cip1).

OBJECTIVE: The Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffolding protein known to regulate ion homeostasis in the kidney and intestine. Previous work showed that EBP50 expression increases after balloon injury in rat carotids. This study was designed to determine the role of EBP50 on vascular smooth muscle cells (VSMC) proliferation and the development of neointimal hyperplasia. METHODS AND RESULTS: Wire injury was performed in wild type (WT) and EBP50 knockout (KO) mice. Two weeks after injury, neointima formation was 80% lower in KO than in WT mice. Proliferation of KO VSMC was significantly lower than WT cells and overexpression of EBP50 increased VSMC proliferation. Akt activity and expression of S-phase kinase protein2 decreased in KO cells resulting in the stabilization of the cyclin-dependent kinase inhibitor, p21(cip1). Consequently, KO cells were arrested in G(0)/G(1) phase. Consistent with these observations, p21(cip1) was detected in injured femoral arteries of KO but not WT mice. No differences in apoptosis between WT and KO were observed. CONCLUSIONS: EBP50 is critical for neointima formation and induces VSMC proliferation by decreasing S-phase kinase protein2 stability, thereby accelerating the degradation of the cell cycle inhibitor p21(cip1).

EBP50 inhibits the anti-mitogenic action of the parathyroid hormone type 1 receptor in vascular smooth muscle cells.

Parathyroid hormone-related protein (PTHrP) and the parathyroid hormone type 1 receptor (PTH1R) are important regulators of vascular remodeling. PTHrP expression is associated to increased proliferation of vascular smooth muscle cells (VSMC). In contrast, signaling via the PTH1R inhibits cell growth. The mechanisms regulating the dual effect of PTHrP and PTH1R on VSMC proliferation are only partially understood. In this study we examined the role of the adaptor protein ezrin-radixin-moesin-binding phosphoprotein (EBP50) on PTH1R expression, trafficking, signaling and control of A10 cell proliferation. In normal rat vascular tissues, EBP50 was restricted to the endothelium with little expression in VSMC. EBP50 expression significantly increased in VSMC following angioplasty in parallel with PTHrP. Interestingly, PTHrP was able to induce EBP50 expression. In the clonal rat aortic smooth muscle cell line A10, EBP50 increased the recruitment of PTH1R to the cell membrane and delayed its internalization in response to PTHrP(1-36). This effect required an intact C-terminal motif in the PTH1R. In naïve A10 cells, PTHrP(1-36) stimulated cAMP production but not intracellular calcium release. In contrast, PTHrP(1-36) induced both cAMP and calcium signaling in A10 cells over-expressing EBP50. Finally, EBP50 attenuated the induction of p27(kip1) and the anti-proliferative effect of PTHrP(1-36). In summary, this study demonstrates the dynamic expression of EBP50 in vessels following injury and the effects of EBP50 on PTH1R function in VSMC. These findings highlight one of the mechanisms leading to increased VSMC proliferation and have important implication in the understanding of the molecular events leading to restenosis.

Induction of human beta-cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6.

OBJECTIVE: Most knowledge on human beta-cell cycle control derives from immunoblots of whole human islets, mixtures of beta-cells and non-beta-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 beta-cell. RESEARCH DESIGN AND METHODS: Immunocytochemistry for the five molecules and their relative abilities to drive human beta-cell replication were examined. Human beta-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse. RESULTS: Human beta-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 beta-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control beta-cells). A single molecule, cdk6, proved to be capable of driving human beta-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 beta-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent beta-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal beta-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human beta-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human beta-cell proliferation and function in vivo.

Endogenous parathyroid hormone-related protein regulates the expression of PTH type 1 receptor and proliferation of vascular smooth muscle cells.

The PTH type 1 receptor (PTH1R) and PTHrP are expressed in vessels, where they contribute to regulating vascular smooth muscle cell (VSMC) function. Elevated PTHrP levels in VSMC are often associated with hyperplasia. In contrast, exogenous PTHrP, acting through the PTH1R, inhibits VSMC proliferation. In this study, we investigated the regulation of PTH1R expression by endogenous PTHrP and the associated effects on VSMC proliferation. Blocking binding of secreted PTHrP fragments to the PTH1R by treatment with either an antagonist or an antibody against PTHrP, and inhibition of PTHrP expression by small interfering RNA significantly increased PTH1R expression. Interestingly, treatment of the cells with a PTHrP analog (Bpa(1)-PTHrP) that activates the PTH1R without inducing its internalization had the same effect on receptor expression. To examine the association between receptor expression and the antiproliferative effect of N-terminal fragments of PTHrP, VSMC were treated with exogenous PTHrP (1-36) acutely and chronically to induce receptor down-regulation. Stimulation of VSMC with exogenous PTHrP (1-36) significantly reduced cell proliferation during the first 18 h of treatment but was no longer effective after 3 d, a time when PTH1R was down-regulated. In contrast, treatment with the noninternalizing agonist Bpa(1)-PTHrP strongly inhibited cell proliferation at all time points. In conclusion, our study show that PTHrP, after its intracellular processing and secretion, promotes down-regulation of the PTH1R in VSMC, thereby regulating cell proliferation in an auto/paracrine fashion. This regulatory mechanism may have important implication during vascular remodeling, in particular in the development of neointima after arterial injury, where PTHrP overexpression occurs.

Survey of the human pancreatic beta-cell G1/S proteome reveals a potential therapeutic role for cdk-6 and cyclin D1 in enhancing human beta-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 beta-cell replication, to determine whether human beta-cell replication can be demonstrated in an in vivo model, and to enhance human beta-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 beta-cells were studied in both in vitro and in vivo models. RESULTS: Multiple differences between murine and human islets occur, most strikingly the presence of cdk-6 in human beta-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 beta-cell replication, both in vitro as well as in vivo, without inducing cell death or loss of function. Using this model, human beta-cell replication can be induced and studied in vivo.

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 enhances engraftment and function of nonhuman primate islets.

OBJECTIVE: Adenoviral delivery of hepatocyte growth factor (HGF) to rodent islets improves islet graft survival and function, markedly reducing the number of islets required to achieve glucose control. Here, we asked whether these prior observations in rodent models extend to nonhuman primate (NHP) islets. RESEARCH DESIGN AND METHODS: NHP islets were transduced with murine (Ad.mHGF) or human (Ad.hHGF) adenoviral HGF (Ad.HGF) at low multiplicity of infection and studied in vitro. To study the function of Ad.HGF-transduced NHP islets in vivo, a renal subcapsular marginal mass islet transplant model was developed in streptozotocin-induced diabetic NOD-SCID mice. RESULTS: Baseline glucose values were 454.7 +/- 11.3 mg/dl (n = 7). Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 +/- 9.7 mg/dl, n = 5). In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0 +/- 6.2 mg/dl, n = 7) for the 6-week duration of the experiment. Unilateral nephrectomy resulted in an immediate return of glucose to baseline diabetic levels. Interestingly, adenoviral DNA, as well as mouse HGF (mHGF) mRNA derived from the adenovirus, were present for 42 days posttransplantation. Surprisingly, transplant of 500 IE with Ad.hHGF, as compared with Ad.mHGF, resulted in only marginal correction of blood glucose, suggesting that human HGF is less efficient than mHGF in this system. CONCLUSIONS: These studies demonstrate that mHGF markedly improves islet transplant outcomes in the highest preclinical species examined to date. HGF has promise as an agent that can improve islet mass and function in transplant models and likely in other models of types 1 and 2 diabetes.

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 beta-cell lines and provide clues as to the central cell cycle regulatory molecules in the beta-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 beta-cells to those in insulinoma cells. Unfortunately, this therapeutic overexpression appears to mildly attenuate beta-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 beta-cells.