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.

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.

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.

Minireview: parathyroid hormone-related protein as an intracrine factor--trafficking mechanisms and functional consequences.

PTH-related protein (PTHrP) was originally discovered as the factor responsible for humoral hypercalcemia of malignancy. PTHrP is produced by most cell types and is a prohormone that gives rise to a family of mature secretory forms arising from posttranslational endoproteolytic cleavage of the initial translation product. Each of these secretory forms of PTHrP is believed to have one or more of its own receptors on the cell surface that mediates the normal paracrine, autocrine, and endocrine actions of PTHrP. Recently, evidence has accumulated that indicates that PTHrP is also able to enter the nucleus and/or the nucleolus and influence cellular events in an intracrine fashion. This review discusses the mechanisms by which PTHrP may gain access to the nucleus/nucleolus and the functional consequences of this nuclear entry by PTHrP.

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.

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.

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.

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.

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.

Prevention of acute ischemic renal failure by targeted delivery of growth factors to the proximal tubule in transgenic mice: the efficacy of parathyroid hormone-related protein and hepatocyte growth factor.

Treatment of acute renal failure (ARF) would be enhanced by identification of factors that accelerate renal recovery from injury. Parathyroid hormone-related protein (PTHrP) and hepatocyte growth factor (HGF) have been shown to stimulate proliferation in proximal nephron-derived cells. For studying the pathophysiologic roles and therapeutic potential of these two factors in ARF, transgenic mice overexpressing PTHrP or HGF in the proximal tubule under the direction of the gamma-glutamyl transpeptidase-I promoter were developed. These mice display (1) abundant expression of the respective transgenes in the kidney; (2) similar PTH type I receptor and HGF receptor (c-met) expression levels in the proximal tubule compared with control littermates; and (3) normal renal morphology, function, and tubule cell proliferation under basal conditions. However, in contrast to control mice, when acute ischemic renal injury was induced, renal function rapidly and dramatically recovered in HGF-overexpressing mice. In addition, 48 h after ischemia, HGF-overexpressing transgenic mice displayed a fourfold increase in tubule cell proliferation and a threefold decrease in apoptotic tubule cell death compared with control mice. In contrast, PTHrP-overexpressing mice responded to either ischemic or folic acid-induced renal damage similarly to control mice. These studies demonstrate that overexpression of PTHrP in the proximal nephron of mice does not seem to provide protection against acute renal injury. In marked contrast, HGF overexpression results in dramatic protection from ischemia-induced ARF, without inducing any apparent alteration in the physiology of the kidney under normal conditions. These studies suggest that HGF, when targeted specifically to the proximal tubule, may have therapeutic potential in providing protection against ischemia-induced renal failure.