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.

Calcium efflux activity of plasma membrane Ca2+ ATPase-4 (PMCA4) mediates cell cycle progression in vascular smooth muscle cells.

We explored the role played by plasma membrane calcium ATPase-4 (PMCA4) and its alternative splice variants in the cell cycle of vascular smooth muscle cells (VSMC). A novel variant (PMCA4e) was discovered. Quantitative real-time-PCR-quantified PMCA4 splice variant proportions differed in specific organs. The PMCA4a:4b ratio in uninjured carotid arteries (∼1:1) was significantly reduced by wire denudation injury (to ∼1:3) by modulation of alternative splicing, as confirmed by novel antibodies against PMCA4a/e and PMCA4b. Laser capture microdissection localized this shift to the media and adventitia. Primary carotid VSMC from PMCA4 knock-out (P4KO) mice showed impaired [(3)H]thymidine incorporation and G1 phase arrest as compared with wild type (P4WT). Electroporation of expression constructs encoding PMCA4a, PMCA4b, and a PMCA4b mutant lacking PDZ binding rescued this phenotype of P4KO cells, whereas a mutant with only 10% of normal Ca(2+) efflux activity could not. Microarray of early G1-synchronized VSMC showed 39-fold higher Rgs16 (NFAT (nuclear factor of activated T-cells) target; MAPK inhibitor) and 69-fold higher Decorin (G1 arrest marker) expression in P4KO versus P4WT. Validation by Western blot also revealed decreased levels of Cyclin D1 and NFATc3 in P4KO. Microarrays of P4KO VSMC rescued by PMCA4a or PMCA4b expression showed reversal of perturbed Rgs16, Decorin, and NFATc3 expression levels. However, PMCA4a rescue caused a 44-fold reduction in AP-2ß, a known anti-proliferative transcription factor, whereas PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca(2+) efflux activity of PMCA4 underlies G1 progression in VSMC and that PMCA4a and PMCA4b differentially regulate specific downstream mediators.

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.

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.

Factors associated with non-adherence among psychiatric patients at a tertiary care hospital, Karachi, Pakistan: a questionnaire based cross-sectional study.

OBJECTIVE: To elucidate predictors of non-adherence among psychiatric patients presenting at a tertiary care hospital of Pakistan, for follow-up with consultant psychiatrist. METHODS: A convenient sampleof psychiatric patients from Aga Khan University Hospital was enrolled between April and May, 2005. An interviewer assisted, standardized questionnaire was used for data collection. Patients with cognitive deficit or psychosis and those presenting for the first time were not included in the study. RESULTS: Out of 128 patients, those with co-morbidity (32.81%) were less adherent than those without comorbidity (p-value:0.002). Adherence among depressed was 61.53%; psychotic was 58.82%; bipolar disorder was 73.91%. Reasons for non-adherence included sedation (30%), medication cost (22%), forgot to take medication (36%); and inability of the physicians to explain timing and dose (92%) or benefit of medication (76%). CONCLUSIONS: Non-adherence is a common and important issue. Treatment cost and co-morbidity should be reviewed in order to keep the medication regime affordable and comprehensible.

Myths and fallacies about epilepsy among residents of a Karachi slum area.

Misconceptions about epilepsy may explain the considerable stigma accompanying it. We aimed to identify such fallacies through questionnaire-based interviews of 487 adult residents of a slum area in Karachi, Pakistan. Of those interviewed, 25% believed that epilepsy was caused by evil spirits, black magic and envy by others - those without a school education were more likely to hold these views (P < 0.05). Perceived complications included impotence and cancer. Shoe-sniffing was considered a treatment modality by 13%. It appears that misconceptions abound regarding epilepsy's causes, complications and methods of treatment. However, those who had received a school education were less likely to link epilepsy with supernatural phenomena.