10 resultados para EPITHELIAL-CELL PROLIFERATION
em Duke University
Resumo:
BACKGROUND: Uterine leiomyomas (fibroids) are benign smooth muscle tumors that often contain an excessive extracellular matrix (ECM). In the present study, we investigated the interactions between human uterine leiomyoma (UtLM) cells and uterine leiomyoma-derived fibroblasts (FB), and their importance in cell growth and ECM protein production using a coculture system. RESULTS: We found enhanced cell proliferation, and elevated levels of ECM collagen type I and insulin-like growth factor-binding protein-3 after coculturing. There was also increased secretion of vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor-2, and platelet derived growth factor A and B in the media of UtLM cells cocultured with FB. Protein arrays revealed increased phosphorylated receptor tyrosine kinases (RTKs) of the above growth factor ligands, and immunoblots showed elevated levels of the RTK downstream effector, phospho-mitogen activated protein kinase 44/42 in cocultured UtLM cells. There was also increased secretion of transforming growth factor-beta 1 and 3, and immunoprecipitated transforming growth factor-beta receptor I from cocultured UtLM cells showed elevated phosphoserine expression. The downstream effectors phospho-small mothers against decapentaplegic -2 and -3 protein (SMAD) levels were also increased in cocultured UtLM cells. However, none of the above effects were seen in normal myometrial cells cocultured with FB. The soluble factors released by tumor-derived fibroblasts and/or UtLM cells, and activation of the growth factor receptors and their pathways stimulated the proliferation of UtLM cells and enhanced the production of ECM proteins. CONCLUSIONS: These data support the importance of interactions between fibroid tumor cells and ECM fibroblasts in vivo, and the role of growth factors, and ECM proteins in the pathogenesis of uterine fibroids.
Resumo:
Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.
Resumo:
Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy.
Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.
Resumo:
We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.
Resumo:
Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.
Resumo:
BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle.
Resumo:
Restenosis continues to be a major problem limiting the effectiveness of revascularization procedures. To date, the roles of heterotrimeric G proteins in the triggering of pathological vascular smooth muscle (VSM) cell proliferation have not been elucidated. betagamma subunits of heterotrimeric G proteins (Gbetagamma) are known to activate mitogen-activated protein (MAP) kinases after stimulation of certain G protein-coupled receptors; however, their relevance in VSM mitogenesis in vitro or in vivo is not known. Using adenoviral-mediated transfer of a transgene encoding a peptide inhibitor of Gbetagamma signaling (betaARKct), we evaluated the role of Gbetagamma in MAP kinase activation and proliferation in response to several mitogens, including serum, in cultured rat VSM cells. Our results include the striking finding that serum-induced proliferation of VSM cells in vitro is mediated largely via Gbetagamma. Furthermore, we studied the effects of in vivo adenoviral-mediated betaARKct gene transfer on VSM intimal hyperplasia in a rat carotid artery restenosis model. Our in vivo results demonstrated that the presence of the betaARKct in injured rat carotid arteries significantly reduced VSM intimal hyperplasia by 70%. Thus, Gbetagamma plays a critical role in physiological VSM proliferation, and targeted Gbetagamma inhibition represents a novel approach for the treatment of pathological conditions such as restenosis.
Resumo:
Activation of CD4+ T cells results in rapid proliferation and differentiation into effector and regulatory subsets. CD4+ effector T cell (Teff) (Th1 and Th17) and Treg subsets are metabolically distinct, yet the specific metabolic differences that modify T cell populations are uncertain. Here, we evaluated CD4+ T cell populations in murine models and determined that inflammatory Teffs maintain high expression of glycolytic genes and rely on high glycolytic rates, while Tregs are oxidative and require mitochondrial electron transport to proliferate, differentiate, and survive. Metabolic profiling revealed that pyruvate dehydrogenase (PDH) is a key bifurcation point between T cell glycolytic and oxidative metabolism. PDH function is inhibited by PDH kinases (PDHKs). PDHK1 was expressed in Th17 cells, but not Th1 cells, and at low levels in Tregs, and inhibition or knockdown of PDHK1 selectively suppressed Th17 cells and increased Tregs. This alteration in the CD4+ T cell populations was mediated in part through ROS, as N-acetyl cysteine (NAC) treatment restored Th17 cell generation. Moreover, inhibition of PDHK1 modulated immunity and protected animals against experimental autoimmune encephalomyelitis, decreasing Th17 cells and increasing Tregs. Together, these data show that CD4+ subsets utilize and require distinct metabolic programs that can be targeted to control specific T cell populations in autoimmune and inflammatory diseases.
Resumo:
T cell activation leads to engagement of cellular metabolic pathways necessary to support cell proliferation and function. However, our understanding of the signal transduction pathways that regulate metabolism and their impact on T cell function remains limited. The liver kinase B1 (LKB1) is a serine/threonine kinase that links cellular metabolism with cell growth and proliferation. In this study, we demonstrate that LKB1 is a critical regulator of T cell development, viability, activation, and metabolism. T cell-specific ablation of the gene that encodes LKB1 resulted in blocked thymocyte development and a reduction in peripheral T cells. LKB1-deficient T cells exhibited defects in cell proliferation and viability and altered glycolytic and lipid metabolism. Interestingly, loss of LKB1 promoted increased T cell activation and inflammatory cytokine production by both CD4(+) and CD8(+) T cells. Activation of the AMP-activated protein kinase (AMPK) was decreased in LKB1-deficient T cells. AMPK was found to mediate a subset of LKB1 functions in T lymphocytes, as mice lacking the α1 subunit of AMPK displayed similar defects in T cell activation, metabolism, and inflammatory cytokine production, but normal T cell development and peripheral T cell homeostasis. LKB1- and AMPKα1-deficient T cells each displayed elevated mammalian target of rapamycin complex 1 signaling and IFN-γ production that could be reversed by rapamycin treatment. Our data highlight a central role for LKB1 in T cell activation, viability, and metabolism and suggest that LKB1-AMPK signaling negatively regulates T cell effector function through regulation of mammalian target of rapamycin activity.
Resumo:
Emerging evidence suggests that microRNAs can initiate asymmetric division, but whether microRNA and protein cell fate determinants coordinate with each other remains unclear. Here, we show that miR-34a directly suppresses Numb in early-stage colon cancer stem cells (CCSCs), forming an incoherent feedforward loop (IFFL) targeting Notch to separate stem and non-stem cell fates robustly. Perturbation of the IFFL leads to a new intermediate cell population with plastic and ambiguous identity. Lgr5+ mouse intestinal/colon stem cells (ISCs) predominantly undergo symmetric division but turn on asymmetric division to curb the number of ISCs when proinflammatory response causes excessive proliferation. Deletion of miR-34a inhibits asymmetric division and exacerbates Lgr5+ ISC proliferation under such stress. Collectively, our data indicate that microRNA and protein cell fate determinants coordinate to enhance robustness of cell fate decision, and they provide a safeguard mechanism against stem cell proliferation induced by inflammation or oncogenic mutation.