Endothelial cell (EC) dysfunction is implicated in cardiovascular diseases, including diabetes. The decrease in nitric oxide (NO) bioavailability is the hallmark of endothelial dysfunction, and it leads to attenuated vascular relaxation and atherosclerosis followed by a decrease in blood flow. In the heart, decreased coronary blood flow is responsible for insufficient oxygen supply to cardiomyocytes and, subsequently, increases the incidence of cardiac ischemia. In this study we investigate whether and how reactive oxygen species (ROS) in mitochondria contribute to coronary endothelial dysfunction in type 2 diabetic (T2D) mice. T2D was induced in mice by a high-fat diet combined with a single injection of low-dose streptozotocin. ACh-induced vascular relaxation was significantly attenuated in coronary arteries (CAs) from T2D mice compared with controls. The pharmacological approach reveals that NO-dependent, but not hyperpolarization- or prostacyclin-dependent, relaxation was decreased in CAs from T2D mice. Attenuated ACh-induced relaxation in CAs from T2D mice was restored toward control level by treatment with mitoTempol (a mitochondria-specific O2(-) scavenger). Coronary ECs isolated from T2D mice exhibited a significant increase in mitochondrial ROS concentration and decrease in SOD2 protein expression compared with coronary ECs isolated from control mice. Furthermore, protein ubiquitination of SOD2 was significantly increased in coronary ECs isolated from T2D mice. These results suggest that augmented SOD2 ubiquitination leads to the increase in mitochondrial ROS concentration in coronary ECs from T2D mice and attenuates coronary vascular relaxation in T2D mice.

Background:The cytoprotective enzyme HO-1 promotes tumor growth. Results: HO-1 down-regulated apoptosis-and autophagy-regulating proteins, and attenuated Rapamycin-and Sorafenibinduced apoptosis and autophagy in renal cancer cells. Conclusion: HO-1 protects cancer cells from chemotherapeutic drug-induced death by down-regulating apoptosis and autophagy. Significance: Inhibition of HO-1 augments efficiency of chemotherapeutic agents to kill cancer cells by promoting both apoptosis and autophagy.

In this study, we find that CD45RO+ memory populations of CD4+ T lymphocytes express the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 at both the mRNA and protein levels. Furthermore, by Western blot analysis, we find that VEGF increases the phosphorylation and activation of ERK and Akt within CD4+CD45RO+ T cells. These VEGF-mediated signaling responses were inhibited by a KDR-specific small interfering RNA in a VEGF receptor-expressing Jurkat T cell line and by SU5416, a pharmacological KDR inhibitor, in CD4+CD45RO+ T cells. We also find that VEGF augments mitogen-induced production of IFN-γ in a dose-dependent manner (p < 0.001) and significantly (p < 0.05) increases directed chemotaxis of this T cell subset. Collectively, our results for the first time define a novel function for VEGF and KDR in CD45RO+ memory T cell responses that are likely of great pathophysiological importance in immunity.

Cancer is an increasing and major problem after solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors. We propose that the effect of calci-

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The development of cancer is a major problem in immunosuppressed patients, particularly after solid organ transplantation. We have recently shown that calcineurin inhibitors (CNI) used to treat transplant patients may play a critical role in the rapid progression of renal cancer. To examine the intracellular signaling events for CNI-mediated direct tumorigenic pathway(s), we studied the effect of CNI on the activation of proto-oncogenic Ras in human normal renal epithelial cells (REC) and renal cancer cells (786-0 and Caki-1). We found that CNI treatment significantly increased the level of activated GTP-bound form of Ras in these cells. In addition, CNI induced the association of Ras with one of its effector molecules, Raf, but not with Rho and phosphatidylinositol 3-kinase; CNI treatment also promoted the phosphorylation of the Raf kinase inhibitory protein and the downregulation of carabin, all of which may lead to the activation of the RasRaf pathway. Blockade of this pathway through either pharmacologic inhibitors or gene-specific small interfering RNA significantly inhibited CNI-mediated augmented proliferation of renal cancer cells. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the Ras-Raf pathway is significantly activated in the tumor tissues of CNI-treated mice. Together, targeting the Ras-Raf pathway may prevent the development/progression of renal cancer in CNI-treated patients. [Cancer Res 2009;69(23):8902-9]

BackgroundEstrogen receptor α (ERα) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERα phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERα phosphorylation sites exist, COS-1 cells expressing human ERα were labeled with [32P]H3PO4 in vivo and ERα tryptic phosphopeptides were isolated to identify phosphorylation sites.ResultsPreviously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERα in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERα as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor.ConclusionThese novel ERα phosphorylation sites represent new means for modulation of ERα activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor.

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