HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.     

AMPK inhibits fatty acid-induced increases in NF-kappaB transactivation in cultured human umbilical vein endothelial cells

The fuel sensing enzyme AMP-activated protein kinase (AMPK) enhances processes that generate ATP when stresses such as exercise or glucose deprivation make cells energy deficient. We report here a novel role of AMPK, to prevent the activation of NF-kappaB in endothelial cells exposed to the fatty acid palmitate or the cytokine TNF-alpha. Incubation of cultured human umbilical vein endothelial cells (HUVEC) with elevated levels of palmitate (0.4mM) increased NF-kappaB reporter gene expression by 2- to 4-fold within 8h and caused a 7-fold increase in VCAM-1 mRNA expression at 24h. In contrast, no increase in reporter gene expression was detected for AP-1, glucocorticoid-, cyclic AMP-, or serum response elements. Similar increases in NF-kappaB activation and VCAM-1 expression were not observed in cells incubated with an elevated concentration of glucose (25mM). The increases in NF-kappaB activation and VCAM-1 expression caused by palmitate were markedly inhibited by co-incubation with the AMPK activator AICAR and, where studied, by expression of a constitutively active AMPK. Likewise, AMPK activation inhibited the increase in NF-kappaB reporter gene expression observed in HUVEC incubated with TNF-alpha. The results suggest that AMPK inhibits the activation of NF-kappaB caused by both palmitate and TNF-alpha. The mechanism responsible for this action, as well as its relevance to the reported anti-atherogenic actions of exercise, metformin, thiazolidinediones, and adiponectin, all of which have been shown to activate AMPK, remains to be determined.     

人免疫缺陷病毒1型TAT突变蛋白及反义TAT RNA对TAT反式激活作用的抑制

Ｔａｔ是人免疫缺陷病毒（ＨＩＶ）基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制ＨＩＶ复制的途径,构建了以ＨＩＶ－１ＬＴＲ（－１５８－＋８０）为启动子的Ｔａｔ ｃＤＮＡ全长反义表达质粒ｐＡＳ－Ｔａｔ,并用已经构建的ＨＩＶ ＬＴＲ－１５８到＋８０为启动子,具有不同突变点的突变Ｔａｔ基因表达质粒,以荧光酶基因为报告基因,共转染Ｊｕｒｋａｔ细胞,结果发现无论是反义Ｔａｔ表达质粒还     

Low shear stress regulates monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha dependent pathway

In regions of a vessel that experience low shear stress and reversing flow patterns, early features in the pathogenesis of atherosclerosis include the accumulation of oxidized LDL (OxLDL) and adhesion of monocytes to endothelial cells (EC). Here we investigated the hypothesis that low shear stress (2 dyn/cm2) and OxLDL are synergistic for enhanced expression of vascular cell adhesion molecule (VCAM-1) and human aortic endothelial cell (HAEC)-monocyte adhesion. This study shows low shear stress can significantly reduce IkappaBalpha levels, activate NF-kappaB, increase the expression of VCAM-1 in HAEC and binding of monocytes. OxLDL itself cannot significantly increase the expression of VCAM-1 in HAEC and binding of monocytes, but through activation of NF-kappaB and degradation of IkappaBalpha induced by low shear stress it can significantly enhance VCAM-1 expression and monocyte adhesion, over that in unmodified LDL or control. These results suggest that low shear stress can regulate monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha-dependent pathway, and that low shear stress together with OxLDL may likely play an important role in atherogenesis.     

HIV-1 Tat-mediated effects on focal adhesion assembly and permeability in brain microvascular endothelial cells

The blood-brain barrier (BBB) is a network formed mainly by brain microvascular endothelial cells (BMECs). The integrity of the BBB is critical for brain function. Breakdown of the BBB is commonly seen in AIDS patients with HIV-1-associated dementia despite the lack of productive HIV infection of the brain endothelium. The processes by which HIV causes these pathological conditions are not well understood. In this study we characterized the molecular mechanisms by which Tat mediates its pathogenic effects in vitro on primary human BMECs (HBMECs). Tat treatment of HBMECs stimulated cytoskeletal organization and increased focal adhesion sites compared with control cells or cells treated with heat-inactivated Tat. Pretreatment with Tat Abs or with the specific inhibitor SU-1498, which interferes with vascular endothelial growth factor receptor type 2 (Flk-1/KDR) phosphorylation, blocked the ability of Tat to stimulate focal adhesion assembly and the migration of HBMECs. Focal adhesion kinase (FAK) was tyrosine-phosphorylated by Tat and was found to be an important component of focal adhesion sites. Inhibition of FAK by the dominant interfering mutant form, FAK-related nonkinase, significantly blocked HBMEC migration and disrupted focal adhesions upon Tat activation. Furthermore, HIV-Tat induced permeability changes in HBMECs in a time-dependent manner. Tat also impaired BBB permeability, as observed in HIV-1 Tat transgenic mice. These studies define a mechanism for HIV-1 Tat in focal adhesion complex assembly in HBMECs via activation of FAK, leading to cytoskeletal reorganization and permeability changes.     

HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication is highly dependent on and modulated by interactions between viral and host cellular factors. Tat protein, encoded by one of the HIV-1 regulatory genes, tat, is essential for HIV-1 gene expression. A number of host cellular factors have been shown to interact with Tat in this process. During our attempts to determine the molecular mechanisms of Tat interaction with brain cells, we isolated a cDNA clone that encodes a novel Tat-interacting protein of 110 kDa or Tip110 from a human fetal brain cDNA library. GenBank BLAST search revealed that Tip110 was almost identical to a previously cloned KIAA0156 gene with unknown functions. In vivo binding of Tip110 with Tat was confirmed by immunoprecipitation and Western blotting, in combination with mutagenesis. The yeast three-hybrid RNA-protein interaction assay indicated no direct interaction of Tip110 with Tat transactivating response element RNA. Nevertheless, Tip110 strongly synergized with Tat on Tat-mediated chloramphenicol acetyltransferase reporter gene expression and HIV-1 virus production, whereas down-modulation of constitutive Tip110 expression inhibited HIV-1 virus production. Northern blot analysis showed that Tip110 mRNA was expressed in a variety of human tissues and cells. Moreover, digital fluorescence microscopic imaging revealed that Tip110 was expressed exclusively in the nucleus, and within a nuclear speckle structure that has recently been described for human cyclin T and CDK9, two critical components for Tat transactivation function on HIV-1 long terminal repeat promoter. Taken together, these data demonstrate that Tip110 regulates Tat transactivation activity through direct interaction, and suggest that Tip110 is an important cellular factor for HIV-1 gene expression and viral replication.