The Journal of biological chemistry
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The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two alpha-helices that sit upon an 8-stranded beta-sheet platform. ⋯ Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility.
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The mechanism(s) by which Smads mediate and modulate the transforming growth factor (TGF)-beta signal transduction pathway in fibrogenesis are not well characterized. We previously showed that Smad3 promotes alpha2(I) collagen gene (COL1A2) activation in human glomerular mesangial cells, potentially contributing to glomerulosclerosis. Here, we report that Sp1 binding is necessary for TGF-beta1-induced type I collagen mRNA expression. ⋯ Using the transactivation domain B of Sp1 fused to the Gal4 DNA binding domain, we show that, in our system, the transcriptional activity of this Sp1 domain is not regulated by TGF-beta1, but it becomes responsive to this factor when Smad3 is coexpressed. Finally, combined Sp1 and Smad3 overexpression induces marked ligand-independent and ligand-dependent promoter activity of COL1A2. Thus, Sp1 and Smad proteins form complexes and their synergy plays an important role in mediating TGF-beta1-induced alpha2(I) collagen expression in human mesangial cells.
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Vascular endothelial growth factor (VEGF) induces adhesion molecules on endothelial cells during inflammation. Here we examined the mechanisms underlying VEGF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in human umbilical vein endothelial cells. VEGF (20 ng/ml) increased expression of ICAM-1, VCAM-1, and E-selectin mRNAs in a time-dependent manner. ⋯ These results suggest that VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs was mainly through NF-kappaB activation with PI 3'-kinase-mediated suppression, but was independent of nitric oxide and MEK. Thus, VEGF simultaneously activates two signal transduction pathways that have opposite functions in the induction of adhesion molecule expression. The existence of parallel inverse signaling implies that the induction of adhesion molecule expression by VEGF is very finely regulated.
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We have analyzed tyrosine phosphorylation associated with retraction of the fibrin clot by washed platelets in purified fibrinogen. Retraction was dependent on integrin alpha(IIb)beta(3), based on absence of retraction of alpha(IIb)beta(3)-deficient thrombasthenic platelets. However, only a subset of alpha(IIb)beta(3)-blocking antibodies or peptides were able to inhibit retraction, suggesting a differential engagement of alpha(IIb)beta(3) in fibrin clot retraction versus aggregation. ⋯ Tyrosine phosphatase activities were found associated with clot retraction using the "in-gel" tyrosine phosphatase assay; however, none were alpha(IIb)beta(3)-dependent. An 85-kDa protein and to a lesser degree "Src" showed the closest dose-dependent correlation between inhibition of tyrosine dephosphorylation and inhibition of retraction. We thus postulate that alpha(IIb)beta(3) engagement in fibrin clot retraction drives, in an actin cytoskeleton-dependent manner, the interaction of tyrosine phosphatases and of the tyrosine-phosphorylated substrates 85-kDa protein and Src, the dephosphorylation of which regulates the force generation and/or transmission required for full contraction of the fibrin matrix.