The signals mediating transforming growth factor β (TGFβ)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFβ-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFβ1-dependent. By immunocytochemistry, TGFβ1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFβ-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFβ-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFβ-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFβ-induced responses to regulate collagen production.

Interendothelial junctions are important regulators of endothelial cell functions such as migration and proliferation, major features in angiogenesis, and endothelial cell monolayer wound healing. 17beta-estradiol regulates these functions in vivo and in vitro and also increases endothelial monolayer permeability as it results from impaired monolayer integrity and intercellular adhesion. We hypothesized that 17beta-estradiol affects these cell adhesion-dependent functions in endothelial cells by targeting the adherens junction complex. Here, we show that 17beta-estradiol increases uterine microvascular endothelial cell monolayer permeability and transiently redistributes interendothelial junction-forming proteins in endothelial cells. Concomitantly, adherens junction proteins are disconnected from the cytoskeleton and alpha-catenin, which links VE-cadherin to the cytoskeleton, is redistributed from the membrane and the adherens junction complex. Furthermore, 17beta-estradiol increased tyrosine phosphorylation of the adherens junction complex. These effects were inhibited by the estrogen receptor antagonist ICI 182,780 but could be provoked using non-cell membrane-permeable 17beta-estradiol-BSA in all cells tested, including EA.hy 926 cells, which have been shown unable to stimulate 17beta-estradiol-dependent gene transcription. Additionally, 17beta-estradiol treatment enhanced the angiogenic effect of vascular endothelial growth factor in an in vitro angiogenesis model, as a potential implication of the adherens junction disruption. Cotreatment with the Src-family kinase inhibitor PP2 prevented the redistribution and phosphorylation of the adherens junction proteins. Taken together, our data show that adherens junctions in endothelial cells are a downstream target of membrane-associated 17beta-estradiol signaling, possibly through Src-family kinases.