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Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface [31 integrin expression, and [beta]1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and [beta]1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-[epsilon]. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-[epsilon], which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-E, thereby enabling vimentin phosphorylation, which is important for [beta]1 integrin activation and cell spreading on collagen. actin; cell guidance; human embryonic kidney cells doi: 10.1152/ajpcell.00323.2009

We characterized the role of protein tyrosine phosphatase (PTP)-[alpha] in focal adhesion (FA) formation and remodeling using wild-type and PTP[alpha]deficient (PTP[[alpha].sup.-/-] ) cells. Compared with wild-type cells, spreading PTP[[alpha].sup.-/-] fibroblasts displayed fewer leading edges and formed elongated [alpha]-actinin-enriched FA at the cell periphery. These features suggest the presence of slowly remodeling cell adhesions and were phenocopied in human fibroblasts in which PTP[alpha] was knocked down using short interfering RNA (siRNA) or in NIH3T3 fibroblasts expressing catalytically inactive (C433S/C723S) PTP[alpha]. Fluorescence recovery after photobleaching showed slower green fluorescence protein-[alpha]-actinin recovery in the FA of PTP[[alpha].sup.-/-] than wild-type cells. These alterations correlated with reduced cell spreading, adhesion, and polarization and retarded contraction of extracellular matrices in PTP[[alpha].sup.-/-] fibroblasts. Activation of Rac1 and its recruitment to FA during spreading were diminished in cells expressing C433S/C723S PTP[alpha]. [Rac1.sup.-/-] cells also displayed abnormally elongated and peripherally distributed FA that failed to remodel. Conversely, expression of constitutively active Rac1 restored normal FA remodeling in PTPe[[alpha].sup.-/-] cells. We conclude that PTP[alpha] is required for remodeling of FA during cell spreading via a pathway involving Rac1. cell spreading; integrins; extracellular matrix; actin cytoskeleton

Cyclosporin A (CsA) inhibits collagen remodeling by interfering with the collagen-binding step of phagocytosis. In rapidly remodeling connective tissues such as human periodontium this interference manifests as marked tissue overgrowth and loss of function. Previous data have shown that CsA inhibits integrin-induced release of [Ca.sup.2+] from internal stores, which is required for the binding step of collagen phagocytosis. Because gelsolin is a [Ca.sup.2+]-dependent actin-severing protein that mediates collagen phagocytosis, we determined whether gelsolin is a CsA target. Compared with vehicle controls, CsA treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. Collagen degradation by phagocytosis in cultured gelsolin wild-type fibroblasts was blocked by CsA, comparable to levels of vehicletreated gelsolin-null fibroblasts. In wild-type cells treated with CsA, collagen binding was similar to that of gelsolin-null fibroblasts transfected with a gelsolin-severing mutant and treated with vehicle. CsA blocked collagen-induced [Ca.sup.2+] fluxes subjacent to bound collagen beads, gelsolin recruitment, and actin assembly at bead sites. CsA reduced gelsolin-dependent severing of actin in wild-type cells to levels similar to those in gelsolin-null fibroblasts. We conclude that CsA-induced accumulation of collagen in the extracellular matrix involves disruption of the actin-severing properties of gelsolin, thereby inhibiting the binding step of collagen phagocytosis. adhesion molecules; fibroblasts; knockout mice; actin

New research suggests that, during tubulointerstitial fibrosis, [alpha]-smooth muscle actin (SMA)-expressing mesenchymal cells might derive from the tubular epithelium via epithelial-mesenchymal transition (EMT). Although transforming growth factor-[beta].sub.1] (TGF-[beta].sub.1]) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-[beta].sub.1]-induced EMT in LLC-[PK.sub.1] cells and examined the role of the small GTPase Rho and its effector, Rho kinase, (ROK) in the ensuing cytoskeletal remodeling and SMA expression. TGF-[beta].sub.1] treatment caused delocalization and downregulation of cell contact proteins (ZO-1, E-cadherin, [beta].sub.1]-catenin), cytoskeleton reorganization (stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-[beta].sub.1] induced a biphasic Rho activation. Stress fiber assembly was prevented by the Rho-inhibiting C3 transferase and by dominant negative (DN) ROK. The SMA promoter was activated strongly by constitutively active Rho but not ROK. Accordingly, TGF-[beta].sub.1]-induced SMA promoter activation was potently abrogated by two Rho-inhibiting constructs, C3 transferase and p190RhoGAP, but not by DN-ROK. Truncation analysis showed that the first CC(A/T)richGG (CArG B) serum response factor-binding cis element is essential for the Rho responsiveness of the SMA promoter. Thus Rho plays a dual role in TGF-[beta].sub.1]-induced EMT of renal epithelial cells. It is indispensable both for cytoskeleton remodeling and for the activation of the SMA promoter. The cytoskeletal effects are mediated via the Rho/ ROK pathway, whereas the transcriptional effects are partially ROK independent. Rho kinase; epithelial-mesenchymal transdifferentiation; transforming growth factor-[beta].sub.1]; kidney proximal tubule cells

Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, [Beta]-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases. leukocyte; inflammation; adherens junctions; adhesion molecules; epithelium; inflammatory mediators; tight junctions

Biochemical and functional characterization of intercellular adhesion and gap junctions in fibroblasts. Am J Physiol Cell Physiol 279: C147-C157, 2000.--Despite their significance in wound healing, little is known about the molecular determinants of cell-to-cell adhesion and gap junctional communication in fibroblasts. We characterized intercellular adherens junctions and gap junctions in human gingival fibroblasts (HGFs) using a novel model. Calcein-labeled donor cells in suspension were added onto an established, Texas red dextran (10 kDa)-labeled acceptor cell monolayer. Cell-to-cell adhesion required [Ca.sup.2+] and was [is greater than]30-fold stronger than cell-to-fibronectin adhesion at 15 min. Electron micrographs showed rapid formation of adherens junction-like structures at ~15 min that matured by ~2-3 h; distinct gap junctional complexes were evident by ~3 h. Immunoblotting showed that HGF expressed [Beta]-catenin and that cadherins and connexin43 were recruited to the Triton-insoluble cytoskeletal fraction in confluent cultures. Confocal microscopy localized the same molecules to intercellular contacts of acceptor and donor cells. There was extensive calcein dye transfer in a cohort of Texas red dextran-labeled cells, but this was almost completely abolished by the gap junction inhibitor [Beta]-glycyrrhetinic acid and the connexin43 mimetic peptide GAP 27. This donor-acceptor cell model allows large numbers ([is greater than][10.sup.5]) of cells to form synchronous cell-to-cell contacts, thereby enabling the simultaneous functional and molecular studies of adherens junctions and gap junctions. adherens junctions; fluorescence dye; cell adhesion

New research suggests that, during tubulointerstitial fibrosis, α,-smooth muscle actin (SMA)-expressing mesenchymal cells might derive from the tubular epithelium via epithelial-mesenchymal transition (EMT). Although transforming growth factor-β[sub l] (TGFβ[sub 1]) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-β[sub 1]-induced EMT in LLC-PK[sub 1] cells and examined the role of the small GTPase Rho and its effector, Rho kinase, (ROK) in the ensuing cytoskeletal remodeling and SMA expression. TGF-β[sub 1] treatment caused delocalization and downregulation of cell contact proteins (ZO-1, E-cadherin, β-catenin), cytoskeleton reorganization (stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-β[sub 1] induced a biphasic Rho activation. Stress fiber assembly was prevented by the Rho-inhibiting C3 transferase and by dominant negative (DN) ROK. The SMA promoter was activated strongly by constitutively active Rho but not ROK. Accordingly, TGF-β[sub 1]-induced SMA promoter activation was potently abrogated by two Rho-inhibiting constructs, C3 transferase and p190RhoGAP, but not by DN-ROK. Truncation analysis showed that the first CC(A/T)richGG(CArG B) serum response factor-binding cis element is essential for the Rho responsiveness of the SMA promoter. Thus Rho plays a dual role in TGF-β[sub 1]-induced EMT of renal epithelial cells. It is indispensable both for cytoskeleton remodeling and for the activation of the SMA promoter. The cytoskeletal effects are mediated via the Rho/ ROK pathway, whereas the transcriptional effects are partially ROK independent. [ABSTRACT FROM AUTHOR]