Integrins and the cytoskeleton: focal adhesion kinase and paxillin.

Increases in intraglomerular pressure are known to organization leading to stress fibre formation, and predispose for the development of glomerular sclerosis. focal adhesion assembly accompany tyrosine phosMesangial cells are exposed to high glomerular capilphorylation of FAK and paxillin. Thus, these structures lary pressures and appear to be a potential primary play important roles in the intracellular signal transtarget within the glomerulus for increased mechanical duction from extracellular stimuli. Because of the stress. We have studied the effect of cyclic stretch on possible involvement of cell to substratum interactions the mesangial cellular function to mimic the condition in the mechanical deformation-induced mesangial cell of mesangial cells in glomerular hypertension, and function alterations, we examined the effect of stretch previously demonstrated that subjecting cultured rat on the integrin aggregation, stress fibre formation, and mesangial cells to the continuous cycles of stretch/ tyrosine phosphorylation of FAK and paxillin. relaxation stimulates, cell proliferation, prostaglandin, Rat cultured mesangial cells were plated on six-well TGF-b, and extracellular matrix (ECM) components plates with a flexible bottom coated with type I collagen production. Translocation of protein kinase C (PKC) (Flexcell Corp., PA). Cells were grown to subconfluactivity to the membrane and increase in calcium ence and rendered quiescent by incubation for 48 h in uptake and total calcium content, expression of several 0.5% FBS medium and exposed to cycles of stretching immediate early genes including c-fos and egr-1, were and relaxation using a computer-driven, vacuumalso observed in the early time course of the stretch. operated, stress-providing instrument (Flexercell Strain The mechanisms by which these cells sense passive Unit 100B; Flexcell Corp.). The condition of stretch/ stretch and transduce it into signals leading to specific relaxation was 9 to 12% elongation and 30 cycles per gene expression have not yet been determined. min. F-actin or b1 integrin was stained using FITCIntegrins are a family of a,b-heterodimeric transphalloidin or anti-b1-integrin antibody. Tyrosine phosmembrane proteins that connect the cytoskeleton to phorylation of FAK and paxillin were studied by the ECM. Recent studies have shown that integrinWestern blotting after immunoprecipitation of cell mediated adhesion regulates a variety of intracellular lysates using phosphotyrosine antibody. After probing events, including induction of cell spreading, cytoskeleFAK, the membrane was stripped and reprobed with tal organization, and activation of phosphoinsitide paxillin. synthesis. Thus integrins mediate a variety of signalling When mesangial cells were subjected to stretch, cascades. The focal adhesions are localized at the sites F-actin staining by FITC-phalloidin showed stress fibre of cell attachment to the ECM and the most concenformation at 3 min reaching a peak at 10 min. This trated sites of tyrosine phosphorylation. The focal stress fibre formation disappeared 30 min after subadhesion consists of a complex of proteins such as jecting to stretch. The staining pattern of b1-integrin p125 focal adhesion kinase (FAK) and paxillin. FAK after 10 min of stretch also changed from diffuse is the focal adhesion tyrosine kinase protein and autostaining before stretch becoming more condensed at phosphorylated with the focal adhesion complex the periphery of the cell, this indicates clustering of assembly. Paxillin, which interacts with other focal b1-integrin by stretching. Angiotensin II, ionomycin, adhesion proteins and the cytoskeleton, such as vincuand activation of PKC with phorbol dibutyrate lin and FAK, is thought to be a substrate of FAK and induced stress fibre formation, but the degree and also phosphorylated with the focal adhesion complex duration of stress fibre formation were weaker and assembly. The focal adhesion complex connects inteshorter than those induced by stretch. We also examgrin with cytoskeletal filaments and thus acts both as ined the effect of a longer time course of stretch on a signalling device and a connection to the cytostress fibre formation. After 24 and 48 h of stretch, skeleton. Many stimuli, such as integrin occupancy condensed stress fibre was observed and the stress with a ligand, induce integrin aggregation, F-actin fibres were aligned with their long axis perpendicular to the direction of stretch. As the mechanisms of this Correspondence and offprint requests to: Takashi Yasada, Department stress fibre formation after a longer stretch time seemed of Medicine 1, St Marianna University School of Medicine, 2-16-1 Sugao Miyamae-ku, Kawasaki-shi, 216-8511, Japan. to be different from those observed in the early time