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. 1999 Jun;5(6):382–392.

TNF-alpha induces actin cytoskeleton reorganization in glomerular epithelial cells involving tyrosine phosphorylation of paxillin and focal adhesion kinase.

S B Koukouritaki 1, E A Vardaki 1, E A Papakonstanti 1, E Lianos 1, C Stournaras 1, D S Emmanouel 1
PMCID: PMC2230436  PMID: 10415163

Abstract

Glomerular permeability for macromolecules depends partially on proper attachment of the glomerular epithelial cells (GEC) to the glomerular basement membrane (GBM). The latter requires integrity of the actin cytoskeleton, which in turn is regulated by specific actin-associated proteins. Since several glomerulopathies characterized by heavy proteinuria are associated with increased glomerular tumor necrosis factor alpha (TNF-alpha) expression, we studied the interaction of TNF-alpha with the actin cytoskeleton of cultured rat GEC. Incubation of GEC with 10 ng/ml TNF-alpha for variable time periods ranging from 15 min to 24 hr demonstrated a marked accentuation and redistribution of actin microfilaments, as shown by direct fluorescence analysis and confocal laser scanning microscopy. Quantitative biochemical determination of the G/total-actin ratio confirmed the above observations. Indeed, this ratio was significantly reduced, indicating substantial polymerization of G-actin and formation of F-actin. Concurrently, TNF-alpha rapidly induced tyrosine phosphorylation of both paxillin and focal adhesion kinase, without affecting the expression levels of these two proteins. In addition, tyrosine phosphorylation of vinculin became evident, indicating involvement of this focal adhesion marker in the observed actin reorganization. Inhibition of tyrosine phosphorylation by genistein prevented the reorganization of the actin cytoskeleton by TNF-alpha. We conclude that TNF-alpha induces substantial reorganization of actin cytoskeleton and focal adhesions. These effects occur simultaneously, with a prompt TNF-alpha-induced tyrosine phosphorylation of paxillin and focal adhesion kinase, indicating that these proteins, known to regulate actin polymerization and formation of focal adhesions, may be directly involved in the mechanism controlling the observed actin redistribution. These findings suggest that the observed TNF-alpha-actin cytoskeleton interactions may relate to the pathogenesis of glomerulopathies with heavy proteinuria, in which increased glomerular expression of TNF-alpha is associated with disturbances in the attachment of podocytes to the GBM.

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