Polymerase chain reaction and focal contact formation indicate integrin expression in mesangial cells

Abstract

Cultured kidney glomerular mesangial (MCs) allow the role of extracellular matrix (ECM) and growth factors in glomerular inflammatory disease to be studied. To investigate the potential of MCs to interact with matrix components, the expression of integrin mRNA in cultured MCs was examined by polymerase chain reaction (PCR), by Northern blotting and by immunofluorescence. In addition, the effect of matrix substrates on mRNA expression was assessed by PCR. Northern blots with cDNA probes to integrin α-chains revealed that MCs expressed α1, α3 and α5 integrin mRNA. α1 and α3 were the major messages. No α2, α4 or α6 were detectable. RT-PCR revealed that α2 and α6 were also expressed at low levels. The control cells, HT1080, expressed α2, α3, α4, α5 and α6 mRNA, and Rugli expressed α1, α3, and α5, supporting previous studies. Immunocytochemistry confirmed that α1β1, α2β1, α3β1 and α5β1 integrins were expressed and that they were concentrated into focal adhesions (α1β1 on type I collagen and laminin; α2β1 on type I collagen; α3β1 on type I collagen, laminin and fibronectin; α5β1 on fibronectin). α6β1 was not detected in focal contacts. Attachment, spreading, and formation of talin and integrin containing focal contacts still occurred when endogenous protein synthesis was blocked with 30 μg·ml-1 cycloheximide. Variation of substrate did not lead to a rapid degradation of integrin α-chain mRNA as assessed by RT-PCR. These results provide a basis for studying the regulation of interactions between MCs and extracellular matrix mediated by integrins.