alpha v integrin regulates TNF-alpha-induced nitric oxide synthesis in ratmesangial cells - possible role of osteopontin

Tumour necrosis factor-alpha (TNF-alpha) induces nitric oxide (NO) synthesi s in rat mesangial cells (MCs). We previously demonstrated that osteopontin (OP), a matrix protein that mainly interacts with the alpha v integrin fam ily, increased time-dependently by TNF-alpha stimulation at gene and protei n levels. The regulation of NO synthesis by integrins or matrix proteins is unclear. Methods. We examined whether integrin, especially alpha v integrin, regulat es NO synthesis in rat MCs and whether OP, an alpha v integrin ligand, has an effect on TNF-alpha-induced NO synthesis. Furthermore, OP and inducible NO synthase (iNOS) gene expression was examined by Northern blotting. Results. TNF-alpha increased NO synthesis in MCs in a time-dependent manner . Synthetic GRGDSP peptide, which is known to inhibit various integrins tha t interact with RGD-containing extracellular matrices, increased TNF-alpha- induced NO levels in a dose-dependent manner. Cyclical RGD peptide, the spe cific inhibitor of alpha v integrin, also exhibited a dose-dependent effect of increasing NO levels, while GRGESP peptide, which has very low affinity to integrins, had no effect. In addition, NO synthesis was found to be sig nificantly reduced when MCs were plated on OF-coated dishes compared to typ e I or IV collagen-coated dishes. Furthermore, anti-OF antibody increased N O synthesis in MCs. iNOS mRNA levels were increased by TNF-alpha, and were abruptly diminished after OP mRNA was significantly induced. Conclusions. The present study demonstrated the involvement of alpha v inte grin in TNF-alpha-induced NO synthesis in rat MCs, and the possible role of OP was suggested in the mechanism. TNF-alpha and extracellular matrices ca n co-operate to regulate the behaviour of MCs at least partly through NO sy nthesis, which may participate in the course of glomerular diseases.