Osteopontin inhibits inducible nitric oxide synthase activity in rat vascular tissue

We tested the hypothesis that osteopontin (OPN) can inhibit the induction of inducible nitric oxide synthase (iNOS) in vascular tissue. iNOS activity was induced in rat thoracic aortas by incubation of the tissue with lipopolysaccharide (LPS) and measured by conversion of L-[H-3]arginine to L-[H-3]citrulline. Addition of greater than or equal to 1 nM recombinant OPN protein significantly reduced the LPS-induced increase in iNOS activity. Western blotting and the RT-PCR were used to determine the effect of LPS with and without OPN on tissue levels of iNOS protein and RNA, respectively. LPS resulted in an increase in iNOS protein and RNA, whereas OPN dose-dependently reduced tissue levels of iNOS activity, protein, and RNA. Mutated OPN proteins, in which the integrin-binding RGD amino acid sequence was deleted or mutated to RGE, resulted in complete and partial loss, respectively, of the ability of OPN to inhibit LPS-induced iNOS activity, implicating integrin binding in the effect. These results indicate that OPN can prevent induction of iNOS in vascular tissue.