Effect of nitric oxide and peroxynitrite on type I collagen synthesis in normal and scleroderma dermal fibroblasts

Nitric oxide (NO) is an important physiological signaling molecule and potent vasodilator. Recently, we have shown abnormal NO metabolism in the plasma of patients with systemic sclerosis (SSc), a disease that features excessive collagen overproduction as well as vascular dysfunction. The current study investigates the effects of NO and peroxynitrite (ONOO–) on secretion of type I collagen by SSc dermal fibroblasts, compared with those from normal dermal fibroblasts (CON) and a dermal fibroblast cell line (AG). Dermal fibroblasts were incubated with NO donors (SNP, DETA-NONOate) with or without the antioxidant ascorbic acid, or ONOO– for 24–72 h. In CON and AG fibroblasts, type I collagen was dose dependently decreased by SNP or DETA-NONOate. However, NO had no effect in SSc fibroblasts. Furthermore, the inhibition of collagen synthesis by NO was reversed by ascorbic acid and was not affected by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanyl cyclase, or 8-bromoguanosine cyclic 3′,5′-monophosphate, a cGMP agonist. SNP also showed a significant up-regulation of matrix metalloproteinase-1 (MMP-1) protein and activity levels, an essential collagenase involved in collagen degradation, in the AG fibroblasts. Additionally, NO-treated fibroblasts had lower prolyl hydroxylase activity, an enzyme important in the post-translational processing of collagen, while there was no effect on total protein levels. There were no significant effects on type I collagen levels when dermal fibroblasts were treated with ONOO–. Taken together, NO inhibits collagen secretion in normal dermal fibroblasts but regulation is lost in SSc fibroblasts, while ONOO– itself is ineffective. NO inhibition of collagen was by cGMP-independent regulatory mechanisms and in part may be due to up-regulation of MMP-1 and/or inhibition of prolyl hydroxylase. These differences may contribute to the observed pathology of SSc.