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

Abstract

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 1 H-[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.