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

Nitric oxide ((NO)-N-.) is an important physiological signaling molecule and potent vasodilator. Recently, we have shown abnormal (NO)-N-. 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)-N-. 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)-N-. 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)-N-. had no effect in SSc fibroblasts. Furthermore, the inhibition of collagen synthesis by (NO)-N-. was reversed by ascorbic acid and was not affected by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1 -one, ail inhibitor of soluble guanyl cyclase, or 8-bromoguanosine cyclic 3,5'-monophosphate, a cGMP agonist. SNP also showed a significant upregulation of matrix metalloproteinase-1 (MMP-1) protein and activity levels, ail essential collagenase involved in collagen degradation, in the AG fibroblasts. Additionally, (NO)-N-.-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)-N-. 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 upregulation of MMP-1 and/or inhibition of prolyl hydroxylase. These differences may contribute to the observed pathology of SSc. (C) 2007 Elsevier Inc. All rights reserved.