PROLYL HYDROXYLATION REGULATES INTRACELLULAR PROCOLLAGEN DEGRADATION IN CULTURED RAT CARDIAC FIBROBLASTS

To determine the regulatory role of prolyl hydroxylation in intracellu lar cardiac procollagen turnover, we examined the effects of prolyl 4- hydroxylase inhibitors (alpha,alpha'-dipyridil, 3,4-dihydroxybenzoic a cid ethyl ester, pyridine 2,4-dicarboxylic acid ethyl ester) and ascor bic acid on procollagen metabolism by cultured, neonatal rat cardiac f ibroblasts. Ascorbate-deficient fibroblasts showed decreased rates of prolyl hydroxylation and total collagen accumulation without a signifi cant reduction in alpha(1)(I) and alpha(1)(III) mRNA levels. The fract ion of newly synthesized procollagens degraded intracellularly was als o substantially increased in ascorbate-deficient cells (50 +/- 7 v 30 +/- 3% in ascorbate-deficient v control fibroblasts; P<0.05). These fi ndings were associated with increased intracellular accumulation of Ty pe I procollagen, enhanced secretion of ''underhydroxylated'' pro alph a(1)(I) polypeptide into the cell culture medium, and decreased extrac ellular Type I collagen deposition, Similar results were obtained:by t reating cells with alpha,alpha'-dipyridil (300 mu M), and 3,4-dihydrox ybenzoic acid ethyl ester (400 mu M) in the presence of ascorbate. A m ajor portion of the enhanced degradation of newly synthesized procolla gens occurred within acidic intracellular compartments as indicated by the inhibition of procollagen degradation by chloroquine (25 mu M). I nhibition of procollagen secretion by colchicine (0.5 mu g/ml) enhance d the diversion to, and subsequent intracellular degradation of underh ydroxylated procollagens in cardiac fibroblast lysosomes, We conclude that inactivation of prolyl 4-hydroxylase increases intracellular accu mulation and intralysosomal degradation of newly synthesized cardiac p rocollagen polypeptides. These observations suggest that procollagen p rolyl hydroxylation may be important in the regulation of collagen acc umulation by cardiac interstitial cells during fibrotic processes in v ivo. (C) 1995 Academic Press Limited