PROLYL HYDROXYLATION REGULATES INTRACELLULAR PROCOLLAGEN DEGRADATION IN CULTURED RAT CARDIAC FIBROBLASTS

To determine the regulatory role of prolyl hydroxylation in intracellular cardiac procollagen turnover, we examined the effects of prolyl 4-hydroxylase inhibitors (alpha,alpha'-dipyridil, 3,4-dihydroxybenzoic acid ethyl ester, pyridine 2,4-dicarboxylic acid ethyl ester) and ascorbic acid on procollagen metabolism by cultured, neonatal rat cardiac fibroblasts. Ascorbate-deficient fibroblasts showed decreased rates of prolyl hydroxylation and total collagen accumulation without a significant reduction in alpha(1)(I) and alpha(1)(III) mRNA levels. The fraction of newly synthesized procollagens degraded intracellularly was also substantially increased in ascorbate-deficient cells (50 +/- 7 v 30 +/- 3% in ascorbate-deficient v control fibroblasts; P<0.05). These findings were associated with increased intracellular accumulation of Type I procollagen, enhanced secretion of ''underhydroxylated'' pro alpha(1)(I) polypeptide into the cell culture medium, and decreased extracellular Type I collagen deposition, Similar results were obtained:by treating cells with alpha,alpha'-dipyridil (300 mu M), and 3,4-dihydroxybenzoic acid ethyl ester (400 mu M) in the presence of ascorbate. A major portion of the enhanced degradation of newly synthesized procollagens occurred within acidic intracellular compartments as indicated by the inhibition of procollagen degradation by chloroquine (25 mu M). Inhibition of procollagen secretion by colchicine (0.5 mu g/ml) enhanced the diversion to, and subsequent intracellular degradation of underhydroxylated procollagens in cardiac fibroblast lysosomes, We conclude that inactivation of prolyl 4-hydroxylase increases intracellular accumulation and intralysosomal degradation of newly synthesized cardiac procollagen polypeptides. These observations suggest that procollagen prolyl hydroxylation may be important in the regulation of collagen accumulation by cardiac interstitial cells during fibrotic processes in vivo. (C) 1995 Academic Press Limited