Effects of puromycin and hydroxynorvaline on net production and intracellular degradation of collagen in human fetal lung fibroblasts.

Amino acid substitutions in collagen that impair folding of the triple helix result in significant increases in intracellular degradation of newly synthesized collagen. We have studied the effects of agents that cause other kinds of defects in collagen: hydroxynorvaline, a threonine analog that interferes with association of pro-alpha chains; and puromycin, an antibiotic that causes premature release of nascent polypeptides. cis-Hydroxyproline and cycloheximide, whose effects on collagen synthesis and degradation have already been studied and reported, were employed as reference compounds. Human fetal lung fibroblasts were used in these experiments. All the agents inhibited total protein production, and all except cycloheximide inhibited percentage collagen production. Intracellular collagen degradation was increased in cultures exposed to puromycin, hydroxynorvaline, and cis-hydroxyproline, but not in cultures exposed to cycloheximide. These results suggest that pro-alpha chains that were either unassociated (due to hydroxynorvaline) or shortened (due to puromycin) were recognized as abnormal and degraded to the same extent as chains that contained cis-hydroxyproline. However, the increases in degradation could not account completely for the decreases in collagen production (except when cis-hydroxyproline was used at low concentrations). These findings indicate that, in addition to rendering newly synthesized procollagen molecules or partial polypeptide chains more susceptible to intracellular degradation, puromycin, hydroxynorvaline, and cis-hydroxyproline significantly inhibited collagen synthesis.

Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts.

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.