Procollagen production and procollagen messenger RNA levels and activity in human lung fibroblasts during periods of rapid and stationary growth.

P Tolstoshev,R.A Berg,S.I Rennard,K.H Bradley,B.C Trapnell,R.G Crystal

doi:10.1016/s0021-9258(19)69735-8

Abstract

The production of procollagen molecules by human diploid fetal lung fibroblasts (HFL-1 cells) remains constant in both rapid and stationary growth phases. However, log phase cells degrade 3-fold more newly synthesized collagen inside the cell prior to secretion than do stationary phase cells. Procollagen mRNA levels, measured by hybridization with a type I procollagen mRNA-specific complementary DNA, are approximately 2-fold higher in confluent cells than in log phase cells. There are no significant differences in the ability of either log phase or confluent HFL-1 cell procollagen mRNA to be translated in an in vitro cell-free translation system. Therefore, the ability of HFL-1 cells to maintain constant collagen production irrespective of the growth status of the cells results from the combined action of a number of regulatory mechanisms, including changes in procollagen mRNA levels, the utilization of procollagen mRNA, and intracellular procollagen degradation.

Highlights

The production of procollagen molecules by human regulate collagen production by fibroblasts, the production diploid fetal lung fibroblasts(HFL-1cells) remains con- and intracellular degradation of collagen were determined in stant in both rapid and stationary gropwhtahses
The number of type I procollagen molecules produced/cell/h calculated from this assay was in excellent agreement with that obtained by the hydroxyproline method
When RNAs from log phase and confluent HFL-1 cells were hybridized to the type I procollagen cDNA probe, a smaller amount of the RNA from confluent cells was required to achieve saturation of the cDNA probe (Fig. 3B), suggesting that confluent cell RNA contained a higher level of procollagen mRNA sequences than did log cell RNA

Summary

THEJOURNALOF BIOLOGICCAHLEMISTRY

The. The high molecular weight RNApresent in the pooled aqueous purified cDNA was used to measure procollagen-specific sequences in phases was precipitated by the addition of 3 volumes of 4 M sodium total RNA using the titration curve analysis of Young et al 118).A acetate,pH 5.0. After 16 h a t -2O"C, theRNA was collected by constant amount (0.2 ng) of cDNA was hybridized with increasing centrifugation (16,000 X g, 30 min, -1O"C), dissolved indistilled water, and reprecipitated by the addition of0.1 volume of 20% potassium acetate, pH6.5, and 2 volumes of 95% (w/v) ethanol.After 4-16 h a t -2O"C, the RNA was againrecovered by centrifugation amounts of test RNAin 2pl of hybridization buffer containing 1mg/ ml of E. coli tRNA carrier, a t 43°C for 83 h. The L-way Student’s t test was used to evaluate the differences between the means of parameters measured for log and confluent cells

RESULTS

RNAlcDNA RATIO

MechanismsControllingProcollagen mRNALevelsin

Confluent in collagen production observed in primary cultures of avian

DISCUSSION