Regulation of the production of secretory proteins: Intracellular degradation of newly synthesized “defective” collagen

Abstract

Confluent cultures of human fetal lung fibroblasts degrade approximately 10% of their newly synthesized collagen within the cell prior to secretion. This basal level of intracellular degradation could not be inhibited by colchicine or cytochalasin B, inhibitors of microtubular and microfilament function, respectively, or by N -tosyl-L-lysine chloromethyl ketone, chloroquine, or NH4C1, inhibitors of Iysosomal enzymes.In contrast, cells in early logarithmic growth degrade approximately 30% of their newly synthesized collagen. This enhanced degradation of collagen in rapidly growing cells could be suppressed by inhibitors of lysosomal proteases and partially inhibited by disrupters of microtubular and microfilament function. A significant prootion of the collagen synthesized by these cultures contained prolyl residues that were incompletely hydroxylated. Because such collagen is "defective" (i.e., not capable of assuming a triple helical conformation), the results suggest that enhanced intracellular degradation may be a mechanism by which cells control the quality of collagen they produce. To test this hypothesis, confluent cells were incubated with the proline analog cis4hydroxyproline; such cells demonstrated en anced collagen degradation that could be inhibited by agents that interfere with lysosomal, microtubular, or microfilament function. Because collagen containing cis4 hydroxyproline cannot form a perfect triple helix, the data are consistent with the concept that defective collagen is recognized by cells and degraded prior to secretion. Thus, the proportion of newly synthesized collagen that undergoes intracellular degradation seems to be modulated, in part, by the conformation of the collagen molecule. Intracellular proteolysis may represent a means by which collagen-producing cells regulate the quality and quantity of collagen available for extracellular function. Although the exact mechanism of intracellular collagen degradation is unknown, the data presented here are consistent with a role for lysosomal proteases in this process. The normal collagen molecule is a triple helical, rod-like structure. Inherent in this structure is the property to polymerize into extracellular fibers that play a major role in defining organ structure and function (1-4). The ability of collagen to form fibers is dependent on its triple helical conformation. To form this helix, each collagen polypeptide chain must have a specific primary sequence, the most important aspect of which is a repetitive Gly-X-Y triplet; approximately 30% of the X residues are proline and 30% of the Y residues are trans-4-hydroxyproline (1)(2)(3)(4)(5). Several studies have demonstrated that, if a sufficient number of Y position residues are not filled with hydroxyproline, the resulting collagen molecule is "defective"-i.e., it cannot form a perfect triple helical structure at body temperature (5). Such defective collagen molecules will not polymerize into normal collagen fibers and thus cannot serve their function in the extracellular mat...

Specifically decreased collagen biosynthesis in scurvy dissociated from an effect on proline hydroxylation and correlated with body weight loss. In vitro studies in guinea pig calvarial bones.

“…It is possible that there is a pool of completely unhydroxylated collagen that is preferentially degraded in bones from scorbutic animals. In our studies, however, as in others (34), the measurement of low molecular weight hydroxyproline as an index of collagen degradation precluded measuring the degradation of completely unhydroxylated collagen. At any rate, this possibility seems unlikely, since the relative rates of collagen production in normal bones treated in vitro with a,a'-dipyridyl remained unchanged, in spite of the presence of an almost completely unhydroxylated collagen pool.…”

Tissue specificity of type I collagen gene expression is determined at both transcriptional and post-transcriptional levels.

“…Chondrocytes were prepared from the ventral vertebral cartilage of 12-day-old chicken embryos, and floating cells were selected after 5 days in culture; this selection procedure removes fibroblast cells, which attach to the substratum (16 (5,8,10,57). This permitted us to determine the relative rates of type I collagen synthesis and to correlate these rates with the amounts of type I collagen mRNAs.…”

Modulation of procollagen gene expression by retinoids. Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures.

“…The reduction in the mRNA levels was even more pronounced than the inhibition of the rate of procollagen production. This observation suggests that the reduction in mRNA levels by alltrans-RA may be accompanied by additional changes at different levels of procollagen gene expression, including the efficiency of message utilization, compartmentalization of the precursor amino acid pools, and intracellular degradation of the newly synthesized polypeptides (29)(30)(31). Northern blot analyses showed that qualitatively there were no changes in mRNA pattern, and both in control and RA-treated cultures three major transcripts corresponding to proa2(I) sequences were present.…”