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Abstract
Procollagen biosynthesis was examined in cultured aortic smooth muscle cells as a function of cell density and 17(beta)-estradiol treatment. With increasing culture densities both assembled procollagens and individual collagen polypeptides of types I and III increased. At all cell densities the production of type III procollagen was inhibited approximately 50% by the addition of physiological concentrations of 17(beta)-estradiol (10('-8)M) while type I procollagen remained essentially unaffected. The processing of procollagen types I and III were also examined. Data obtained utilizing a previously undescribed micromethod of CNBr peptide mapping for collagen suggested that both procollagen types are processed sequentially by removal of the amino extension peptide followed by the removal of the carboxyl extension peptide. {('14)C}-proline pulse-chase experiments indicated that the complete processing of type I procollagen occurs more rapidly than type III. By three hours 50% of the type I procollagen synthesized during a one hour pulse was fully processed. In contrast, type III procollagen was rapidly converted to its processing intermediate and only slowly converted to fully processed collagen molecules. To probe the mechanism by which procollagen synthesis was regulated, as a function of cell density, the relative concentrations of procollagen mRNAs were determined using a rabbit reticulocyte cell-free translation system. Type I and III pro-(alpha) chains could be identified as products of the system. The cell-free produced pro-(alpha) chains displayed ratios which were compatible with the cellular levels of procollagen synthesis. At saturating mRNA concentrations the both chains of type I procollagen tend to translate with a higher efficiency than the (alpha)1(III) chain mRNA. This suggests that type I procollagen mRNA competes favorably under limiting initiation conditions over type III. All three procollagen mRNA concentrations were found to significantly increase as a function of cell density. These result indicates that the level of procollagen synthesis was regulated at a pretranslational step.
