Ascorbate-generated endogenous extracellular matrix affects cell protein synthesis in calf aortic smooth muscle cells.

Abstract

Ascorbate supplementation of cultured fetal calf aortic smooth muscle cells leads to increased deposition of extracellular matrix proteins and stimulation of cellular protein synthesis (E. Schwartz et al., J cell biol 92 (1983) 462) [7]. In the present study, we have investigated this phenomenon at the level of gene expression. Cells were grown for three weeks on tissue culture plastic with or without ascorbate (50 micrograms/ml). When compared to controls, cells grown in presence of ascorbate had twice as much poly(A+) RNA per microgram of total RNA, and ascorbate led to a 50% increase in [35S]methionine incorporation when the total RNA was translated in the reticulocyte lysate system. SDS-PAGE revealed no change in the protein pattern under the two conditions. "Northern" hybridization revealed a two- to fivefold increase in the sequence content of beta-actin, alpha-tubulin and type I pro alpha 1-collagen in total RNA of ascorbate-supplemented cells, but no difference was observed in the mRNA sequence content for the three specific proteins when equal amounts of poly(A+) RNA from ascorbate and control cells were hybridized with the three cloned cDNAs. To evaluate the effect of an exogenous matrix, cells were also plated on collagen gels. RNA isolated from cells grown on collagen without added ascorbate exhibited translational activity and mRNA sequence content similar to cells grown with ascorbate on tissue culture plastic. In contrast, no differences from controls were found in cells grown for one week in the presence of ascorbate, at which time no significant deposition of collagen occurs in the extracellular matrix. These results suggest that the stimulation in protein synthesis in fetal calf smooth muscle cells supplemented with ascorbate is associated with an increase in the proportion of poly(A+) RNA in the total RNA pool, and that the production of an endogenous collagen-rich matrix in the presence of ascorbate may be the basis for these pretranslational changes.