Ascorbate induction of collagen synthesis as a means for elucidating a mechanism of quantitative control of tissue-specific function.

Abstract

Ascorbic acid displays the characteristics of an ideal inducer of tissue-specific function in primary avian tendon cells in culture. It is a highly specific, potent stimulator of collagen synthesis, it demonstrates slow reversible kinetics, and it has no effect on growth rate of the cultured cells. Kinetic analysis of ascorbate induction of collagen synthesis was used to determine the critical steps in this complex biosynthetic pathway. Full hydroxylation of the proline residues in collagen, although probably a necessary step for collagen induction, was in itself not sufficient for achieving either increased secretion or increased synthesis. On the other hand, an increase in secretion rate, which required both the presence of ascorbate and a high cell density, did correlate with the later stimulation in procollagen production. The process of procollagen secretion, therefore, meets the minimal requirements for the rate-limiting step. The fact that the cells maintained a large pool of intracellular procollagen despite changes in the rates of translation or secretion led us to postulate a possible feedback between the level of the internal procollagen pool and the rate of procollagen synthesis.The quantitative expression of tissue-specific function is precisely regulated within the organism, but this regulation can be readily lost when cells are cultured (2, 6, 10, 35). In primary avian tendon (PAT) cells, the ability to maintain in ovo levels of collagen synthesis (30% of total protein synthesized [11]) is directly related to the cell culture environment (35,36,37). Of the many factors that influence cells in culture through poorly understood mechanisms, in this paper we have focused on ascorbic acid for several reasons. Ascorbic acid serves as a clear example of how a small molecule can influence the protein synthetic machinery and the level of post-translational modifications in eucaryotic cells. In PAT cells, ascorbic acid shifts the level of protein synthesis devoted to collagen from 5 to 10% to 20 to 30% (35) and greatly increases the hydroxylation of proline residues in collagen. This modulation becomes especially significant since ascorbate deprivation in vivo, i.e., scurvy, is characterized by the same quantitative and qualitative response as that observed in PAT cells in culture (3).Additionally, although the general mechanism of ascorbate action appears to be straightfort Present address: The Jackson Laboratory, Bar Harbor, ME 04609.

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.

“…In both the bone and cell cultures, however, ascorbate was required for normal proline hydroxylation. In a few instances, there was a specific increase in the relative rate of collagen production by ascorbate under well-defined culture conditions, and it has been suggested that this occurs independently of the role of ascorbate in hydroxylation (14,50). It is not clear whether this modulation of collagen production by ascorbate in cell culture systems occurs by the same mechanism operative in vivo.…”

Role of procollagen mRNA levels in controlling the rate of procollagen synthesis.

“…On the other hand, PAT cells at low cell density or in a medium deficient in ascorbate will produce only 12% procollagen (22,26). This shift has previously been shown to be specific to procollagen production; the rate of noncollagen protein synthesis is identical in induced and uninduced cells (26).…”

“…In the presence of 0.3 mM a,a'-dipyridyl there was a specific two-to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.Primary avian tendon (PAT) cells at high cell density (>4 x 104 cells per cm2) and in the presence of ascorbate (50 p.g/ml) allocate half of their protein production to procollagen (22,26). On the other hand, PAT cells at low cell density or in a medium deficient in ascorbate will produce only 12% procollagen (22,26).…”

“…Primary avian tendon (PAT) cells at high cell density (>4 x 104 cells per cm2) and in the presence of ascorbate (50 p.g/ml) allocate half of their protein production to procollagen (22,26). On the other hand, PAT cells at low cell density or in a medium deficient in ascorbate will produce only 12% procollagen (22,26).…”

“…PAT cells were isolated from 16-day chicken embryos by a modification (22) of the Dehm and Prockop (8) procedure. PAT cells were grown in F12 medium (10) with 0.2% fetal calf serum in 25-cm2 flasks as described previously (24,26).…”