Role of ascorbic acid in the regulation of proliferation in osteoblast-like MC3T3-El cells

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Abstract
Proliferation of osteoblast-like MC3T3-El cells was minimal in serum-free Eagle's minimum essential medium (MEM) but was enhanced by about 3.5-fold in serum-free (alpha)-modification of MEM ((alpha)-MEM). By adding back each of the extra constituents present in (alpha)-MEM to MEM, it was found that ascorbic acid was responsible for the sustained proliferation of MC3T3-El cells without serum. Ascorbic acid also stimulated the synthesis of collagen and increased the hydroxyproline content of MC3T3-El cell cultures markedly. Inhibitors of collagen synthesis, L-azetidine-2-carboxylic acid, cis-4-hydroxyproline, and 3,4-dehydroproline, almost completely eliminated the stimulatory effect of ascorbic acid on DNA synthesis of MC3T3-El cells. The dose response of the effect of L-azetidine-2-carboxylic acid on the hydroxyproline content closely paralleled that on DNA synthesis of MC3T3-El cells. Furthermore, a 10 times higher concentration of proline, which competes with L-azetidine-2-carboxylic acid for the incorporation into procollagen molecules, reversed the inhibition of DNA synthesis by L-azetidine-2-carboxylic acid. These results are consistent with the assumption that the stimulatory effect of ascorbic acid on the proliferation of MC3T3-El cells is mediated through its effect on the synthesis of collagen or some related protein. Furthermore, a fibronectin attachment peptide, GRGDTP, that competes with matrix proteins for specific binding to cell surface adhesion receptors also inhibited the stimulation of proliferation by ascorbic acid almost completely. It is suggested that ascorbic acid stimulates osteoblast proliferation through its effect on the synthesis of collagen or some related protein and that such a matrix protein interacts with cell surface adhesion receptors to cause the stimulation of proliferation in osteoblasts. The mechanism of how such a protein affects the proliferation of osteoblasts remains to be clarified.
Funding Information
  • Scientific Research from the Ministry of Education, Science and Culture
  • Ministry of Health and Welfare of Japan