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Collagen Synthesis in Human Skin Fibroblasts: Effects of Ascorbate, -Ketoglutarate and Ferrous Ion on Proline Hydroxylation¹

Department of Biochemistry, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514

The intracellular rate of proline hydroxylation and collagen-hydroxyproline formation was investigated in human diploid skin fibroblasts in tissue culture. In testing the cofactors for proline hydroxylase, it was found that collagen-hydroxyproline synthesis is dependent upon defined ranges of concentration of sodium ascorbate, -ketoglutarate and ferrous ion in supplemented medium. When sodium ascorbate in concentrations from 150 to 500 µg/ml was added to cultures during the logarithmic growth phase, toxic effects consisting of increased granularity, and rounding up and detachment of cells were noted. Intracellular hydroxylation of proline was significantly affected by the frequency of medium change containing sodium ascorbate. The maximal extent of proline hydroxylation in the monolayer was observed with changes of medium containing 50 µg/ml ascorbate every two days after confluency of cells was reached, whereas in cultures subjected to daily changes of medium containing 50 µg/ml ascorbate, the degree of proline hydroxylation in the cells never exceeded the level observed in control cultures without added ascorbate. The increase in cellular proliferation and concomitant decrease in collagen-hydroxyproline accumulation in the monolayers subjected to daily changes of medium with 50 µg/ml ascorbate, in contrast to cultures treated similarly every 2 days, suggests that the state of confluency of fibroblasts in culture plays an important role in regulation of the activity of proline hydroxylase.

KEY WORDS: • collagen synthesis • fibroblasts • tissue culture • ascorbic acid

¹ Supported in part by grants from the Easter Seal Research Foundation and National Institute of Child Health and Human Development (HD-03110), and by Project 236, Health Services and Mental Health Administration, DHEW. We should also like to acknowledge a General Research Support Award (5-SO1-FR-05406) from the National Institutes of Health, a University Research Council Grant, an American Cancer Society Institutional Grant (IN15), a National Science Foundation Equipment Grant (GB-4577), and a Research Career Development Award (5-K3-AM-5058) from the National Institute of Arthritis and Metabolic Diseases (GKS). Support was also rendered through a training grant (5T-01-GM-00404-08) and by a predoctoral fellowship (1-FO1-GM-41, 423-01) from the National Institute of General Medical Sciences (BRS).

Manuscript received 12 July 1971.

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