ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 226, No. 2, October 15, pp. 681-686, 1983 Ascorbate Deficiency Results in Decreased Collagen Production: Under- Hydroxylation of Proline Leads to Increased Intracellular Degradation RICHARD A. BERG,’ BEAT STEINMANN: STEPHEN I. RENNARD, AND RONALD G. CRYSTAL Pulmonary Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland, 20205 Received May 2, 1983 Collagen production by cultured human lung fibroblasts was examined when the cells were made deficient in ascorbate. Cells grown in the absence of ascorbate produced 30% less collagen during a 6-h labeling period than cells incubated with as little as 1 pg/ml ascorbate during the labeling period. Cells grown without ascorbate produced under-hydroxylated collagen which was subject to increased intracellular degradation from a basal level of 16% to an enhanced level of 49% of all newly synthesized collagen. The likely mechanism for increased intracellular degradation is the inability of under- hydroxylated collagen to assume a triple-helical conformation causing it to be susceptible to intracellular degradation. Measurement of collagen production by enzyme linked immunoassay (ELISA) using antibodies directed against triple-helical determinants of collagen showed that both types I and III collagens were affected. In contrast, another connective tissue component, fibronectin, was not affected. Analysis by ELISA showed a greater decrease in collagen production than did analysis by the collagenase method, suggesting that some non-helical collagen chains (detected by collagenase but not by ELISA) were secreted in the absence of ascorbate. These results provide a mechanism to account, in part, for the deficiency of collagen in connective tissues which occurs in a state of ascorbate deficiency. Collagen is the major extracellular pro- tein in connective tissues in the body, and the level of collagen production is, there- fore, of critical importance in maintaining tissue integrity. Lung fibroblasts in tissue culture produce a constant amount of col- lagen both during the log phase of growth and after the cells are confluent (1,2). Nev- ertheless, fibroblasts are capable of mod- ulating their production of collagen in var- ious situations (3-6). Such regulation oc- 1 To whom correspondence should be addressed: Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Rutgers Medical School, Piscataway, New Jersey 08854. ‘Present address: Division of Metabolism, De- partment of Pediatrics, University of Zurich, Kin- derspital, Streinwiesstrasse 75, CH 8032 Zurich, Switzerland. curs at many levels including transcription (7,8), the rate of translation of messenger RNA (9), and also through changes in posttranslational modifying reactions (for review see Refs. (10, 11)). There are many observations in the lit- erature indicating that collagen production is decreased in cultured cells grown in the absence of ascorbate (12, 13). The mech- anism for the reduced amount of collagen produced in ascorbate deficiency is un- known. Specifically, it is unknown whether collagen synthesis is decreased (eg., reg- ulated at the transcriptional or transla- tional levels) or whether collagen degra- dation is increased (es., regulated at a post- translational level). Although there are recent studies suggesting that collagen synthesis is reduced in ascorbate deficiency (14, 15), an alternate hypothesis, i.e., that 681 0003-9861/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in anyformreserved.