Inhibition of prolyl hydroxylation during collagen biosynthesis in human skin fibroblast cultures by ethyl 3,4-dihydroxybenzoate.
The enzymatically catalyzed formation of 4-hydroxyproline plays a key role in the intracellular biosynthesis of collagen, since a critical number of 4-hydroxyprolyl residues is required for synthesis and secretion of triple-helical procollagen molecules under physiologic conditions. The enzyme catalyzing the conversion of prolyl residues to 4-hydroxyproline, prolyl 4-hydroxylase, requires ferrous ion, alpha-ketoglutarate, and ascorbate for its activity. 3,4-Dihydroxybenzoic acid has been known to act as potent competitive inhibitor of purified prolyl 4-hydroxylase with respect to one or several of the cofactors or cosubstrates of the enzyme. 3,4-Dihydroxybenzoic acid, however, is a poor inhibitor of prolyl hydroxylation in intact cells, probably due to its polarity not allowing it to enter the cells. In this study, several hydrophobic modifications of 3,4-dihydroxybenzoic acid were tested in human skin fibroblast cultures for their efficacy to inhibit the synthesis of 4-hydroxyproline. The results indicated that the ethyl ester of 3,4-dihydroxybenzoic acid was an efficient inhibitor of prolyl hydroxylation in fibroblast cultures, with Ki of approximately 0.4 mM. Ethyl 3,4-dihydroxybenzoate had little, if any, effect on the hydroxylation of lysyl residues, and it did not affect total protein synthesis or DNA replication in these cells. To test the hypothesis that ethyl 3,4-dihydroxybenzoate might serve as a potential antifibrotic agent, its efficacy in inhibiting prolyl hydroxylation in scleroderma fibroblasts was also tested. The results indicated that the synthesis of 4-hydroxyproline in scleroderma cell cultures was similarly reduced by ethyl 3,4-dihydroxybenzoate. Thus, structural analogs of the cofactors or cosubstrates of prolyl 4-hydroxylase, such as ethyl 3,4-dihydroxybenzoate tested here or its further modifications, may serve as inhibitors of posttranslational hydroxylation of prolyl residues also in vivo. These compounds could potentially provide a novel means of reducing collagen deposition in tissues in fibrotic diseases, such as scleroderma.
Majamaa
K
K
Department of Medicine, UCLA School of Medicine, Torrance.
Sasaki
T
T
Uitto
J
J
D002478:Cells, Cultured; D006801:Humans; D062385:Hydroxybenzoates / Q000494:pharmacology*; D006909:Hydroxyproline / Q000096:biosynthesis*; D011347:Procollagen / Q000096:biosynthesis*; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors*; D012595:Scleroderma, Systemic / Q000378:metabolism; D012867:Skin / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship
D062385:Hydroxybenzoates; D011347:Procollagen; C018063:ethyl protocatechuate; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline