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0022-202X/80/7505-0404$02.00/0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 75:404-407, 1980 Vol. 75, No. 5 Copyright © 1980 by The Williams & Wilkins Co. Printed in U.S.A. Prolyl and Lysyl Hydroxylase Activities of Human Skin Fibroblasts: Effect of Donor Age and Ascorbate SAOOD MuRAD, PH.D., ARUNTHATHY SIVARAJAH, B.Sc., AND SHELDON R. PINNELL, M.D. Division of Dermatology, Department of Medicine, Dulle University Medical Center, Durham, North Carolina, U.S.A. Prolyl and lysyl hydroxylase activities in cultures of MATERIALS AND METHODS human skin fibroblasts from fetal to 94-yr-old donors Fetal human skin fibroblasts were obtained from the American Type were measured. In contrast to earlier studies with whole Culture Collection, Rockville, Maryland. Fibroblasts from donors of all skin, neither prolyl nor lysyl hydroxylase activity was other ages were obtained from the Human Genetic Mutant Cell Re­ found related to donor age. Prolyl hydroxylase activity pository, Camden, New Jersey. They were split at 1:3 ratio in Dul­ increased 3- to 6-fold when cell extracts were incubated becco's modified Eagle's medium (Grand Island Biological Company, with ascorbate and other hydroxylation cofactors before Grand Island, New York) supplemented with 10% heat inactivated assay. A similar increase in prolyl hydroxylase activity fetal calf serum (Irvine Scientific, Santa Ana, California) and main­ occurred when cells were incubated with ascorbate. Ly­ tained at 37°C in a humid atmosphere of 5% carbon diox.ide/95% air. On the 7th day the cells were harvested with 0.5% trypsin/0.54 syl hydroxylase activity remained unaltered under these M[ethylenedinitrilo]-tetraacetate and counted in a hemocytometer. conditions. The cell suspension was centrifuged at 500 Xg for 5 min and the pellet was washed 4 times with Hanks' balanced salt solution (without cal­ cium, magnesium, bicarbonate, and glucose), each wash step being Collagen is the major extracellular protein of connective followed by centrifugation. The pellet was then suspended in a medium tissues and its metabolism has been the subject of many age (1 ml per 5 x 106 cells) containing 0.2 M NaCI, 0.1 M glycine, 50 ILM related studies [reviewed in reference 1]. The biosynthesis of dithiothreitol, 20 mM Tris-(hydroxymethyl) aminomethane (pH 7.5), collagen is of particular interest in this regard as it involves, in and 0.1% Triton X-100. The cells were subjected 3 times to quick addition to the polypeptide synthesis, certain intra- and extra­ freezing and thawing. The lysate was centrifuged at 27,000 x g for 1 hr cellular modifications that are essential for collagen maturation and the supernatant was used for enzyme assays. [2]. Two of the intracellular modifications are hydroxylation of Prolyl and lysyl hydroxylase activities were measured by methods basfd on tritium release [16,17]. The substrate for prolyl hydroxylase prolyl and lysyl residues, giving rise to important reaction war, unhydroxylated procollagen containing [4- 3H]-L-proline and for products. Hydroxyproline is believed to stabilize the collagen lyf.yl hydroxylase was that containing [4,5-3H]-L-lysine [18]. The reac­ triple helix [3] and hydroxylysine is the site for glycosylation tion mixture (1.5 ml) contained either 127,000 cpm of prolyl substrate and intermolecular crosslinking in collagen [2]. The formation or 280,000 cpm of lysyl substrate, 50 mM tris-(hydroxymethyl)-amino­ of hydroxyproline and hydroxylysine in procollagen is catalyzed methane (pH 7.8), 0.5 mM a-ketoglutaric acid, 2 mM sodium ascorbate, by separate enzymes, prolyl [EC 1.14.11.2] and lysyl [EC 0.1 mM dithiothreitol, 0.1 mg per ml catalase, 1.5 mg per ml bovine 1.14.11.4] hydroxylases, which share the same cofactors [3]. serum albumin, and cell extract (approximately 100 ILg of protein for Prolyl hydroxylase occurs in inactive (monomeric) and active prolyl hydroxylase and 200 ILg of protein for lysyl hydroxylase). Incu­ (tetrameric) forins and is considered to play a key regulatory bation time was 1 hr for prolyl hydroxylase and 2 hr for lysyl hydrox­ ylase. Incubation temperature was 30°C. The reactions were terminated role in collagen biosynthesis [1,3]. by addition of 0.1 ml of 50% trichloroacetic acid and the tritiated water Age dependent changes in prolyl and lysyl hydroxylase activ­ formed was vacuum distilled in a custom made multisample apparatus. ities of human tissue have been reported [ 4-6]. Both enzyme One milliliter of the distillate was mixed with 10 ml of Aquasol-2 (New activities were high in fetal skin and rapidly declined with age England Nuclear Corporation, Boston, Massachusetts) and its radio­ until maturity. However, it is not clear from these studies if the activity was determined in an Intertechnique liquid scintillation spec­ aging effect reflects a decrease in the number of fibroblasts or trometer (efficiency 36%). Under these conditions assays without en­ in the enzyme activity per fibroblast, the cell mainly responsible zyme gave 50 to 100 cpm; the values with enzyme were at least 10 times for collagen production. The interpretation of these results is higher. Three or more cultures were pooled for measurement of enzyme further complicated by technical difficulties encountered in activities. Values from duplicate assays on the same cell extract agreed within 2%. The rate of reaction was found linearly related to the amount handling skin specimen for enzyme studies. These considera­ of cell extract up to approximately 400 ILg of protein for prolyl hydrox­ tions prompted us to measure prolyl and lysyl hydroxylase ylase and 300 }lg of protein for lysyl hydroxylase. activities in human skin fibroblasts from donors of various ages. Enzyme activation was performed either by incubating the cells with In the course of this study it became necessary to explore ascorbate or by preincubating the cell extracts with complete reaction possible activation of prolyl hydroxylase by ascorbate admin­ mixture minus substrate. The stimulated prolyl hydroxylase activity istration. Such an activation has been observed in some types was measured with half the usual amount of cell extract (approximately of cells [7-10] but not in others [11,12], and for one cell type 50 ILg of protein) in order to ensure that the activity fell on the linear different assay methods have given conflicting results [7,13, portion of the rate versus enzyme concentration curve. 14]. Moreover, in human skin fibroblasts ascorbate has been Protein was assayed using Bio-Rad kit with bovine gamma globulin found to depress prolyl hydroxylase activity [15). The present as the standard [19]. report provides evidence for preferential stimulation of prolyl RESULTS hydroxylase activity by ascorbate in cultured human skin fibro­ blasts. In human -skin fibroblasts prolyl hydroxylase activity was stimulated by ascorbate in a time dependent manner, reaching Manuscript received February 26, 1980; accepted for publication a maximum by hour 3 at which time the ascorbate-treated cells May 27 , 1980 . had 3 times the activity of the untreated cells (Fig 1). As This work was supported by grants AM-17123 and AM-07093 from expected, lysyl hydroxylase activity was not affected by ascor­ the National Institutes of Health and from the Howard Hughes Medical Institute Laboratories of Duke University Medical Center. bate treatment. The observed stimulation ofprolyl hydroxylase This is publication number 56 from the Dermatological Research activity by ascorbate is in apparent conflict with the reported Laboratories of Duke University Medical Center. depression of this activity in human skin fibroblasts [15]. How­ Reprint requests to: Sheldon R. Pinnell, M.D., Box 3135, Duke ever, in this study the cells were cultured with ascorbate; the University Medical Center, Durham, NC 27710. long-term effect of ascorbate on prolyl hydroxylase activity may 404 Nov. 1980 ASCORBATE AND AGE EFFECTS ON COLLAGEN HYDROXYLASES 405 75 ,.., c:: b <l.> PH >< 0 ElO -~ a. 0.. u C7' ::1.. 'E5o 0.. u ""0 <l.> ~ E Q.) ~ -0 ~ 3: ~ <l.> ""0 "025 Q.) 3: -0 ""0 <l.> -~ I-- LH -0 LH ~ ......... a~--~1----~2~--~3----~ Preincubation Time (hr) 1 2 3 4 FIG 2. Time course of activation of prolyl and lysyl hydroxy lases in Ascorbate Treatment ( hr) cell extracts. Extracts of human skin fibroblasts.from a 15-yr-old male Duration of were incubated with complete reaction mixture minus substrate for varying times at the indicated temperatures. Substrates were then FIG 1. Time course of activation of prolyl and lysyl hydroxy lases in added and incubations were continued at 30°C for additional! and 2 hr intact cells. Confluent cultures of human skin fibroblasts from a 15-yr­ for prolyl and lysyl hydroxylases, respectively. Other details are given old male were incubated for varying times with serum containing in Materials and Methods section. medium supplemented with 0.25 mM sodium ascorbate. A set of cultures was not exposed to ascorbate and this is referred to as "zero time." TABLE I. Cofactor requirement for activation ofprolyl hydroxylase Cells were then harvested and prolyl and lysyl hydroxylase activities in in cell extracts their extracts were measured as described in Materials and Methods Prolyl section. hydroxylase Additions during preincubation activity be different from the short-term effect observed in the present % study. None 100 Prolyl hydroxylase activity was also stimulated in cell ex­ Complete system" 361 (334)b tracts when these were preincubated with ascorbate and other Complete system minus ascorbate 86 hydroxylation cofactors. The time course of this activation is Complete system minus a-ketoglutaric acid 71 shown in Fig 2. The activation was complete in 2 hr at 37°C Complete system minus ferrous sulfate 445 (24l)b and nearly so in 4 hr at 30°C. As in intact cells, lysyl hydroxylase Complete system minus catalase 226 activity remained unchanged in cell extracts when these were Complete system minus bovine serum albumin 327 similarly preincubated with hydroxylation cofactors.

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