Regulation of Collagen Synthesis

0022-202X/82/7903-073s$02.00/0

Vol. 79, Supplement 1 Printed in U.S.A.

THE ,JOURNAL OF INVESTIGATIVE DERMATOLOGY, 79:73s-76s, 1982

Copyright © 1982 by The Williams & Wilkins Co.

Regulation of Collagen Synthesis SHELDON R.

PINNELL, M.D.

Division of Dermatology, Department of Medicine, Dulle University

Collagen synthesis is a complex orchestration of intra­ cellular and extracellular events. In addition to synthesis of the polypeptide chains more than a dozen modifica­ tions of the molecule occur; most of these are enzymatic and specific for collagen. Regulational control of colla­ gen synthesis promises to be equally complex. Examples are described to 4 specific regulatory influences. Ascor­ bic acid markedly stimulates collagen synthesis without affecting synthesis of other proteins. This effect appears to be unrelated to its cofactor roles for hydroxylation of lysine and proline. Glucocorticoids at J-tM concentration specifically inhibit collagen synthesis. Tissues treated with glucocorticoids have diminished levels of mRNA for collagen. During collagen synthesis the aminoter­ minal propeptide of procollagen is cleaved by a specific protease. This peptide appears to be a feedback inhibitor of collagen synthesis. This effect can be demonstrated in cells and in cell-free synthesizing systems. A membrane receptor system may permit the peptide to be recognized and subsequently act as a translational control mecha­ nism. Viral transformation of fibroblasts results in selec­ tively decreased synthesis of collagen. Levels of cyto­ plasmic and nuclear mRNA are likewise selectively di­ minished consistent with transcriptional control.

The regulation of collagen synthesis promises to be complex since many post-translational modifications are necessary for the synthesis of the final product. In addition the simultaneous synthesis of several species of collagen must be orchestrated at the cellular leveL Thus regulation could be operational at many levels of synthesis. These include gene selection, transcription, messenger splicing, translation, hydroxylation of proline and lysine, glycosylation of hydroxylysine, glycosylation of propep­ tides, helix formation, translocation, secretion, cleavage of pro­ peptides, fibril formation, cross linking, intracellular and extra­ cellular degradation, and aging modifications. Many environ­ mental factors, hormones, drugs, vitamins, minerals and other substances have been reported to influence collagen production [review, reference 1]. A comprehensive review is beyond the scope of this manuscript. Instead I have chosen 4 representative regulation systems in which specific effects on collagen synthe­ sis have been clearly established. Although regulation studies have been carried out in whole animals, organ culture, primary, and continuous cell culture, as well as cell-free systems each has its advantages and disadvan­ tages. Human skin fibroblasts in culture are convenient for studying cellular regulation. Although not immortal, these cells can be studied through 30 or more replications. The amount of collagen synthesized by these cells is appreciably greater than continu­ ous fibroblast lines and with proper attention to culture condi­ tions [2,3] relative collagen synthesis approaches that achieva­ ble in primary cell culture. Supported in part by grant 5 ROI AM-17128 from the National Institutes of Health and the Westwood Pharmaceuticals Inc. This is publication number 122 from the Dermatological Research Laboratories of Duke University Medical Center. Reprint requests to: Sheldon

R. Pinnell, M.D., Division of Derma­

tology, Department of Medicine, Duke University Medical Center, Durham, NC 27710.

Medical Center, Durham, North Carolina, U.S.A.

Certain disadvantages in using human skin fibroblasts in collagen regulation experiments need to be understood. Human skin fibroblasts in culture are a heterogenous population of cells. Although growth characteristics of fibroblasts explanted from papillary and reticular dermis are different, relative col­ lagen synthesis and qualitative collagen synthesis are similar [4]. Relative collagen synthesis is cell density dependent and is only maximal at confluent densities [2]. In order to carry out regulatory experiments it would be desirable to have a chemically defined media. Growth of human skin fibroblasts has not been achieved in serum-free media although cells can be maintained at a stable density in 0.5% dialyzed calf serum for many days. Human skin fibroblasts synthesize several types of collagen including types I, III, and V [4]. Although this may be an advantage for studying type­ specific or linked regulation, it could be a disadvantage for other studies. Collagen synthesis in most studies has been measured by determination of hydroxyproline or digestion with purified clos­ tridial collagenase [2]. When hydroxyproline is used to measure collagen it is assumed that hydroxylation of proline is stable. Since relative hydroxylation of proline could be a variable in regulatory systems, determination of collagen synthesis inde­ pendent of hydroxylation is desirable. Collagenase digestion has been demonstrated to be a reliable technique for the measure­ ment of collagen if care is taken to inhibit nonspecific proteo­ lytic activity [2]. SPECIFIC STIMULATION OF COLLAGEN SYNTHESIS BY ASCORBIC ACID That ascorbic acid is essential for collagen synthesis has been known for years and is evident from the well-known connective tissue disturbances that occur in scurvy. Its importance in hydroxylation of proline to form hydroxyproline and hydrox­ ylation of lysine to form hydroxylysine have been thought to account for its primary influence [5]. Since hydroxyproline is essential for collagen helix formation [6] and subsequent cellu­ lar secretion [7] and hydroxylysine is essential for collagen intermolecular crosslinking [8], inadequate levels of ascorbate might be expected to interfere with normal collagen production. Recently, however, it has become apparent that ascorbate has a specific primary stimulatory effect on collagen synthesis, one that is apparently unrelated to hydroxylation [9,10]. In confluent human skin fibroblasts ascorbate was capable of markedly stimulating total collagen synthesis without influenc­ ing noncollagen protein synthesis. Maximal stimulation oc­ curred at L-ascorbic acid levels as low as 30 flM (Fig 1) and the effect steadily increased for at least 4 days (Fig 2). During this interval collagen synthesis was stimulated fourfold and relative collagen synthesis was boosted from 9% to 32% of total protein synthesis (Fig 3). These levels are appreciably higher than those usually achieved in human skin fibroblast culture and represent the results that can be achieved when culture condi­ tions are maximized. Quite surprisingly levels of prolyl hydroxylase activity were diminished although lysyl hydroxylase activity increased under the influence of ascorbate [11]. The ascorbate stimulation of collagen synthesis appears not to be related to hydroxylation because relative hydroxylation of lysine in collagen synthesized by skin fibroblasts in the presence and absence of ascorbate was similar. In addition, although hydroxylation of proline was 73s

Vol. 79, Supplement

PINNELL

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not understood, it required a confluent cellular density [6] and

100

could be demonstrated in the presence of dialyzed calf serum from 0.5% to 20% concentration (Pinnell SR: unpublished ob­ servations).

Collagen

80

SPECIFIC INHIBITION OF COLLAGEN SYNTHESIS BY GLUCOCORTICOIDS Although quite effective as therapeutic agents, the clinical use of glucocorticoids is tempered by undesirable side effects. Many of these are related to connective tissue and include

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growth retardation, cutaneous atrophy, osteoporosis and re­ tarded wound healing. Although a selective effect on collagen

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synthesis has been suspected, unequivocal experimental evi­

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dence has been difficult to accumulate. Many earlier studies measured the effect of glucocorticoids on hydroxyproline ac­ cumulation but these effects could have resulted from under­ estimation of collagen synthesis resulting from inhibition of

Noncollagen Protein

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prolyl hydroxylase activity by glucocorticoids [12,13] and sub­ sequent synthesis of underhydroxylated collagen. The most convincing studies have been carried out in human skin fibro­ blasts in which the synthesis of collagen was selectively de­

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creased over that of noncollagen protein [14-16]. In these studies however the amount of protein synthesis devoted to collagen was small. Since ascorbate-stimulated cells devote appreciably more

FIG 1. The effect of L-ascorbic acid on collagen synthesis. Human skin fibroblasts at confluent density were exposed to L-ascorbic acid for a total of 72 hr. Fresh ascorbate was added each day. Cells were '

protein synthesis to collagen we studied the specific effects of

incorporated into collagen and noncollagen protein was determined

noncollagen protein. Cells received ascorbate (100 /LM) as well

pulse labeled the final 6 hr with [2,3-IH1L-proline. The radioactivity after digestion with clostridial collagenase.

glucocorticoids on collagen synthesis in association with ascor­ bate stimulation. Figure 4 shows the effect of hydrocortisone valerate at varying concentrations on synthesis of collagen and

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FIG 2. The effect of time on the stimulation by L-ascorbic acid of collagen synthesis. Human skin fibroblasts at confluent density were exposed for varying times to

100 /LM L-ascorbic acid. All cultures were

pulse labeled the final 6 hr with [2,3-'Hl-L-proline. The radioactivity incorporated into collagen and noncollagen protein was determined

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after digestion with clostridial collagenase.

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Days lowered in the absence of ascorbate, it was unchanged once ascorbate was present despite the continued increase in relative collagen synthesis and decline in prolyl hydroxylase activity [9]. Although the level of regulation of the ascorbate effect is

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The effect of L-ascorbic acid on relative collagen synthesis.

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formula which takes into account the relative abundance of proline in collagen in comparison to other proteins [2].

July

REGULATION OF COLLAGEN SYNTHESIS

1982

as glucocorticoid for 3 days. Collagen synthesis was slightly increased at 10-9 M. Similar increases were observed with dex­ amethasone and fluocinonide. Striking inhibition of collagen synthesis was observed at higher concentrations. Noncollagen protein synthesis remained relatively unchanged. When fibro­ blasts were exposed to hydrocortisone valerate (1 P.M) over 3 days relative collagen synthesis decreased from 34% to 17% (Fig 5). Similar results were observed with dexamethasone and fluocinonide. Thus glucocorticoids at low concentration specif­ ically and dramatically diminished collagen synthesis while having no effect on noncollagen protein synthesis. Rokowski, Sheehy and Cutroneo further examined the effect of glucocorticoids on collagen biosynthesis [17). They prepared ribosomes from skin and lung of control rats and rats injected with triamcinolone. In a cell free wheat germ translation system they demonstrated a selective reduction in collagen synthesis on membrane-bound polysomes from glucocorticoid-treated an­ imals. These studies further support a selective effect of gluco­ corticoids on collagen synthesis which can be demonstrated at the level of mRNA. CONTROL OF COLLAGEN SYNTHESIS BY AMINO­ TERMINAL EXTENSION PEPTIDES Feedback inhibition of collagen synthesis has been recently demonstrated by the globular portion of amino-terminal exten­ sion propeptides of procollagen. Peptides derived from the aminoterminal regions of type I and III calf procollagens spe­ cifically inhibited procollagen synthesis in calf and human

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- Log Hydrocortisone Valerate (M) FIG 4. The effect of hydrocortisone valerate on collagen synthesis . Human skin fibroblasts were incubated for 72 hr with varying concen­ trations of hydrocor tisone valerate and 100 liM L-ascorbic acid . The media was changed daily. All cultures were pulse labeled the final (; hr

with

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L - proline . The ra dioac ti vi t y incorporated into col lagen

and noncollagen protein collagenase.

was determined after digestion with clostridial

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Days 5. The effect of hydrocortisone valerate on collagen synthesis. 100 liM L­ ascorbic acid for 72 hr and 1 liM hydrocortisone valerate for varying in tervals. All cultures were pulse labeled the final (; hr with [2,3-1Hl L­ proline. The radioactivity incorporated into collagen and noncollagen protein was d ete rmi ned after diges tion w ith c los tridial c o llagena se . FIG

Human skin fibroblasts at confluent density were exposed to

fibroblast culture without affecting synthesis of other proteins [18). Types I and III propeptides were equally inhibitory for type I procollagen synthesis; type III procollagen synthesis was not measured. The effect was specific since peptides prepared from the helical region of the collagen molecule were incapable of exerting the effect. The type I propeptide was prepared from dermatosparactic calf skin and was derived from the pro 0'1 (I) chain; its molecular weight was 10,700 daltons. The type III propeptide was prepared from fetal calf skin and had a molec­ ular weight of 45,000 daltons. These results support previous observations in fibroblasts derived from patients with type VII Ehlers-Danlos syndrome in which conversion of procollagen to collagen was defective. Procollagen synthesis in these fibro­ blasts was markedly increased [19). The peptides were also capable of inhibiting procollagen synthesis in a cell free synthesizing system [20). Synthesis of pro 0'1 (I) and pro O'AI) by rat calvarial mRNA in a reticulocyte lysate was specifically inhibited by the propeptides but not by collagen helical peptides. The propeptides had little effect on translation of mRNAs for globin and rat liver vitellogenin. The effect of the propeptides on type II collagen synthesis was more complex [21). The type I propeptide was capable of inhibiting type II procollagen synthesis by chick sternal mRNA in a reticulocyte lysate. The type III propeptide however had no effect. The type I propeptide on the other hand was incap­ able of inhibiting type II procollagen synthesis when added to chick sternal chondrocytes or fetal calf chondrocytes in culture. In addition no effect was demonstrated on chondrocytes allowed to dedifferentiate to produce predominantly type I collagen or on procollagen synthesis by chick tendon fibroblasts. The data supports a specific translational control for these peptides in pro collagen synthesis. Cellular access may be limited by a specific recognition of the propeptide by the cell. REGULATION OF COLLAGEN SYNTHESIS BY VIRAL TRANSFORMATION Perhaps the best studied system with respect to regulation of collagen synthesis is viral transformation. When fibroblasts are transformed by tumor virus a number of changes occur. These include alterations in cellular growth, adhesive properties, cel­ lular shape and synthesis of discrete proteins. Although synthe­ sis of most proteins is unaltered, synthesis of type I collagen is markedly diminished [22-25). Moreover the level of collagen

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PINNELL

synthesis appears to be related to the degree of transformation [26]. The availability of cloned cDNA for chick pro al(l) and pro aAI) has made it possible to measure by hybridization tech­ niques the levels of mRNA specific for collagen. In chick embryo fibroblasts transformed with Rous sarcoma virus both total and nuclear collagen mRNA were about 5-fold lower than levels in normal cells [27,28]. Moreover, the changes were similar for mRNA specific for pro adI) and pro a2(1) i.e. mRNA levels for the two type I collagen chains were coordinately regulated. Using a strain of Rous sarcoma virus containing a temperature sensitive mutation in the pp 60"" gene, this transformation effect on transcription of collagen genes could be shown to be related to the pp 60"'c protein kinase [28]. Avvedimento et al prepared genomic pro a2(1) DNA clones containing introns and used these to specifically measure nu­ clear RNA for pro a2(I) [29]. In chick embryo fibroblasts infected with Rous sarcoma virus they were able to demonstrate a marked reduction in primary transcript mRNA for pro a2(1) suggesting a primary transcriptional control of type I collagen formation following transformation. Thus transcriptional con­ trol of collagen synthesis is an important result of cellular transformation and may be critical for maintaining the trans­ formed state. Although regulation of collagen synthesis promises to be complex, tools are now becoming available to understand these changes at a molecular level. REFERENCES

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of! he Skin. Edited by LA Goldsmith. New York, Oxford Univer­ sity Press, 1982, in press Freiberger H, Grove D, Sivaraj ah A, Pinnell SR: Procollagen I synthesis in human skin fibroblast: Effect of culture conditions on biosyn thesis. J Invest Dermatol 75:425-430, 1980 Booth BA, Polak KL, Uitto J: Collagen biosynthesis by hum an skin fibroblasts I. Optimization of the culture conditions for synthesis of type I and type III procollagens. Biochim Biophys Acta 607:145-160,1980 Tajima S, Pinnell SR: Collagen synthesis by human skin fibroblasts in culture. Studies of fibroblasts explanted from papillary and reticular dermis. J Invest Dermatol, 1981, in press Cardinale GJ, Udenfriend S: Prolyl hydroxylase. Adv Enz ymol 41:245-300, 1974 Ramachandran GN, Ramakrishnan C: Molecular structure, Bio­ chemistry of Collagen. Edited by GN Ramachandran, AH Reddi. New York, Plenum Press, 1976, pp 45-84 Prockop D,], Berg RA, Kivnikko K I, Uitto J: Intracellular steps in the biosynthesis of collagen in Biochemistry of Collagen. Edited by GN Ramachandran, AH Reddi. New York, Plenum Press, 1 976, pp 163-273 Light ND, Bailey AJ: Molecular struc tur e and stabilization of the collagen fibre in Biology of Collagen. Editors A Viidik , J Vuust, New York, Academic Press, 1980, pp 15-38 Murad S, Grove D, Lindberg KA , Reynolds G, Sivarajah A, Pinnell SR: Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci USA 78:2879-2882, 1981 Schwarz RI, Mandell RB, Bissell M,l: Ascorbate induction of col­ la gen synthesis as a means for elucidating a mechanism for

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quantitative control of tissue-specific function. Mol Cell BioI 1:843-853, 1981 Murad S, Sivarajah A, Pinnell SR: Regulation of prolyl and l ysyl hydroxylase activities in cultured human skin fibrob l asts by ascorbic acid. Biochem Biophys Res Commun 101:868-875, 1981 Counts, DF, Rojas FJ, Cutroneo KR: Glucocorticoids decrease prolyl hydroxylase activity without the cellular accumulation of underhydroxylated collagen. Mol Pharmacol 15:99-107, 1979 Benson SC, LuValle PA: Inhibition of lysyl oxidase and prolyl hydroxylase activity in glucocorticoid treated rats. Biochem Bio· phys Res Commun 99:557-562,1981 Russell JD, Russell SB, Trupin KM: Differential effects of hydro­ cortisone on both growth and collagen metabolism of human fibroblasts from normal and keloid tissue. J Cell Physiol 97:221-230, 1978 Ponec M, Kempenaar JA, Van Der Meulen-Van Harskamp GA, Bachra BN: Effects of glucocorticosteroids in cultured human skin fibroblasts IV. Specific decrease in the synth e sis of collagen but no effect on its hydroxylation. Biochem Pharmacol 28:2777 -2783, 1979 McCoy BJ, Diegelmann RF, Cohen IK: In vitro inhibition of cell growth, collagen synthesis, and prolyl hydroxylase activity by triamcinolone acetonide (40750). Proc Soc Exp Bioi Med 163:216-222, 1980 Rokowski RJ, She ehy J, Cutroneo KR: Glucocorticoid-mediated selective reduction of functioning collagen messenger ribonucleic acid. Arch Biochem Biophys 210:74-81, 1981 Wiestner M, Krieg T, Horlein D, Glanville RW, Fietzek P, !\!luller PK: Inhib iting effect of procollagen pep tides on collagen biosyn­ thesis in fibroblast cultures. J Bioi Chern 254:7016-7023, 1979 Lichtenstein JR, Martin GR, Kohn KD, Byers PH, McKusick VA: Defect in conversion of procollagen to collagen in a form of the Ehlers-Danlos syndrome. Science 182:298-299, 1973 Paglia LM, W ilcz ek J, Diaz de Leon L, Martin GR, Horlcin D, Mulle r PK: Inhibition of procollagen cell-free synthesis by amino­ terminal extension peptides. Biochemistry 18:5030-5034, J 979 Paglia LM, Wiestner M, Duchene M, Ouellette LA, Horlein D, Martin GR, Muller PK: Effects of procollagen peptides on the translation of type II collagen messenger ribonucleic acid and on collagen biosynthesis in chondrocytes. Biochemistry 20:35233527, 1981 Green H, Todaro GJ, Goldberg B: Collagen synthesis in fibroblasts transformed by oncogenic viruses. Nature 209:916-917, 19()6 Hata RI, Peterkofsky B: Specific changes in the collagen pheno­ types of BALB 3T3 cells as a result of transformation by sa rcoma viruses or a chemical carcinogen. Proc Natl Acad Sci USA 74:2933-2937, 1977 Levinson W, Bhatnagar RS, Liu TZ: Loss of ability to synthesize collagen in fibroblasts transformed by Rous sarcoma virus . .J Nat! Canc Inst 55:807-810, 1975 Schwarz RI, Farson DA, Soo W-J, Bissell MJ: Primary avian tendon cells in culture: an improved system for understanding malignant transformation. J Cell Bioi 79:672-679, 1978 Sandmeyer S, Smith R, Kiehn D, Bornstein P: Correia! ion of collagen synthesis and procollagen messenger RNA levels with transformation in rat embryo fibroblasts. Can Res 41:8:10-838, 1981 Adams SL, Alwine JC, de Crombrugghe B, Pastan I: Use of recom­ binant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts. J Bioi Chern 254:4935-4938, 1979 Sandmey er S, Gallis B, Bornstein P: Coordinate transcriptional regulation of type I procollagen genes by Rous sarcoma virus. J Bioi Chern 256:5022-5028, 1981 Avvedimento E, Yamada Y, Lovelace E, Vogeli G, de Crombllrgghe B, Pastan I: Decrease in the levels of nuclear RNA precursors for alpha 2 collagen in Rous sarcoma virus transformed fibroblasts. Nucleic Acids Research 9:1123-1131, 1981