Modulation of collagen production by human dermal fibroblasts by all

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Modulation of collagen production by human dermal fibroblasts by all-trans-retinoic acid and transforming growth factor-fi. MICHAEL J. RAXWORTHY,* WILLIAM ...
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Modulation of collagen production by human dermal fibroblasts by all-trans-retinoicacid and transforming growth factor-fi MICHAEL J. RAXWORTHY,* WILLIAM J. CUNLlFFEt and EDWARD J. WOOD* *Department of Biochemistry, University of Leeds, Leeds LS2 YJT, U.K. and ?Department of Dermatology, Leeds General Infirmary, Leeds LSI 3EX, U.K. All-trans-retinoic acid ( R A ) has a number of effects on the skin and on skin cells in culture. In epidermis it modulates the expression of certain keratin polypeptides in both normal and malignant keratinocytes (Fuchs & Green, 1981; Kopan et al., 1987; Raxworthy et af., 1988). In dermis it modulates the synthesis of collagen (Oikarinen et af., 1985) and glycosaminoglycans (Priestley, 1987) by fibroblasts. In addition, several growth factors and cytokines have been shown to influence the production of extracellular matrix components by dermal fibroblasts and recent reports have implicated transforming growth factor-beta (TGF-j3) in the wound repair process (see Sporn et al., 1987). We have studied the effects of TGF-j3 and RA on collagen production by cultured human dermal fibroblasts. Human dermal fibroblasts were isolated from foreskins obtained at routine surgical circumcision. Dermis was separated from epidermis (Raxworthy et af.,1987) and incubated with collagenase (0.5 mg/ml) for 18 h at 37"C, and fibroblasts were grown in monolayer culture in Dulbecco's modified Eagle's medium (DMEM) and used at passages 4-12. Fibroblasts were plated in 24-well tissue culture dishes at 7.5 x lo4 cells/well and grown in medium containing fetal bovine serum (10%, v/v) for 24 h. The medium was then changed to serum-free DMEM for 24 h and finally to serumfree medium containing ascorbic acid (50 pg/ml), ~-[2,3'Hlproline (8.3 pCi/ml) and RA ( 10-x-10-4 M in ethanol or dimethyl sulphoxide) and incubation continued for 20 h. Collagen production in the culture medium was measured by following the incorporation of [ 3H]proline into bacterialcollagenase-sensitive protein (Peterkofsky & Diegelmann, 197 1) using chromatographically purified collagenase (form 111, Advance Biofactures Corp., Lynbrook, NY ). Control cultures were treated with vehicle only. To investigate the effects of TGF-j3, human dermal fibroblasts were exposed to TGF-Bl (R & D Systems, Minneapolis, MN) at concentrations of 0.33-10 ng/ml. Conditions were the same as those described for RA treatment. RA modulated collagen production in a biphasic manner (Fig. la).Low concentrations ( < lo-' M ) stimulated production by almost 300% compared with controls, but concentrations above M were markedly inhibitory. Concentrations between lo-' and 10-' M had no significant effect on collagen production. The observed stimulation was not due to an effect of RA on dermal fibroblast proliferation. M, RA was strongly inhibitory to However, at 1 W sand cell proliferation indicating that the apparent inhibition of collagen production at these concentrations was related to a cytotoxic effect of RA (data not shown). At concentrations of 0.33-5 ng/ml TGF-j3 stimulated collagen production in two strains of dermal fibroblasts. There was, however, a marked difference in sensitivity (Fig. 1b). The apparent biphasic modulation of collagen production by RA contrasts with the findings of Oikarinen et al. (1985) who observed a dose-dependent inhibition in procollagen production at > M-RA,but no stimulation at < M. Abbreviations used: RA, all-trans-retinoic acid; TGF-/?, transforming growth factor-/?; DMEM, Dulbecco's modified Eagle's medium.

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Fig. 1. Effect of all-trans-retinoic acid and TGF-j3 on collagen production by human dermal fibroblasts in monolayer culture Fibroblasts were exposed to the test substance for 20 h and collagen production was measured as described in the text. Each point is the mean of at least two experiments which were themselves conducted in triplicate. ( a )Effect of RA on fibroblast strain C96 (donor age, 13 years); ( b )effect of TGFj3 using strains C96 (continuous line) and A61 (donor age, 16 years; dotted line). 1989

628th MEETING, GALWAY A biphasic modulation does, however, agree with recent observations reported with other synthetic retinoids (Godt et al., 1988). Furthermore, RA was reported to exert a biphasic effect on glycosaminoglycan secretion by normal human dermal fibroblasts (Priestley, 1987) and human bronchial epithelial cells in vitro (Wu & Wu, 1986). However, our findings indicate that the inhibition of production of extracellular matrix components may be due partly to the cytotoxicity of RA at high concentrations. The stimulation of collagen production was not accompanied by increased cell proliferation and occurred at physiological RA concentrations ( 10-8-10-6 M, Vahlquist et al., 1982; Kim et al., 1987). Although a marked difference in responsiveness to TX3F-p was observed with the two strains of human dermal fibroblasts used, the maximal stimulation of collagen production was recorded at physiological concentrations (i.e. 1-5 ng/ml, Cromack et al., 1987). It will be interesting to compare the effects of RA and growth factors such as TX3F-B on collagen production by fibroblasts grown within a collagen lattice rather than in monolayer culture. This work was supported by the Wellcome Trust.

405 Cromack, D. T., Sporn, M. B., Roberts, A. B., Merino, M. J., Dart, L. D. & Norton, J. A. (1987)J. Surg. Res. 42,622-628 Fuchs, E. & Green, H. (1981)Cell 25,617-625 Godt, C., Muller, K. P., Stadler, R. & Orfanos, C. E. (1 988) J. Invesf. Dermatol. 91,4 17 Kim, K. H., Stellbach, V., Javors, J. & Fuchs, E. ( 1 987) J. Cell Biol. 105,3039-305 1 Kopan, R., Traska, G. & Fuchs, E. (1987) J. Cell Biol. 105,427-440 Oikarinen, H., Oikarinen, A. I., Tan, E. M. L., Abergel, R. P., Meeker, C. A,, Chu, M. L., Prockop, D. J. & Uitto, J. (1985) J. Clin. Invest. 15, 1545-1553 Peterkofsky, B. & Diegelmann, R. (1971) Biochemistry 10,988-994 Priestley, G. C. (1987)Brit. J. Dermatol. 117,575-583 Raxworthy, M. J., Cunliffe, W. J. & Wood, E. J. ( 1 987) Biochem. Soc. Trans. 15,519-520 Raxworthy, M. J., Holland, D. B., Cunliffe, W. J. & Wood, E. J. (1988)Biochem. SOC.Trans. 16,327-328 Sporn, M. B., Roberts, A. B., Wakefield, L. M. & de Crombrugge, B. (1987)J.CellBiol. 105,1039-1045 Vahlquist, A,, Lee, J. B., Michaelsson, G. & Rollman, 0. (1982) J. Invest. Dermatol. 19,94-97 Wu, R. & Wu, M. M. J. (1986)J. CeNPhysiol. 127,73-82 Received 30 September 1988

Inhibitors of adenosine diphosphate ribosyl transferase suppress the induction of ornithine decarboxylase DESMOND B. JOHNSON: MELVIN M. MARKOWITZ,* RONALD W. PERO,*t PHILIP E. JOSEPH* and DANIEL G. MILLER* *PreventiveMedicine Institutelstrang Clinic, 55 East 34 Street, New York, NY 10016, U.S.A.and t University of Lund, Division of Molecular Ecogenetics, Wallenberg Laboratory, Box 7031,22007 Lund 7, Sweden Ornithine decarboxylase (ODC) and adenosine diphosphate ribosyl transferase (ADPRT ) are enzymes involved in different ways in the processing of DNA and in protein synthesis (Tabor & Tabor, 1984; Ueda & Hayaishi, 1985). In this report, the interdependence of these two enzyme activities is explored. It has been reported that inhibition of ADPRT also inhibits thymidine incorporation into DNA, i.e. an S phase event. It was also shown that thymidine incorporation was more extensively reduced when ADPRT inhibitors were included in cell cultures before the addition of mitogen, rather than 24 h after that addition (Durkacz et al., 1980). Therefore, inhibitors of ADPRT are likely to affect processes before S phase. We show that one such process affected is the induction of ODC. In human cells (Rittling el al., 1986; Kaczmarek et al., 1987) and bovine lymphocytes (White et al., 1987),synthesis of ODC mRNA, and induction of ODC are G , events. Mononuclear leucocytes were prepared from whole blood as previously described (Pero & Sjogren, 1982). Blood plasma was centrifuged, and the platelet-depleted plasma was used subsequently in culture media. Cells (4 x loh)were cultured in RPMI medium supplemented with 20% (v/v) platelet-depleted plasma, at a cell density of 2 x lo6 cells/ml, with added phytohaemagglutinin (PHA; Gibco, 20 pl/ml of culture) for 20 h at 37°C. ODC was assayed by a modification of the procedure of Djurhus (1981),using strong cationexchange paper (Whatman P-81,14 mm x 50 mm) to absorb [3H]putrescine. Assay of ADPRT was adapted from the Abbreviations used: ODC, ornithine decarboxylase; ADPRT, adenosine diphosphate ribosyl transferase; PHA, phytohaemagglutinin.

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procedure of Berger ( 1978) with modifications previously described (Pero et al., 1983). The influence of inhibitors of ADPRT on ODC induction by PHA was tested by including nicotinamide, benzamide and caffeine in cultures of mononuclear cells. These compounds markedly suppressed the induction of ODC (Table 1).The effects of such compounds were further examined by adding nicotinamide and caffeine to cell cultures at different times relative to addition of PHA. Most complete inhibition was achieved when inhibitors were added at the same time as mitogen: inhibition by caffeine (6 mM) was 92% when added with PHA, 83% when added 1 h later and 74% when added 3 h after PHA. While increases in ADPRT during cell cycling have been well documented, there is general agreement that the enzyme activity is low in quiescent cells. According to one report, there is no enzyme synthesis at least within 8 h of exposure to mitogen (Scovasi et al., 1987). It is also reported that, in pig lymphocytes treated with PHA, there was, overall, a 2.9fold increase in ADPRT, with one-quarter of this increase

Table 1. Suppression of ODC induction Mononuclear cells were cultured in the presence of the compounds shown. O D C activity was determined after 20 h. ODC

Inhibitor (mM)

n

("/. control fs.E.M.)

Nicotinamide 10.0

5

5.6k4.1

Benzamide 1.o 2.0 10.9

2 3 4

80.0 f 7.8 62.0 f 9.9 17.0 f 8.3

1 1 2

46.0 76.0 8.0 f 4.2

Caffeine 0.6 1.5 2.5