Journal of Dermatological

Science, 2 (199 1) 45-49 45

Elsevier

DESC 00059

Effect of tretinoin on collagen gel contraction induced by mouse 3T3 fibroblasts Takeshi Kono, Tsukasa Tanii, Masayoshi Furukawa, Taniguchi, Masamitsu Ishii, Toshio Hamada and Department

of Dermatology,

Laboratory,

Osaka City University Medical School, Abeno-ku, Osaka, Japan and ’ Developmental Biology

Zoological Institute, Faculty of Science, Hiroshima

(Received

Key words:

Collagen

Nobuyuki Mizuno, Shoji ’ Katsutoshi Yoshizato

gel culture;

6 December

1989; accepted

Collagen, type I; Balb/3T3 cells; 12-0-Tetradecanoylphorbol-13-acetate

University, Naka-ku, Hiroshima, Japan

25 July 1990)

Cell growth; (TPA)

Tretinoin

(all-trans-retinoic

acid);

Abstract

Balb/3T3 fibroblasts were cultured in type I collagen gel and the effects of tretinoin (all-trans-retinoic acid) were examined on cell growth and the gel contraction produced by cells. Cell proliferation was suppressed and the degree of gel contraction was enhanced by the addition of 10 -’ and 10 -6 M tretinoin. Growth and gel contractility of transformed cells derived from the Balb/3T3 cells were not influenced by this agent. Addition of 12-0-tetradecanoylphorbol ester, which is known to antagonize tretinoin in several biological processes, enhanced gel contraction synergistically with tretinoin. These results suggest that tretinoin influences cell-to-collagen interactions.

Introduction

Three-dimensional culture of fibroblasts in collagen gel was first reported by Elsdale and Bard [ 11. Since then, fibroblast-populated collagen gel has come to be recognized as an in vitro reconstituted dermis model [2-S]. Bell et al. [ 61 reported the phenomenon of gel contraction induced by fibroblasts, which is considered to provide an in vitro model 6f wound contracture

Correspondence to: Takeshi Kono, Department of Dermatology, Osaka City University Medical School, l-5-7, Asahimachi, Abeno-ku, Osaka 545, Japan.

0923-181 l/91/$03.50

0 1991 Elsevier Science Publishers

[ 6-81 and a convenient system for studying cellto-collagen interactions [ 8,9]. Tretinoin (all-truns-retinoic acid) and other retinoids are usually used clinically in the treatment of acne or psoriasis. Recently, tretinoin has been applied to the treatment of keloids [lo], photo-aged skin, and wrinkles [ 1 l-131. Though its effects on the biological and biochemical reactions of keratinocytes have been studied intensively, basic data concerning the effects on connective tissue remain inadequate. In the present study, we investigated the influences of tretinoin on cell-to-collagen interactions by examining the effects of this reagent on cell growth and gel contraction using Balb/3T3 fibroblasts. In

B.V. (Biomedical

Division)

46

addition, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which is known to antagonize tretinoin in several biological processes [ 14-171. Materials and Methods Cell culture

The established normal libroblastic cell line, Balb/3T3 clone A31, its spontaneously transformed variant, Balb/3T12-3, and the SV40 virustransformed line, 3T3-B-SV40, were all purchased from Dainippon Pharmaceutical Co. (Osaka, Japan). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Grand Island, NY) supplemented with 10% (v/v) fetal calf serum (FCS, GIBCO) in a moist atmosphere of 5% CO,/95% air at 37 “C. Preparation of collagen gel Fibroblasts cultured on plastic dishes were harvested when the monolayer of the cells reached about 70-90% confluency. Prior to use the cells were briefly treated with a 0.25 y0 trypsin solution (GIBCO), washed twice, then counted and diluted with medium to the desired concentrations. Fibroblasts were embedded three-dimensionally in hydrated collagen gel as previously described [3-5, 18-201. In brief, 0.3% (w/v) pepsinized type I collagen solution (prepared from porcine tendon, Nitta Gelatine Co., Osaka, Japan), concentrated DMEM, HEPES/NaOH solution for neutralization, the cell suspension, and FCS were mixed rapidly at a temperature below 4 ’ C. The final collagen concentration was 1.75 mg/ml and the initial cell density was lo5 cells/ml. Amounts of 5 ml or 1 ml of the mixture were then poured into 60-mm or 35-mm bacteriological dishes (Falcon) and cultured at 37 “C. Either tretinoin (Sigma) or TPA (Sigma) was added to the collagen mixture and medium at the indicated concentrations. The solvents used were ethanol for tretinoin and dimethylsulfoxide for

TPA. For the control experiments, was added at the same volume.

solvent alone

Measurement of gel diameter The gel diameter was measured every 24 h by placing the dishes on a ruler, and the mean diameters in three directions were recorded. Assay of cell growth At indicated times, the collagen gels in the 35-mm dishes were digested by collagenase (4 mg/ml, Sigma, type IV) at 37 ’ C for 40 min and the cells released were collected by centrifugation at 1,000 rpm for 5 min. The cells were resuspended in a 0.04% trypan blue solution in phosphate-buffered saline and the viable cells were counted with a hemocytometer. Results Effect of tretinoin on gel contraction Balb/3T3 libroblasts produced contraction of the collagen gel so that the gels floated in the medium. The time course of gel contraction with or without tretinoin is shown in Fig. 1. The extent

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Fig. 1. Effects of tretinoin on the collagen gel contraction produced by Balb/3T3 fibroblasts. Tretinoin was added at concentrations of 10e6 M (0) and lo-’ M (A). (O), control. Each point represents the average of three cultures (+ SD).

47

of gel contraction was increased by tretinoin, with the gel surface area ratio compared to the control gel after 7 days of culture being 68.1 and 54.6% for 10 - ’ and 10 - 6 M tretinoin, respectively (mean area at 10 _ 6 M versus that at 10 - ’ M ; P -C 0.01). At a concentration of 10 - ’ M, no significant difference was observed from the control in the gel contraction. Effect of tretinoin on cell growth The kinetics of cell proliferation are shown in Fig. 2. Growth in the collagen gel was markedly inhibited by the addition of tretinoin. The cell numbers after 7 days of culture were reduced to 75.6 % at a tretinoin concentration of 10 - ’ M and to 53.5 y0 at 10 - 6 M compared with the control (mean cell number at 10 - 6 M versus that at lo-’ M; P < 0.05). No significant difference from the control was observed in the cell numbers at lo-‘M. Effects of tretinoin on gel contraction and growth with transformed lines Figure 3 shows the time course of gel contraction produced by the tibroblastic cells of 2 transformed lines derived from Balb/3T3 (the

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Fig. 3. Effects of tretinoin on collagen contraction produced by transformed fibroblastic cells. (0), 3T3-B-SV40 cells. ( x ), Balb/3T12-3 cells. Tretinoin was added at the concentration of 10m6 M (---). (-), control. Each point represents the average of three cultures ( f SD).

Balb/3T12-3 and 3T3-B-SV40 cell lines). No effect was observed in either line on gel contraction (Fig. 3) or cell growth (data not shown) with lo-’ or lop6 M tretinoin.

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Days Fig. 2. Effects of tretinoin on the growth of Balb/3T3 fibroblasts in collagen gel. Tretinoin was added at concentrations of 1O-6 M (0) and lO_‘M (A). (0), control. Each point represents the average of three cultures ( f SD).

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Fig. 4. Effects of tretinoin and TPA on collagen gel contraction produced by Balb/3T3 tibroblasts. Tretinoin (10m6 M) alone (A) or both tretinoin (10e6 M) and TPA (lo-’ M) were added ( x ). (0), control. Each point represents the average of three cultures ( + SD).

48

Effect of TPA on gel contraction Addition of TPA alone did not influence gel contraction at 10 - ’ M, but it enhanced contraction at 10 - 6 M to a degree similar to that produced by the same concentration of tretinoin. The simultaneous addition of both TPA (10 - ’ M) and tretinoin (10 - 6 M) enhanced contraction more strongly than did tretinoin alone (lop6 M) (Fig. 4). Cells used in the experiment shown in Fig. 1 were harvested at about 70% confluency and those shown in Fig. 4 at about 90% confluency. As the contractility was influenced by the state of cells harvested, the degrees of contraction at 10 - 6 M tretinoin were slightly different between those in Figs. 1 and 4. Cell growth was not influenced at these concentrations of TPA (data not shown). Discussion We have previously reported the enhancement of collagen gel contraction by tretinoin in normal human dermal fibroblasts [ 201. Recently, Adams et al. have reported similar results using various retinoids [ 211. As with other cellular biological events, the sensitivity of normal human fibroblasts to tretinoin varies with the donor, donor’s age, number of passages and so on. Therefore, we studied a well-established and commonly used normal fibroblastic cell line, Balb/3T3 clone A3 1. Enhancement of collagen gel contraction by tretinoin was shown (Fig. 1). Though the mechanism underlying collagen contraction is still unclear [22], the phenomenon is regarded as a reflection of cell-to-collagen interactions [6-91. Accordingly, the present results suggest that tretinoin influences cell-to-collagen interactions. On the other hand, cell growth was suppressed by tretinoin (Fig. 2). Buttle et al. [ 91 have reported that poor contraction was produced by transformed lines which can grow well in collagen gel. There is possibly a reverse correlation between gel contraction and proliferative potential. Several other authors have reported that tretinoin induces cell differentiation and enhances differentiating function in transformed or tumor

cell lines [ 14, 16, 171. Steinberg et al. [8], Buttle et al. [ 91 and our group [ 231 have reported on the correlation of the extent of cell differentiation and contractility. And, further, we have recently, demonstrated the tretinoin-induced enhancement of gel contraction in murine melanoma cells [ 241. However, two transformed lines derived from Balb/3T3 cells did not show enhanced gel contraction with tretinoin (Fig. 3). The cause of this differential response is unclear. It is possible that the mode of response to tretinoin in transformed tibroblasts differs from those in other tumor cells. As Jetten et al. [25] have suggested, there may be a deficiency of cell receptors for retinoids in Balb/3T3 cell lines transformed by the SV40 virus. The effects of retinoids on tibroblasts have been reported with respect to cell growth, morphological changes, glycoprotein or collagen synthesis and cell attachment [25-281. TPA has been shown to antagonize some of the actions of tretinoin described above [ 14-171. However, contrary to our expectations, TPA enhanced gel contraction and appeared to act synergistically with tretinoin (Fig. 4). In the process of gel contraction, some mechanism may exert, in which tretinoin and TPA do not antagonize each other. These problems warrant further investigation. Though it is still obscure which effects of tretinoin relate to gel contraction, the present results suggest the influence of tretinoin on cell-tocollagen interactions of fibroblasts.

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