Vol. 40, No. 1, September 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Poges

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1 Vol. 40, No. 1, September 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Poges BISINDOLYLMALEIMIDE INHIBITS THE PMA-INDUCED DOWN-REGULATION OF COLLAGEN SYNTHESIS IN FIBROBLASTS Rang-Woon Park, In-San Kim,* and Joon-Seung Jo Department of Biochemistry, School of Medicine, Kyungpook National University, 101 Dongin-dong, Jung-gu, Taegu , Korea Received June 28, 1996 Summary : We assessed the role of protein kinase C (PKC) in the regulation of collagen synthesis. Two PKC activators, Phorbol 12-myristate 13-acetate (PMA) and 1-oleyl 2-acetyl-sn-glycerol (OAG), decreased the relative rate of collagen synthesis in a dose- and time-dependent manner in fibroblasts, whereas an inactive phorbol ester, 4a-phorbol didecanoate failed to affect the collagen synthesis. In PKC-depleted cells both PMA and OAG were unable to inhibit collagen synthesis. Bisindolylmaleimide, a specific inhibitor of PKC, completely abrogated PMA-induced inhibition of collagen synthesis m a dose-dependent fashion while two other PKC inhibitors with low specificity, H7 and staurosporin failed to block PMA effect on collagen synthesis. The results provide evidence that collagen synthesis is regulated through the signal pathway involving PKC activation. Key words : Protein kinase C, Collagen, Bisindolylmaleimide Introduction Studies have shown that collagen metabolism can be modulated by variety of factors including cytokines, hormones, and growth factors (1, 2). Understanding how these factors affect fibroblast proliferation and collagen metabolism is critical for elucidating the mechanism by which the fibrogenic response is initiated, maintained, or inhibited. The flow of information upon cell stimulation by a ligand can be traced through a cascade of regulatory reactions that involve a number of specific enzymes, regulatory proteins, and other regulatory factors. Previously we and other groups reported that phorbol myristate acetate (PMA), a well-known and *Corresponding address : TEL, ; Fax, i /96/ /0 Copyright 1996 by Academic Press Australia. All rights of reproduction in any form reserved.

2 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL potent activator of protein kinase C (PKC), inhibited collagen production in fibroblasts (3-5). However, it is not clear whether the effects of PMA on collagen synthesis in fibroblasts reflect the changes in the state of PKC activity since PMA frequently exerts its biological effects via PKC-independent mechanisms (6, 7). Since the potent and specific inhibitor is required to study the role of PKC in biological processes, we used a recently introduced PKC inhibior, bisindolylmaleimide GF X to investigate the possible role of PKC in the PMA-induced down-regulation of collagen synthesis in fibroblasts. Bisindolylmaleimide interacts with ATP-binding site of PKC and has great potency and excellent specificity compared with the other PKC inhibitors described previously (8). The present study provides evidence that PMA decreases the collagen synthesis in fibroblasts through the signal transduction pathway involving the activation of PKC. Materials and Methods Cell Culture: The human embryonic lung fibroblasts (HEL299) and human skin fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM)(Gibco/BRL, NY) containing 10 % fetal bovine serum (FBS). NIH3T3 cells were maintained in DMEM containing 10% calf serum and osteoblastic MC3T3-E1 cells were maintained in u-minimum essential medium containing 10% FBS. Collagen and Noncollagen Protein Synthesis: Cells were inoculated in 24-well tissue culture plates at 1 x 105 (HEL299 and HSF30) or 5 x 104 (NIH3T3 and MC3T3-E1) cells per well. Protein synthesis was assayed in confluent cultures under serum-free conditions. They were labeled with [ah]proline (12.4 Ci/mmol) for the last 3 or 6 h in the presence of 1% bovine serum albumin and 50 ~g/ml of ascorbic acid with or without testing agents. The incorporation of the labeled amino acid into collagenase-digestible protein (CDP) and noncollagen protein (NCP) were assayed as previously described (9). The relative rate of collagen production as a percentage of total protein production was calculated by the following formula: dpm in CDP x 100 (dpm in NCP) x 5.4 dpm in CDP Northern Blot Analysis: Total cellular RNA was isolated with acid guanidine thiocyanate-phenol-chloroform (10). Ten ~g of RNA was fractionated on a 0.8%. agarose-formaldehyde gel, then transferred onto a nitrocellulose membrane. The membrane was hybridized with [32p]-labeled cdna probes-hf677, human ctl(i)procollagen (11) and phcgap, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (12). 102

3 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Results and Discussion Exposure of quiescent human lung fibroblasts, HEL299 to PMA ranging from 1 to 1,000 ng/ml for 6 h resulted in a dose-dependent inhibition of [3H]proline incorporation into CDP. These changes were not the result of an overall decrease in protein synthesis since NCP synthesis was actually increased by the treatment of PMA in a dose-dependent manner. Therefore, PMA over the same concentration range reduced collagen synthesis expressed as a percentage of total protein synthesis. The dose-dependent inhibitory effects of PMA on collagen synthesis were also observed in mouse fibroblast-like cells (NIH3T3), mouse osteoblast-like cells (MC3T3-E1), and primary human skin fibroblasts (HSF30) (Table 1). The time course of collagen synthesis following treatment of HEL299 cells with PMA showed that collagen synthetic rate decreased in 100 ng/ml of PMA-treated cells for the first 3 h of incubation followed by a gradual increase and was higher than that of untreated cells at 18 h and 24 h, and then declined to the level of untreated cells at 48 h. The increased collagen synthetic rates after 18 h of incubation with PMA were due to the increased tdtal protein synthetic activity since PMA stimulated NCP synthetic rate. Thus, the percent collagen rapidly decreased by 3 h, maximally by 6 h, and then gradually returned to the level of untreated cells (Fig. 1). This time-course pattern of collagen synthetic activity was well consistent with the steady state levels of al(i)procollagen mrna by Northern blot analysis (data not shown). It is of interest that collagen synthetic rate and mrna levels for al(i)procollagen recovered gradually to the control level despite the continued presence of PMA. It is unclear whether it results from down-regulation of PKC or from other intracellular mechanism obscuring the PMA-induced inhibition of collagen synthesis. Since cycloheximide had little or no effect on basal level of al(i)procollagen mrna and did not affect PMA-mediated decrease in the al(i)procollagen mrn.~ level as shown in Figure 2, protein synthesis evidently is not required for the PMA effect indicating that collagen synthesis in fibroblasts can be modulated by preexisting proteins which are posttranscriptionally modified by PMA, possibly through the PKC-mediated pathway. 103

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Table 1. Effect of PMA on CDP and NCP synthesis in various cell lines Cell Concentration (ng/ml) CDP NCP % collagen (percent of control) HEL299 NIH3T3 HSF30 MC3T3-E Confluent ce~s were treated with PMA and labeled with 2 LICi/ml of [ H]proline for 6 h. Data are expressed as the percentage of control. It is needed to determine whether a certain effect of PMA is PKC-dependent since PMA has been shown to produce some of its effects through PKC-independent mechanisms, for examples, the activating phospholipase D in HL-60 granulocytes (6) and inducing collagenase production in chondrocytes (7). Here, by the following experiments, we revealed that the PMA-induced inhibition of collagen synthesis in fibroblasts is PKC-dependent. Oleylacetylglycerol (OAG), a synthetic analogue of diacylglycerol which is a well-known endogenous PKC activator, inhibited collagen synthesis in a dose-dependent manner, while 4a-phorbol didecanoate (4a-PDD), an inactive phorbol ester which is not able to activate PKC failed to inhibit collagen synthesis in the same concentration range used for PMA (Table 2). The result implies that the inhibitory effect of PMA 104

5 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 5OO0 Z ~0 CL, 2000 I000 0 I I F I I I I I! Time (hours) "~Z 12OOO C:~ b~ :L BOO Z 0 I I I 1 I! I I I 0 5 to Time (hours) o 6 ~ 4 O ~ 2 0 I I I I I I I I I Time (hours) Fig. 1. Time response experiment for the effect of PMA on CDP, NCP, and percent collagen synthesized in HEL299 cells. Confluent cells were treated ~ith PMA (100 ng/ml) for the indicated time and labeled with 2 ~Ci/ml of [ H]proIine for the last 3 h. Each value represents mean + SD of six measurements. C), control; 0, PMA-treated. I05

6 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL CONT PMA "H" PMA x+chx Fig. 2. Effect of PMA on steady-state levels of al(i)procollagen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mrnas in the presence or absence of cycloheximide (CHX) in HEL299 cells. Confluent cells were exposed to PMA (100 ng/ml) in the presence of absence of CHX (10 Ilg/ml) for 6 h. A Northern blot of total RNA revealed 4.8 and 5.8 kb mrnas for the al(i)procollagen and 1.3 kb mrna for GAPDH. Table 2. Effect of OAG and 4a-PDD on CDP and NCP synthesis by HEL299 cells Concentration CDP NCP % collagen (ng/ml) (percent of control) OAG 4a-PDD Confluent cell~ were treated with OAG or zla-pdd and labeled with ['H]proline for 6 h. Data are expressed as the percentage of control. OAG, oleylacetylglycerol; 4a-PDD, 4a-phorbol didecanoate. on collagen synthesis is not a nonspecific structural effect but an effect related to PKC activation. Since phorbol esters stimulate arachidonic acid and prostaglandin production in many cells (13-15) we treated cells with indomethacin to prevent endogenous prostaglandins, but no apparant change was detected in the PMA-induced inhibition of collagen synthesis (data not shown). It is well established that treating cells with high concentration of phorbol esters for extended period results in degradation of PKC, leading to abrogation of PKC-specific reponses (16-18). PKC depletion studies 106

7 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL have been used by a variety of investigators to study involvement of this second messenger pathway in many biological systems. In this study, pretreatment of HEL299 cells with 1 pg/ml of PMA for 36 h, which resulted in a decrease of more than 90 /6o of PKC activity in the cells (data not shown), abolished the inhibiting effect of an additional PMA (100 ng/ml) or OAG (500 ng/ml) on collagen synthesis (Fig. 3). In order to further substantiate the role of PKC in modulating collagen synthesis, we tried to demonstrate the effect of PKC inhibitor preventing the PMA-induced down-regulation of collagen synthesis. Unfortunately neither H7 nor staurosporine could block the PMA effect on collagen (data not shown). However, it is very unlikely that PKC is not involved in the regulation of collagen synthesis in fibroblasts. Because H7 and staurosporine are not very specific for PKC and both are very toxic to cells especially in the case of long-term treatment, they do not appear to be ideal for the study of PKC-specific responses (19, 20). Bisindolylmaleimide is a newly introduced protein kinase inhibitor. It inhibits PKC activity via the ATP-binding site just like its structural analog, staurosporine. But unlike staurosporine, which has inhibitory effect for broad spectrum of protein kinases and cytotoxic effect, bisindolylmaleimide selectively inhibits PKC including PKC isofc,xns a, [3I, td ta0 o 6 5 D 4 Q) 3 o 2 1 ControIPMA OAGControIPMA OAG PMA pretreated Fig. 3. Effect of PKC depletion on PMA-induced inhibition of collagen synthesis in HEL299 cells. Confluent cells were exposed to PMA (1 ~g/ml) or vehicle for 36 h, extensively washed and then treated with PMA (100 nfih/ml) or oleyl acetyl glycerol (500ng/ml) for 6 h. Two ~Ci/ml of ]proline were added for the last 6 h. Each value represents mean + SD of six measurements. 107

8 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Table 3. Effect of bisindolylmaleimide on PMA-induced inhibition of collagen synthesis in IIEL299 cells Cell treatment Concentration % collagen (~am) No addition PMA alone BIM alone PMA + BIM I Confluent cultures were pretreated with bisindolylmaleimide (BIM) for 10 min. prior to the addition of PM~ (100 ng/ml). The cells were incubated and labeled with ['H]proline for 6 h. Data are expressed as mean + SD for six measurements. [~II, and "t as well as it seemed to be suitable for both in vivo and in vitro uses (8). We pretreated cells with bisindolylmaleimide ( I~M) for 10 minutes prior to the addition of PMA (100 ng/ml) and then measured collagen synthesis. At these concentrations it was reported that bisindolylmaleimide completely inhibited the phosphorylation of p47 and p20 in human platelets and the mitogen-stimulated DNA synthesis in Swiss 3T3 cells (8). As shown in Table 3, bisindolylmaleimide apparently blocked the PMA-induced inhibition of collagen synthesis in a dose-dependent manner while bisindolylmaleimide alone at each concentration did not affect collagen synthesis. A complete block was achieved at a concentration of 0.5 IJM bisindolylmaleimide. In conclusion, these results strongly suggest that PKC plays a major role in PMA-induced inhibition of collagen synthesis in fibroblasts. With our previous report that a protein phosphatase inhibitor, okadaic acid could also modulate collagen synthesis in fibroblasts (9), the present results 108

9 Vol. 40, No. 1, ] 996 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL indicate that a number of phosphorylation-dephosphorylation processes are implicated in the regulation of collagen synthesis in fibroblasts. Acknowledgements We are grateful to Dr. Benoit de Chrombrugghe for helpful discussions. This work was supported by a grant from the Korea Research Foundation of Ministry of Education. References 1. Freundlich, B., Bomalaski, J.S., Neison, E., and Jimenez, S.A. (1986) Immunol. Today 7, Slack, J.L., Liska, K.J., and Bornstein, P. (1993) Am. J. Med. GeneL 45, Delclos, K.B., and Blumberg, P.M. (1979) Cancer Res. 39, Goldstein, R.H., Fine, A., Farnsworth, L.J., Poliks, C., and Polgar, P. (1990) J. Biol. Chem. 265, Jo, J.-S., Cho, Y.-S., Choi, J.-Y., and Kim, I.-S. (1991) Korean J. Biochem. 23, Billah, M.M., Pai, J.-K., Mullmann, T.J., Egan, R.W., and Siegel, M.I. (1992) J. Biol. Chem. 264, Conquer, J.A., Kandel, R.A., and Cruz, T.F. (1992) Biochim. Biophys. Acta 1134, Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovsky, J. (1991) J. Biol. Chem. 266, Kim, I.-S., Park, R.-W., Sohn, K.-Y., and Jo, J.-S. (1994) Biochem. Biophys. Res. Commun. 199, Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, Chu, M.L., Myers, J.C., Bernard, M.P., Ding, J.F., and Ramirez, F. (1982) Nucleic Acids Res. 10, Tso, J.Y., Sun, X.-H., Kao, T.-H., Reece, K.S., and Wu, R. (1985) Nucleic Acids Res. 13, Burch, R.M., Ma, A.L., and Axelrod, J. (1988) J. Biol. Chem. 263, Halenda, $2., Zavoico, G.B., and Feinstein, M.B. (1985) J. Biol. Chem. 260, Parker, J., Daniel, L.W., and Waite, M. (1987) J. Biol. Chem. 262, Adams, J.C., and Gullick, W.J. (1989) Biochem. J. 257, Blackshear, P.J. (1988) Am. J. Med. Sci. 296, Young, S., Parker, P.J., Ullrich, A., and Stabel, S. (1987) Biochem. J. 244, Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, Ruegg, U.T., and Burgess, G.M. (1989) Trends Pharmacol. Sci. 10,