Proc. NatI. Acad. Sci. USA Vol. 88, pp. 7180-7184, August 1991 Medical Sciences
Transforming growth factor ,8 stimulates urokinase-type plasminogen activator and DNA synthesis, but not prostaglandin E2 production, in human synovial fibroblasts (arthritis/dssue remodelng/differentiation/retinoids/interleukin 1)
JOHN A. HAMILTON*, DIANA S. PICCOLI, TALI LEIZER, DEBRA M. BUTLER, MARYANN CROATTO, AND A. KEITH M. ROYSTON Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
Communicated by J. F. A. P. Miller, April 2S, 1991 (received for review January 8, 1991)
fibroblasts could be a source of u-PA activity in rheumatoid lesions, and its increased expression by these cells could be important in understanding the pathogenesis of such lesions (3, 7, 15). It has been proposed by us that the enhanced PA activity in these cells, as a result of the action of monocytemacrophage cytokines, could be one manifestation of the expression of an immature, invasive, "tumor-like" phenotype, which could help to explain some of the features of the erosive synovial "pannus" of rheumatoid joints (3, 15). Transforming growth factor ( (TGF-(3), which was originally identified by its ability to cause phenotypic transformation of rat fibroblasts (16, 17), is produced by. many cell types, including monocyte-macrophages, platelets, and bone cells (18, 19). TGF-p has been detected in rheumatoid synovial fluids and may therefore have a role in this disease (20). It has recently been suggested that TGF-,8, perhaps in an autocrine manner, can induce a more differentiated phenotype in synovial fibroblast-like cells as evidenced by slower colony growth, increased collagen production, and decreased collagenase expression (4, 21); it was also suggested that in vivo these changes would promote scarring and reparative processes in arthritic synovial connective tissues and inhibit cartilage and bone destruction in surrounding tissues (21). We show here that TGF-f3 can enhance u-PA activity and DNA synthesis, but not prostaglandin E2 (PGE2) production, in human synovial fibroblast-like cells and, as a result, propose that TGF-,B may have, in fact, an alternative function in arthritic connective tissues-i.e., an involvement in synovial cell migration and hyperplasia, as well as breakdown of neighboring tissue by these cells.
Transforming growth factor ,B (TGF-fi) is ABSTRACT usually associated with matrix formation and tissue repair; in contrast, cellular expression of the serine proteinase, urokinase-type plasiunogen activator (u-PA) is often correlated with tissue remodeling, as well as with cell migration and transformation. We report here that purified recombinant human TGF-(3 (.300 pg/ml) can stimulate rapidly (within 2 h) the u-PA activity of nonrheumatoid synovial fibroblast-like cells. As for interleukin 1 (IL-1), u-PA mRNA levels are raised in response to TGF-(8, but unlike IL-1, no increase in prostaglandin E2 levels occurs. In contrast to a number of other examples in the literature, in which these two cytokines have opposing actions, TGF-(8 can potentiate the action of optimal concentrations of IL-1 in enhancing u-PA expression. These effects of TGF-3 are similar to those of all-tnns-retinoic acid. In addition, synovial fibroblast DNA synthesis was stimulated by TGF-f3. Because TGF-,B has been detected in the synovia of patients with rheumatoid arthritis and has been shown to reduce the collagenase levels and proliferation of synovial fibroblast-like cells, it has been proposed by others to be involved beneficially in the reparative processes occurring in arthritic lesions. However, on the basis of our fings, we propose alternative functions for this cytokine-namely, roles in the destructive events as well as in the synovial hyperplasia observed in rheumatoid joints.
Activated synovial fibroblast-like cells have been implicated in the invasive properties of the inflamed synovium of rheumatoid lesions (1-4). It is likely that their properties in such lesions are being modified by locally generated cytokines (3, 4); it has been shown, for example, that interleukin 1 (IL-1) elicits a number of responses in such cells (5-9). Plasmin is a broad spectrum serine protease, which is formed by cleavage of plasminogen by either of the two highly specific serine proteases, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (tPA). As well as having a role in fibrinolysis, plasmin has been invoked as being important in the processes of connective tissue turnover and remodeling, inflammation, and cell migration (10, 11). PA activity has also been correlated with neoplastic transformation and tumor metastasis (10, 11). t-PA is likely to be the PA mainly responsible for plasmin generation during fibrinolysis, while many studies indicate that u-PA is primarily responsible for plasmin generation in the degradation of extracellular matrix and basement membranes. Elevated PA levels have been found in rheumatoid tissue explant cultures and synovial fluids compared to their noninflamed equivalents (12, 13). Also, plasmin has been shown to degrade cartilage (14). Cytokine-activated synovial
MATERIALS AND METHODS Synovial Cell Culture. Human synovial cell explant cultures were established from nonrheumatoid donors as described (15). The passaged synovial fibroblast-like cells were plated overnight at 5 x 104 cells per 0.5 ml per well in 48-well
plates (Costar) in a-modified minimal essential medium (aMEM) (Commonwealth Serum Laboratories, Parkville, Victoria, Australia) supplemented with 10% (vol/vol) heatinactivated (56°C; 30 min) fetal calf serum (FCS) (Flow Laboratories). After washing with phosphate-buffered saline (pH 7.4), cytokine samples were added to a-MEM containing 1% (vol/vol) plasminogen-depleted FCS. Supernatants were collected after 24 h and assayed for PA activity. Measurement of PA Activity. This was measured in 50 ,ul of synovial cell conditioned medium as plasminogen-dependent Abbreviations: FCS, fetal calf serum; IL-1, interleukin 1; PGE2, prostaglandin E2; TGF-,B, transforming growth factor ,; u-PA, urokinase-type plasminogen activator. *To whom reprint requests should be addressed.
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Medical Sciences: Hamilton et al.
fibrinolytic activity (15); values obtained were compared to urokinase standards. PGE2 Assay. The PGE2 levels in synoviocyte culture supernatants were determined by RIA (Seragen) (5). Detection of mRNA in Synovial Cells. Total RNA was prepared as described (22, 23) by the method of Chirgwin et al. (24). All of the RNA from the cultures was fractionated on a formaldehyde-containing 1% agarose gel prior to transfer to GeneScreenPlus nylon membrane (DuPont). The filter was hybridized overnight at 60°C in a standard hybridization buffer containing >2 x 106 cpm per ml of 32P-labeled u-PA cDNA. Hybridization was essentially as described (22), except that it was performed at 45°C. Synovial Cell DNA Synthesis. As described (9), synovial fibroblasts were plated at 1.5 x 104 cells in 0.7-cm-diameter tissue culture wells (Linbro-Flow Laboratories) in a-MEM (Commonwealth Serum Laboratories), containing 10o heatinactivated FCS and allowed to adhere for 6 h. The cells were then placed in a-MEM containing 1% (vol/vol) acid-treated plasminogen-depleted FCS for 24 h to reduce the background DNA synthesis and allow PA activity to be measured. Agents were added and cultures continued for a further 72 h, the cells being pulsed at 48 h with [3H]thymidine. The cultures were terminated and cellular DNA synthesis was assessed by the amount of radiolabel incorporated into DNA on glass fiber filters (9). Reagents. Purified recombinant human TGF-p (Genentech) was used and had an ID50 of -50 pg/ml for inhibition of growth of the CCL64 mink epithelial cells (from A. Roberts, National Cancer Institute, Bethesda, MD) (25). Another sample of purified human platelet TGF-f3 (from A. Roberts and M. Sporn) gave results similar to those reported. Other reagents used were obtained from the following sources: purified recombinant human IL-la (specific activity, 5.7 x 107 units/mg; 1 unit/ml; -1 pM) (P. Lomedico, Hoffmann-La Roche, Nutley, NJ) (5); turkey anti-human TGF-f8 and normal turkey sera (A. Roberts); all-transretinoic acid (Hoffmann-La Roche); PGE2 (Advanced Magnetics, Cambridge, MA); actinomycin D, cycloheximide, and indomethacin (Sigma).
RESULTS
TGF-/3 Stimulates Human Synovial Fibroblast u-PA Expression. In Fig. 1, a dose-response curve for the effect of TGF-,( on human synovial fibroblast supernatant PA activity is presented. The first detectable increase in PA activity occurred at TGF-,B levels of :0.3 ng/ml. The cytokine could be
Proc. Natl. Acad. Sci. USA 88 (1991)
7181
Table 1. Effect of anti-TGF-/3 antibody on TGF-,B-stimulated u-PA activity PA activity, Agent(s) added Ploug U/ml Control 0.09 ± 0.01 0.03 ± 0.01 Anti-TGF-,8 antiserum Control serum 0.07 ± 0.01 0.20 ± 0.01 TGF-,8 0.03 ± 0.004* TGF-,( + anti-TGF-,3 antiserum TGF-/3 + control serum 0.32 ± 0.05 Synovial cells (from donor F.K., passage 4), cultured in triplicate as described, were incubated with TGF-f3 (1.25 ng/ml) in anti-TGF-,3 turkey antiserum or control turkey serum, both diluted 1:2000, for 24 h at 370C. Supernatants were collected and assayed for PA activity. Results are expressed as mean values (±SEM). *P < 0.05 compared with TGF-13-treated group.
shown to be active on fibroblasts from 19 other donors. Minor changes occurred when cell lysates were studied (data not shown). Antiserum to human TGF-13 blocked the action of TGF-f3 (Table 1). When the increase in u-PA activity was measured with time, the first detectable change was observed =2 h after cytokine addition (Fig. 2). Data for the kinetics of the response to IL-la are included for comparison. It can be seen that the early increase occurs at a time similar to that found for TGF-,3 but the effect is transient; this result was obtained with a number of lines (see below). We have shown previously by SDS/zymography and antibody blocking that the type of PA detected in control cell supernatants and those treated with mononuclear cell conditioned medium and all-trans-retinoic acid was, in fact, u-PA (26); the same approach has indicated that the PA measured in the TGF-/3-treated cells was also u-PA (data not shown). Consistent with these findings, it is shown in Fig. 3 that u-PA mRNA levels increased in response to TGF-,f. The result in Fig. 3 suggests that u-PA gene transcription might be stimulated by TGF-,3; in support of this notion is the observation (Table 2) that actinomycin D, an inhibitor of transcription, inhibited the increase in u-PA expression. The inhibition by cycloheximide also suggests that new u-PA protein was being translated and not simply being released from intracellular stores. There was no cell-associated accumulation of u-PA activity in the presence of either of these drugs (data not shown). Effect of Coaddition of TGF-. and IL-1. We next determined what happened when different concentrations of TGF-f3 were combined with an optimal concentration of
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