Changes in Cell Shape Correlate with Collagenase Gene Expression in Rabbit Synovial Fibroblasts 1662

Induction of the neutral proteinase, collagenase, is a marker for a specific switch in gene expression observed in rabbit synovial fibroblasts . A variety of agents, including 120-tetradecanoylphorbol-13-acetate, cytochalasins B and D, trypsin, chymotrypsin, poly(2hydroxyethylmethacrylate), and trifluoperazine induced this change in gene expression . Induction of collagenase by these agents was always correlated with a marked alteration in cell morphology, although the cells remained adherent to the culture dishes . The amount of collagenase induced was positively correlated with the degree of shape change produced by a given concentration and, to some extent, with the duration of treatment . Altered cell morphology was required only during the first few hours of treatment with inducing agents ; after this time collagenase synthesis continued for up to 6 d even when agents were removed and normal flattened cell morphology was regained . All agents that altered cell morphology also produced a characteristic switch in protein secretion phenotype, characterized by the induction of procollagenase (M r 53,000 and 57,000) and a neutral metalloproteinase (Mr 51,000), which accounted for approximately 25% and 15% of the protein secreted, respectively . Secretion of another neutral proteinase, plasminogen activator, did not correlate with increased collagenase secretion . In contrast, synthesis and secretion of a number of other polypeptides, including the extracellular matrix proteins, collagen and fibronectin, were concomitantly decreased . That changes in cell shape correlated with a program of gene expression manifested by both degradation and synthesis of extracellular macromolecules may have broad implications in development, repair, and pathologic conditions . The neutral proteinase, collagenase, which specifically degrades interstitial collagens, is induced by a variety ofagents, including the phorbol diester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)' (2, 10), cytochalasin B (CB) (23), and proteolytic enzymes (52), as well as by phagocytosis (54) . We have demonstrated that TPA triggers an alteration in gene expression in rabbit synovial fibroblasts that is marked by induction of several secreted proteins, including collagenase and a wide-spectrum neutral metalloproteinase, and by reduction of the synthesis of other secreted proteins, including 'Abbrevations used in this paper. CB, cytochalasin B ; DME, Dulbecco's modified Eagle's medium ; EGF, epidermal growth factor ; kd, kilodalton ; PA, plasminogen activator; polyHEMA, poly(2-hydroxyethylmethacrylate) ; preproCL, preprocollagenase; proCL, procollagenase ; TFP, trifluoperazine; TPA, 12-O-tetradecanoylphorbol-13acetate. collagen and fibronectin (2) . When we analyzed this switch in gene expression by following the induction of collagenase by TPA, several characteristics of this phenomenon were evident (2). Collagenase induction required only a brief exposure to TPA, and no detectable collagenase was produced until 6-12 h after addition of TPA. Once begun, collagenase synthesis continued for >48 h after TPA was removed . Translatable mRNA for collagenase was present only in induced cells . Finally, concentrations ofTPA that induced collagenase always caused alterations in cell morphology. In the present study we have used other agents reported to alter cell morphology to test the hypothesis that changes in cell shape play an important role in initiating the switch in gene expression observed with TPA treatment (2). We now report that TPA, CB, and proteinases, as well as two additional agents, poly(2hydroxyethylmethacrylate) (polyHEMA) and trifluoperazine (TFP), induce changes in cell shape in rabbit synovial fibroTHE JOURNAL OF CELL BIOLOGY " VOLUME 98 MAY 1984 1662-1671 C The Rockefeller University Press 0021-9525/84/05/1662/10 $1 .00 on July 9, 2017 jcb.rress.org D ow nladed fom blasts that are correlated with induction of this specific secretion phenotype . MATERIALS AND METHODS Except for the following, all methods used were as described in the accompanying paper (2) . Cell Culture : Rabbit synovial fibroblasts were cultured in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum as described previously (2) . For most experiments, cells (-10' cells per well) plated in 16-mm culture dishes (Costar, Boston, MA 24-well) were incubated at 37°C for 16-48 h, and then placed in serum-free DME supplemented with 0.2% lactalbumin hydrolysate for treatment with various agents . For some experiments, cells were incubated at 37°C for various periods of time in the continuous presence of inducing agents ; in other experiments, inducing agents were included for the first 6-12 h of incubation only, after which the cells were returned to serum-free medium for collection of secreted proteins. Sodium ascorbate (50,g/ml) was added to some cultures to facilitate collagen synthesis. TPA (Sigma Chemical Co., St. Louis, MO) was dissolved at I-5 mg/ml in ethanol ; CB (Sigma Chemical Co .) at l mg/ml in dimethyl sulfoxide; TFP (Sigma Chemical Co .) at 3 mM in phosphate-buffered saline ; and monensin (Eli Lilly and Co., Indianapolis, IN) at 2 .5 mM in DME containing I% ethanol. All stock solutions were stored at -20°C until used. To study the effects of culturing cells on polyHEMA (Hydron Corp ., New Brunswick, NJ) (19), plates were prepared by dissolving polyHEMA (12% wt/ vol) in 95% ethanol overnight at 37°C and then diluting this stock solution to the appropriate concentration with 95% ethanol and adding 200 ul/well to 16mm culture wells. The plates were dried on a leveling platform at 25°C for 23 d. Final polyHEMA coating ranged from 501,200,ug/cm2. Fibroblasts were trypsinized, plated into coated wells in DME-10% fetal bovine serum, and incubated for 18-24 h at 37°C before being washed with saline and placed in serum-free medium . Cell morphology was monitored by phase microscopy, and the degree of morphologic change after various treatments was scored from 0-4 on an arbitrary scale . For scanning electron microscopy, cells plated onto 12-mmdiam glass coverslips coated with polyHEMA were fixed and processed as described previously (3). Plasminogen Activator Assay: Plasminogen activator (PA) activity was measured with an "'I-labeled fibrin substrate in the presence or absence of 6 nM purified bovine plasminogen (5, 50) . Each well contained 2-5 x 10°cpm and 20 pg/ml fibrin substrate. l U of activity releases 5% (1 kg) of the available substrate per hour . Release of labeled peptides in the absence of plasminogen was always <5%, indicating that the activity reported was PA.