INDEPENDENT REGULATION OF COLLAGENASE, 72-KDA PROGELATINASE, AND METALLOENDOPROTEINASE INHIBITOR EXPRESSION IN HUMAN-FIBROBLASTS BY TRANSFORMING GROWTH FACTOR-BETA

Abstract

The effects of transforming growth factor-.beta.(TGF-.beta.) on fibroblast collagenolytic activity were investigated to determine if modulation of matrix metalloendoproteinase activity could augment the stimulation of connective tissue formation by TGF-.beta.. Quiescent human fibroblast cultures were incubated in the continuous presence of 1.0 ng/ml (40 pM) TGF-.beta. in culture medium supplemented with 0.2% (v/v) serum and containing [35S]methionine. Aliquots of conditioned cell culture media, harvested daily for 4 days, were processed individually to separate procollagenase and a 72-kDa progelatinase from metalloendoproteinase inhibitor (TIMP) and plasminogen activator inhibitor (PAI-1) using tandem minicolumns of heparin- and gelatin-Sepharose. The fractionated 54-kDa procollagenase was quantitated, after p-amino-phenylmercuric acetate activation, by functional assays using soluble [14C]glycine-labeled collagen as substrate. In cultures treated with TGF-.beta., procollagenase expression was progressively decreased (.apprx.50% on day 1, .apprx.75% on day 2) to undetectable levels on days 3 and 4. This decrease occurred despite a 1.6-fold increase in the synthesis of total secreted protein. Contrasting the effect on procollagenase, TGF-.beta. increased the synthesis of a 72-kDa progelatinase (characterized as a matrix neutral metalloproteinase and likely to be MMP-2) up to 1.8-fold, as determined by quantitation of affinity-purified radiolabeled protein and by enzymography. TIMP biosynthesis was analyzed by immunoprecipitation and quantitated by functional assays for biologically active TIMP following fractionation of the conditioned media. During the first 24 h TGF-.beta. had little apparent effect on TIMP activity in the medium although the TIMP mRNA transcript was induced 1.3-1.4-fold. Subsequently, TIMP levels were increased 1.7-fold relative to control cells on day 4. This was accompanied by a 2.4-fold increase in TIMP mRNA, indicating that the regulation of TIMP mRNA and protein levels may be a secondary response to TGF-.beta.. In comparison, the synthesis of the Mr 48,000 PAI-1, analyzed by [35S] methionine labeling and immunoprecipitation, was elevated >10-fold by TGF-.beta. at all time points with the highest levels occurring at day 2. Thus, the effects of TGF-.beta. on procollagenase, 72-kDa progelatinase, TIMP, and PAI-1 were selective and showed temporal differences. The suppression of procollagenase and the stimulation of TIMP and PAI-1 synthesis are consistent with the promotion of connective tissue matrix formation by TGF-.beta.; whereas the stimulation of 72-kDa progelatinase synthesis may reflect an important function for this neutral metalloendoproteinase in the removal of abnormal or unfolded collagen from wound sites and from newly synthesized extracellular matrix.