Independent regulation of collagenase, 72-kDa progelatinase, and metalloendoproteinase inhibitor expression in human fibroblasts by transforming growth factor-beta.
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 (approximately 50% on day 1, approximately 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 greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)
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C M
CM
Medical Research Council Group in Peridontal Physiology, University of Toronto, Ontario, Canada.
Wrana
J L
JL
Sodek
J
J
D002352:Carrier Proteins / Q000096:biosynthesis; D002478:Cells, Cultured; D017364:Collagenases; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000378:metabolism*; D003012:Microbial Collagenase / Q000096:biosynthesis* / Q000378:metabolism; D008970:Molecular Weight; D012333:RNA, Messenger / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases; D015291:Transforming Growth Factors / Q000494:pharmacology*
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; C033135:gelatin binding protein; D015291:Transforming Growth Factors; D017364:Collagenases; D008666:Metalloendopeptidases; C022440:procollagenase; D020778:Matrix Metalloproteinase 2; D003012:Microbial Collagenase