EXPRESSION OF 72-KDA GELATINASE (MMP-2) COLLAGENASE (MMP-1) AND TISSUE METALLOPROTEINASE INHIBITOR (TIMP) IN PRIMARY PIG SKIN FIBROBLAST-CULTURES DERIVED FROM RADIATION-INDUCED SKIN FIBROSIS

In addition to producing matrix degradation for normal tissue remodeli ng and repair, matrix metalloproteinases (MMPs) are also involved in v arious pathologic processes. MMPs and the tissue inhibitor of MMPs (TI MP) were investigated in primary cultures of pig fibroblasts from radi ation-induced dermal fibrosis and compared to normal dermal fibroblast s. The free gelatinolytic, collagenolytic, and caseinolytic activities secreted into the culture medium were evaluated against specific H-3 denatured collagen type I, native helical collagen, and casein alpha, respectively. The 72- and 68-kilodalton (kDa) forms of type IV collage nase were investigated by protease zymography and quantified by semiau tomated image analysis. Transcription of the interstitial collagenase (MMP-1) and TIMP genes was studied by Northern hybridization analysis. Results revealed that in fibrotic fibroblasts, the amount of MMP-1 mR NA was greatly reduced to undetectable levels whereas the amount of TI MP mRNA was increased fourfold compared to controls. Functional assays using specific H-3 substrates demonstrated an overall decrease in fre e MMP activities. Concomitantly, catheptic collagenolytic activity dec reased in fibrotic fibroblast extracts compared to controls. These res ults indicate that in addition to accumulating large amounts of collag en, proteoglycans, and fibronectin, pig fibroblasts from radiation-ind uced dermal fibrosis also promote connective tissue matrix formation b y repressing MMP-1 and stimulating TIMP expression at the transcriptio nal level, and by reducing overall free MMP and catheptic collagenolyt ic activities at the post-transcriptional level. In contrast, enzymogr aphy assays and automated image analysis demonstrated no significant c hange in the 72-kDa type IV collagenase activity of fibrotic pig skin fibroblasts. This opposite regulation of 72-kDa collagenase type IV to that of MMP-1 seems to indicate that it has a specific role in remode ling the extracellular matrix during wound healing, fibrogenesis, and angiogenesis.