EXPRESSION OF 72-KDA GELATINASE AND TIMP-2 IN EARLY AND LATE PASSAGE HUMAN FIBROBLASTS

The expression and regulation of 72-kDa gelatinase and TIMP-2 were exa mined in cultures of early and late passage human fibroblasts. In cont rast to collagenase expression, which was low in early passage cells a nd highly expressed in late passage fibroblasts, 72-kDa gelatinase mRN A expression was enhanced only slightly in late passage cultures and 7 2-kDa gelatinase protein expression was similar in early and late pass age cultures. In contrast to published reports of decreased TIMP-1 exp ression, TIMP-2 mRNA and protein were increased in late passage cells. Exposure to Il-1 alpha increased the steady-state level of 72-kDa gel atinase mRNA by 3X in early passage cells but had no effect on late pa ssage cells. Although Il-1 alpha had no significant effect on TIMP-2 m RNA or on expression of 72-kDa gelatinase, in either early or late pas sage cells, Il-1 alpha increased the level of a TIMP-2-72-kDa gelatina se complex. Using monoclonal antibodies to TIMP-2 and to 72-kDa gelati nase we detected two forms of TIMP-2. One form was complexed to 72-kDa gelatinase and migrated with an apparent molecular weight of 72 kDa a nd the other migrated at the expected molecular weight of 21 kDa. Auto radiography in conjunction with Western analysis confirmed that in the late passage cell medium the newly synthesized 72-kDa gelatinase TIMP -2 complex was increased even though the expression of 72-kDa gelatina se did not change. The present results establish that the regulation o f 72-kDa gelatinase and TIMP-2 in early and late passage cultures of h uman fibroblasts are different from collagenase and TIMP-1 regulation. Further, they establish that in late passage cultures the activity of 72-kDa gelatinase may be regulated through the formation of a denatur ation-resistant complex. (C) 1994 Academic Press, Inc.