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 examined in cultures of early and late passage human fibroblasts. In contrast to collagenase expression, which was low in early passage cells and highly expressed in late passage fibroblasts, 72-kDa gelatinase mRNA expression was enhanced only slightly in late passage cultures and 72-kDa gelatinase protein expression was similar in early and late passage cultures. In contrast to published reports of decreased TIMP-1 expression, TIMP-2 mRNA and protein were increased in late passage cells. Exposure to Il-1 alpha increased the steady-state level of 72-kDa gelatinase mRNA by 3x in early passage cells but had no effect on late passage cells. Although Il-1 alpha had no significant effect on TIMP-2 mRNA or on expression of 72-kDa gelatinase, in either early or late passage cells, Il-1 alpha increased the level of a TIMP-2-72-kDa gelatinase complex. Using monoclonal antibodies to TIMP-2 and to 72-kDa gelatinase 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 and the other migrated at the expected molecular weight of 21 kDa. Autoradiography 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 gelatinase did not change. The present results establish that the regulation of 72-kDa gelatinase and TIMP-2 in early and late passage cultures of human 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 denaturation-resistant complex.
Department of Biological Sciences, State University of New York at Albany 12222.
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Zeng
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Millis
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D016922:Cellular Senescence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018093:Gelatinases / Q000096:biosynthesis* / Q000302:isolation & purification; D015870:Gene Expression; D005786:Gene Expression Regulation; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D019716:Tissue Inhibitor of Metalloproteinase-2
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011506:Proteins; D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases