INDUCTION OF COLLAGENASE AND STROMELYSIN GENE-EXPRESSION BY MECHANICAL INJURY IN A VASCULAR SMOOTH MUSCLE-DERIVED CELL-LINE

We describe a novel system for studying the production of matrix metal loproteinases types I and III (collagenase and stromelysin) by a vascu lar smooth muscle cell line (Rb-1 cells) in response to mechanical inj ury. Highly confluent Rb-1 cells are disrupted by passing a plastic co mb around the plate to clear a series of circumferential paths, which are bordered by two ridges of displaced cells. Over the next 24 hours, cells migrate into the cleared areas. Northern blot analysis demonstr ates the accumulation of mRNAs for collagenase and stromelysin during this process, although they are undetectable prior to injury. Cotreatm ent with all-trans-retinoic acid (10(-6) M) markedly decreases the lev el of mRNAs induced by injury, whereas dexamethasone (10(-7) M) Causes only a slight reduction. In situ hybridization studies for stromelysi n mRNA and immunohistochemical staining for collagenase protein on pla tes of injured cells showed the highest levels of stromelysin mRNA in cells in the ridges left by the injury; lower levels were observed in some cells migrating into the clear region. The same pattern of expres sion was observed when cells were stained with antiserum to collagenas e protein. Nuclear run-on assays demonstrated increases in transcripti on of stromelysin and collagenase genes following injury. Transient tr ansfection of cells with a vector containing the luciferase gene drive n by a wild-type promoter comprising 1.8 kb of the 5'-flanking region of the rabbit collagenase gene showed increased activity associated wi th injury. We conclude that: (1) mechanical injury is associated with induction of mRNAs for the metalloproteinases collagenase and stromely sin, (2) retinoic acid effectively antagonizes this responses, and (3) the increase in steady-state mRNA levels is, at least in part, transc riptionally mediated. Thus our data suggest a role for mechanical forc es in initiating the changes in gene expression in vascular smooth mus cle cells following arterial injury in vivo. (C) 1993 Wiley-Liss, Inc.