CHARACTERIZATION OF A NEW HUMAN LIVER MYOFIBROBLAST CELL-LINE - TRANSCRIPTIONAL REGULATION OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-I BY TRANSFORMING-GROWTH-FACTOR BETA-1

Myofibroblasts (MF) are a major effector cell type in liver fibrogenes is, where they are thought to derive from the activation of hepatic st ellate cells. Cultured human MF, grown from liver explants, retain mos t of the in vivo characteristics of liver MF but are in limited supply . A continuous MF cell line would therefore be valuable in studying hu man liver fibrogenesis. For this purpose, we sought to immortalize hum an liver MF with polyoma virus large T antigen. MF were obtained from explants of human liver and transfected with a plasmid containing the coding sequence of polyoma virus large T antigen. This procedure yield ed an actively growing cell line, designated GREF-X, which did not exp ress large T antigen. Nevertheless, this cell line has been passaged r epeatedly for almost 1 year and is thus likely immortalized. The morph ology of GREF-X resembles that of primary liver MF. These cells have a doubling time of approximately 72 hours and are density-inhibited, an d their growth is serum-dependent. Moreover, GREF-X cells do not grow in soft agar or induce tumors in nude mice, suggesting that they are n ot transformed. They stain positively for MF markers, such as smooth m uscle a-actin and vimentin; express collagens type I, IV, V, and VI, f ibronectin, and laminin; and secrete matrix-metalloproteinase-2. In ad dition, GREF-X cells are able to take up and esterify [H-3]retinol, su ggesting that they actually derive from hepatic stellate cells. Finall y, these cells respond to transforming growth factor-beta 1, a major m ediator of liver fibrogenesis, by increasing secretion of fibronectin and plasminogen activator-inhibitor type 1. Transient transfection exp eriments showed that plasminogen activator-inhibitor type 1 regulation , by transforming growth factor-beta 1, was transcriptional. We believ e, therefore, that GREF-X would be a useful tool for studying the path ophysiology and pharmacology of liver fibrogenesis.