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 fibrogenesis, where they are thought to derive from the activation of hepatic stellate cells. Cultured human MF, grown from liver explants, retain most of the in vivo characteristics of liver MF but are in limited supply. A continuous MF cell line would therefore be valuable in studying human liver fibrogenesis. For this purpose, we sought to immortalize human 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 yielded an actively growing cell line, designated GREF-X, which did not express large T antigen. Nevertheless, this cell line has been passaged repeatedly for almost 1 year and is thus likely immortalized. The morphology of GREF-X resembles that of primary liver MF. These cells have a doubling time of approximately 72 hours and are density-inhibited, and 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 not transformed. They stain positively for MF markers, such as smooth muscle a-actin and vimentin; express collagens type I, IV, V, and VI, fibronectin, and laminin; and secrete matrix-metalloproteinase-2. In addition, GREF-X cells are able to take up and esterify [ 3 H]retinol, suggesting that they actually derive from hepatic stellate cells. Finally, these cells respond to transforming growth factor-/31, a major mediator of liver fibrogenesis, by increasing secretion of fibronectin and plasminogen activator-inhibitor type 1. Transient transfection experiments showed that plasminogen activator-inhibitor type 1 regulation, by transforming growth factor-/31, was transcriptional. We believe, therefore, that GREF-X would be a useful tool for studying the pathophysiology and pharmacology of liver fibrogenesis.