c-Myb modulates transcription of the alpha-smooth muscle actin gene in activated hepatic stellate cells.

Expression of alpha-smooth muscle actin (alpha-SMA) defines the phenotype of activated (myofibroblastic) hepatic stellate cells. These cells, but not quiescent stellate cells, have a high level of alpha-SMA and c-Myb expression, as well as increased c-Myb-binding activities to the proximal alpha-SMA E box. Therefore, we analyzed the role of c-Myb in alpha-SMA transcription and stellate cell activation. Activated primary rat stellate cells displayed a high expression of the -724 and -271 alpha-SMA/luciferase (LUC) chimeric genes, which contain c-Myb binding sites (-223/-216 bp). Alpha-SMA/LUC minigenes with mutation (-219/-217 bp), truncation (-224 bp), or deletion (-191 bp) of the c-Myb binding site were not efficiently transcribed. Transfection of wild-type c-Myb into quiescent stellate cells, which do not express endogenous c-Myb, induced a approximately 10-fold stimulation of -724 alpha-SMA/LUC expression. Conversely, expression of either a dominant-negative c-Myb basic domain mutant (Cys(43) --> Asp) or a c-Myb antisense RNA blocked transcription from the -724 alpha-SMA/LUC or -271 alpha-SMA/LUC in activated cells. Moreover, transfection of c-myb antisense, but not sense, RNA inhibited both expression of the endogenous alpha-SMA gene and stellate cell activation, whereas transfection of c-myb stimulated alpha-SMA expression in quiescent stellate cells. These findings suggest that c-Myb modulates the activation of stellate cells and that integrity of the redox sensor Cys(43) in c-Myb is required for this effect.