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.
Buck
M
M
Department of Medicine, Veterans Affairs Medical Center, San Diego, California 92161, USA.
Kim
D J
DJ
Houglum
K
K
Hassanein
T
T
Chojkier
M
M
D000199:Actins / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics / Q000502:physiology*; D016372:RNA, Antisense; D051381:Rats; D017207:Rats, Sprague-Dawley; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D000199:Actins; D020598:Proto-Oncogene Proteins c-myb; D016372:RNA, Antisense