UV irradiation activates JNK and increases alphaI(I) collagen gene expression in rat hepatic stellate cells.
Hepatic stellate cells (HSCs) become activated into myofibroblast-like cells during the early stages of hepatic injury associated with fibrogenesis. The subsequent dysregulation of alphaI(I) collagen gene expression is a central pathogenetic step during the development of cirrhosis. Our recent study in rat HSCs (Davis, B. H., Chen, A., and Beno, D. (1996) J. Biol. Chem. 271, 11039-11042) found that ERK1,2 activation might be required for maximal alphaI(I) collagen gene expression. However, the role of the parallel JNK cascade in regulating alphaI(I) collagen gene expression was unknown. In this study, we initially found that UV irradiation of HSCs activated JNK but not ERK1,2. Furthermore, UV irradiation increased endogenous alpha I(I) collagen mRNA abundance and stimulated alpha I(I) collagen gene transcription in HSCs. The effect of the activation of JNK and Jun on alpha I(I) collagen gene expression was further evaluated via transfection of chloramphenicol acetyltransferase reporter plasmids with various sizes of truncated 5' upstream promoter sequence (UPS) of the alphaI(I) collagen gene. This revealed that dominant negative transcription factor JUN suppressed alpha I(I) collagen gene transcription in HSCs maintained in media with 20% serum and constitutively activated JUN increased alphaI(I) collagen gene transcription in HSCs cultured in media with 0.4% serum. UV activated JNK utilized a distal GC box in the 5'-UPS of the collagen gene to regulate gene transcription. This observation was confirmed by site-directed mutagenesis. In co-transfection experiments, the col-chloramphenicol acetyltransferase reporter with a mutagenized GC box was not suppressed by dn-JUN and was not stimulated by activated JUN or by UV irradiation. Southwestern blotting analyses and gel shift assays with basic transcription element-binding protein antiserum suggested that the GC box was bound by basic transcription element-binding protein, a recently described DNA-binding protein. In conclusion, the current study combined with our previous report suggests that ERK1,2 and JNK cascades regulate alphaI(I) collagen expression in HSCs through different regions of the 5'-UPS of the gene. The distal GC box in the 5'-UPS of the alphaI(I) collagen gene may play a central role in receiving extracellular signals through the JNK pathway.
Chen
A
A
Gastroenterology Section, Department of Medicine, University of Chicago Medical Center, Chicago, Illinois 60637, USA.
Davis
B H
BH
D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003094:Collagen / Q000235:genetics*; D017931:DNA Primers; D004789:Enzyme Activation; D048031:JNK Mitogen-Activated Protein Kinases; D008099:Liver / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects*; D008297:Male; D020928:Mitogen-Activated Protein Kinases; D051381:Rats; D017207:Rats, Sprague-Dawley; D014466:Ultraviolet Rays; D015854:Up-Regulation
D017931:DNA Primers; D003094:Collagen; D015500:Chloramphenicol O-Acetyltransferase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases