UVB irradiation-induced activator protein-1 activation correlates with increased c-fos gene expression in a human keratinocyte cell line.
The effects of UVB irradiation on transcription factor activator protein-1 (AP-1) DNA binding and AP-1 transactivation were studied in a human keratinocyte cell line, HaCaT. UVB-induced AP-1 binding to a consensus AP-1 binding site was observed by gel shift assays with maximum stimulation at 12 h after UVB irradiation. A promoter region of the human collagenase-1 gene containing the same AP-1 binding sequence linked to a luciferase reporter gene was stably transfected into HaCaT cells. UVB irradiation significantly increased luciferase activity in these stably transfected cells, with maximum activity observed at 24 h after UVB irradiation. c-Fos and Jun D were identified by antibody clearing assays as the main components of the bound AP-1 complexes. Inhibition of transcription with actinomycin D and inhibition of protein synthesis with cycloheximide significantly abrogated the effect of UVB on AP-1 DNA binding, indicating that transcription and translation were required for AP-1 activation. Northern and Western analyses revealed a correlation between increased AP-1 activity and accumulation of c-fos mRNA and c-Fos protein after UVB irradiation. UVB irradiation increased c-fos transcription in HaCaT cells stably transfected with a plasmid containing the human c-fos promoter driving a luciferase reporter gene. These results suggest that increased c-fos expression may play an important role in UVB-induced AP-1 activation in HaCaT cells.
Chen
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W
Department of Radiation Oncology, Arizona Cancer Center, College of Medicine, University of Arizona, Tucson, Arizona 85724, USA.
Borchers
A H
AH
Dong
Z
Z
Powell
M B
MB
Bowden
G T
GT
D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004307:Dose-Response Relationship, Radiation; D005786:Gene Expression Regulation / Q000528:radiation effects*; D017930:Genes, Reporter; D016762:Genes, fos / Q000528:radiation effects*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism / Q000528:radiation effects*; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000378:metabolism / Q000528:radiation effects*; D014158:Transcription, Genetic / Q000187:drug effects / Q000528:radiation effects; D015533:Transcriptional Activation / Q000528:radiation effects; D014466:Ultraviolet Rays
D016760:Proto-Oncogene Proteins c-fos; D018808:Transcription Factor AP-1; D003609:Dactinomycin; D003513:Cycloheximide; D008156:Luciferases