Modulation of UVB-induced and basal cyclooxygenase-2 (COX-2) expression by apigenin in mouse keratinocytes: Role of USF transcription factors

Apigenin is a bioflavonoid with chemopreventive activity against UV‐ or chemically‐induced mouse skin tumors. To further explore the mechanism of apigenin's chemopreventive activity, we determined whether apigenin inhibited UVB‐mediated induction of cyclooxygenase‐2 (COX‐2) expression in mouse and human keratinocytes. Apigenin suppressed the UVB‐induced increase in COX‐2 protein and mRNA in mouse and human keratinocyte cell lines. UVB radiation of keratinocytes transfected with a mouse COX‐2 promoter/luciferase reporter plasmid resulted in a threefold increase in transcription from the promoter, and apigenin inhibited the UV‐induced promoter activity at doses of 5–50 µM. Transient transfections with COX‐2 promoter deletion constructs and COX‐2 promoter constructs containing mutations in specific enhancer elements indicated that the effects of UVB required intact Ebox and ATF/CRE response elements. Electrophoretic mobility shift assays with supershifting antibodies were used to identify USF‐1, USF‐2, and CREB as proteins binding to the ATF/CRE‐Ebox responsive element of the COX‐2 promoter. Keratinocytes co‐transfected with the COX‐2 luciferase reporter and a USF‐2 expression vector, alone or in combination with a USF‐1 expression vector, exhibited enhanced promoter activity in both UVB‐irradiated and nonirradiated cultures. However, COX‐2 promoter activity was inhibited in keratinocytes co‐transfected with USF‐1 alone. Finally, we present data showing that the suppressive effect of apigenin on COX‐2 expression could be reversed by co‐expression of USF‐1 and USF‐2. These results suggest that one pathway by which apigenin inhibits COX‐2 expression is through modulation of USF transcriptional activity. © 2006 Wiley‐Liss, Inc.