Modulation of the expression of the invasion-suppressor CRMP-1 by cyclooxygenase-2 inhibition via reciprocal regulation of Sp1 and C/EBPalpha.
Collapsin response mediator protein-1 (CRMP-1) controls neural development and axonal growth but also acts as a cancer invasion suppressor. In this study, we investigated the transcriptional regulation of CRMP-1 expression. Using a serial deletion strategy, we identified a basal promoter region between nucleotides -100 and -180 in the 5' flanking region of CRMP-1 (nucleotides -1,920 to +50) that contains multiple putative Sp1 and C/EBPalpha sites. Site-directed mutagenesis and deletion analysis revealed that the two C/EBPalpha sites, from nucleotides -122 to -133 and from nucleotides -101 to -113, are the most important regulatory elements. Gel-shift and antibody supershift assays showed that Sp1 protein was also present at this C/EBPalpha site, which overlaps with a Sp1 site. Overexpression of Sp1 decreased CRMP-1 promoter activity and protein expression, whereas overexpression of C/EBPalpha produced the opposite effect. Chromatin immunoprecipitation assays confirmed that Sp1 and C/EBPalpha compete for binding at the overlapping C/EBPalpha and Sp1 sites and reciprocally regulate CRMP-1 expression. Overexpression of cyclooxygenase-2 (COX-2) decreased CRMP-1 mRNA and protein expression. Conversely, the COX-2 inhibitor, celecoxib, induced a dose-dependent increase in CRMP-1 expression. COX-2 inhibition also decreased Sp1-DNA complex formation and inhibited cell invasion. We conclude that transcription of the invasion suppressor, CRMP-1, is reciprocally regulated at the promoter region by C/EBPalpha and Sp1. COX-2 inhibitors increase CRMP-1 expression by inhibiting Sp1-DNA complex formation and enhancing DNA binding of C/EBPalpha at the promoter.
Wu
Cheng-Chung
CC
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Lin
Jau-Chen
JC
Yang
Shuenn-Chen
SC
Lin
Chiu-Wen
CW
Chen
Jeremy J W
JJ
Shih
Jin-Yuan
JY
Hong
Tse-Ming
TM
Yang
Pan-Chyr
PC
D001483:Base Sequence; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D015854:Up-Regulation / Q000235:genetics; D000098855:Collapsin Response Mediator Protein 1
D022763:CCAAT-Enhancer-Binding Protein-alpha; D051546:Cyclooxygenase 2; D004273:DNA, Neoplasm; D009419:Nerve Tissue Proteins; D016329:Sp1 Transcription Factor; C102924:CRMP1 protein, human; D000098855:Collapsin Response Mediator Protein 1; C496540:PTGS2 protein, human