Modulation of the expression of the invasion-suppressor CRMP-1 by cyclooxygenase-2 inhibition via reciprocal regulation of Sp1 and C/EBP alpha

摘要

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/EBP alpha sites. Site-directed mutagenesis and deletion analysis revealed that the two C/EBP alpha 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/EBP alpha site, which overlaps with a Sp1 site. Overexpression of Sp1 decreased CRMP-1 promoter activity and protein expression, whereas overexpression of C/EBP alpha produced the opposite effect. Chromatin immunoprecipitation assays confirmed that Sp1 and C/EBP alpha compete for binding at the overlapping C/EBP alpha 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/EBP alpha and Sp1. COX-2 inhibitors increase CRMP-1 expression by inhibiting Sp1-DNA complex formation and enhancing DNA binding of C/EBP alpha at the promoter.