Identification And Functional Analysis Of Nuclear Proteins Binding To The Repressor Element In The Mouse Cyclooxygenase-2 Promoter

Cyclooxygenase-2 (COX-2) is a key enzyme that catalyzes theconversion of arachidonic acid to prostaglandins. It plays an importantrole in inflammation, carcinogenesis, and the development of diabetes.Although COX-2 gene regulation is deeply and widely investigated, someimportant mechanism underlying its transcriptional regulation is stillunknown. In this study, we identified a novel repressor element locatedbetween nucleotides -655/-632 of the mouse COX-2 promoter throughdeletion and mutation analysis in the pancreaticβ-cells RINm5F. Therewere three kinds of nuclear proteins or protein complexes binding to thiselement, as three shifted bands appeared in electrophoretic mobility shiftassays (EMSA). These proteins were purified on the basis of theirspecific binding to the inhibitory element, and then subjected to massspectrometry analysis. One of the proteins was identified as anon-POU-domain-containing, octamer-binding protein (Nono). Nonoparticipated in the formation of two slower shifted bands in EMSA, asunlabeled octamer consensus probe attenuated the formation of the slowest and intermediate shifted bands, but had no effect on the fastestshifted band. Overexpression of Nono in RINm5F cells significantlyinhibited the wild type COX-2 promoter activity but had no effect on themutant type promoter in which the element was mutated. These findingsidentified an inhibitory element located in the nucleotides from-655 to-632 of the COX-2 promoter for the first time. Nono bound to thiselement and inhibited the COX-2 promoter activity in RINm5F cells. Ourresults also suggested that the element was cell-specific, as mutation ofthe element did not alter COX-2 promoter activity in other cells. Ourstudy provides important information on COX-2 regulation and mayprovide clues for the clinical treatment of diseases where COX-2 ishighly expressed.