C/EBPβ and Its Binding Element Are Required for NFκB-induced COX2 Expression Following Hypertonic Stress
Journal of Biological Chemistry
NFB plays a critical role mediating COX2 expression in renal medullary interstitial cells (RMICs). The trans-activating ability of NFB can be modified by another nuclear factor C/EBP␤ that can physically bind to NFB and regulate its activity. Because the COX2 promoter also contains a C/EBP␤ site adjacent to the NFB site, the present study examined whether these two transcription factors cooperate to induce COX2 expression following hypertonic stress. Hypertonicity markedly induced COX2
... n in cultured medullary interstitial cells by immunoblot analysis. The tonicity-induced COX2 expression was suppressed by mutant IB (IBm) that blocks NFB activation, demonstrating that tonicity-induced COX2 expression depends on NFB activation. However, mutation of the NFB site in the COX2 promoter failed to abolish tonicity-induced COX2 reporter activity. IB kinase-1 (IKK1) significantly induced COX2-luciferase activity by 2.3-fold (n ؍ 10, p < 0.01); mutation of the NFB site also failed to abolish IKK1-stimulated COX2 reporter activity (86 ؎ 3.1% of wild type, p > 0.05, n ؍ 4). Interestingly, mutation of the C/EBP␤ site of the COX2 gene significantly reduced both IKK1 and hypertonicityinduced COX2 reporter activity (p < 0.01). To further examine the potential role of C/EBP␤ in tonicity-induced COX2 expression, a dominant negative C/EBP␤-p20 was transduced into RMICs. C/EBP␤-p20 markedly suppressed hypertonic (550 mOsm) induction of COX2 (immunoblot) to a similar extent as IBm. No additional suppression was observed when both NFB and C/EBP␤ were simultaneously blocked by IBm and C/EBP␤-p20. Interestingly, IKK-induced COX2 expression was not only blocked by IBm, but also completely abolished by C/EBP␤-p20. Further studies demonstrated physical association of C/EBP␤ to NFB p65 by coimmunoprecipitation. Importantly, this interaction between C/EBP␤ and NFB was greatly enhanced following hypertonic stress. These studies indicate C/EBP␤ is required for the transcriptional activation of COX2 by NFB, suggesting a dominant role for the C/EBP␤ pathway in regulating induction of RMIC COX2 by hypertonicity.