C/EBPβ and its binding element are required for NFκB-induced COX2 expression following hypertonic stress

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