C/EBPbeta and its binding element are required for NFkappaB induced COX2 expression  following hypertonic stress

doi:10.1074/jbc.M411134200

C/EBPbeta and its binding element are required for NFkappaB induced COX2 expression following hypertonic stress

Jing Chen, Min Zhao, Reena Rao, Hiroyasu Inoue, and Chuan-Ming Hao

Department of Medicine and Nephrology, Vanderbilt University, Nashville, TN 37232

Corresponding Author: chuanming.hao{at}vanderbilt.edu

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. Since 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 determined by immunoblot in RMICs. The tonicity induced COX2 expression was suppressed by mutant I $kappa$ B that blocks NF $kappa$ B activation, demonstrating 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 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 co-immunoprecipitation. 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.

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