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