Co-operative functions between nuclear factors NFκB and CCAT/enhancer-binding protein-β (C/EBP-β) regulate the IL-6 promoter in autocrine human prostate cancer cells†

BACKGROUND IL-6 is a growth and survival factor for prostate cancer cells through autocrine pathways. Here, we have systematically examined the transcriptional regulation mechanisms of IL-6 in autocrine prostate cancer cells. METHODS RT-PCR and immunohistochemical staining were used to determine IL-6 production in the cells. Serial mutant IL-6 promoter luciferase reporters were generated and their transcriptional activities were examined. The transcription factors involved in IL-6 regulation were identified with super-shift EMSA. Overexpression of NFκB p65 and C/EBP-β, and blockade of NFκB with IκBα or CAPE were performed to demonstrate the cooperation between NFκB p65 and C/EBP-β in activation of IL-6. RESULTS Transcription factor regulatory sites IL6-NFκB, IL6-C/EBP, IL6-CREB, and IL6-AP1, are responsive to constitutively activated IL-6 production in autocrine prostate cancer cell lines. Among these sites, IL6-AP1 and IL6-C/EBP appear most important, while IL6-NFκB shows the least effect for IL-6 promoter activity as determined by mutant IL-6 promoter luciferase reporter assay. Nevertheless, nuclear factor NFκB is activated and required. Such activation is minimally dependent upon the IL6-NFκB site, occurring through cooperation with other transcription factors that bind the IL-6 promoter. Cooperation between NFκB p65 and C/EBP-β did not require a functional IL6-NFκB binding site. CONCLUSIONS These data support a unique role for NFκB p65 as the primary trigger in autocrine production of IL-6 in prostate cancer cells. Furthermore, we describe a novel transcriptional activation mechanism for NFκB that is independent of its regulatory binding site, occurring through cooperation with other transcription factors that facilitate the neighboring regulatory site. © 2004 Wiley-Liss, Inc.