Interleukin-6 Production by Human Bladder Tumor Cell Lines is Up-Regulated by Bacillus Calmette-Guerin Through Nuclear Factor-κB and Ap-1 Via an Immediate Early Pathway

Abstract

The expression of interleukin-6 (IL-6) by normal and malignant urothelium in response to bacillus Calmette-Guerin (BCG) may have direct and indirect effects on the antitumor activity of BCG. We evaluated the molecular signaling pathway through which BCG induces IL-6 expression in human transitional cell carcinoma lines. We evaluated IL-6 messenger RNA and protein expression by human transitional carcinoma cell lines in response to BCG. Pharmacological inhibition of protein synthesis was used to determine if BCG mediated IL-6 induction occurred via an immediate-early or delayed pathway. We used 5' deletion analysis and site directed mutagenesis to identify BCG responsive regions in the human IL-6 promoter. Electrophoretic mobility shift assays were done to assess nuclear translocation of the putative signaling proteins AP-1 and nuclear factor-kappaB (NF-kappaB) in response to BCG. BCG increased IL-6 messenger RNA and protein in a time and dose dependent manner. IL-6 induction by BCG occurred via an immediate-early response. Promoter analysis identified 2 areas in the -1,200 to 14, 5' region of the IL-6 gene, which when deleted were associated with significant losses of absolute or BCG responsive activity. Site specific mutation of putative AP-1 or NF-kappaB elements associated with each region demonstrated that these elements were necessary but not sufficient for BCG induced IL-6 transcription. Gel mobility shift assays showed that AP-1 and NF-kappaB were induced in response to BCG exposure. Our results show that BCG induced IL-6 expression by human transitional cell neoplasms occurs as an immediate-early gene pathway that requires NF-kappaB and AP-1.