Interleukin-6 production by human bladder tumor cell lines is up-regulated by bacillus Calmette-Guérin through nuclear factor-kappaB and Ap-1 via an immediate early pathway.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Division of Urology and Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Fang-Hong
FH
Chen
Scott A
SA
Crist
Guang-Jian
GJ
Zhang
Yoshiki
Y
Iwamoto
William A
WA
See
D001500:BCG Vaccine / Q000494:pharmacology*; D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000276:immunology*; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D017781:Genes, Immediate-Early / Q000187:drug effects*; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured / Q000276:immunology*; D015854:Up-Regulation / Q000187:drug effects; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000276:immunology*
D001500:BCG Vaccine; D015850:Interleukin-6; D016328:NF-kappa B; D012333:RNA, Messenger; D018808:Transcription Factor AP-1