Regulation of interleukin-6 gene expression by pro-inflammatory cytokines in a colon cancer cell line

The two carcinoma cell lines HeLa and HTM-29 show different behaviour in terms of interleukin-6 (IL-6) production. Analyses of secreted IL-6 by ELISA and of IL-6 mRNA by reverse transcription-PCR revealed that, whereas HeLa cells produced high levels of IL-6 in response to tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta, the HTM-29 cell line failed to produce both IL-6 protein and mRNA. Nevertheless, the transcription factors nuclear factor-kappa B (NF-kappa B) and NF-IL6, the main factors involved in IL-6 gene transcriptional activation by cytokines, were activated in both cell lines after treatment with TNF-alpha or IL-1 beta. In order to verify that the lack of IL-6 expression in HTM-29 cells was not due to an endogenous IL-6 gene deficiency or to IL-6 mRNA instability, we carried out transient transfection assays with an IL-6 promoter-reporter construct. Strong activation of the IL-6 promoter by cytokines could be observed in HeLa cells, whereas no induction could be detected in cytokine-treated HTM-29 cells. These cytokines induced a very strong stimulation of NF-kappa B-mediated transcription in HeLa cells transfected with a kappa B luceriferase reporter construct, whereas no induction could be detected in cytokine-stimulated HTM-29 cells. Thus IL-6 promoter repression in HTM-29 cells probably results from a failure of cytokine-activated NF-kappa B to exert its transactivating activities. Western blotting experiments demonstrated that the lack of NF-kappa-B-mediated transcription was not due to increased expression of I kappa B (inhibitor of NF-kappa B) proteins in HTM-29 cells. Co-transfection experiments with the kappa B Luc reporter construct and the CBP [CREB (cAMP response element binding protein) binding protein] expression vector showed that the impairment in NF-kappa B-dependent transcription did not result from a deficiency in the co-activator CBP. Interestingly,both NF-kappa B-mediated transcription and IL-6 promoter activation could be restored in HTM-29 cells by transfection with RelA. Furthermore, CBP could have a significant synergistic effect on exogenous RelA-mediated transcription. Since sequencing of the endogenous relA gene did not reveal any mutation, it is likely that repression of NF-kappa B-mediated transcription results from negative cross-talk between NF-kappa B and another nuclear factor specifically expressed or regulated by TNF-alpha in HTM-29 cells.