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. 2000 Aug 1;349(Pt 3):765–773. doi: 10.1042/bj3490765

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

S Legrand-Poels 1, S Schoonbroodt 1, J Piette 1
PMCID: PMC1221203  PMID: 10903137

Abstract

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-1beta, the HTM-29 cell line failed to produce both IL-6 protein and mRNA. Nevertheless, the transcription factors nuclear factor-kappaB (NF-kappaB) 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-1beta. 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-kappaB-mediated transcription in HeLa cells transfected with a kappaB 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-kappaB to exert its transactivating activities. Western blotting experiments demonstrated that the lack of NF-kappaB-mediated transcription was not due to increased expression of IkappaB (inhibitor of NF-kappaB) proteins in HTM-29 cells. Co-transfection experiments with the kappaB Luc reporter construct and the CBP [CREB (cAMP response element binding protein) binding protein] expression vector showed that the impairment in NF-kappaB-dependent transcription did not result from a deficiency in the co-activator CBP. Interestingly, both NF-kappaB-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-kappaB-mediated transcription results from negative cross-talk between NF-kappaB and another nuclear factor specifically expressed or regulated by TNF-alpha in HTM-29 cells.

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Selected References

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