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-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.

[1]  深山 正久.  p300 Gene Alterations in Colorectal and Gastric Carcinomas , 1997 .

[2]  W. Fiers,et al.  Recombination signal sequence binding protein Jkappa is constitutively bound to the NF-kappaB site of the interleukin-6 promoter and acts as a negative regulatory factor , 1997, Molecular and cellular biology.

[3]  T. Libermann,et al.  Activation of interleukin-6 gene expression through the NF-kappa B transcription factor , 1990 .

[4]  M. Merville,et al.  Regulation of NF-κB activity by IκB-related proteins in adenocarcinoma cells , 1999, Oncogene.

[5]  S. Akira,et al.  Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[6]  L. Baldini,et al.  Identification of a tumor-associated mutant form of the NF-κB RelA gene with reduced DNA-binding and transactivating activities , 1997, Oncogene.

[7]  M. Merville,et al.  CBP and histone deacetylase inhibition enhance the transactivation potential of the HOXB7 homeodomain-containing protein , 1999, Oncogene.

[8]  V. Bours,et al.  A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site , 1992, Molecular and cellular biology.

[9]  M. Billah,et al.  Interleukin-10 inhibits interleukin-8 production in human neutrophils. , 1994, Blood.

[10]  N. Perkins,et al.  Transcriptional Cross Talk between NF-κB and p53 , 1999, Molecular and Cellular Biology.

[11]  D. Thomas,et al.  Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B , 1995, Molecular and cellular biology.

[12]  C. Ito,et al.  Three NF-χB sites in the IχB-α promoter are required for induction of gene expression by TNFα , 1994 .

[13]  S. Akira,et al.  Genomic structure of the murine IL-6 gene. High degree conservation of potential regulatory sequences between mouse and human. , 1988, Journal of immunology.

[14]  N. Mackman,et al.  Role of cyclic AMP response element-binding protein in cyclic AMP inhibition of NF-kappaB-mediated transcription. , 1997, Journal of immunology.

[15]  T. Okamoto,et al.  Identification of a Novel Inhibitor of Nuclear Factor-κB, RelA-associated Inhibitor* , 1999, The Journal of Biological Chemistry.

[16]  M J May,et al.  NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. , 1998, Annual review of immunology.

[17]  R. Cortese,et al.  The two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBP delta/NF-IL6 beta, are induced by IL-6 to promote acute phase gene transcription via different mechanisms. , 1993, Nucleic acids research.

[18]  G. Haegeman,et al.  p38 and Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase Pathways Are Required for Nuclear Factor-κB p65 Transactivation Mediated by Tumor Necrosis Factor* , 1998, The Journal of Biological Chemistry.

[19]  J. J. Chen,et al.  The papillomavirus E6 proteins. , 1998, Biochimica et biophysica acta.

[20]  J. Stankova,et al.  Leukotriene B4 enhances interleukin-6 (IL-6) production and IL-6 messenger RNA accumulation in human monocytes in vitro: transcriptional and posttranscriptional mechanisms. , 1992, Blood.

[21]  Thorsten Heinzel,et al.  A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors , 1996, Cell.

[22]  R. Janknecht,et al.  Transcription. A growing coactivator network. , 1996, Nature.

[23]  J. Gauldie,et al.  The acute phase response. , 1994, Immunology today.

[24]  G. Haegeman,et al.  The Nuclear Factor-κB Engages CBP/p300 and Histone Acetyltransferase Activity for Transcriptional Activation of the Interleukin-6 Gene Promoter* , 1999, The Journal of Biological Chemistry.

[25]  P. Sassone-Corsi,et al.  A multiple cytokine- and second messenger-responsive element in the enhancer of the human interleukin-6 gene: similarities with c-fos gene regulation , 1989, Molecular and cellular biology.

[26]  S. Akira,et al.  Cytokine signal transduction , 1994, Cell.

[27]  Ralf Janknecht,et al.  Transcriptional control: Versatile molecular glue , 1996, Current Biology.

[28]  J. Elias,et al.  IL-1 and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA. , 1990, Journal of immunology.

[29]  R. Janknecht,et al.  Versatile molecular glue. Transcriptional control. , 1996, Current biology : CB.

[30]  M. Meisterernst,et al.  Interaction of the COOH-terminal Transactivation Domain of p65 NF-κB with TATA-binding Protein, Transcription Factor IIB, and Coactivators (*) , 1995, The Journal of Biological Chemistry.

[31]  S. Ghosh,et al.  Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. , 1998, Molecular cell.

[32]  S. Akira,et al.  A nuclear factor for IL‐6 expression (NF‐IL6) is a member of a C/EBP family. , 1990, The EMBO journal.

[33]  D. Papanicolaou,et al.  The Pathophysiologic Roles of Interleukin-6 in Human Disease , 1998, Annals of Internal Medicine.

[34]  M. Mallardo,et al.  DNA damaging agents increase the stability of interleukin-1 alpha, interleukin-1 beta, and interleukin-6 transcripts and the production of the relative proteins. , 1994, Journal of Biological Chemistry.