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Published online before print September 28, 2007

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(Journal of Leukocyte Biology. 2008;83:190-199.)
© 2008 by Society for Leukocyte Biology

The interaction with Sp1 and reduction in the activity of histone deacetylase 1 are critical for the constitutive gene expression of IL-1 in human melanoma cells

Kazuaki Enya^*, Hidetoshi Hayashi^*, Takemasa Takii^*, Nobumichi Ohoka^*, Shinya Kanata^*, Takashi Okamoto and Kikuo Onozaki^*,1

^* Departments of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, and
Molecular and Cellular Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan

¹ Correspondence: Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan. E-mail: konozaki{at}phar.nagoya-cu.ac.jp

A375-6 human melanoma cells are sensitive to the antiproliferative effect of IL-1. After a long period of culturing, we have obtained cells resistant to IL-1. The resistant clone A375-R8 constitutively produced IL-1. In this study, we identified a sequence, CGCC, located at –48 to –45 upstream of the transcription start site, to be essential for the constitutive IL-1 gene activation. Specificity protein 1 (Sp1) and Sp3 bound to the nucleotide containing the sequence. Although the binding level to the nucleotide and expression level of Sp1 and Sp3 are comparable in A375-R8 and A375-6 cells, transactivation activity of Sp1 is higher in A375-R8 cells as compared with A375-6 cells. Sp3 could not transactivate the IL-1 promoter. These results suggest that Sp1 but not Sp3 is important for IL-1 gene activation. Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), greatly augmented the IL-1 promoter activity in A375-6 cells to the level comparable with that in A375-R8 cells. TSA also induced IL-1 mRNA expression in A375-6 cells. Sp1 and Sp3 bound to HDAC1 in A375-R8 and A375-6 cells. The chromatin immunoprecipitation assay revealed the binding of Sp1 and HDAC1 to the promoter region of the IL-1 gene. The activities of HDAC bound to Sp1 and Sp3, and that of HDAC1 was lower in A375-R8 cells as compared with A375-6 cells. These results indicate that the reduction in the activity and interaction of HDAC1 with Sp1 are critical for the constitutive IL-1 gene expression.

Key Words: HDAC • trichostatin A • chromatin immunoprecipitation