Involvement of NF-κB in the regulation of S100A6 gene expression in human hepatoblastoma cell line HepG2

https://doi.org/10.1016/S0006-291X(03)01199-9Get rights and content

Abstract

S100A6 (calcyclin) is an acidic calcium binding protein with two EF-hand motifs and overexpressed in several tumors including intrahepatic carcinoma. TNFα, a strong NF-κB activator required for hepatocyte proliferation during liver regeneration, triggered the expression of S100A6 mRNA in human hepatoblastoma cell line HepG2. Transient expression of NF-κB (p65) increased S100A6 promoter activity and expression of inhibitor of NF-κB (IκBα) decreased TNFα-induced S100A6 promoter activity. To confirm the involvement of NF-κB in S100A6 promoter activation, we analyzed serially deleted promoter constructs of the S100A6 gene by luciferase reporter assay and found a NF-κB-responsive DNA fragment at the position between −584 and −361. Electrophoretic mobility shift assays showed that TNFα induced p65 binding to a potential NF-κB binding site at −460/−451. Furthermore, treatment of cells with CAPE (caffeic acid phenethyl ester), a specific NF-κB (p65) inhibitor, decreased NF-κB binding and promoter activity. These results suggest that NF-κB transcription factor contributes to the activation of S100A6 gene expression in response to TNFα in HepG2 cells.

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Materials and methods

Cell cultures. Human liver cell lines, HepG2, PLC/ARF/5, Hep3B, and Chang were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Life Technologies, Gaithersburg, MD, USA) at 37 °C in a 5% CO2 air environment.

Northern blot analysis and RT-PCR. HepG2 cells were stimulated with TNFα (0.5, 2, and 5 ng/ml, Sigma Chemical, St. Louis, MA, USA) for 24 h and total RNA was isolated from

S100A6 is differentially expressed in liver cell lines

To study the regulation of S100A6 gene in hepatocytes, we carried out Northern blot analysis on several liver cell lines cultured in DMEM with 10% FBS (Fig. 1). S100A6 mRNA was highly expressed in Chang and Hep3B cells but barely detected in HepG2 and PLC/ARF/5 cells. The expression levels of S100A6 in these cell lines were accordant with their cell growth rates; Chang and Hep3B had higher growth rate than other liver cell lines (data not shown). This result is in agreement with the previous

Acknowledgements

This study was supported in part by grants of Korea Ministry of Science and Technology (KGS2010212, KGS2110311, and NTM0020213).

References (26)

  • U. Brinck et al.

    Differential expression of calcyclin and its accessible ligands in various types of cutaneous tumors

    J. Dermatol. Sci.

    (1995)
  • W. Lesniak et al.

    Upstream stimulatory factor is involved in the regulation of the human calcyclin (S100A6) gene

    Biochim. Biophys. Acta

    (2000)
  • C.W. Heizmann

    The multifunctional S100 protein family

    Methods Mol. Biol.

    (2002)
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