Biochemical and Biophysical Research Communications
Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNFα in human epidermal keratinocytes (HaCaT)
Section snippets
Materials and methods
Reagents. Human recombinant TNFα was purchased from R&D systems (Tokyo, Japan). Prednisolone (PSL) was obtained from Sigma–Aldrich, (Tokyo, Japan). PD98059 and U0126 (MEK-1/ERK inhibitors), SC-514 (IKKβ inhibitor), SB203580 (p38 MAPK inhibitor), wortmannin (PI3 kinase inhibitor), and SP600125 (JNK inhibitor) were purchased from Calbiochem (Tokyo, Japan).
Cells and culture conditions. HaCaT cells were kindly provided by Dr. Norbert E. Fusenig (German Cancer Research Centre, Heidelberg, Germany).
TNFα reduces PSL-dependent transactivation of pGRE-luc and an endogenous gene in HaCaT cells
We investigated the effect of TNFα on GC-induced transactivation in HaCaT cells. PSL (100 ng/ml) treatment for 4 h induced relative luciferase activity in HaCaT cells transiently transfected with pGRE-luc and pRL-TK plasmids (Fig. 1A). This transactivation was significantly reduced by a pre-treatment of cells with TNFα (0.1–10 ng/ml) in a dose-dependent manner. Statistically significant differences were observed when TNFα concentrations were higher than 1 ng/ml (p < 0.05 and p < 0.01 at TNFα
Discussion
In the current study, we investigated the effect of TNFα on GC sensitivity of HaCaT, a human epidermal keratinocyte cell line. Data showed that pre-treatment of cells with TNFα suppressed PSL-induced transactivation of pGRE-luc and GILZ mRNA expressions in dose-dependent manners. This GC insensitivity was not accompanied by an increase in GRβ or a decrease in GRα mRNA expression, but was significantly recovered by pre-treatments of cells with an MEK-1/ERK specific inhibitor (PD98059 or U0126).
Acknowledgments
We thank Dr. Norbert E. Fusenig for generously providing the HaCaT cell line. We also thank Mr. Kamogawa, Uchida, Nakamura, and Taniguchi for their technical assistance. This study was supported by a grant from the Japan Research Foundation for Clinical Pharmacology.
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