Objective: To explore the change of transcription activity and expression of PPARbeta in the apoptotic HaCaT keratinocytes induced by TNF-alpha.
Methods: HaCaT keratinocytes were exposed to different concentration TNF-alpha for 24 hours. Apoptotic morphological changes and percentage of apoptotic nuclei were assayed with Hoechst 33258 staining. Activities of Caspase-3 were analyzed with Caspase Colorimetric Assay Kit after HaCaT keratinocytes were exposed to TNF-alpha (10 and 20 ng/ml) for indicated durations. The expression of PPARbeta in HaCaT keratinocytes treated with TNF-alpha was observed by Western-blot and RT-PCR. Electrophoretic mobility shift assays demonstrated a impermanency increase in PPARbeta binding activity with DNA. Furthermore, luciferase assay system were employed to analyze PPARbeta transcription activity.
Results: The apoptosis of HaCaT keratinocytes treated with different concentration TNF-alpha for 24 hours was increased by Hoechst 33258 stained, and fluorescent microscopy showed apoptotic cells with condensed chromatin. The nuclear apoptotic percentage were (12 +/- 3)%, (32 +/- 4)%, (57 +/- 5)%, respectively, in HaCaT keratinocytes exposed to TNF-alpha (5, 10, 20 ng/ml) for 24 hours. The activation of Caspase-3 were enhanced in HaCaT keratinocytes treated with TNF-alpha (10 or 20 ng/ml) for indicated durations (P < 0.01). The expression of PPARbeta protein significantly increased in HaCaT keratinocytes treated with TNF-alpha (10 ng/ml) for 12 and 24 hours. After exposure to different concentration of TNF-alpha for 24 hours, Western-blot analysis demonstrated to augment the expression of PPARbeta in HaCaT keratinocytes. RT-PCR testified the expression of PPARbeta mRNA is markedly increased in HaCaT keratinocytes treated with TNF-alpha (10,20 ng/ml) for 3 hours and 6 hours. PPARbeta-DNA binding was assessed by EMSA using a PPARbeta response element (PPRE) and nuclear extracts prepared from HaCaT keratinocytes treated for 30 minutes and 60 minutes with 10 ng/ml of TNF-alpha demerstrated TNF-alpha enhanced PPARbeta DNA binding activity. Furthermore, luciferase assay system obtained TNF-alpha increased PDK1 activity through an PPARbeta-dependent pathway.
Conclusion: TNF-alpha could increase the expression and transcription activity of PPARbeta in HaCaT keratinocytes.
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