Title: The role of PPARβ in the apoptotic HaCaT keratinocytes induced by TNF-α
Abstract: Objective To explore the change of transcription activity and expression of PPARβ in the apoptotic HaCaT keratinocytes induced by TNF-α.Methods HaCaT keratinocytes were exposed to different concentration TNF-α for 24 hours.Apoptotic morphological changes and percentage of apoptotic nuclei were assayed with Hoeehst 33258 staining.Activities of Caspase-3 were analyzed with Caspase Colorimetric Assay Kit after HaCaT keratinoeytes were exposed to TNF-α(10 and 20 ng/ml)for indicated durations.The expression of PPARβ in HaCaT keratinoeytes treated with TNF-α was observed by Western- blot and RT-PCR.Eleetrophoretie mobility shift assays demonstrated a impermanency increase in PPARβ binding activity with DNA.Furthermore,lueiferase assay system were employed to analyze PPARβ transcription activity.Results The apoptosis of HaCaT keratinocytes treated with different concentration TNF-α 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-α(5,10,20 ng/ml)for 24 hours.The activation of Caspase-3 were enhanced in HaCaT keratinocytes treated with TNF-α(10 or 20 ng/ml)for indicated durations(P0.01).The expression of PPARβ protein significantly increased in HaCaT keratinoeytes treated with TNF-α(10 ng/ml)for 12 and 24 hours.After exposure to different concentration of TNF-α for 24 hours,Western-blot analysis demonstrated to augment the expression of PPARβ in HaCaT keratinocytes.RT-PCR testified the expression of PPARβ mRNA is markedly increased in HaCaT keratinocytes treated with TNF-α(10,20 ng/ml)for 3 hours and 6 hours.PPARβ- DNA binding was assessed by EMSA using a PPARβ response element(PPRE)and nuclear extracts prepared from HaCaT keratinocytes treated for 30 minutes and 60 minutes with 10 ng/ml of TNF-α demerstrated TNF-α enhanced PPARβ DNA binding activity.Furthermore,luciferase assay system obtained TNF-α increased PDK1 activity through an PPARβ-dependent pathway.Conclusion TNF-α could increase the expression and transcription activity of PPARβ in HaCaT keratinocytes.
Publication Year: 2007
Publication Date: 2007-01-01
Language: en
Type: article
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