[The role of PPARbeta in the apoptotic HaCaT keratinocytes induced by TNF-alpha].
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.


CONCLUSIONS
TNF-alpha could increase the expression and transcription activity of PPARbeta in HaCaT keratinocytes.
Liang
Peng-fei
PF
Department of Burns and Plastic Surgery, Xiangya Hospital, Central South University, Changsha 410008, China.
Jiang
Bi-mei
BM
Zhang
Pi-hong
PH
Yang
Xing-hua
XH
Long
Jian-hong
JH
Huang
Xiao-yuan
XY
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002151:Calorimetry / Q000379:methods; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D048068:PPAR-beta / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D048068:PPAR-beta; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D020169:Caspases