Involvement of mitogen-activated protein kinases and protein kinase C in regulation of antioxidant response element activity in human keratinocytes.
Antioxidant response element (ARE) is a unique cis-acting regulatory sequence located in the upstream regions of many genes encoding anticarcinogenic/antioxidant proteins. Induction of ARE dependent genes plays an important role in protection of cells against oxidative damage. However, the signaling mechanism(s) involved in regulating transcription of ARE dependent gene expression has not been clearly defined. In this study, we identified protein kinases that are involved in regulation of ARE activity by using specific pharmacological inhibitors of protein kinases in engineered human HaCaT keratinocytes, which stably express the ARE-driven green fluorescent protein (GFP) as a reporter. When HaCaT/GFP cells were treated with tert-butylhydroquinone (tBHQ), a well-known ARE activator, GFP expression was up-regulated in time and dose dependent manner, indicating that tBHQ activates the ARE in these cells. Treatment of cells with SB202190 (a specific inhibitor of p38), staurosporine (a wide-spectrum inhibitor of PKC) or rottlerin (a specific inhibitor of PKCdelta) all augmented ARE activation by tBHQ. These results suggest that p38 and PKC, especially PKCdelta, play inhibitory roles in ARE activation in human keratinocytes. Furthermore, UVB irradiation minimally affects the basal ARE activity but significantly suppresses tBHQ induced ARE activation, indicating that UVB irradiation interrupts tBHQ signaling. Interestingly, treatment of HaCaT/GFP cells with SP600125 (a specific inhibitor of JNK) could reverse UVB mediated suppression of ARE activation by tBHQ. This suggests that the suppressive effect of UVB on ARE activation by tBHQ is mediated by a JNK pathway(s). These findings provide useful information for developing novel strategies for skin cancer chemoprotection through ARE activation.
Zhu
Ming
M
Translational Genomics Research Institute, Phoenix, AZ, USA.
Zhang
Yuesheng
Y
Bowden
G Tim
GT
D000098:Acetophenones / Q000494:pharmacology; D000873:Anthracenes / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D001578:Benzopyrans / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D020218:Response Elements; D012235:Rhodamines / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000098:Acetophenones; D000873:Anthracenes; D000975:Antioxidants; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D007093:Imidazoles; D011725:Pyridines; D012235:Rhodamines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C432165:pyrazolanthrone; C022027:lissamine rhodamine B; C018855:2-tert-butylhydroquinone; C085746:rottlerin; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019311:Staurosporine; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole