Molecular mechanisms of p21 and p27 induction by 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, involved in antiproliferation of human umbilical vascular endothelial cells.
We previously reported that 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, inhibits the proliferation of human umbilical vascular endothelial cells (HUVECs; Juan et al., 2006, Eur J Pharmacol 530: 1-8). Herein, pretreatment of HUVECs with p21 or p27 small interfering (si)RNA reduced 3MC-induced elimination of [(3)H]thymidine incorporation, demonstrating their essential roles in the antiproliferation of HUVECs. The molecular mechanisms of p21 and p27 involved in the antiproliferative effects of 3MC were elucidated in this study. 3MC time- and concentration-dependently increased p21 and p27 levels, and decreased the protein level of CDK2 with no apparent alteration of p53. Interestingly, 3MC-mediated p21 and p27 inductions were eliminated by resveratrol, an AhR antagonist, suggesting their AhR dependency, further confirmed by AhR siRNA. Among the relevant pathways, p38MAPK activation sustained the levels of p21 and p27 induced by 3MC, which was eliminated by AhR antagonists and N-acetylcysteine (NAC), an antioxidant. 3MC concentration-dependently enhanced not only the consensus dioxin-responsive element (DRE)-driven luciferase activity, but also the binding activity of the AhR to the putative DRE derived from the p21 and p27 promoters. A deletion of the DRE (-285/-270) in p21 (-2,300/+8) only partially alleviated the 3MC-induced luciferase activity unless NAC was added, suggesting that there may be a DRE-independent mechanism associated with oxidative stress. However, a deletion of the DRE (-660/-645) in p27 (-1,358/-100) almost completely abrogated the activation. Our study demonstrated that both the functional DRE and the phosphorylation of p38MAPK are essential for the induction of p21 and p27, resulting in the antiproliferative action of 3MC in HUVECs.
Pang
Pai-Huei
PH
Department of Ophthalmology, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
Lin
Ying-Hsi
YH
Lee
Yi-Hsuan
YH
Hou
Hsing-Han
HH
Hsu
Sung-Po
SP
Juan
Shu-Hui
SH
D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008748:Methylcholanthrene / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists*; D020218:Response Elements; D000077185:Resveratrol; D017384:Sequence Deletion; D013267:Stilbenes / Q000494:pharmacology; D013997:Time Factors; D014316:Tritium / Q000378:metabolism; D014470:Umbilical Cord / Q000166:cytology* / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
D050759:Cyclin-Dependent Kinase Inhibitor p21; D018336:Receptors, Aryl Hydrocarbon; D013267:Stilbenes; D014316:Tritium; D050760:Cyclin-Dependent Kinase Inhibitor p27; D008748:Methylcholanthrene; D008156:Luciferases; D048051:p38 Mitogen-Activated Protein Kinases; D000077185:Resveratrol