G-protein-coupled OX1 orexin/hcrtr-1 hypocretin receptors induce caspase-dependent and -independent cell death through p38 mitogen-/stress-activated protein kinase.
We have investigated the signaling of OX(1) receptors to cell death using Chinese hamster ovary cells as a model system. OX(1) receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca(2+) elevation. The classical mitogen-activated protein kinase (MAPK) pathways, ERK and p38, were strongly activated by orexin-A. p38 was essential for induction of cell death, whereas the ERK pathway appeared protective. A pathway often implicated in the p38-mediated cell death, activation of p53, did not mediate the cell death, as there was no stabilization of p53 or increase in p53-dependent transcriptional activity, and dominant-negative p53 constructs did not inhibit cell demise. Under basal conditions, orexin-A-induced cell death was associated with compact chromatin condensation and it required de novo gene transcription and protein synthesis, the classical hallmarks of programmed (apoptotic) cell death. However, though the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (Z-VAD-fmk) fully inhibited the caspase activity, it did not rescue the cells from orexin-A-induced death. In the presence of Z-VAD-fmk, orexin-A-induced cell death was still dependent on p38 and de novo protein synthesis, but it no longer required gene transcription. Thus, caspase inhibition causes activation of alternative, gene transcription-independent death pathway. In summary, the present study points out mechanisms for orexin receptor-mediated cell death and adds to our general understanding of the role of G-protein-coupled receptor signaling in cell death by suggesting a pathway from G-protein-coupled receptors to cell death via p38 mitogen-/stress-activated protein kinase independent of p53 and caspase activation.
Ammoun
Sylwia
S
Department of Neuroscience, Unit of Physiology, Uppsala University, Sweden.
Lindholm
Dan
D
Wootz
Hanna
H
Akerman
Karl E O
KE
Kukkonen
Jyrki P
JP
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D049109:Cell Proliferation; D002843:Chromatin / Q000737:chemistry; D004396:Coloring Agents / Q000494:pharmacology; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D008301:Maleimides / Q000494:pharmacology; D064446:Orexin Receptors; D011493:Protein Kinase C / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000737:chemistry* / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D002843:Chromatin; D004396:Coloring Agents; D016895:Culture Media, Serum-Free; C578630:HCRTR1 protein, human; D007211:Indoles; D008301:Maleimides; D064446:Orexin Receptors; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases; C022616:thiazolyl blue; D001973:Bromodeoxyuridine; C070515:bisindolylmaleimide I; D002118:Calcium