Delineation of signalling pathway leading to antioxidant-dependent inhibition of dexamethasone-mediated muscle cell death.
The molecular mechanism of the cell death-promoting effect of dexamethasone (Dex) was studied during myogenesis (10 days) in L6 muscle cells by making use of several indices such as cell viability (protein synthesis, mitochondrial respiration), mortality (DNA fragmentation, chromatin condensation, structural modifications) and immunocytochemical studies [hydrogen peroxide, m-calpain (calpain 2)]. Dex initially (2 nM) stimulated protein synthesis (P < 0.001), but a further increase (20 nM) did not stimulate, whereas a higher dose (200 nM) inhibited formation of cellular proteins (P < 0.001). The latter, apparently, resulted from impaired cell viability (P < 0.001). From the day 4, structural changes featuring cell death were observed. Antioxidants [sodium ascorbate (ASC), catalase (CAT) or N-acetyl-L-cysteine (NAC)] as well as the inhibition of transcription and translation by actinomycin D abrogated Dex-induced cell death (P < 0.001). Using a fluorescent probe (DCFH-DA) we directly corroborated the working hypothesis of the mediating role of H2O2 in the reduction of cell viability by the excess of glucocorticoids. We also found that tPKC, PLCgamma, PLA2 were required to induce Dex-dependent cell death since inactivation of tPKC by H7 completely abolished the cytotoxic effect of Dex, while the blockade of PLCgamma and PLA2 by U 73122 partially abolished the effect. Cell death was triggered by Ca2+ influx necessary to activate m-calpain since it was reversed by the calcium chelator EGTA or m-calpain inhibitor ALLN but not EDTA nor ALLM. However, cell viability impaired by Ca2+ ionophore A 23187 (P < 0.001) was neither reversed by EGTA, nor EDTA, nor caspase-3 blocker--Ac DEVD CHO, nor ALLN, nor antioxidants--ASC, NAC, CAT. Specific caspase-3 inhibitor Ac DEVD CHO also did not rescue cells from Dex-induced cell death (P < 0.001), in contrast to m-calpain inhibitor--ALLN. Taken together, these findings suggest that reactive oxygen species inhibit protein synthesis and amplify m-calpain-dependent proteolysis. The events that led to the death of L6 muscle cells most likely resulted from Dex-mediated repression of antioxidative defences on the genomic level.
Orzechowski
Arkadiusz
A
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland. orzechowski@alpha.sggw.waw.pl
Jank
Michał
M
Gajkowska
Barbara
B
Sadkowski
Tomasz
T
Godlewski
Michał Marek
MM
Ostaszewski
Piotr
P
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000235:genetics; D020013:Calcium Signaling / Q000502:physiology; D002154:Calpain / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D004247:DNA / Q000032:analysis; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D006023:Glycoproteins / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D009025:Morpholines / Q000494:pharmacology; D032342:Muscle Cells / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D024510:Muscle Development / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*
D000975:Antioxidants; D002867:Chromones; D006023:Glycoproteins; D009025:Morpholines; D011506:Proteins; C039304:calpain inhibitors; D003609:Dactinomycin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D003907:Dexamethasone; D004247:DNA; D006861:Hydrogen Peroxide; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; C087857:protein kinase C gamma; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C449000:m-calpain