The role of endoplasmic reticular Ca(2+) stores in cell viability and tumor necrosis factor-alpha production of the murine macrophage RAW 264.7 cell line.
Thapsigargin (TG), an endoplasmic reticular (ER) Ca(2+)-ATPase inhibitor, can increase the intracellular calcium concentration and then deplete the TG-sensitive intracellular Ca(2+) pool. In this study, we investigated the effects of TG on cell viability and tumor necrosis factor-alpha (TNF-alpha) production in the murine macrophage RAW 264.7 cell line. We found that treatment with TG (10-800 nM) induced apoptosis in RAW 264.7 cells in a dose-dependent manner (IC(50), 200 nM). Lipopolysaccharide (LPS, 1 microg/ml) markedly potentiated low concentrations of TG (10-75 nM) in inducing apoptosis (IC(50), 20 nM) as revealed by the DNA ladder. Polymycin B (an LPS receptor antagonist) inhibited the cytotoxic effect induced by LPS plus TG. Although TG, A23187 and ionomycin all definitely increased intracellular Ca(2+) concentrations, neither A23187 nor ionomycin mimicked TG in inducing apoptotic events in LPS-activated RAW 264.7 cells. Moreover, the production of TNF-alpha induced by LPS was profoundly potentiated by TG but not by A23187 or by ionomycin. We conclude from these combined results that TG-sensitive ER Ca(2+) stores play a pivotal role in modulating cell viability and TNF-alpha production. The mutual potentiation between the LPS receptor signaling pathway and the depletion of ER Ca(2+) stores implies the existence of cross-talk between these multiregulatory mechanisms in this murine macrophage RAW 264.7 cell line.
Chen
Y J
YJ
Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Lin-Shiau
S Y
SY
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000096:biosynthesis; D002460:Cell Line; D002470:Cell Survival; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D006360:Heat-Shock Proteins; D015759:Ionomycin / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D018832:Molecular Chaperones / Q000096:biosynthesis; D011112:Polymyxin B / Q000494:pharmacology; D020239:Receptor Cross-Talk; D019284:Thapsigargin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D018832:Molecular Chaperones; D014409:Tumor Necrosis Factor-alpha; C018551:glucose-regulated proteins; D000001:Calcimycin; D015759:Ionomycin; D019284:Thapsigargin; D011112:Polymyxin B