title: Thapsigargin activates Ca²+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells.
abstract: The ER Ca²+ sensor STIM1 and the Ca²+ channel Orai1 are key players in store-operated Ca²+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca²+ entry by about 50-60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca²+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca²+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PKCeta is required for Ca²+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca²+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca²+ entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca²+ entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3.
authors.affiliation: Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Antigny
authors.forename: Hélène
authors.initials: H
authors.lastname: Jousset
authors.forename: Stéphane
authors.initials: S
authors.lastname: König
authors.forename: Maud
authors.initials: M
authors.lastname: Frieden
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D016257:Fura-2 / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000071740:ORAI1 Protein; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D010703:Phorbol Esters; D034741:RNA, Small Interfering; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D019284:Thapsigargin; D019061:src-Family Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2