title: Chorein Sensitive Orai1 Expression and Store Operated Ca2+ Entry in Rhabdomyosarcoma Cells.
abstract: BACKGROUND
  Chorein, a protein encoded by VPS13A (vacuolar protein sorting-associated protein 13A), is defective in chorea acanthocytosis, a rare disease characterized by acanthocytosis of red blood cells and neuronal cell death with progressive hyperkinetic movement disorder, cognitive dysfunction, behavioral abnormalities and chronic hyperkalemia. Chorein is highly expressed in ZF rhabdomyosarcoma cells and counteracts apoptosis of those cells. Chorein is effective in part by interacting with and fostering stimulation of phosphoinositide-3-kinase (PI3K)-p85-subunit. PI3K dependent signaling includes the serum and glucocorticoid inducible kinase SGK1. The kinase activates NFκB with subsequent up-regulation of the Ca2+ channel subunit Orai1, which accomplishes store operated Ca2+ entry (SOCE). Orai1 and SOCE have been shown to confer survival of tumor cells. The present study thus explored whether chorein impacts on Orai1 expression and SOCE.
  
  
  METHODS
  In rhabdomyosarcoma cells chorein, Orai1, NFκB and SGK1 transcript levels were quantified by RT-PCR, Orai1 protein abundance by Western blotting, FACS analysis and confocal laser microscopy, [Ca2+]i utilizing Fura-2 fluorescence, and SOCE from the increase of [Ca2+]i following store depletion with extracellular Ca2+ removal and inhibition of the sarcoendoplasmatic reticular Ca2+ ATPase with thapsigargin.
  
  
  RESULTS
  The mRNA coding for chorein was most abundant in drug resistant, poorly differentiated human ZF rhabdomyosarcoma cells. Chorein silencing significantly decreased Orai1 transcript levels and Orai1 protein expression, as well as SGK1 and NFκB transcript levels. SOCE in ZF rhabdomyosarcoma cells was significantly blunted by chorein silencing, Orai1 inhibitor 2-APB (50 µM), SGK1 inhibitor EMD638683 (50 µM, 10 h) and NFκB inhibitor wogonin (50 µM, 24 h).
  
  
  CONCLUSIONS
  Chorein is a stimulator of Orai1 expression and thus of store operated Ca2+ entry. The effect may involve SGK1 and NFκB.
authors.affiliation: Department of Cardiology, Vascular Medicine and Physiology, University of Tuebingen, Germany.
authors.forename: Willi
authors.initials: W
authors.lastname: Yu
authors.forename: Sabina
authors.initials: S
authors.lastname: Honisch
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schmidt
authors.forename: Jing
authors.initials: J
authors.lastname: Yan
authors.forename: Evi
authors.initials: E
authors.lastname: Schmid
authors.forename: Saad
authors.initials: S
authors.lastname: Alkahtani
authors.forename: Abdullah A
authors.initials: AA
authors.lastname: AlKahtane
authors.forename: Saud
authors.initials: S
authors.lastname: Alarifi
authors.forename: Christos
authors.initials: C
authors.lastname: Stournaras
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D001549:Benzamides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002648:Child; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D016257:Fura-2 / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D000071740:ORAI1 Protein / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012208:Rhabdomyosarcoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D001549:Benzamides; D002118:Calcium; D016257:Fura-2; D006834:Hydrazines; D017874:Immediate-Early Proteins; D016328:NF-kappa B; D000071740:ORAI1 Protein; D017346:Protein Serine-Threonine Kinases; D033921:Vesicular Transport Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C569030:EMD 638683; C510746:ORAI1 protein, human; C429654:VPS13A protein, human