title: Regulation of apoptosis in HL-1 cardiomyocytes by phosphorylation of the receptor tyrosine kinase EphA2 and protection by lithocholic acid.
abstract: OBJECTIVE
  Heart failure and atrial fibrillation are associated with apoptosis of cardiomyocytes, suggesting common abnormalities in pro-apoptotic cardiac molecules. Activation of the receptor tyrosine kinase EphA2 causes apoptosis in vitro, and dysregulation of EphA2-dependent signalling is implicated in LEOPARD and Noonan syndromes associated with cardiomyopathy. Molecular pathways and regulation of EphA2 signalling in the heart are poorly understood. Here we elucidated the pathways of EphA2-dependent apoptosis and evaluated a therapeutic strategy to prevent EphA2 activation and cardiac cell death.
  
  
  METHODS
  EphA2 signalling was studied in an established model of doxazosin-induced apoptosis in HL-1 cells. Apoptosis was measured with TUNEL assays and as cell viability using a formazan method. Western blotting and siRNA for EphA2 were also used.
  
  
  RESULTS
  Apoptosis induced by doxazosin (EC(50)  = 17.3 μM) was associated with EphA2 activation through enhanced phosphorylation (2.2-fold). Activation of pro-apoptotic downstream factors, phospho-SHP-2 (3.9-fold), phospho-p38 MAPK (2.3-fold) and GADD153 (1.6-fold) resulted in cleavage of caspase 3. Furthermore, two anti-apoptotic enzymes were suppressed (focal adhesion kinase, by 41%; phospho-Akt, by 78%). Inactivation of EphA2 with appropriate siRNA mimicked pro-apoptotic effects of doxazosin. Finally, administration of lithocholic acid (LCA) protected against apoptosis by increasing EphA2 protein levels and decreasing EphA2 phosphorylation.
  
  
  CONCLUSIONS
  EphA2 phosphorylation and activation of SHP-2 are critical steps in apoptosis. Reduction of EphA2 phosphorylation by LCA may represent a novel approach for future anti-apoptotic treatment of heart failure and atrial fibrillation.
authors: Department of Cardiology, Medical University Hospital, Heidelberg, Germany.
authors: J
authors: J
authors: Jehle
authors: I
authors: I
authors: Staudacher
authors: F
authors: F
authors: Wiedmann
authors: Pa
authors: P
authors: Schweizer
authors: R
authors: R
authors: Becker
authors: Ha
authors: H
authors: Katus
authors: D
authors: D
authors: Thomas
mesh_terms: D058668:Adrenergic alpha-1 Receptor Antagonists; D000818:Animals; D000959:Antihypertensive Agents; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D017292:Doxazosin; D008095:Lithocholic Acid / Q000494:pharmacology*; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D020011:Protective Agents / Q000494:pharmacology*; D036104:Receptor, EphA2 / Q000378:metabolism*
chemical_list: D058668:Adrenergic alpha-1 Receptor Antagonists; D000959:Antihypertensive Agents; D020011:Protective Agents; D008095:Lithocholic Acid; D036104:Receptor, EphA2; D017292:Doxazosin