title: Ethanol inhibits γ-secretase proteolytic activity in vascular smooth muscle cells.
abstract: BACKGROUND
  Ethanol (EtOH) inhibits Notch-mediated vascular smooth muscle cell (SMC) proliferation, an event that is key in vessel remodeling and atherogenesis. The object of this study was to determine whether EtOH inhibits Notch signaling in SMC at the level of γ-secretase, a protease that in concert with α-secretase catalyzes the release of the intracellular domain of the Notch receptor necessary for signaling.
  
  
  METHODS
  Human coronary artery SMCs (HCASMCs) were treated with a recombinant soluble Notch ligand, Delta-like ligand 4 (DLL4) (2 μg/ml), or transfected with a constitutively active Notch 1 intracellular domain (N1ICD), in the absence or presence of EtOH. EtOH (25 mM) treatment inhibited DLL4-stimulated CBF-1/RBP-Jk-dependent promoter activity (determined by luciferase assay) and downstream target gene HRT-3 mRNA levels. In contrast, EtOH had no effect on N1ICD-driven CBF-1/RBP-Jk-dependent promoter activity or HRT-3 expression.
  
  
  RESULTS
  These data suggest that EtOH inhibits Notch signaling at, or prior to, Notch intracellular domain (NICD) generation. γ-Secretase activity was determined in solubilized membrane preparations from HCASMC treated with/without EtOH (25 mM) or the γ-secretase inhibitor DAPT (20 μM) using (i) a fluorometric assay and (ii) Western blot detection of cleavage products using a Flag-tagged Notch-based substrate, N100Flag. EtOH inhibited basal and DLL4-stimulated γ-secretase activity, and SMC growth to a similar extent as DAPT, whereas it had no effect on α-secretase (TACE/ADAM17) activity also determined by fluorometric assay. Moreover, EtOH treatment inhibited the expression of caveolin-1, a lipid raft protein implicated in regulating γ-secretase activity, and altered its cellular distribution in HCASMC.
  
  
  CONCLUSIONS
  EtOH inhibits Notch signaling in vascular SMCs at the level of γ-secretase activity, possibly by affecting lipid raft function. Such a response might be expected to result in attenuation of pathologic vessel remodeling and thus may contribute to moderate alcohols' cardioprotective effects.
authors.affiliation: Department of Surgery, University of Rochester Medical Center, Rochester, New York.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Hatch
authors.affiliation: Department of Surgery, University of Rochester Medical Center, Rochester, New York.
authors.forename: David
authors.initials: D
authors.lastname: Morrow
authors.affiliation: Department of Surgery, University of Rochester Medical Center, Rochester, New York.
authors.forename: Weimin
authors.initials: W
authors.lastname: Liu
authors.affiliation: Vascular Health Research Centre, Dublin City University, Dublin, Ireland.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Cahill
authors.affiliation: Department of Surgery, University of Rochester Medical Center, Rochester, New York.
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Redmond
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology*; D059748:Proteolysis / Q000187:drug effects*; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D051880:Receptors, Notch; D000431:Ethanol; D053829:Amyloid Precursor Protein Secretases
keywords: Alcohol; Atherosclerosis; Notch; Vascular Smooth Muscle Cells; γ-Secretase