title: Metabotropic glutamate receptor 5 mediates phosphorylation of vascular endothelial cadherin and nuclear localization of β-catenin in response to homocysteine.
abstract: Elevated plasma homocysteine (Hcy) is an independent risk factor for vascular disease and stroke in part by causing generalized endothelial dysfunction. A receptor that is sensitive to Hcy and its intracellular signaling systems has not been identified. β-catenin is a pleiotropic regulator of transcription and cell function. Using a brain microvascular endothelial cell line (bEnd.3), we tested the hypothesis that Hcy causes receptor-dependent nuclear translocation of β-catenin. Hcy increased phosphorylation of Y731 on vascular endothelial cadherin (VE-cadherin), a site involved in coupling β-catenin to VE-cadherin. This was blocked by inhibition of either metabotropic glutamate receptor 5 (mGluR5) or ionotropic glutamate receptor (NMDAr) and by shRNA knockdown of mGluR5. Expression of these receptors was confirmed by flow cytometry, immunohistochemistry, and western blotting. Directed pharmacology with specific agonists elucidated a signaling cascade where Hcy activates mGluR5 which activates NMDAr with subsequent PKC activation and uncoupling of the VE-cadherin/β-catenin complex. Moreover, Hcy caused a shift in localization of β-catenin from membrane-bound VE-cadherin to the cell nucleus, where it bound DNA, including a regulatory region of the gene for claudin-5, leading to reduced expression of claudin-5. Nuclear localization, DNA binding of β-catenin, and reduced claudin-5 expression were blocked by inhibition of mGluR5. Knockdown of mGluR5 expression with shRNA also rescued claudin-5 expression from the effects of Hcy treatment. These data uniquely identify mGluR5 as a master switch that drives β-catenin nuclear localization in vascular endothelium and regulates cell-cell coupling in response to elevated Hcy levels. These studies dissect a pharmacological opportunity for developing new therapeutic strategies in HHcy.
authors.affiliation: Department of Biological Sciences, Idaho State University, Pocatello, ID 83209-8007, United States. bearshaw@isu.edu
authors.forename: Richard S
authors.initials: RS
authors.lastname: Beard
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Reynolds
authors.forename: Shawn E
authors.initials: SE
authors.lastname: Bearden
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism*; D002448:Cell Adhesion; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D062507:Claudin-5; D057167:Claudins / Q000235:genetics / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020868:Gene Silencing; D006710:Homocysteine / Q000378:metabolism*; D020138:Hyperhomocysteinemia / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D051379:Mice; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D064529:Receptor, Metabotropic Glutamate 5; D058468:Receptors, Ionotropic Glutamate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D051176:beta Catenin / Q000378:metabolism*; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D062507:Claudin-5; D057167:Claudins; D004791:Enzyme Inhibitors; D006710:Homocysteine; D064529:Receptor, Metabotropic Glutamate 5; D058468:Receptors, Ionotropic Glutamate; D018094:Receptors, Metabotropic Glutamate; D051176:beta Catenin; D000099152:Cadherin 5; C491511:Cldn5 protein, mouse; C578936:Grm5 protein, mouse