title: Heat shock protein 90 is an essential molecular chaperone for CB2 cannabinoid receptor-mediated signaling in trabecular meshwork cells.
abstract: OBJECTIVE
  To examine the interaction of heat shock protein 90 (Hsp90) with the CB2 cannabinoid receptor in trabecular meshwork (TM) cells and to investigate the roles of Hsp90 in CB2 receptor-mediated cell signaling and actin cytoskeleton remodeling.
  
  
  METHODS
  Coimmunoprecipitation experiments and western blot analyses, using specific anti-CB2 and anti-Hsp90 antibodies, were performed to study the interaction of Hsp90 with the CB2 receptor in TM cells. An antiphospho-extracellular-signal-regulated kinases 1/2 (ERK1/2) antibody was used to detect the CB2 receptor-mediated phosphrylation of ERK1/2. In cytoskeleton studies, Alexa Fluor 488-labeled phalloidin staining was used to examine actin filaments of TM cells. PD98059, a specific inhibitor of the ERK1/2 pathway, was used to evaluate the role ERK1/2 pathway in CB2 receptor-mediated actin cytoskeleton changes. Geldanamycin, an inhibitor of Hsp90, was used to investigate the roles of Hsp90 in CB2 receptor-mediated ERK1/2 phosphorylation and actin cytoskeleton remodeling.
  
  
  RESULTS
  The interaction of Hsp90 with the CB2 receptor was established in TM cells with coimmunoprecipitation experiments and western blot analyses. Treatment of TM cells with geldanamycin significantly inhibited the interaction of Hsp90 with the CB2 receptor. Disruption of the CB2/Hsp90 interaction by treating TM cells with geldanamycin inhibited CB2 receptor-mediated ERK1/2 phosphorylation, as well as actin cytoskeleton remodeling. Furthermore, treatment of TM cells with PD98059 profoundly attenuated CB2 receptor-mediated actin cytoskeleton changes.
  
  
  CONCLUSIONS
  The data from this study establish a specific interaction between Hsp90 and the CB2 receptor in TM cells. In addition, the current study demonstrates that by interacting with the CB2 receptor, Hsp90 plays an important role as a molecular chaperone in CB2 receptor-mediated cell signaling and actin cytoskeleton rearrangement in TM cells.
authors.affiliation: Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: He
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Kumar
authors.forename: Zhao-Hui
authors.initials: ZH
authors.lastname: Song
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D043885:Receptor, Cannabinoid, CB2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013552:Swine; D046509:Tissue Culture Techniques; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D005419:Flavonoids; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D047428:Protein Kinase Inhibitors; D043885:Receptor, Cannabinoid, CB2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; C001277:geldanamycin