title: Obligatory roles of filamin A in E-cadherin-mediated cell-cell adhesion in epidermal keratinocytes.
abstract: BACKGROUND
  Extracellular Ca(2+) (Cao(2+))-induced E-cadherin-mediated cell-cell adhesion plays a critical role in promoting differentiation in epidermal keratinocytes. Our previous studies show that the calcium-sensing receptor (CaR) regulates keratinocyte cell-cell adhesion and differentiation via Rho A-mediated signaling. CaR forms a protein complex with Rho A, guanine nucleotide exchange factor Trio, and a cytoskeletal actin-binding protein, filamin A, at the cell-cell junctions in response to elevated Cao(2+) levels. Filamin A has the ability to interact directly with CaR, Trio, and Rho and mediate CaR-dependent signaling events.
  
  
  OBJECTIVE
  This study was conducted to investigate the roles of filamin A and Trio in regulating Cao(2+)-induced Rho activation and intercellular adhesion.
  
  
  METHODS
  Expression of filamin A and Trio in keratinocytes was inhibited by siRNA. Its effects on Cao(2+)-dependent junction formation and adhesion complex formation were evaluated by fluorescence immunostaining and immunoprecipitation. Endogenous Rho activity and expression of keratinocyte differentiation markers were also examined. The significance of the physical interactions of filamin A with Trio and Rho was assessed in dominant-negative inhibition studies.
  
  
  RESULTS
  Inhibiting filamin A expression blocked the formation of CaR-Rho A-Trio-E-cadherin protein complex. Knockdown of filamin A or Trio inhibited Cao(2+)-induced membrane localization and activation of Rho A, formation of the E-cadherin-catenin adhesion complex, and keratinocyte terminal differentiation. Expressing dominant-negative peptides disruptive to the endogenous filamin-Trio, filamin-Rho, and CaR-filamin interactions suppressed the formation of adherens junctions.
  
  
  CONCLUSIONS
  Through physical interactions with CaR, Trio and Rho, filamin A generates a scaffold for organizing a signaling complex that promotes E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.
authors.affiliation: Endocrine Unit, Veteran Affairs Medical Center and University of California, San Francisco, CA, USA. Electronic address: chia-ling.tu@ucsf.edu.
authors.forename: Chia-Ling
authors.initials: CL
authors.lastname: Tu
authors.affiliation: Endocrine Unit, Veteran Affairs Medical Center and University of California, San Francisco, CA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: You
mesh_terms: D015703:Antigens, CD; D015820:Cadherins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D051177:Catenins / Q000378:metabolism; D002448:Cell Adhesion; D002454:Cell Differentiation; D002478:Cells, Cultured; D004817:Epidermis / Q000378:metabolism*; D064448:Filamins / Q000502:physiology*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; D051177:Catenins; C578663:FLNA protein, human; D064448:Filamins; D034741:RNA, Small Interfering; D054460:rho-Associated Kinases; D020744:rac GTP-Binding Proteins; D002118:Calcium
keywords: AJ; Ca(o)(2+); CaR; Calcium-sensing receptor; E-cadherin; Filamin; GEF; Keratinocyte differentiaiton; PI3K; Rho A; Trio; adherens junction; calcium-sensing receptor; extracellular Ca(2+); guanine nucleotide exchange factor; phosphatidylinositol 3-kinase; siRNA; small interfering RNA