title: β-catenin regulates parathyroid hormone/parathyroid hormone-related protein receptor signals and chondrocyte hypertrophy through binding to the intracellular C-terminal region of the receptor.
abstract: OBJECTIVE
  To investigate the underlying mechanisms of action and functional relevance of β-catenin in chondrocytes, by examining the role of β-catenin as a novel protein that interacts with the intracellular C-terminal portion of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor type 1 (PTHR-1).
  
  
  METHODS
  The β-catenin-PTHR-1 binding region was determined with deletion and mutagenesis analyses of the PTHR1 C-terminus, using a mammalian two-hybrid assay. Physical interactions between these 2 molecules were examined with an in situ proximity ligation assay and immunostaining. To assess the effects of gain- and loss-of-function of β-catenin, transfection experiments were performed to induce overexpression of the constitutively active form of β-catenin (ca-β-catenin) and to block β-catenin activity with small interfering RNA, in cells cotransfected with either wild-type PTHR1 or mutant forms (lacking binding to β-catenin). Activation of the G protein α subunits G(αs) and G(αq) in the cells was determined by measurement of the intracellular cAMP accumulation and intracellular Ca(2+) concentration, while activation of canonical Wnt pathways was assessed using a TOPflash reporter assay.
  
  
  RESULTS
  In differentiated chondrocytes, β-catenin physically interacted and colocalized with the cell membrane-specific region of PTHR-1 (584-589). Binding of β-catenin to PTHR-1 caused suppression of the G(αs)/cAMP pathway and enhancement of the G(αq)/Ca(2+) pathway, without affecting the canonical Wnt pathway. Inhibition of Col10a1 messenger RNA (mRNA) expression by PTH was restored by overexpression of ca-β-catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of β-catenin (via the Cre recombinase) in chondrocytes from β-catenin-floxed mice. Mutagenesis analyses to block the binding of β-catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers.
  
  
  CONCLUSIONS
  β-catenin binds to the PTHR-1 C-tail and switches the downstream signaling pathway from G(αs)/cAMP to G(αq)/Ca(2+), which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.
authors.affiliation: University of Tokyo, Tokyo, Japan.
authors.forename: Fumiko
authors.initials: F
authors.lastname: Yano
authors.forename: Taku
authors.initials: T
authors.lastname: Saito
authors.forename: Naoshi
authors.initials: N
authors.lastname: Ogata
authors.forename: Toshiko
authors.initials: T
authors.lastname: Yamazawa
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Iino
authors.forename: Ung-il
authors.initials: UI
authors.lastname: Chung
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawaguchi
mesh_terms: D002118:Calcium / Q000378:metabolism*; D049108:Cell Enlargement / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010281:Parathyroid Hormone / Q000494:pharmacology*; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects / Q000502:physiology; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
chemical_list: D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D051153:Wnt Proteins; D051176:beta Catenin; D000242:Cyclic AMP; D002118:Calcium