RRC ID 62127
著者 Yano F, Saito T, Ogata N, Yamazawa T, Iino M, Chung UI, Kawaguchi H.
タイトル 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.
ジャーナル Arthritis Rheum
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.
CONCLUSION: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.
巻・号 65(2)
ページ 429-35
公開日 2013-2-1
DOI 10.1002/art.37779
PMID 23124878
MeSH Calcium / metabolism* Cell Enlargement / drug effects Chondrocytes / cytology Chondrocytes / drug effects Chondrocytes / metabolism* Cyclic AMP / metabolism HEK293 Cells HeLa Cells Humans Parathyroid Hormone / pharmacology* Receptor, Parathyroid Hormone, Type 1 / genetics Receptor, Parathyroid Hormone, Type 1 / metabolism* Signal Transduction / drug effects Signal Transduction / physiology* Wnt Proteins / genetics Wnt Proteins / metabolism Wnt Signaling Pathway / drug effects Wnt Signaling Pathway / physiology beta Catenin / genetics beta Catenin / metabolism*
リソース情報
ヒト・動物細胞 ATDC5(RCB0565) HeLa(RCB0007) 293(RCB1637)