ß-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 ß-catenin in chondrocytes, by examining the role of ß-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 ß-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 ß-catenin, transfection experiments were performed to induce overexpression of the constitutively active form of ß-catenin (ca-ß-catenin) and to block ß-catenin activity with small interfering RNA, in cells cotransfected with either wild-type PTHR1 or mutant forms (lacking binding to ß-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, ß-catenin physically interacted and colocalized with the cell membrane-specific region of PTHR-1 (584-589). Binding of ß-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-ß-catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of ß-catenin (via the Cre recombinase) in chondrocytes from ß-catenin-floxed mice. Mutagenesis analyses to block the binding of ß-catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers. CONCLUSION: ß-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.