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

Objective. To investigate the underlying mechanisms of action and functional relevance of beta-catenin in chondrocytes, by examining the role of beta-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 beta-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 beta-catenin, transfection experiments were performed to induce overexpression of the constitutively active form of beta-catenin (ca-beta-catenin) and to block beta-catenin activity with small interfering RNA, in cells cotransfected with either wild-type PTHR1 or mutant forms (lacking binding to beta-catenin). Activation of the G protein alpha subunits G(alpha s) and G(alpha q) in the cells was determined by measurement of the intracellular cAMP accumulation and intracellular Ca2+ concentration, while activation of canonical Wnt pathways was assessed using a TOPflash reporter assay. Results. In differentiated chondrocytes, beta-catenin physically interacted and colocalized with the cell membrane-specific region of PTHR-1 (584-589). Binding of beta-catenin to PTHR-1 caused suppression of the G(alpha s)/cAMP pathway and enhancement of the G(alpha q)/Ca2+ pathway, without affecting the canonical Wnt pathway. Inhibition of Col10a1 messenger RNA (mRNA) expression by PTH was restored by overexpression of ca-beta-catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of beta-catenin (via the Cre recombinase) in chondrocytes from beta-catenin-floxed mice. Mutagenesis analyses to block the binding of beta-catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers. Conclusion. beta-catenin binds to the PTHR-1 C-tail and switches the downstream signaling pathway from G(alpha s)/cAMP to G(alpha q)/Ca2+, which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.