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 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 Ca²⁺ 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/Ca²⁺ 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/Ca²⁺, which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.