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.

为了研究ss-连环蛋白在软骨细胞中的作用机制，通过检测β-连环蛋白作为一种与甲状旁腺激素/甲状旁腺激素相关蛋白1型受体(PTHR-1)细胞内C末端相互作用的新蛋白的作用，采用哺乳动物双杂交方法，通过对PTHR1C-末端的缺失和突变分析，确定了SS-连环蛋白-PTHR-1结合区域。用原位邻近连接实验和免疫染色检测这两个分子之间的物理相互作用。为了评估SSPTHR1-连环蛋白功能的获得和丧失的影响，在与野生型β或突变形式(与SS-连环蛋白缺乏结合)共转染的细胞中，进行了诱导组成活性形式的ss-连环蛋白(ca-ss-catenin)过表达以及用小干扰RNA阻断ss-连环蛋白活性的实验。G蛋白α亚基G(αS)和G(αQ)在CE…中的激活在分化的软骨细胞中，ss-catenin与PTHR-1(584-589)的细胞膜特异区相互作用并共定位。β-连环蛋白与α-1结合后，可抑制G(αS)/cAMP通路，增强G(WnT Q)/Ca(2+)通路，但不影响典型的WnT通路。甲状旁腺激素对COL10a1信使核糖核酸表达的抑制可通过高表达的ca-β-catenin恢复，甚至在阻断典型的Wnt途径后也可恢复，而通过Cre重组酶敲除ss-catenin可进一步降低ss-catenin诱导的小鼠软骨细胞COL10a1mRNA的表达。结论：ss-catenin与PTHR-1C-Tail结合，将下游信号通路从G(αS)/cAMP转换为G(αQ)/Ca(2+)，这可能是通过PTH/PTHrP信号调节软骨细胞肥大的一种可能机制。较少

To investigate the underlying mechanisms of action and functional relevance of ss-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).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 ofssβ-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 ce … More lls 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.In differentiated chondrocytes, ss-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 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.As a 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 thecanonical Wnt pathway. Less