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β-catenin在软骨细胞中的作用机制和功能相关性，通过检测β-catenin作为与甲状旁腺激素（PTH）/PTH-related protein（PTHrP）1型受体（PTHR-1）的细胞内C-末端部分相互作用的新蛋白的作用，通过PTHR-1 C-末端的缺失和突变分析确定β-catenin-PTHR-1结合区域，使用哺乳动物双杂交测定。这2个分子之间的物理相互作用进行了检查，在原位邻位连接试验和免疫染色。为了评估β-连环蛋白功能获得和丧失的影响，在与野生型PTHR 1或突变体形式（缺乏与β-连环蛋白的结合）共转染的细胞中进行转染实验，以诱导组成型活性形式的β-连环蛋白（ca-ss-连环蛋白）的过表达，并用小干扰RNA阻断β-连环蛋白活性。细胞中G蛋白α亚基G（αs）和G（αq）的活化 ...更多信息 在分化的软骨细胞中，β-连环蛋白与PTHR-1（584-589）的细胞膜特异性区域物理相互作用并共定位。β-catenin与PTHR-1的结合导致G（αs）/cAMP途径的抑制和G（αq）/Ca（2+）途径的增强，而不影响经典Wnt途径。PTH对Col 10a 1信使RNA（mRNA）表达的抑制可通过过度表达ca-β-catenin恢复，即使在阻断经典Wnt通路后也是如此，并且在ss-catenin-floxed小鼠的软骨细胞中敲除ss-catenin（通过Cre重组酶）可进一步降低Col 10a 1 mRNA表达。结论：ss-catenin与PTHR-1的C-尾结合，使下游信号通路由G（αs）/cAMP转变为G（αq）/Ca（2+），这可能是通过PTH/PTHrP信号途径调控软骨细胞肥大的一种机制。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