Department of Basic Medical Sciences, Bone Physiopathology Laboratory, Universidad San Pablo-CEU, Alcorcón, Madrid, Spain.
Departamento de Ciencias Médicas Básicas, Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, Campus Monteprincipe, Alcorcón, Madrid, Spain.
J Cell Physiol. 2020 Oct;235(10):7356-7369. doi: 10.1002/jcp.29636. Epub 2020 Feb 10.
Mechanical stimulation of primary cilia in osteocytes and osteoblasts has been proposed as a mechanism that participates in bone cell survival and skeletal remodeling. Among different signaling pathways stimulated by primary cilia, the hedgehog signaling pathway has been associated with the regulation of bone development. Parathyroid hormone (PTH)-related protein (PTHrP) signaling through PTH 1 receptor (PTH1R) also regulates bone cell survival and remodeling and has been associated with the hedgehog pathway during skeletal development. We hypothesize that primary cilia and PTH1R concomitantly regulate bone remodeling and cell survival and aim to describe the mechanisms that mediate these effects in osteocytes and osteoblasts. Colocalization of PTH1R with primary cilia was observed in control and PTHrP-stimulated MLO-Y4 osteocytic and MC3T3-E1 osteoblastic cells. Activation of PTH1R by PTHrP increased cell survival, osteoblast gene expression (osteocalcin, runt-related transcription factor 2, and bone alkaline phosphatase) and the expression of the hedgehog transcription factor Gli-1 in osteocytes and osteoblasts. These effects were abrogated by small interfering RNAs for the primary cilia protein IFT88 or by a primary cilia specific inhibitor (chloral hydrate). Preincubation of MLO-Y4 osteocytic and MC3T3-E1 osteoblastic cells with the Gli-1 antagonist GANT61 inhibited PTHrP prosurvival actions but did not affect PTHrP-induced overexpression of osteogenic genes. Mechanical stimulation by fluid flow increased the number of primary cilia-presenting cells in osteocytes and osteoblasts. We propose that PTH1R activation induces prosurvival actions via primary cilia- and Gli-1-dependent mechanism and modulates osteogenic responses via a primary cilia-dependent and Gli-1-independent pathway in osteocytes and osteoblasts.
骨细胞和成骨细胞中初级纤毛的机械刺激被认为是参与骨细胞存活和骨骼重塑的机制之一。在初级纤毛刺激的不同信号通路中， hedgehog 信号通路与骨发育的调节有关。甲状旁腺激素（PTH）相关蛋白（PTHrP）通过 PTH1 受体（PTH1R）的信号转导也调节骨细胞的存活和重塑，并与骨骼发育过程中的 hedgehog 通路有关。我们假设初级纤毛和 PTH1R 同时调节骨重塑和细胞存活，并旨在描述在骨细胞和成骨细胞中介导这些效应的机制。在对照和 PTHrP 刺激的 MLO-Y4 成骨细胞和 MC3T3-E1 成骨细胞中观察到 PTH1R 与初级纤毛的共定位。PTHrP 激活 PTH1R 增加了细胞存活、成骨基因表达（骨钙素、 runt 相关转录因子 2 和骨碱性磷酸酶）以及 hedgehog 转录因子 Gli-1 在成骨细胞和骨细胞中的表达。这些作用被针对初级纤毛蛋白 IFT88 的小干扰 RNA 或初级纤毛特异性抑制剂（水合氯醛）消除。预先孵育 MLO-Y4 成骨细胞和 MC3T3-E1 成骨细胞用 Gli-1 拮抗剂 GANT61 抑制了 PTHrP 的促生存作用，但不影响 PTHrP 诱导的成骨基因过表达。流体流动的机械刺激增加了成骨细胞和骨细胞中存在初级纤毛的细胞数量。我们提出，PTH1R 的激活通过初级纤毛和 Gli-1 依赖性机制诱导促生存作用，并通过初级纤毛依赖性和 Gli-1 非依赖性途径调节成骨反应在成骨细胞和骨细胞中。
