Signaling of extracellular inorganic phosphate up-regulates cyclin D1 expression in proliferating chondrocytes via the Na+/Pi cotransporter Pit-1 and Raf/MEK/ERK pathway
Signaling of extracellular inorganic phosphate up-regulates cyclin D1 expression in proliferating chondrocytes via the Na+/Pi cotransporter Pit-1 and Raf/MEK/ERK pathway
复制标题
DOI：
10.1016/j.bone.2010.08.006
复制
发表时间：
2010-11-01
期刊：
影响因子：
4.1
通讯作者：
Ozono, Keiichi
中科院分区：
文献类型：
--
作者：
Kimata, Masaaki;Michigami, Toshimi;Ozono, Keiichi
随着软骨细胞成熟，细胞外环境中无机磷酸盐（Pi）的浓度增加。结果表明，生长板增殖区开始积累，肥大区积累最多。虽然据报道细胞外Pi参与成熟软骨细胞的凋亡和矿化，但其在增殖软骨细胞中的作用仍不清楚。在这里，我们研究了这一作用，利用ATDC 5，软骨细胞分化的细胞模型。在增殖的ATDC 5细胞中，我们发现细胞周期蛋白D1的表达上调，碱性磷酸酶（ALP）的表达下调，响应于细胞外Pi的增加在24 h内。此外，增加细胞外Pi诱导的Raf/MEK/ERK途径的激活，并与MEK抑制剂PD 98059的治疗取消了对细胞周期蛋白D1和ALP的表达的影响，表明细胞外Pi通过Raf/MEK/ERK途径调节这些基因的表达。与其上调细胞周期蛋白D1的表达一致，细胞外Pi促进ATDC 5细胞的增殖。磷酸化钠/磷酸盐（Na+/Pi）协同转运蛋白抑制剂膦甲酸（PFA）的治疗，废除了Raf/MEK/ERK通路的激活和细胞外Pi增加诱导的基因表达。敲低III型Na+/Pi协同转运蛋白Pit-1降低了ATDC 5细胞对细胞外Pi增加的反应性。有趣的是，增加的细胞外Pi诱导成纤维细胞生长因子受体底物2 α（FRS 2 α）的磷酸化，这也被Pit-1表达的敲低所抵消。在从小鼠肋骨笼中分离的原代软骨细胞中，细胞外Pi的增加诱导ERK 1/2的磷酸化和细胞周期蛋白D1和ALP的表达的改变，这两者都被取消与PFA治疗。这些结果表明，细胞外Pi的信号传导由Pit-1和FRS 2 α介导，并导致Raf/MEK/ERK通路的激活和细胞周期蛋白D1表达的增加，这有利于未成熟软骨细胞的增殖。(C)2010年爱思唯尔公司All rights reserved.
As chondrocytes mature, the concentration of inorganic phosphate (Pi) increases in the extracellular milieu. It was demonstrated that the progressive accumulation of Pi started from the proliferative zone and peaked in the hypertrophic zone of growth plate. Although extracellular Pi is reported to be involved in the apoptosis and mineralization of mature chondrocytes, its role in proliferating chondrocytes remains unclear. Here we investigated this role utilizing ATDC5, an established cell model of chondrocytic differentiation. In proliferating ATDC5 cells, we found that the expression of cyclin D1 was up-regulated, and that of alkaline phosphatase (ALP) was down-regulated in response to an increase in extracellular Pi within 24 h. Moreover, an increase in extracellular Pi-induced activation of the Raf/MEK/ERK pathway, and treatment with a MEK inhibitor PD98059 abolished the effects on the expression of cyclin D1 and ALP, indicating that extracellular Pi regulates the expression of these genes through the Raf/MEK/ERK pathway. Consistent with its up-regulation of cyclin D1 expression, the extracellular Pi facilitated the proliferation of ATDC5 cells. Treatment with phosphonoformic acid (PFA), an inhibitor of sodium/phosphate (Na+/Pi) cotransporters, abrogated the activation of the Raf/MEK/ERK pathway and gene expression induced by the increase in extracellular Pi. Knocking down of the type III Na+/Pi cotransporter Pit-1 diminished the responsiveness of ATDC5 cells to the increase in extracellular Pi. Interestingly, the increased extracellular Pi induced the phosphorylation of fibroblast growth factor receptor substrate 2 alpha (FRS2 alpha), which was also cancelled by knocking down of the expression of Pit-1. In primary chondrocytes isolated from mouse rib cages as well, increased extracellular Pi induced the phosphorylation of ERK1/2 and alterations in the expression of cyclin D1 and ALP, both of which were abolished by treatment with PFA. These results suggest that signaling by extracellular Pi is mediated by Pit-1 and FRS2 alpha, and leads to activation of the Raf/MEK/ERK pathway and increased expression of cyclin D1, which facilitates the proliferation of immature chondrocytes. (C) 2010 Elsevier Inc. All rights reserved.