Chloride-hydrogen antiporters ClC-3 and ClC-5 drive osteoblast mineralization and regulate fine-structure bone patterning in vitro.
Chloride-hydrogen antiporters ClC-3 and ClC-5 drive osteoblast mineralization and regulate fine-structure bone patterning in vitro.
复制标题
氯化物 - 氢抗抗植物CLC-3和CLC-5驱动成骨细胞矿化，并在体外调节精细结构骨图案。
DOI：
10.14814/phy2.12607
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发表时间：
2015-11
影响因子：
2.5
通讯作者：
Blair HC
中科院分区：
文献类型：
--
作者：
Larrouture QC;Nelson DJ;Robinson LJ;Liu L;Tourkova I;Schlesinger PH;Blair HC
成骨细胞形成上皮样层，其紧密连接将骨基质与细胞外液分开。在矿物质沉积过程中，羟基磷灰石中的钙和磷酸盐沉淀会每摩尔 Ca+2 释放 0.8 摩尔 H+。因此，矿物形成需要酸输出。我们检查了支持成骨细胞矢量矿物质沉积的离子运输。之前我们确定 Na/H 交换剂 1 和 6 在分泌性成骨细胞基底外侧表面高度表达并中和大量酸负荷。 Na/H 交换调节因子 1 (NHERF1) 是一种 pdz 组织蛋白，存在于矿化成骨细胞基底外侧表面。我们假设必须存在从基质到成骨细胞胞质的高容量质子运输以支持酸转胞吞作用以进行矿物质沉积。矿化成骨细胞中的基因筛选显示氯质子逆向转运蛋白 ClC-3 和 ClC-5 的显着表达。抗体定位显示 ClC-3 和 ClC-5 出现在面向骨基质的顶端分泌表面和埋藏骨细胞的膜中。令人惊讶的是，Clcn3−/− 小鼠仅有轻度的矿化紊乱。然而，Clcn3−/− 成骨细胞的 ClC-5 表达有较大的代偿性增加。 Clcn3−/− 成骨细胞在体外以引人注目且新颖的小梁模式矿化；野生型成骨细胞形成骨结节。在 Clcn3−/− 小鼠的间充质干细胞中，慢病毒 ClC-5 shRNA 产生了 Clcn3−/−、ClC-5 敲低细胞，并通过蛋白质印迹和 PCR 进行了验证。在野生型或 Clcn3−/− 细胞矿化良好的条件下，这些细胞的成骨细胞不产生矿物质。我们得出的结论是，由氯-质子交换介导的受调节的酸输出对于驱动正常的骨矿化至关重要，并且 CLC 转运蛋白还调节骨的精细图案。
Osteoblasts form an epithelium-like layer with tight junctions separating bone matrix from extracellular fluid. During mineral deposition, calcium and phosphate precipitation in hydroxyapatite liberates 0.8 mole of H+ per mole Ca+2. Thus, acid export is needed for mineral formation. We examined ion transport supporting osteoblast vectorial mineral deposition. Previously we established that Na/H exchangers 1 and 6 are highly expressed at secretory osteoblast basolateral surfaces and neutralize massive acid loads. The Na/H exchanger regulatory factor-1 (NHERF1), a pdz-organizing protein, occurs at mineralizing osteoblast basolateral surfaces. We hypothesized that high-capacity proton transport from matrix into osteoblast cytosol must exist to support acid transcytosis for mineral deposition. Gene screening in mineralizing osteoblasts showed dramatic expression of chloride–proton antiporters ClC-3 and ClC-5. Antibody localization showed that ClC-3 and ClC-5 occur at the apical secretory surface facing the bone matrix and in membranes of buried osteocytes. Surprisingly, the Clcn3−/− mouse has only mildly disordered mineralization. However, Clcn3−/− osteoblasts have large compensatory increases in ClC-5 expression. Clcn3−/− osteoblasts mineralize in vitro in a striking and novel trabecular pattern; wild-type osteoblasts form bone nodules. In mesenchymal stem cells from Clcn3−/− mice, lentiviral ClC-5 shRNA created Clcn3−/−, ClC-5 knockdown cells, validated by western blot and PCR. Osteoblasts from these cells produced no mineral under conditions where wild-type or Clcn3−/− cells mineralize well. We conclude that regulated acid export, mediated by chloride–proton exchange, is essential to drive normal bone mineralization, and that CLC transporters also regulate fine patterning of bone.