Department of Pathology, University of Pittsburgh, and Veterans Affairs Medical Center, Pittsburgh, PA, USA.
Lab Invest. 2010 Oct;90(10):1533-42. doi: 10.1038/labinvest.2010.120. Epub 2010 Jun 21.
Osteoclast activity is central to balanced bone turnover to maintain normal bone mass. A specialized osteoclast attachment to bone localizes acid secretion to remove bone mineral; in some cases, attachment is functionally impaired despite normal attachment proteins. The inositol-1,4,5-trisphosphate receptor-1 (IP3R1) is an intracellular calcium channel required for regulation of reversible osteoclast attachment by nitric oxide (NO), an important regulator of both normal and pathological bone degradation. In studies using human osteoclasts produced in vitro, we found that IP3R1 binds an endosomal isoform of the IP3R-associated cGMP-dependent kinase substrate (IRAG). IRAG is a substrate of cGMP-dependent kinase-1 (PKG1) and binds the PKG1 isoform PKG1β, which was the predominant form of PKG1 in human osteoclasts. Western blots of IRAG were consistent with NO-dependent serine phosphorylation of IRAG. An additional effect of PKG1β activity in osteoclasts was disassociation of IP3R1-IRAG complexes, as shown by analysis of IP3R1 complexes and by localization of the proteins within cells. IP3R1-IRAG complexes were stabilized by PKG or Src antagonists, Src activity being a requirement for IP3R1 calcium release downstream of PKG. IP3R1-mediated calcium release regulates cellular detachment in part through the calcium-dependent proteinase μ-calpain. In osteoclasts with IRAG suppressed by siRNA, activity of μ-calpain was increased relative to cells with normal IRAG, and regulation of μ-calpain by NO was lost. Furthermore, cells deficient in IRAG detached easily from substrate and had smaller attached diameters and randomly distributed podosomes, although IRAG knockdown did not affect cell viability. Our results indicate that IRAG is required for PKG1β-regulated cyclic calcium release during motility, and that disruption of the IP3R1-IRAG calcium regulation system is a novel cause of dysfunctional osteoclasts unrelated to defects in attachment proteins or acid secretion.
破骨细胞的活性对于维持正常骨量的平衡骨转换至关重要。破骨细胞与骨的特殊附着将酸分泌到骨矿物质中；在某些情况下，尽管附着蛋白正常，但附着功能受损。肌醇 1,4,5-三磷酸受体 1（IP3R1）是一种细胞内钙通道，对于一氧化氮（NO）调节可逆性破骨细胞附着是必需的，NO 是正常和病理性骨降解的重要调节剂。在使用体外产生的人类破骨细胞进行的研究中，我们发现 IP3R1 与 IP3R 相关的 cGMP 依赖性激酶底物（IRAG）的内体同工型结合。IRAG 是 cGMP 依赖性激酶-1（PKG1）的底物，并且与 PKG1 同工型 PKG1β结合，PKG1β是人类破骨细胞中 PKG1 的主要形式。IRAG 的 Western blot 与 IRAG 的 NO 依赖性丝氨酸磷酸化一致。PKG1β 在破骨细胞中的另一种作用是 IP3R1-IRAG 复合物的解离，这可以通过分析 IP3R1 复合物和蛋白质在细胞内的定位来证明。PKG 或 Src 拮抗剂稳定了 IP3R1-IRAG 复合物，而 Src 活性是 PKG 下游 IP3R1 钙释放所必需的。IP3R1 介导的钙释放通过钙依赖性蛋白酶 μ-钙蛋白酶调节细胞脱落，在部分通过钙依赖性蛋白水解酶 μ-钙蛋白酶调节细胞脱落。在用 siRNA 抑制 IRAG 的破骨细胞中，μ-钙蛋白酶的活性相对于具有正常 IRAG 的细胞增加，并且 NO 对 μ-钙蛋白酶的调节丧失。此外，IRAG 缺陷的细胞容易从基质上脱落，附着直径较小，并且有随机分布的足突，尽管 IRAG 敲低不影响细胞活力。我们的结果表明，IRAG 是 PKG1β 调节的运动过程中环状钙释放所必需的，并且破坏 IP3R1-IRAG 钙调节系统是一种与附着蛋白或酸分泌缺陷无关的功能失调性破骨细胞的新原因。
