Dataset Information

Necessity of inositol (1,4,5)-trisphosphate receptor 1 and mu-calpain in NO-induced osteoclast motility.

ABSTRACT: In skeletal remodeling, osteoclasts degrade bone, detach and move to new locations. Mechanical stretch and estrogen regulate osteoclast motility via nitric oxide (NO). We have found previously that NO stimulates guanylyl cyclase, activating the cGMP-dependent protein kinase 1 (PKG1), reversibly terminating osteoclast matrix degradation and attachment, and initiating motility. The PKG1 substrate vasodilator-stimulated protein (VASP), a membrane-attachment-related protein found in complexes with the integrin alphavbeta3 in adherent osteoclasts, was also required for motility. Here, we studied downstream mechanisms by which the NO-dependent pathway mediates osteoclast relocation. We found that NO-stimulated motility is dependent on activation of the Ca(2+)-activated proteinase mu-calpain. RNA interference (RNAi) showed that NO-dependent activation of mu-calpain also requires PKG1 and VASP. Inhibition of Src kinases, which are involved in the regulation of adhesion complexes, also abolished NO-stimulated calpain activity. Pharmacological inhibition and RNAi showed that calpain activation in this process is mediated by the inositol (1,4,5)-trisphosphate receptor 1 [Ins(1,4,5)P(3)R1] Ca(2+) channel. We conclude that NO-induced motility in osteoclasts requires regulated Ca(2+) release, which activates mu-calpain. This occurs via the Ins(1,4,5)P(3)R1.

SUBMITTER: Yaroslavskiy BB

PROVIDER: S-EPMC2976040 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Necessity of inositol (1,4,5)-trisphosphate receptor 1 and mu-calpain in NO-induced osteoclast motility.

Yaroslavskiy Beatrice B BB Sharrow Allison C AC Wells Alan A Robinson Lisa J LJ Blair Harry C HC

Journal of cell science 20070801 Pt 16

In skeletal remodeling, osteoclasts degrade bone, detach and move to new locations. Mechanical stretch and estrogen regulate osteoclast motility via nitric oxide (NO). We have found previously that NO stimulates guanylyl cyclase, activating the cGMP-dependent protein kinase 1 (PKG1), reversibly terminating osteoclast matrix degradation and attachment, and initiating motility. The PKG1 substrate vasodilator-stimulated protein (VASP), a membrane-attachment-related protein found in complexes with t ...[more]

PMID: 17690304

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Project description: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.

Dataset Information

Necessity of inositol (1,4,5)-trisphosphate receptor 1 and mu-calpain in NO-induced osteoclast motility.

Publications

Necessity of inositol (1,4,5)-trisphosphate receptor 1 and mu-calpain in NO-induced osteoclast motility.

Similar Datasets

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