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Measles virus nucleocapsid protein modulates the Signal Regulatory Protein-?1 (SIRP?1) to enhance osteoclast differentiation in Paget's disease of bone.

ABSTRACT: Paget's disease of bone (PDB) is a chronic localized bone disorder in an elderly population. Environmental factors such as paramyxovirus are implicated in PDB and measles virus nucleocapsid protein (MVNP) has been shown to induce pagetic osteoclasts (OCLs). However, the molecular mechanisms underlying MVNP stimulation of OCL differentiation in the PDB are unclear. We therefore determined the MVNP regulated gene expression profiling during OCL differentiation. Agilent microarray analysis of gene expression identified high levels of SIRP?1 (353-fold) expression in MVNP transduced human bone marrow mononuclear cells stimulated with RANKL. Real-time PCR analysis further confirmed that MVNP alone upregulates SIRP?1 mRNA expression in these cells. Also, bone marrow mononuclear cells derived from patients with PDB showed high levels of SIRP?1 mRNA expression compared to normal subjects. We further show that MVNP increases SIRP?1 interaction with DAP12 adaptor protein in the presence and absence of RANKL stimulation. shRNA knockdown of SIRP?1 expression in normal human bone marrow monocytes decreased the levels of MVNP enhanced p-Syk and c-Fos expression. In addition, SIRP?1 knockdown significantly decreased MVNP stimulated dendritic cell-specific transmembrane protein (DC-STAMP) and connective tissue growth factor (CTGF) mRNA expression during OCL differentiation. Furthermore, we demonstrated the contribution of SIRP?1 in MVNP induced OCL formation and bone resorption. Thus, our results suggest that MVNP modulation of SIRP?1 provides new insights into the molecular mechanisms which control high bone turnover in PDB.

SUBMITTER: Sundaram K

PROVIDER: S-EPMC5558424 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Measles virus nucleocapsid protein modulates the Signal Regulatory Protein-β1 (SIRPβ1) to enhance osteoclast differentiation in Paget's disease of bone.

Sundaram Kumaran K Sambandam Yuvaraj Y Shanmugarajan Srinivasan S Rao D Sudhaker DS Reddy Sakamuri V SV

Bone reports 20160614

Paget's disease of bone (PDB) is a chronic localized bone disorder in an elderly population. Environmental factors such as paramyxovirus are implicated in PDB and measles virus nucleocapsid protein (MVNP) has been shown to induce pagetic osteoclasts (OCLs). However, the molecular mechanisms underlying MVNP stimulation of OCL differentiation in the PDB are unclear. We therefore determined the MVNP regulated gene expression profiling during OCL differentiation. Agilent microarray analysis of gene ...[more]

PMID: 28840181

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Project description:Paget's disease of bone (PDB) is characterized by abnormal osteoclasts with unique characteristics that include increased sensitivity of osteoclast progenitors to 1,25(OH)2 D3 , receptor activator of NF-?B ligand (RANKL), and TNF-?; increased osteoclast numbers; and increased expression of IL-6 and several transcription factors. We recently reported that measles virus nucleocapsid protein (MVNP) plays a key role in the development of these abnormal osteoclasts. MVNP can induce the pagetic osteoclast phenotype in vitro and in vivo in TRAP-MVNP transgenic mice. However, the molecular mechanisms by which MVNP generates pagetic osteoclasts have not been determined. TANK-binding kinase 1 (TBK1) and I?B kinase-? (IKK?) are IKK family members that complex with MVNP and activate both IRF3 and NF-?B pathways. MVNP increases the amount of TBK1 protein in bone marrow monocytes (BMM). Interestingly, we found that RANKL increased TBK1 and IKK? early in osteoclast differentiation, suggesting a possible role in normal osteoclastogenesis. However, only TBK1 is further increased in osteoclasts formed by TRAP-MVNP BMM owing to increased TBK1 protein stability. TBK1 overexpression induced IL6 promoter reporter activity, and elevated endogenous IL6 mRNA and p65 NF-?B, TAF12, and ATF7 proteins in several cell lines. Overexpression of TBK1 was insufficient to induce pagetic osteoclasts from WT BMM but synergized with MVNP to increase pagetic osteoclast formation from TRAP-MVNP BMM. BX795 inhibition of TBK1 impaired MVNP-induced IL-6 expression in both NIH3T3 cells and BMM, and shRNA knockdown of Tbk1 in NIH3T3 cells impaired IL-6 secretion induced by MVNP and decreased TAF12 and ATF7, factors involved in 1,25(OH)2 D3 hypersensitivity of pagetic osteoclasts. Similarly, Tbk1 knockdown in BMM from TRAP-MVNP and WT mice specifically impaired development of the MVNP-induced osteoclast pagetic phenotype. These results demonstrate that TBK1 plays a critical role in mediating the effects of MVNP on osteoclast differentiation and on the expression of IL-6, a key contributor to the pagetic osteoclast phenotype.

Project description:Measles virus plays an important role as an environmental factor in the pathogenesis of Paget's disease (PD). Previous studies have shown that IL-6 is increased in the bone marrow of Paget's patients and that measles virus nucleocapsid protein (MVNP) induces IL-6 secretion by pagetic osteoclasts. Further, IL-6 plays a critical role in the development of pagetic osteoclasts and bone lesions induced by PD, but the mechanisms regulating IL-6 production by MVNP remain unclear. Our current studies revealed that MVNP expression in osteoclast precursors down-regulated Sirt1 mRNA and protein, a negative regulator of NF-?B activity, which is a key factor for IL-6 expression. MVNP expression in NIH3T3 cells also elevated Il-6 transcription and impaired the expression of Sirt1 mRNA both under basal conditions and upon activation of the Sirt1 upstream regulator FoxO3 by LY294002 (a PI3K/AKT inhibitor). Luciferase activity assays showed that constitutively active FoxO3 abolished the repressive effect of MVNP on reporters driven by either FoxO3 response elements or the Sirt1 promoter. Further, protein stability assays revealed that FoxO3 was degraded more rapidly in MVNP-expressing cells than in control cells following the addition of cycloheximide. Similarly, co-transfection of MVNP and FoxO3 into HEK293 cells demonstrated that MVNP decreased the protein levels of over-expressed FoxO3 in a dose-dependent manner. Treatment with the proteasome inhibitor, MG132, blocked the MVNP-triggered decrease of FoxO3, and the treatment with the serine/threonine phosphatase inhibitor, calyculin A, revealed that MVNP increased phosphorylation of FoxO3. Further, over-expression of Sirt1 or treatment with the Sirt1 activator resveratrol blocked the increase in Il-6 transcription by MVNP. Finally, resveratrol reduced the numbers of TRAP positive multi-nuclear cells in bone marrow cultures from TRAP-MVNP transgenic mice to wild type levels. These results indicate that MVNP decreases FoxO3/Sirt1 signaling to enhance the levels of IL-6, which in part mediate MVNP's contribution to the development of Paget's disease.

Project description:BACKGROUND:Signal Regulatory Protein ? (SIRP?) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRP? is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRP?. RESULTS:We obtained crystals of SIRP? by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRP? and SIRP?. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRP? and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRP? and SIRP? but not SIRP?. Comparison of the three domain structures of SIRP? and SIRP? showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRP? in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRP? were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRP?: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd?=?1.2 +/- 0.3 ?M). CONCLUSION:The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRP? stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Dataset Information

Measles virus nucleocapsid protein modulates the Signal Regulatory Protein-?1 (SIRP?1) to enhance osteoclast differentiation in Paget's disease of bone.

Publications

Measles virus nucleocapsid protein modulates the Signal Regulatory Protein-β1 (SIRPβ1) to enhance osteoclast differentiation in Paget's disease of bone.

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