Both viral and genetic factors have been implicated in the pathogenesis of Paget's disease (PD). However, the molecular mechanisms responsible for their effect on osteoclast (OCL) formation and activity in PD are unclear. It is our hypothesis that measles virus nucleoprotein (MVNP) mediates its effects in OCL precursors predominantly through increased NFicB signaling via activation of the two kB kinase (IKK)-related kinases IKKe (also called IKK-/) and TANK-binding kinase 1 (TBK1) and increased levels of RIP1 and TRAF2. This results in upregulation of IL-6 and a vitamin D receptor coactivator TAFM-17 and increases OCL formation. MVNP also drives multi-potential cells towards the OCL lineage through increased expression and activation of NFATd. The p62P392L mutation linked to PD enhances the effects of MVNP by further increasing NFxB signaling in response to TNF-ct and RANKL as well as increasing p38 MAPK activation in response to RANKL, TNF-alpha and 1,25-(OH)2D3. In support of this hypothesis, we recently found that MVNP expression increases RIP1, TRAF2, and p65 NFicB resulting in enhanced constitutive activation of NFicB, and increased NFicB responsivity to TNF-alpha and RANKL. We also found that MVNP increases NFATd and synergistically activates NFATcl-driven gene transcription, with either NFATd 'or RANKL, and induces IL-6 and TAFn-17. In addition, the p62p392L mutation activates NFicB and increases the sensitivity of OCL precursors to RANKL and TNF-alpha. MVNP also interacts with IKKe and TBK1 to activate NFKB but their role in PD are unknown. In this project we will: 1. Determine if MVNP activation of NFKB and induction of pagetic OCL formation results from alterations of the canonical pathway and/or utilization of an alternative pathway that involves activation of IKKepsilon/TBK1 and the effects of co-expression of MVNP and p62P392L on these pathways. 2. Determine if NFKB activation by MVNP is sufficient for induction of IL-6 or if other mechanisms are also necessary, and the effects of co-expression of p62R392L and MVNP on the molecular mechanlsm(s) identified above. 3. Determine if the synergy of MVNP with NFATd is due to modulation of NFATd activation or of a cofactor of NFATd. The studies of the molecular mechanisms responsible for MVNP and p62p392L effects on OCL formation outlined above' will provide important insight into the pathogenesis of PD and are directly linked to the cellular and in vivo studies proposed in Project 2,
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