Understanding the pathogenesis of Paget's Disease and the role that paramyxovirus may play is severely hampered by the lack of an in vivo model. Recently, we cloned and sequence-analyzed a full-length Measles Virus Nucleocapsid (MVNP) cDNA from bone marrow cells from a Paget's patient. The MVNP gene contained several sense mutations, which constituted 1% of the nucleotide sequence. Interestingly, partial sequence analysis of MVNP cDNAs isolated from three other patients from different geographic locales revealed similar mutations. However, the pathologic significance of these mutations, in terms of persistence of the virus or induction of abnormal osteoclast (OCL) formation and activity in Paget's disease is unknown. It is our hypothesis that the mutated MVNP expressed in OCL from Paget's patients participates in protein interactions and induces gene expression that result in pagetic OCL development. These effects cannot be mimicked by nucleocapsids from other paramyxovirus or non-paramyxovirus. Furthermore, specific structural motifs present within the MVNP gene are responsible for development of pagetic OCL. Thus, expression of the MVNP gene in OCL in the setting of increased OCL activity is sufficient for development of Paget's disease. Therefore, to test this hypothesis we will: (1.A) determine the effects of mutant MVNP expression on normal OCL formation and activity. If differences are noted, the mutations present in the pagetic MVNP will be sequentially introduce into the Edmonston strain MVNP gene to identify which mutations or structural motifs may be responsible for these differences; (1.B) determine the effects of nucleocapsid proteins derived from other paramyxoviruses, such as Canine Distemper Virus (CDV), or Respiratory Syncytial Virus (RSV) and the non-paramyxovirus Influenza on osteoclastogenesis; (1.C) determine if IL-6 is induced by the various nucleocapsid proteins (MVN, CDV, RS influenza) and if IL-6 is in part responsible for development of the pagetic OCL; (2) identify the OCL-specific gene products that interact with MVNP. As part of these studies, the expression of candidate genes identified above will be confirmed in pagetic bone marrow cells by RT-PCR. These candidate genes will then be over-expressed in normal OCL precursors to determine their capacity to induce the pagetic phenotype in OCL; (3) determine if constitutive expression of the MVNP gene from a patient with Paget's disease in cells of the OCL lineage in transgenic mice (TRAP-MVNP) is sufficient to induce pagetic-like bone lesions; (3.A) determine if treatment of these mice chronically for 5 days per month with parathyroid hormone-related protein (PTHrP) or RANK ligand (RANKL) to enhance OCL formation is required to develop pagetic lesions in these animals; (3.B) determine if local overproduction of RANK ligand by marrow stromal cells in these mice results in development of localized Paget's disease; (3.C) determine if marrow cells from TRAP-MVNP mice can form OCLs in vitro, which have features of pagetic OCLs, and if IL-6 expression is critical to this process; and (4) determine if chronic overproduction of IL-6 is in part responsible for development of the pagetic lesions, by breeding TRAP-MVNP mice either to transgenic mice in which IL-6 expression is targeted to cells in the OCL lineage (TRAP-IL-6) or to IL-6 knock-out mice.
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