The osteoclast is the cell that mediates bone destruction and is a major contributor to healthy bone turnover. The integrity of bone metabolism in individuals is intimately associated with tight regulation of osteoclast development and function. Dysregulation of osteoclastogenesis has been linked to common diseases such as osteoporosis and periarticular bone destruction associated with rheumatoid arthritis, as well as periprosthetic osteolysis. These conditions are devastating for patients and place a significant burden on the healthcare system. Therefore, it is critical to understand the molecular signals and pathways that drive osteoclastogenesis in order to develop new and effective ways to combat these diseases. NF-kappaB is a family of transcription factors whose activity orchestrates the cellular inflammatory program, and its function is required for osteoclastogenesis. Extracellular signals such as RANKL, TNF- alpha, LPS, and M-CSF stimulate NF-kappaB activity through the activation of the l-kappaBalpha Kinase (IKK) complex, comprised of IKK-alpha, IKK-beta, and IKK-gamma/NEMO. IKK-alpha activity is important for activation of the non-classical NF-kappaB pathway, and its function is considered less important in osteoclastogenesis than that of IKK-beta. IKK-beta is a kinase whose activity is required for activation of the classical NF-kappaB pathway. A tissue-specific knockout mouse for IKK-beta in osteoclasts demonstrates an ovewhelming defect in osteoclastogenesis, but the precise role that this molecule plays is not fully understood. Furthermore, the essential components of the IKK-beta molecule which mediate osteoclastogenesis have not been described. The proposed project has three specific aims: 1) Identify mutations in IKK-beta which affect the activity of the molecule and describe mechanism of this effect. Site directed mutagenesis will be used to create novel mutant IKK-beta constructs, and their activity will be characterized biochemically by kinase assays, electromobility shift assays, co-ip assays, and luciferase assays. 2) Determine the effect of these mutations in the development of the osteoclast. These mutant constructs will be subcloned into viral vectors used to infect primary bone marrow macrophages to determine their effect on osteoclastogenesis. An osteoclast- specific knockout for IKK-beta will also be used to test these constructs for rescue of osteoclastogenesis. 3)Characterize the effect of these mutant forms of IKK-beta in the pathogenesis of inflammatory osteolysis. Mouse models for inflammatory arthritis and periprosthetic osteolysis will be infected with viruses containing the mutant IKK-beta constructs to determine the effect of the IKK2 mutations in the setting of disease. ? ? ?
| Darwech, Isra; Otero, Jesse E; Alhawagri, Muhammad A et al. (2010) Tyrosine phosphorylation is required for IkappaB kinase-beta (IKKbeta) activation and function in osteoclastogenesis. J Biol Chem 285:25522-30 |
| Otero, Jesse E; Dai, Simon; Alhawagri, Muhammad A et al. (2010) IKKbeta activation is sufficient for RANK-independent osteoclast differentiation and osteolysis. J Bone Miner Res 25:1282-94 |
| Otero, Jesse E; Dai, Simon; Foglia, Domenica et al. (2008) Defective osteoclastogenesis by IKKbeta-null precursors is a result of receptor activator of NF-kappaB ligand (RANKL)-induced JNK-dependent apoptosis and impaired differentiation. J Biol Chem 283:24546-53 |
