LNX2 Regulates Osteoclastogenesis In Vitro Through Numb/Notch Pathway

BACKGROUND Skeletal homeostasis in adults is maintained by a constant process called bone remodeling, in which old or damaged bone is removed by hematopoietic-derived osteoclasts followed by the deposit of new bone from mesenchymal-derived osteoblasts. Imbalance in these two processes leads to either loss of bone mass, in the case of osteoporosis, rheumatoid arthritis, and Paget’s disease of the bone, or to accumulation of disorganized bone mass, as in osteopetrosis and osteosclerosis. The generation, lifespan, and activity of osteoclasts and osteoblasts are regulated by systemic hormones as well as autocrine and paracrine cytokines and growth factors that are released from the bone matrix, secreted by neighboring cells in the bone microenvironment, or are anchored in the membrane of these cells.The Notch signaling pathway is evolutionarily conserved and plays a critical role in the determination of cell fate during embryonic development and postnatal tissue homeostasis. Notch signaling regulates proliferation, differentiation and apoptosis in a cell-cell contact dependent manner. Four Notch receptors and seven ligands have been identified in mammals so far. Both Notch receptors and ligands are single-span transmembrane proteins localized at the cell surface. Interaction of Notch receptor-ligand triggers two sequential proteolytic cleavages: first by a metalloproteinase ADAM(a disintegrin and metalloproteinase) and the second by a γ-secretase complex. As a result, the Notch intracellular domain(NICD) is released from the plasma membrane and translocates to the nucleus where it binds to the transcription factor CSL(CBF1/RBPJκ, Su, and LAG1) and induces the expression of Notch target genes, including members of Hes and Hey families.Recent genetic studies in human and mice have reinforced an essential role of Notch signaling in bone development and homeostasis. in vivo studies, using mouse models with gain- and loss-of-function of Notch signaling in osteoblast lineage cells, have revealed that Notch stimulates proliferation in osteoblast precursors but inhibits their final differentiation to mature osteoblasts.Nonetheless, in vitro studies have suggested that Notch signaling may exert opposite effects on osteoblastic cells to those described in vivo. Deletion of Notch receptors in osteoclast precursor cells enhances osteoclast formation and bone resorption, suggesting an inhibitory role of Notch in osteoclastogenesis.On the other hand, activation of Notch signaling, especially Notch2, promotes osteoclast differentiation.Numb, a membrane-associated adaptor protein critical for cell-fate determination, inhibits Notch signaling through the regulation of Notch endocytosis/recycling and the ubiquitin/proteasome degradation of NICD. Numb binds to the Notch receptor and components of the Clathrin-dependent endocytic complexes. Numb also helps to recruit several E3 ubiquitin ligases to the membrane-tethered Notch1 receptor and promote Notch1 degradation.LNX(ligand of numb protein X) 1 and 2 are Ring finger and PDZ domain containing E3 ubiquitin ligases, sharing extensive homology with each other. LNX1 and LNX2 bind to Numb and foster its ubiquitylation, leading to proteasome-dependent degradation of Numb. LNX proteins may enhance Notch signaling by lowering the level of Numb. The expression of LNX and Numb, and whether they regulate the Notch pathway in osteoclasts have not been examined and reported.The experiment is divided into three parts:THE FIRST PARTObjective:To determine the expression level of LNX induced changes in the differentiation of osteoclasts.Methods: We use the methods of inducing bone marrow mononuclear macrophage by Rankl in vitro,and check the foramation and activity of osteoclasts.Then we detect gene expression of LNX in the osteoclast differention and formation.Results: The methods of inducing bone marrow mononuclear macrophage by Rankl in vitro can generate osteoclasts with bone absorption ability. During osteoclast differentiation,both LNX1 and LNX2 gene level increased,while the expression level of LNX2 was significantly higher then LNX1.Conclusion: LNX2 may be for osteoclast differentiation and played an important role in regulating.THE SECOND PARTObjective: To observe the effect of silencing LNX2 on the differentiation and function of osteoclasts.Try to understand the possible mechanisms of LNX2 regulation of osteoclast differentiation and function.Methods: We construct lentivirus containing LNX2 sh RNA, then we transfect lentivirus to bone marrow mononuclear macrophage and induce them to osteoclast differentiation. Effects on the differentiation and function of osteoclasts are observed after silencing the LNX2 gene. Western Blot, RT-PCR, immunofluorescence are studied of LNX2 related Numb protein expression and the changes of Notch signal pathway.Results: After silencing the LNX2, differentiation and function of osteoclasts were effectively depressed,osteoclast markergene and protein level decreased; the silence of LNX2 made the Numb protein level increased, immunofluorescence showed Numb expression was significantly elevated with a large number of distribution around the cell membrane and nucleus. NICD1 and NICD2 expression levels were reduced at the same time. Immunofluorescence showed that Notch2 significantly was reduced with decreased expression level in the perinuclear distribution.Hes1 gene,downstream of Notch signaling pathways was also down-regulated.Conclusion:LNX2 is a key protein required for osteoclast differentation.LNX2 regulate osteoclast in vitro through Numb/Notch Pathway.THE THIRD PARTObjective: To observe the effect of overexpression of LNX2 on the differentiation and function of osteoclasts,try to understand of the possible mechanisms.Methods:We construct retrovirus containing the full-length LNX2 DNA, and transfect retrovirus to bone marrow mononuclear macrophages. Then we induce them to osteoclast differentiation.Effects on the differentiation and function of osteoclasts are observed after silencing the LNX2.Western Blot,RT-PCR,immunofluorescence are studied of LNX2 related Numb protein expression.Results: Overexpressing full-length LNX2, osteoclasts differentiation were partially inhibited, but the inhibition degree is far lower than the silence of LNX2 inhibited, osteoclast markers and protein levels decreased. The expression level of Numb protein was partially up-regulated.Conclusion: Overexpression of full-length LNX2 up-regulate Numb level and down-regulate osteoclast differentiation.Final Conclusion: LNX2 regulate osteoclast in vitro through Numb/Notch Pathway, indicating that LNX2 is a key protein required for osteoclast differentation. Overexpression of LNX2 up-regulated the Numb level, osteoclasst differentiation is partial inhibited, There are no mechanistic explanations for these results at the moment. Future work will be required to identify LNX2 interacting proteins and substrates in osteoclast lineage cells.