The Role Of Transcription Factor RBP-J In Osteoclast Differentiation And Function

Its amazing that the bones of our human ancestors were well preservedoutside the body, but inside the body bone is remodeled at such a high rate.Approximately10%of the total bone content is replaced in adult humans peryear. Bone remodeling needs a delicate balance between bone-formingosteoblasts and bone-resorbing osteoclasts. Many diseases are caused byabnormal osteoclastogenesis, Such as erosive inflammatory arthritis, periodontaldiseases, metabolic bone diseases and more often is osteoporosis. Sounderstanding the molecular mechanisms underlying osteoclastogenesis iscritical to understand the molecular basis for the pathogenesis of these bonediseases with increased osteoclast activity. This will enrich our knowledge aboutthe prevention and treatment of these diseases. Osteoclasts are multinucleated giant cells that differentiate fromhematopoietic stem cells (HSCs) of the monocyte/macrophage lineage.Osteoclasts formation and maturation are under the control of macrophagecolony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand(RANKL). RANKL is a member of the tumor necrosis factor (TNF) family ofcytokines, and its expression is controlled by several bone-resorbing factorsincluding interleukin-1(IL-1), IL-6, and TNF-а. M-CSF support the survivaland proliferation of osteoclast precursor cells. RANKL is essential for thedifferentiation and activation of these cells.Notch signaling pathway is a highly conserved pathway that is involved incell fate determinations, cell proliferation,differentiation and apoptosis duringembryonic and postnatal stages. In mammals, when the Notch receptors(Notch1-4) are activated by the binding of the Notch ligands (Jagged1, Jagged2,and Delta-like (Dll)1,3, and4), the Notch intracellular domain (NICD) iscleaved by the proteinase complex containing γ-secretase and translocates to thenucleus. The NICD subsequently interacts with transcription factor RBP-J andMastermind-like proteins and initiates the expression of target genes such as theHes family basic helix-loop-helix members. So RBP-J is the key integrator ofNotch signaling. It has been reported that Notch signaling passway is involvedin bone remodeling. Although RBP-J is the key integrator of all four mammalianNotch receptors, the function of RBP-J in osteoclasts has rarely been explored.To clarify the role of RBP-J in osteoclasts differentiation and function, weused conditional alleles to genetically remove RBP-J in the bone marrow and inthe macrophages respectively. The main results are as follows:1. RBP-J floxed mice were mated with the Mx-Cre transgenic mice. In theadult Mx-Cre RBP-J+/f(WT) mice and Mx-Cre RBP-Jf/f(KO) mice, the expression of Cre was induced by the injection of the interferon (IFN)-α inducerpoly (I)–poly (C). After6-8weeks of the induction, the deletion was almostcomplete in the bone marrow. We find that the trabecular bone mass wasdecreased in KO mice. Furthermore, the amount of osteoclasts increased in thebone marrow. To exclude the possibility that the decreased bone mass was dueto decreased osteoblastic activity, we examined the mineral apposition rate(MAR) by using tetracycline and calcein double-labeling experiments. Theresult showed that the activity of osteoblasts were normal. These findingsdemonstrated that RBP-J deletion could promote osteoclast differentiation andlead to a phenomenon of osteopenia.2. To clarify the phenomenon of osteopenia caused by RBP-J deletion wasexclusively in macrophage, we mated RBP-J floxed mice with the LysM-Cretransgenic mice. Cre recombinase was driven by the myeloid-specific LysMpromoter, so the deletion of RBP-J was only restricted to macrophages.Osteopenia also happened in the LysM-Cre RBP-Jf/f(KO) mice and the numbersof osteoclast was also increased in the bone marrow of KO mice. These resultsshowed that RBP-J deletion in macrophages could really influence thedifferentiation of osteoclasts.3. In vitro,we cultured BMMs isolated from LysM-Cre RBP-Jf/f(KO) miceand control mice with50ng/ml M-CSF and50ng/ml RANKL. We found thatBMMs of KO mice differentiated into more osteoclasts and the activity ofosteoclasts was also enhance. To determine the molecular mechanisms of RBP-Jfunction in this context, we examined these key transcriptional regulators ofosteoblast differentiation and maturation. We found that TRAP expression wassignificantly increased in KO mice. All these results showed that RBP-J was anegatively regulator of osteoclasts differentiation. In conclusion, we used conditional alleles to genetically remove RBP-J inthe bone marrow and in the macrophages respectively. We found that thenumber of osteoclast in the bone marrow of KO mice was increased and itsactivity was also enhanced, so we could see osteopenia in the femurs of KOmice. TRAP gene expression was also significantly increased due to RBP-Jdeletion. Together our results showed that RBP-J was a negatively regulator inthe differentiation and function of osteoclasts. This will be helpful to thediscovery of novel molecular therapeutic approaches for these diseases.