Summary Osteoclasts resorb bone via the ruffled border whose complex folds are generated by secretory lysosome fusion with bone-apposed plasma membrane. Lysosomal fusion with the plasmalemma results in acidification of the resorptive microenvironment and release of CatK to digest the organic matrix of bone. The means by which secretory lysosomes are directed to fuse with the ruffled border are enigmatic. We show that proteins essential for autophagy including Atg5, Atg7, Atg4B and LC3, are important for generating the osteoclast ruffled border, the secretory function of osteoclasts and bone resorption in vitro and in vivo. Further, Rab7 which is required for osteoclast function, localizes to the ruffled border in an Atg5-dependent manner. Thus, autophagy proteins participate in polarized secretion of lysosomal contents into the extracellular space by directing lysosomes to fuse with the plasma membrane. These findings are in keeping with a putative link between autophagy genes and human skeletal homeostasis.

In this study, we establish that the tyrosine kinase Syk is essential for osteoclast function in vitro and in vivo. Syk−/− osteoclasts fail to organize their cytoskeleton, and, as such, their bone-resorptive capacity is arrested. This defect results in increased skeletal mass in Syk−/− embryos and dampened basal and stimulated bone resorption in chimeric mice whose osteoclasts lack the kinase. The skeletal impact of Syk deficiency reflects diminished activity of the mature osteoclast and not impaired differentiation. Syk regulates bone resorption by its inclusion with the αvβ3 integrin and c-Src in a signaling complex, which is generated only when αvβ3 is activated. Upon integrin occupancy, c-Src phosphorylates Syk. αvβ3-induced phosphorylation of Syk and the latter's capacity to associate with c-Src is mediated by the immunoreceptor tyrosine-based activation motif (ITAM) proteins Dap12 and FcRγ. Thus, in conjunction with ITAM-bearing proteins, Syk, c-Src, and αvβ3 represent an essential signaling complex in the bone-resorbing osteoclast, and, therefore, each is a candidate therapeutic target.

A quantitative genetic model relates the genotypic value of an individual to the alleles at the loci that contribute to the variation in a population in terms of additive, dominance, and epistatic effects. This partition of genetic effects is related to the partition of genetic variance. A number of models have been proposed to describe this relationship: some are based on the orthogonal partition of genetic variance in an equilibrium population. We compare a few representative models and discuss their utility and potential problems for analyzing quantitative trait loci (QTL) in a segregating population. An orthogonal model implies that estimates of the genetic effects are consistent in a full or reduced model in an equilibrium population and are directly related to the partition of the genetic variance in the population. are many ways to define a QTL model, thus additive, A similar argument has been made for epistasis (Chevdominance, and epistatic effects. The models compared erud and Routman 1995). On the one hand, we have by Van Der Veen (1959) are all based on genotypic the model proposed by Hayman and Mather (1955) values only, so to speak. and discussed in length in Mather and Jinks (1982),The purpose of modeling QTL, of course, is to provide a way to summarize and interpret the differences between the genotypic values and also the genetic varia-1

Lipopolysaccharide (LPS) modulates bone resorption by augmentation of osteoclastogenesis. It increases in osteoblasts the production of RANKL, interleukin (IL)-1, prostaglandin E 2 (PGE 2 ), and TNF-␣, each known to induce osteoclast activity, viability, and differentiation. We examined the role of direct interactions of LPS with osteoclast precursors in promoting their differentiation. To this end, we have used bone marrow mononuclear cell preparations in the absence of osteoblasts or stromal cells. We found that LPS does not induce osteoclast differentiation in these cells. Moreover, the inclusion of LPS blocked the osteoclastogenic activity of RANKL. However, LPS is a potent inducer of osteoclastogenesis in RANKL-pretreated cells, even if present in the absence of exogenous RANKL. Osteoprotegerin (OPG) does not affect the stimulatory phase of LPS modulation of osteoclastogenesis, ruling out involvement of endogenous RANKL. LPS induces the expression of TNF-␣ and IL-1␤ in osteoclast precursors, regardless if they were or were not pretreated with RANKL. These two cytokines induced osteoclast differentiation in RANKL-pretreated cells. To examine if these cytokines mediate LPS effect in an autocrine mechanism, we measured the effect of their neutralization on LPS osteoclastogenic activity. Although neutralization of IL-1␤ did not affect LPS activity, a marked inhibition was observed when TNF-␣ was neutralized. However, TNF-␣ expression was increased also in conditions in which LPS inhibited RANKL osteoclastogenic activity. We found that LPS reduces the expression of RANK and macrophage colony-stimulating factor (M-CSF) receptor. In summary, LPS impacts on osteoclastogenesis also via its interactions with the precursor cells. LPS inhibits RANKL activity by reducing the expression of RANK and M-CSF receptor and stimulates osteoclastogenesis in RANKL-pretreated cells via

NOTCH signaling is a key regulator of cell fate decisions in prenatal skeletal development and is active during adult tissue renewal. In addition, its association with neoplasia suggests that it is a candidate therapeutic target. We find that attenuated NOTCH signaling enhances osteoclastogenesis and bone resorption in vitro and in vivo by a combination of molecular mechanisms. First, deletion of Notch1-3 in bone marrow macrophages directly promotes their commitment to the osteoclast phenotype. These osteoclast precursors proliferate more rapidly than the wild type in response to macrophage colony-stimulating factor and are sensitized to RANKL and macrophage colonystimulating factor, undergoing enhanced differentiation in response to low doses of either cytokine. Conforming with a role for NOTCH in this process, presentation of the NOTCH ligand JAGGED1 blunts the capacity of wild-type bone marrow macrophages to become osteoclasts. Combined, these data establish that NOTCH suppresses osteoclastogenesis via ligand-mediated receptor activation. Although NOTCH1 and NOTCH3 collaborate in regulating osteoclast formation, NOTCH1 is the dominant paralog. In addition, NOTCH1 deficiency promotes osteoclastogenesis indirectly by enhancing the ability of osteoblast lineage cells to stimulate osteoclastogenesis. This is achieved by decreasing the osteoprotegerin/RANKL expression ratio. Thus, NOTCH1 acts as a net inhibitor of bone resorption, exerting its effect both directly in osteoclast precursors and indirectly via osteoblast lineage cells. These observations raise caution that therapeutic inhibition of NOTCH signaling may adversely accelerate bone loss in humans.NOTCH signaling is an evolutionarily conserved pathway that profoundly impacts mammalian development by regulating survival, proliferation, and cell fate decision in a context-dependent manner. It contributes to tissue maintenance and/or renewal in the adult intestine (1), skin (2), hematopoietic system (3), mammary epithelium (4), and central nervous system (5) and can either promote (6 -9) or suppress (10) cancer.There are four NOTCH receptors (NOTCH1-4) and at least seven NOTCH ligands (JAGGED1, JAGGED2, DLL1 (Deltalike1), DLL3, DLL4, and DNER (11) and contactin/F3/NB-3 (12)) in mice and humans. The receptors and ligands are singlepass transmembrane proteins expressed on the surface of adjacent cells. Activation of NOTCH signaling requires cell/cell contact because ligand binding to specific epidermal growth factor-like repeats in the extracellular domain of NOTCH receptors must induce a conformational change (13), most likely by trans-endocytosis (14), to expose the juxtamembrane region to cleavage by ADAM metalloproteases. The exposed N terminus is recognized by ␥-secretase (15), which cleaves NOTCH again within its transmembrane domain. This cleavage releases the NOTCH intracellular domain (NICD), 2 which translocates to the nucleus, where it associates with the DNAbinding protein CSL and other transcriptional coactivators. This complex is responsible for t...

NF-B inducing kinase (NIK) is required for osteoclastogenesis in response to pathologic stimuli, and its loss leads to functional blockade of both alternative and classical NF-B caused by cytoplasmic retention by p100. We now show that deletion of p100 restores the capacity of NIK-deficient osteoclast (OC) precursors to differentiate and normalizes RelB and p65 signaling. Differentiation of NIK؊/؊ precursors is also restored by overexpression of RelB, but not p65. Additionally, RelB؊/؊ precursors fail to form OCs in culture, and this defect is rescued by re-expression of RelB, but not by overexpression of p65. To further support the role of RelB in OCs, we challenged RelB؊/؊ mice with TNF-␣ in vivo and found a diminished osteoclastogenic response. We then examined tumor-induced osteolysis in both RelB؊/؊ and NIK؊/؊ mice by using the B16 melanoma model. Growth of tumor cells in the bone marrow was similar to WT controls, but the absence of either RelB or NIK completely blocked the tumor-induced loss of trabecular bone. Thus, the alternative NF-B pathway, culminating in activation of RelB, has a key and specific role in the differentiation of OCs that cannot be compensated for by p65.bone ͉ metastasis ͉ receptor activator of NF-B ligand

Summary We examined the mechanism by which M-CSF regulates the cytoskeleton and function of the osteoclast, the exclusive bone resorptive cell. We show that binding of M-CSF to its receptor c-Fms generates a signaling complex comprising phosphorylated DAP12, an adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) and the non-receptor tyrosine kinase Syk. c-Fms tyrosine 559, the exclusive binding site of c-Src, is necessary for regulation of DAP12/Syk signaling. Deletion of either of these molecules yields osteoclasts that fail to reorganize their cytoskeleton. Retroviral transduction of null precursors with wild type or mutant DAP12 or Syk reveals that the SH2 domain of Syk and the ITAM tyrosine residues and transmembrane domain of DAP12 mediate M-CSF signaling. Our data provide genetic and biochemical evidence that uncovers, an epistatic signaling pathway linking the receptor tyrosine kinase c-Fms to the immune adaptor DAP12 and the cytoskeleton.

Significant advances have been made in the discovery of genes affecting bone mineral density (BMD); however, our understanding of its genetic basis remains incomplete. In the current study, genome-wide association (GWA) and co-expression network analysis were used in the recently described Hybrid Mouse Diversity Panel (HMDP) to identify and functionally characterize novel BMD genes. In the HMDP, a GWA of total body, spinal, and femoral BMD revealed four significant associations (−log10P>5.39) affecting at least one BMD trait on chromosomes (Chrs.) 7, 11, 12, and 17. The associations implicated a total of 163 genes with each association harboring between 14 and 112 genes. This list was reduced to 26 functional candidates by identifying those genes that were regulated by local eQTL in bone or harbored potentially functional non-synonymous (NS) SNPs. This analysis revealed that the most significant BMD SNP on Chr. 12 was a NS SNP in the additional sex combs like-2 (Asxl2) gene that was predicted to be functional. The involvement of Asxl2 in the regulation of bone mass was confirmed by the observation that Asxl2 knockout mice had reduced BMD. To begin to unravel the mechanism through which Asxl2 influenced BMD, a gene co-expression network was created using cortical bone gene expression microarray data from the HMDP strains. Asxl2 was identified as a member of a co-expression module enriched for genes involved in the differentiation of myeloid cells. In bone, osteoclasts are bone-resorbing cells of myeloid origin, suggesting that Asxl2 may play a role in osteoclast differentiation. In agreement, the knockdown of Asxl2 in bone marrow macrophages impaired their ability to form osteoclasts. This study identifies a new regulator of BMD and osteoclastogenesis and highlights the power of GWA and systems genetics in the mouse for dissecting complex genetic traits.