Osteoclasts (OCs) are bone-resorbing multinuclear giant cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. Although many molecules are known to contribute to OC differentiation, RANKL chief among them, the mechanisms controlling the recruitment and homing of OC precursors (OPs) to the bone surface have not been elucidated. Here we report that sphingosine-1-phosphate (S1P) controls the movement of OPs between the blood and their site of differentiation. Cells with the properties of OPs express functional S1P1 receptors and exhibit positive chemotaxis along an S1P gradient in vitro. Intravital two-photon imaging of bone tissues revealed that a potent S1P1 agonist, SEW2871, stimulated motility of OP-containing monocytoid populations in vivo. OC/monocyte (CD11b) lineage-specific conditional S1P1 knockout mice showed osteoporotic changes due to increased OC attachment to bone surface, suggesting a crucial role of the S1P-S1P1 system in recirculation of OPs to blood where S1P levels are high. Furthermore, treatment with the S1P1 agonist FTY720 relieved ovariectomy-induced osteoporosis in mice by facilitating recirculation of OP-containing cell populations and reducing the number of mature OCs attached to the bone surface. This study provides evidence that S1P controls the migratory behavior of OPs, dynamically regulating bone mineral homeostasis, and identifies a critical control point in osteoclastogenesis that may be promising as a therapeutic target.

Cathepsin K (CTSK) is secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption. Global deletion of Ctsk in mice decreases bone resorption, leading to osteopetrosis, but also increases the bone formation rate (BFR). To understand how Ctsk deletion increases the BFR, we generated osteoclast-and osteoblast-targeted Ctsk knockout mice using floxed Ctsk alleles. Targeted ablation of Ctsk in hematopoietic cells, or specifically in osteoclasts and cells of the monocyte-osteoclast lineage, resulted in increased bone volume and BFR as well as osteoclast and osteoblast numbers. In contrast, targeted deletion of Ctsk in osteoblasts had no effect on bone resorption or BFR, demonstrating that the increased BFR is osteoclast dependent. Deletion of Ctsk in osteoclasts increased their sphingosine kinase 1 (Sphk1) expression. Conditioned media from Ctsk-deficient osteoclasts, which contained elevated levels of sphingosine-1-phosphate (S1P), increased alkaline phosphatase and mineralized nodules in osteoblast cultures. An S1P 1,3 receptor antagonist inhibited these responses. Osteoblasts derived from mice with Ctsk-deficient osteoclasts had an increased RANKL/OPG ratio, providing a positive feedback loop that increased the number of osteoclasts. Our data provide genetic evidence that deletion of CTSK in osteoclasts enhances bone formation in vivo by increasing the generation of osteoclast-derived S1P.

IntroductionThe osteoclast, the exclusive bone resorptive cell, is derived from hematopoietic stem cells through the common myeloid progenitor to the colony-forming unit for granulocytes and macrophages to the colony-forming unit for macrophages and into the osteoclast lineage. 1 Osteoclast differentiation, function, and survival are regulated by several exogenous cytokines (macrophage colonystimulating factor [M-CSF], receptor activator of nuclear factor B ligand [RANKL], tumor necrosis factor ␣, interleukin-1, and interleukin-6) and hormones (sex steroids, parathyroid hormone, vitamin D, insulinlike growth factor-1, calcitonin, and prostaglandins) through several transcription factors activities identified by studies in genetically engineered mice. 2,3 The transcription factor c-Fos plays an important role in osteoclastogenesis because mice lacking c-Fos develop osteopetrosis. 2-4 c-Fos is not required for normal osteoprogenitor development but is required for osteoclastogenesis. [2][3][4] Nuclear factor of activated T cells cytoplasmic 1 (NFATc1) is also a critical transcription factor that was discovered as an early RANKL-inducible gene by gene expression profiling after RANKL stimulation. 3 NFATc1 molecules act as cofactors with activator protein-1 (AP-1) composed of Fos/Jun proteins to bind to regulatory cis DNA elements, 5 and osteoclastspecific markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K have multiple sites recognized by NFATc1 as well as its partner AP-1. 5 MicroRNAs (miRs), which regulate gene expression, are transcribed as primary miRs (pri-miRs) containing a 5Ј-cap structure and poly(A) tail that are processed to produce the mature miRs. 6 Two nuclease enzymes, the nuclear RNaseIII Drosha and the cytosolic RNaseIII Dicer, are known to act sequentially to trim the miRs to mature form. 6 Hundreds of miR genes have been identified in the human genome, and it is estimated that one-third of protein-coding genes are regulated by miRs. Hence, miRs constitute one of the most abundant classes of gene-regulatory molecules in animals, and they are implicated in almost every biologic process, including development timing, cell differentiation, cell proliferation, cell death, metabolic control, transposon silencing, and antiviral defense. 6 Emerging evidence indicates that Dicer-generated miRs play important roles in osteoblastogenesis, chondrocyte proliferation and differentiation, and osteoclastogenesis. [7][8][9][10] Recent studies have shown that Ն 12 miRs, miR-26a, miR-125b, miR-133, miR-135, miR-29a, miR-141, miR-200a, miR-210, miR-29, miR-378, miR-2861, and miR-206, are implicated in osteoblast differentiation. [11][12][13][14][15][16][17][18][19][20] In particular, miR-2861 was identified as a novel miR expressed in mouse osteoblasts promoting osteoblast differentiation by suppressing expression of histone deacetylase 5 at the posttranslational level and contributing to bone formation. 19 Moreover, mutation of miR-2861 causes osteoporosis in humans, suggesting that miR-2861...

The spontaneous mouse grey-lethal (gl) mutation is responsible for a coat color defect and for the development of the most severe autosomal recessive form of osteopetrosis. Using a positional cloning approach, we have mapped and isolated the gl locus from a approximately 1.5 cM genetic interval. The gl locus was identified in a bacterial artificial chromosome (BAC) contig by functional genetic complementation in transgenic mice. Genomic sequence analysis revealed that the gl mutation is a deletion resulting in complete loss of function. The unique approximately 3 kb wild-type transcript is expressed primarily in osteoclasts and melanocytes as well as in brain, kidney, thymus and spleen. The gl gene is predicted to encode a 338-amino acid type I transmembrane protein that localizes to the intracellular compartment. Mutation in the human GL gene leads to severe recessive osteopetrosis. Our studies show that mouse Gl protein function is absolutely required for osteoclast and melanocyte maturation and function.

Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-alpha (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2Akita mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage specific TNF-α deficient mice (CD11bCre/TNF-αFlox/Flox). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and BUN, histopathologic changes and kidney macrophage recruitment compared to diabetic TNF-αFlox/Flox control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.

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Transcription of the mouse alpha-fetoprotein gene is activated in the developing fetal liver and gut and repressed in both tissues shortly after birth. With germline transformation in mice, a cis-acting element was identified upstream of the transcription initiation site of the alpha-fetoprotein gene that was responsible for repression of the gene in adult liver. This negative element acts as a repressor in a position-dependent manner.

To develop specific conditional gene ablation in the hematopoietic myeloid-osteoclast lineage, transgenic mice expressing Cre recombinase under the control of the CD11b promotor were generated on the C57BL/6 background. The cellular specificity of Cre activity following recombination was quantified in the Z/EG reporter transgenic mice by FACS analysis with lineage-specific markers and EGFP coexpression. A high degree of recombination, as evidenced by EGFP-positive cells, was demonstrated in macrophages and granulocytes of bone marrow and spleen by the presence of double-positive cells CD11b/EGFP and Gr1/EGFP, respectively. Interestingly, the peritoneal macrophage population showed almost complete DNA recombination at large. Most important, mature osteoclast cells derived from the double transgenic bone marrow and spleen progenitors were EGFP-positive. Hence, these CD11b-Cre mice will provide a unique tool to unravel novel gene function and activities involved during osteoclast and macrophage differentiation and maturation processes.