ADAM8 enhances osteoclast precursor fusion and osteoclast formation in vitro and in vivo.

ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate-resistant acid phosphatase (TRAP)-ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP-ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone-resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF-κB, Erk, and Akt signaling compared with wild-type (WT) littermates. This increased bone-resorbing capacity per OCL was associated with increased levels of p-Pyk2 and p-Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF-α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF-α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease.

Treatment for the lumbosacral soft tissue defect after spine surgery.

The lumbosacral area is one of the most frequently operated spine regions because of the prevalence of disease in that area. Although a lumbosacral soft tissue defect after surgery due to inflammation and other causes is rare, such soft tissue defects are difficult to treat. Therefore, suitable methods for treating lumbosacral soft tissue defects are necessary. Therefore, this study introduces a case-treated with a transverse lumbosacral rotational flap.

A 4 bp deletion mutation in DLX3 enhances osteoblastic differentiation and bone formation in vitro.

A 4 base-pair deletion mutation in the Distal-less 3 (DLX3) gene is etiologic for Tricho-Dento-Osseous syndrome (TDO). A cardinal feature of TDO is an increased thickness and density of bone. We tested the effects of the DLX3 gene mutation responsible for TDO on the osteoblastic differentiation of preosteoblastic MC3T3E1 cells and multipontent mesenchymal C2C12 cells. Differential expression analysis of C2C12 cells transfected with wild type DLX3 or mutant DLX3 was performed and desmin gene expression, an early myoblastic differentiation marker in mesenchymal cells, was evaluated by RT-PCR, western blot analysis, and desmin promoter transcriptional activity. Transfection of wild type DLX3 into MC3T3E1 and C2C12 cells increased alkaline phosphatase-2 activity, mineral deposition, and promoter activities of the osteocalcin and type 1 collagen genes compared to empty vector transfected cells. Transfection of mutant DLX3 into these cells further enhanced alkaline phosphatase activity, mineral deposition, and osteocalcin promoter activities, but did not further enhance type 1 collagen promoter activity. Transfection of mutant DLX3 into C2C12 cells markedly down regulated desmin gene expression, and protein expression of desmin and MyoD, while increasing protein expression of osterix and Runx2. These results demonstrate that the DLX3 deletion mutation associated with TDO enhances mesenchymal cell differentiation to an osteoblastic lineage rather than a myoblastic lineage by changing the fate of mesenchymal cells. This DLX3 mutation also accelerates the differentiation of osteoprogenitor cells to osteoblasts at later stages of osteogenesis.

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1.

ECF-L is a novel autocrine stimulator of osteoclast (OCL) formation that enhances the effects of 1,25-(OH)2D3 and RANK ligand (RANKL) and is increased in inflammatory conditions such as rheumatoid arthritis. ECF-L acts at the later stages of OCL formation and does not increase RANKL expression. Thus, its mechanism of action is unclear. Therefore, RAW 264.7 cells and M-CSF-dependent murine bone marrow macrophage (MDBM) cells were treated with RANKL and/or with recombinant ECF-L expressed as a Fc fusion protein (ECF-L-Fc) to determine their effects on NF-kappaB, AP-1 and JNK activity, and on the expression of the adhesion molecules that have been implicated in OCL formation. These parameters were measured by semiquantitative and PCR and Western blot analysis. In addition, the role of ICAM-1 was further assessed by treating normal mouse marrow cultures with ECF-L-Fc and 10(-10) M 1,25-(OH)2D3 in the presence or absence of a blocking ICAM-1 antibody or treating marrow cultures from ICAM-1 knockout mice with ECF-L and 1,25-(OH)2D3. ECF-L-Fc by itself only modestly increased NF-kappaB binding and JNK activity in RAW 264.7 cells, which was further enhanced by RANKL. In contrast, ECF-L-Fc increased LFA-1alpha and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10(-10) M 1,25-(OH)2D3 and ECF-L. Furthermore, ECF-L did not increase OCL formation in marrow cultures from ICAM-1 knockout mice. Taken together, these results demonstrate that ECF-L enhances RANKL and 1,25-(OH)2D3-induced OCL formation by increasing adhesive interactions between OCL precursors through increased expression of ICAM-1 and LFA-1.

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

UNLABELLED: We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity. INTRODUCTION: Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity. MATERIALS AND METHODS: Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis. RESULTS: Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice. CONCLUSIONS: These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing ICAM-1 expression.

Eosinophil chemotactic factor-L (ECF-L) is a novel stimulator of osteoclast (OCL) formation that acts at the differentiation/fusion stage of OCL formation, and is a cofactor for RANK ligand (RANKL). We examined the effects of ECF-L on the intracellular signaling pathways utilized by RANKL, and on the expression of ICAM-1/LFA-1 to determine its mechanism of action. RAW 264.7 and bone marrow cells were treated with RANKL and/or ECF-L Fc protein to determine their effect on NF-kappaB and AP-1 activity. ECF-L by itself only modestly increased NF-kappaB binding and JNK activity in RAW 264.7 cells, which were further enhanced by RANKL. In contrast, ECF-L Fc increased LFA-1alpha and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10(-10) M 1,25-(OH)2D3, and ECF-L Fc. Furthermore, ECF-L Fc did not enhance OCL formation by ICAM-1 knockout (KO) cells. Increased expression of ICAM-1 by ECF-L appears to be critical for its effects on OCL formation.

Mutant tamm-horsfall glycoprotein accumulation in endoplasmic reticulum induces apoptosis reversed by colchicine and sodium 4-phenylbutyrate.

As a consequence of uromodulin gene mutations, individuals develop precocious hyperuricemia, gout, and progressive renal failure. In vitro studies suggest that pathologic accumulation of uromodulin/Tamm-Horsfall glycoprotein (THP) occurs in the endoplasmic reticulum (ER), but the pathophysiology of renal damage is unclear. It was hypothesized that programmed cell death triggered by accumulation of misfolded THP in the ER causes progressive renal disease. Stably transfected human embryonic kidney 293 cells and immortalized thick ascending limb of Henle's loop cells with wild-type and mutated uromodulin cDNA were evaluated to test this hypothesis. Immunocytochemistry, ELISA, and deglycosylation studies indicated that accumulation of mutant THP occurred in the ER. FACS analyses showed a significant increase in early apoptosis signal in human embryonic kidney 293 and thick ascending limb of Henle's loop cells that were transfected with mutant uromodulin constructs. Colchicine and sodium 4-phenylbutyrate treatment increased secretion of THP from the ER to the cell membrane and into the culture media and significantly improved cell viability. These findings indicate that intracellular accumulation of THP facilitates apoptosis and that this may provide the pathologic mechanism responsible for the progressive renal damage associated with uromodulin gene mutations. Colchicine and sodium 4-phenylbutyrate reverse these processes and could potentially be beneficial in ameliorating the progressive renal damage in uromodulin-associated kidney diseases.

IL-3 is a potential inhibitor of osteoblast differentiation in multiple myeloma.

Bone destruction in multiple myeloma is characterized both by markedly increased osteoclastic bone destruction and severely impaired osteoblast activity. We reported that interleukin-3 (IL-3) levels are increased in bone marrow plasma of myeloma patients compared with healthy controls and that IL-3 stimulates osteoclast formation. However, the effects of IL-3 on osteoblasts are unknown. Therefore, to determine if IL-3 inhibits osteoblast growth and differentiation, we treated primary mouse and human marrow stromal cells with IL-3 and assessed osteoblast differentiation. IL-3 inhibited basal and bone morphogenic protein-2 (BMP-2)-stimulated osteoblast formation in a dose-dependent manner without affecting cell growth. Importantly, marrow plasma from patients with high IL-3 levels inhibited osteoblast differentiation, which could be blocked by anti-IL-3. However, IL-3 did not inhibit osteoblast differentiation of osteoblastlike cell lines. In contrast, IL-3 increased the number of CD45+ hematopoietic cells in stromal-cell cultures. Depletion of the CD45+ cells abolished the inhibitory effects of IL-3 on osteoblasts, and reconstitution of the cultures with CD45+ cells restored the capacity of IL-3 to inhibit osteoblast differentiation. These data suggest that IL-3 plays a dual role in the bone destructive process in myeloma by both stimulating osteoclasts and indirectly inhibiting osteoblast formation.

MIP-1alpha utilizes both CCR1 and CCR5 to induce osteoclast formation and increase adhesion of myeloma cells to marrow stromal cells.

OBJECTIVES: Macrophage inflammatory protein-1alpha (MIP-1alpha), an osteoclast (OCL) stimulatory factor produced by primary multiple myeloma (MM) cells, increases bone destruction and tumor burden in murine models of MM. Several chemokine receptors (CCR1, CCR5, and CCR9) mediate the effects of MIP-1alpha. In this study, we determined which of these mediates the effects of MIP-1alpha on human OCL formation and myeloma cells. METHODS: We employed RT-PCR analysis, neutralizing antibodies to CCR1 and CCR5 as well as a CCR1-specific antagonist and OCL formation assays to identify the MIP-1alpha receptors involved in MIP-1alpha's effects on myeloma cells and OCL formation. RESULTS: RT-PCR analysis demonstrated that both CCR1 and CCR5 were expressed by highly purified human OCL precursors, myeloma cell lines, and purified marrow plasma cells from MM patients. Neutralizing antibodies to CCR1 or CCR5 inhibited MIP-1alpha-induced OCL formation. Furthermore, monocyte chemotactic protein-3 (MCP-3), which binds CCR1 but not CCR5 and the CCR1-specific antagonist, BX471, markedly inhibited OCL formation stimulated with MIP-1alpha. Anti-CCR1, anti-CCR5, or BX471 also inhibited the upregulation of beta1 integrin mRNA in myeloma cells induced by MIP-1alpha, as well as the adherence of myeloma cells to stromal cells and IL-6 production by stromal cells in response to myeloma cells. CONCLUSION: These data demonstrate that MIP-1alpha utilizes either CCR1 or CCR5 for its effects on OCL formation and myeloma cells, and that blocking either CCR1 or CCR5 inhibits OCL formation and myeloma cell adhesion to stromal cells.

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.

MIP-1 alpha and myeloma bone disease.

Figure 5 is a proposed model for MIP-1alpha's effects on myeloma bone disease. MIP-1alpha is produced by myeloma cells and directly stimulates OCL formation. In addition MIP-1alpha enhances adhesive interactions between myeloma cells and marrow stromal cells increasing expression of RANKL and IL-6, which further increase bone destruction and tumor burden. The recent evidence from our group and others lead to the conclusion that MIP-1alpha is an important mediator in the debilitating bone destruction in multiple myeloma. Blocking MIP-1alpha expression may have profound effects on myeloma cell growth, homing, and bone destruction in this in vivo model of myeloma. These data suggest that antagonists that decrease MIP-1alpha activity in vivo or blocking MIP-1alpha signaling by neutralizing its receptor may provide therapeutic alternatives for treating patients with myeloma to decrease both their tumor burden and bone destruction.

IL-3 expression by myeloma cells increases both osteoclast formation and growth of myeloma cells.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) gene expression is abnormally regulated in multiple myeloma (MM) owing to imbalanced expression of the acute myeloid leukemia-1A (AML-1A) and AML-1B transcription factors. We hypothesized that the increased expression ratios of AML-1A to AML-1B also induced abnormal expression of other hematopoietic and bone-specific genes that contribute to the poor prognosis of MM patients with high levels of MIP-1 alpha. We found that interleukin-3 (IL-3) was also induced by the imbalanced AML-1A and AML-1B expression in myeloma. IL-3 mRNA levels were increased in CD138+ purified myeloma cells with increased AML-1A-to-AML-1B expression from MM patients, and IL-3 protein levels were significantly increased in freshly isolated bone marrow plasma from MM patients (66.4 +/- 12 versus 22.1 +/- 8.2 pg/mL; P = .038). IL-3 in combination with MIP-1 alpha or receptor activator of nuclear factor-kappa B ligand (RANKL) significantly enhanced human osteoclast (OCL) formation and bone resorption compared with MIP-1 alpha or RANKL alone. IL-3 stimulated the growth of interleukin-6 (IL-6)-dependent and IL-6-independent myeloma cells in the absence of IL-6, even though IL-3 did not induce IL-6 expression by myeloma cells. These data suggest that increased IL-3 levels in the bone marrow microenvironment of MM patients with imbalanced AML-1A and AML-1B expression can increase bone destruction and tumor cell growth.

Eosinophil chemotactic factor-L (ECF-L): a novel osteoclast stimulating factor.

UNLABELLED: Screening a cDNA library enriched for genes expressed in OCLs identified ECF-L. ECF-L enhanced OCL formation without increasing RANKL levels. Anti-ECF-L inhibited RANKL-induced OCL formation. These results support a potent role of ECF-L in osteoclastogenesis. INTRODUCTION: To investigate the molecular mechanisms that control osteoclastogenesis, we developed an immortalized osteoclast (OCL) precursor cell line that forms mature OCLs in the absence of stromal cells and used it to form pure populations of OCLs. MATERIALS AND METHODS: Polymerase chain reaction (PCR) selective cDNA subtraction was used to identify genes that are highly expressed in mature OCLs compared with OCL precursors employing OCL and OCL precursors derived from this cell line. RESULTS: Eosinophil chemotactic factor-L (ECF-L), a previously described chemotactic factor for eosinophils, was one of the genes identified. Conditioned media from 293 cells transfected with mECF-L cDNA, or purified ECF-L Fc protein, increased OCL formation in a dose-dependent manner in mouse bone marrow cultures treated with 10(-10) M 1,25(OH)2D3. OCLs derived from marrow cultures treated with ECF-L conditioned media formed increased pit numbers and resorption area per dentin slice compared with OCLs induced by 1,25(OH)2D3 (p < 0.01). Addition of an antisense S-oligonucleotide to mECF-L inhibited OCL formation in murine bone marrow cultures treated only with 10(-9) M 1,25(OH)2D3 compared with the sense S-oligonucleotide control. Time course studies demonstrated that ECF-L acted at the later stages of OCL formation, and chemotactic assays showed that mECF-L increased migration of OCL precursors. mECF-L mRNA was detectable in mononuclear and multinucleated cells by in situ hybridization. Interestingly, a neutralizing antibody to ECF-L blocked RANKL or 10(-9) M 1,25(OH)2D3-induced OCL formation in mouse bone marrow cultures, although ECF-L did not induce RANKL expression. CONCLUSIONS: These data show ECF-L is a previously unknown factor that is a potent mediator of OCL formation, which acts at the later stages of OCL formation and enhances the effects of RANKL.