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Objective We have previously reported that stimulation of mouse bone marrow-derived macrophages with tumor necrosis factor (TNF) and interleukin-6 (IL-6) induces differentiation of osteoclast-like cells. We undertook this study to clarify the characterization and function of human TNF and IL-6-induced osteoclasts using peripheral blood collected from patients with rheumatoid arthritis (RA) and healthy donors. Methods Peripheral blood monocytes were cultured with a combination of TNF and IL-6, TNF alone, IL-6 alone, or with RANKL, and their bone resorption ability was evaluated. Expression levels of NFATc1, proinflammatory cytokines, and matrix metalloproteinase 3 were analyzed. The effects of NFAT inhibitor and JAK inhibitor were examined. Furthermore, the relationship between the number of TNF and IL-6-induced osteoclasts or RANKL-induced osteoclasts differentiated from peripheral blood mononuclear cells (PBMCs) in patients with RA and the modified total Sharp score (mTSS) or whole-body bone mineral density (BMD) was examined. Results Peripheral blood monocytes stimulated with a TNF and IL-6-induced osteoclasts were shown to demonstrate the ability to absorb bone matrix. Cell differentiation was not inhibited by the addition of osteoprotegerin. Stimulation with a combination of TNF and IL-6 promoted NFATc1 expression, whereas the NFAT and JAK inhibitors prevented TNF and IL-6-induced osteoclast formation. Expression levels of IL1β, TNF, IL12p40, and MMP3 were significantly increased in TNF and IL-6-induced osteoclasts, but not in RANKL-induced osteoclasts. The number of TNF and IL-6-induced osteoclasts differentiated from PBMCs in patients with RA positively correlated with the mTSS, whereas RANKL-induced osteoclast numbers negatively correlated with the whole-body BMD of the same patients. Conclusion Our results demonstrate that TNF and IL-6-induced osteoclasts may contribute to the pathology of inflammatory arthritis associated with joint destruction, such as RA.

Background Previously, we reported that novel osteoclast-like cells (OLCs) were induced, both in vitro and in vivo, from mouse bone marrow-derived macrophages (BMMs) by addition of a combination of TNFα and IL-61. Recently, O’Brien et al showed that TNF/IL-6 can drive osteoclastogenesis in BMMs from RANK-deficient mice2. Objectives We aimed to examine the differentiation of OLCs, which were induced by a combination of TNFα and IL-6 from human peripheral blood mononuclear cells (PBMCs) and CD14 +monocytes and to identify differences in molecular expression patterns between OLCs and conventional osteoclasts. Furthermore, we identified OLCs and osteoclasts on the bone tissue of the joint in patients with rheumatoid arthritis (RA). Methods PBMCs and CD14 +monocytes from healthy volunteers and/or RA patients were stimulated with TNFα and IL-6 or RANKL. Quantitative RT-PCR was used to measure mRNA expression levels of RANK, cathepsin K, calcitonin receptor, and dendritic cell-specific transmembrane protein. Prepared undecalcified tibial bone from 6 RA or osteoarthritis (OA) patients undergoing joint surgery were stained by tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemistry with anti-RANK antibody, expression of which were analysed. Osteoclasts and OLCs were identified as multinucleated TRAP+/RANK +cells and TRAP+/RANK- cells, respectively, adherent to the bone surface. Results The number of OLCs treated with a combination of TNFα and IL-6 from PBMCs or CD14 +monocytes in RA patients was significantly increased compared to that in healthy volunteers. Expression levels of RANK mRNA was clearly up-regulated in osteoclasts, and was obviously down-regulated in OLCs compared to that in osteoclast precursors. In cancellous bone, the number of TRAP+/RANK +osteoclasts was significantly increased in RA patients compared to that in OA patients. Interestingly, numerous TRAP+/RANK- OLCs were present in the cancellous bone of RA patients, while almost none were observed in the cancellous bone of OA patients. Conclusions The combination of TNFα and IL-6 strongly induced the differentiation of OLCs from PBMCs or CD14 +monocytes in RA patients. OLCs was characterised with TRAP+/RANK- multinucleated cells, which can distinguish conventional TRAP+/RANK +multinucleated osteoclasts. TRAP+/RANK- OLCs also were present in the bone tissue of RA patients. These results suggest that conventional osteoclasts and novel OLCs could be involved in the pathogenic mechanisms of inflammatory bone destruction such as RA. References [1] Yokota K, Sato K, Miyazaki T, Kitaura H, Kayama H, Miyoshi F, Araki Y, Akiyama Y, Takeda K, Mimura T. Combination of tumor necrosis factor α and interleukin-6 induces mouse osteoclast-like cells with bone resorption activity both in vitro and in vivo. Arthritis Rheumatol 2014Jan;66(1):121–9. [2] O’Brien W, Fissel BM, Maeda Y, Yan J, Ge X, Gravallese EM, Aliprantis AO, Charles JF. RANK-Independent Osteoclast Formation and Bone Erosion in Inflammatory Arthritis. Arthritis Rheumatol.2016Dec;68(12):2889–2900. Disclosure of Interest None declared

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic ossification in muscle tissues. Constitutively activated mutants of a bone morphogenetic protein (BMP) receptor, ALK2, have been identified in patients with FOP. Recently, a novel ALK2 mutation, L196P, was found in the most benign case of FOP reported thus far. In the present study, we examined the biological activities of ALK2(L196P) in vitro. Over-expression of ALK2(L196P) induced BMP-specific activities, including the suppression of myogenesis, the induction of alkaline phosphatase activity, increased BMP-specific luciferase reporter activity, and increased phosphorylation of Smad1/5 but not Erk1/2 or p38. The activities of ALK2(L196P) were higher than those of ALK2(G356D), another mutant ALK2 allele found in patients with FOP and were equivalent to those of ALK2(R206H), a typical mutation found in patients with FOP. ALK2(L196P) was equally or more resistant to inhibitors in comparison to ALK2(R206H). These findings suggest that ALK2(L196P) is an activated BMP receptor equivalent to ALK2(R206H) and that ALK2(L196P) activity may be suppressed in vivo by a novel molecular mechanism in patients with this mutation.

Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of beta-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3beta activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3beta-dependent but beta-catenin-independent mechanism.

Finding a means to ameliorate and prevent bone destruction is one of the urgent issues in the treatment of rheumatoid arthritis. Recent studies revealed bone-resorbing osteoclasts to be essential for arthritic bone destruction, but to date there has been scarce experimental evidence for the underlying mechanism of the bone-protective effect of antirheumatic drugs. Here we examined the effects of one or a combination of disease-modifying antirheumatic drugs (DMARDs) on osteoclast differentiation to provide a cellular and molecular basis for their efficacy against bone destruction. The effects on osteoclast precursor cells and osteoclastogenesis-supporting cells were distinguished by two in vitro osteoclast culture systems. Methotrexate (MTX), bucillamine (Buc) and salazosulphapyridine (SASP) inhibited osteoclastogenesis by acting on osteoclast precursor cells and interfering with receptor activator of NF-kappaB ligand (RANKL)-mediated induction of the nuclear factor of activated T cells (NFAT) c1. MTX and SASP also suppressed RANKL expression on osteoclastogenesis-supporting mesenchymal cells. Interestingly, the combination of three antirheumatic drugs exerted a marked inhibitory effect on osteoclastogenesis even at a low dose at which there was much less of an effect when administered individually. These results are consistent with the reported efficacy of combined DMARDs therapy in humans and suggest that osteoclast culture systems are useful tools to provide an experimental basis for the bone-protective effects of antirheumatic drugs.

Skeletal mineralization is an important step regulating the mechanical properties of the calcified tissues, but molecular events underlying mineralization still remain elusive. We examined the role of extracellular signal-regulated kinase (Erk) pathways in matrix mineralization of osteogenic cells both in vitro and in vivo. Matrix mineralization by preosteocytic MLO-A5 cells and osteoblastic MC3T3-E1 cells was increased by either PD98059 Mek inhibitor treatment or adenovirus vector-mediated dominant negative Ras (RasDN) expression and was suppressed by Erk activation by platelet-derived growth factor (PDGF) treatment or constitutively active Mek1 (MekCA) expression. Administration of adenovirus vectors carrying RasDN gene onto the calvaria of 1-day-old mice increased the mineralization of the tissues, while that of the MekCA adenovirus suppressed it. These results suggest that the Erk pathway is a negative regulator of the matrix mineralization both in vitro and in vivo.

Objective Synovial fibroblasts (SFs) produce matrix-degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs. Methods MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6. Results The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs. Conclusion Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6–induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs.

The receptor activator of NF-kappaB ligand (RANKL), a critical regulator of osteoclastogenesis, is synthesized as a membrane-anchored protein and cleaved into a soluble form by ectodomain shedding. We developed an assay system to identify molecules regulating the RANKL shedding. Using this system, we found that a splice variant of Ca2+-promoted Ras inactivator (CAPRI), deltaCAPRI, which is expressed in primary osteoblasts, promoted the RANKL shedding. The wild type CAPRI is a member of the Ras GTPase-activating protein (GAP) family and suppresses Ca2+-dependent Ras activation, whereas deltaCAPRI, which lacks one exon in the GAP-related domain, activated the Ras pathway. Overexpression of deltaCAPRI or a constitutive active form of Ras up-regulated the expression level of matrix-metalloproteinase 14 (MMP14), which directly cleaves the ectodomain of RANKL, whereas Erk activation by expressing the constitutive active Mek1 did not affect the MMP14 expression or RANKL shedding. These results suggest that deltaCAPRI is a possible regulator of RANKL shedding by modulating MMP14 expression through Ras signaling cascades other than the Erk pathway.

Schwann cells are the myelinating glia of the peripheral nervous system, and their development is regulated by various growth factors, such as neuregulin, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). However, the mechanism of intracellular signaling pathways following these ligand stimuli in Schwann cell differentiation remains elusive. Here, we demonstrate that in cultured Schwann cells, neuregulin and PDGF suppressed the expression of myelin-associated protein markers, whereas IGF-I promoted it. Although these ligands activated common downstream signaling pathways [i.e., extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)-Akt pathways], the profiles of activation varied among ligands. To elucidate the function of these pathways and the mechanisms underlying Schwann cell differentiation, we used adenoviral vectors to selectively activate or inactivate these pathways. We found that the selective activation of Erk pathways suppressed Schwann cell differentiation, whereas that of PI3K pathways promoted it. Furthermore, lithium chloride, a modulator of glycogen synthase kinase-3β (GSK-3β) promoted Schwann cell differentiation, suggesting the involvement of GSK-3β as a downstream molecule of PI3K-Akt pathways. Selective activation of PI3K pathways in Schwann cells by gene transfer also demonstrated increased myelination inin vitroSchwann cell-DRG neuron cocultures andin vivoallogenic nerve graft experiments. We conclude that signals mediated by PI3K-Akt are crucial for initiation of myelination and that the effects of growth factors are primarily dependent on the balance between Erk and PI3K-Akt activation. Our results also propose the possibility of augmenting Schwann cell functions by modulating intracellular signals in light of future cell therapies.

Osteoclasts are cells of monocyte/macrophage origin that erode bone matrix: regulation of their differentiation is central to the understanding of the pathogenesis and treatment of bone diseases such as osteoporosis. Signalling by RANKL (receptor activator of NF-kappaB ligand), also known as Tnfsf11, is essential for the induction of osteoclast differentiation, and it must be strictly regulated to maintain bone homeostasis. But it is not known whether RANKL signalling to the cell interior is linked to any regulatory mechanisms. Here we show that RANKL induces the interferon-beta (IFN-beta) gene in osteoclast precursor cells, and that IFN-beta inhibits the differentiation by interfering with the RANKL-induced expression of c-Fos, an essential transcription factor for the formation of osteoclasts. This IFN-beta gene induction mechanism is distinct from that induced by virus, and is dependent on c-Fos itself. Thus an autoregulatory mechanism operates-the RANKL-induced c-Fos induces its own inhibitor. The importance of this regulatory mechanism for bone homeostasis is emphasized by the observation that mice deficient in IFN-beta signalling exhibit severe osteopenia (loss of bone mass) accompanied by enhanced osteoclastogenesis. Our study places the IFN-beta system in a new context, and may offer a molecular basis for the treatment of bone diseases.

Bone is a highly vascularized organ, thus angiogenesis is a vital process during bone remodeling. However, the role of vascular systems in bone remodeling is not well recognized. Here we show that netrin-4 inhibits osteoclast differentiation in vitro and in vivo. Co-cultures of bone marrow macrophages with vascular endothelial cells markedly inhibited osteoclast differentiation. Adding a neutralizing antibody, or RNA interference against netrin-4, restored in vitro osteoclast differentiation. Administration of netrin-4 prevented bone loss in an osteoporosis mouse model by decreasing the osteoclast number. We propose that vascular endothelial cells interact with bone in suppressing bone through netrin-4.

Accumulating evidence indicates that epigenetic aberrations have a role in the pathogenesis of rheumatoid arthritis (RA). However, reports on histone modifications are as yet quite limited in RA. Interleukin (IL)-6 is an inflammatory cytokine which is known to be involved in the pathogenesis of RA. Here we report the role of histone modifications in elevated IL-6 production in RA synovial fibroblasts (SFs). The level of histone H3 acetylation (H3ac) in the IL-6 promoter was significantly higher in RASFs than osteoarthritis (OA) SFs. This suggests that chromatin structure is in an open or loose state in the IL-6 promoter in RASFs. Furthermore, curcumin, a histone acetyltransferase (HAT) inhibitor, significantly reduced the level of H3ac in the IL-6 promoter, as well as IL-6 mRNA expression and IL-6 protein secretion by RASFs. Taken together, it is suggested that hyperacetylation of histone H3 in the IL-6 promoter induces the increase in IL-6 production by RASFs and thereby participates in the pathogenesis of RA.

We examined the production of substance P (SP) in synovial fibroblasts derived from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Immunoreactive SP was observed in non-stimulated RA fibroblasts. The expression of beta-preprotachykinin-A (beta-PPT-A) mRNA was confirmed by reverse transcription-polymerase chain reaction analysis. SP contents in culture medium were increased by treatment of RA fibroblasts with transforming growth factor-beta (TGFbeta) (10 ng/ml). Levels of SP release were elevated at 12 h after TGFbeta stimulation whereas the expression of beta-PPT-A mRNA was enhanced at 3 h. Furthermore, SP production in response to TGFbeta was dose-dependently enhanced by basic fibroblast growth factor (bFGF). OA fibroblasts also significantly released SP in the presence of TGFbeta (10 ng/ml) plus bFGF (50 ng/ml). These results suggest that SP produced by synovial fibroblasts may participate in joint diseases.

Bone resorption is regulated by the immune system, where T-cell expression of RANKL (receptor activator of nuclear factor (NF)-kappaB ligand), a member of the tumour-necrosis factor family that is essential for osteoclastogenesis, may contribute to pathological conditions, such as autoimmune arthritis. However, whether activated T cells maintain bone homeostasis by counterbalancing the action of RANKL remains unknown. Here we show that T-cell production of interferon (IFN)-gamma strongly suppresses osteoclastogenesis by interfering with the RANKL-RANK signalling pathway. IFN-gamma induces rapid degradation of the RANK adapter protein, TRAF6 (tumour necrosis factor receptor-associated factor 6), which results in strong inhibition of the RANKL-induced activation of the transcription factor NF-kappaB and JNK. This inhibition of osteoclastogenesis is rescued by overexpressing TRAF6 in precursor cells, which indicates that TRAF6 is the target critical for the IFN-gamma action. Furthermore, we provide evidence that the accelerated degradation of TRAF6 requires both its ubiquitination, which is initiated by RANKL, and IFN-gamma-induced activation of the ubiquitin-proteasome system. Our study shows that there is cross-talk between the tumour necrosis factor and IFN families of cytokines, through which IFN-gamma provides a negative link between T-cell activation and bone resorption. Our results may offer a therapeutic approach to treat the inflammation-induced tissue breakdown.

Spinal cord injury in adult mammals results in little axonal regeneration, although the mechanism of regeneration failure still remains elusive. Recent research has revealed that activation of the extracellular-signal-regulated kinases (ERKs) plays an important role in the neurite outgrowth. In the present study, we constructed a replication-defective adenovirus vector carrying mutated form of MEK1 (CA-MEK virus), which constitutively activate ERK pathway, and investigated its effect on thoracic spinal cord injury model in young adult rats as well as neurite outgrowth in vitro. In rat pheocromocytoma cell line PC12 cells, CA-MEK virus infection induced sustained activation of ERKs and stimulated neurite outgrowth in the absence of neurotrophic factors. In rat spinal cord transection model, injection of CA-MEK virus into the completely transected spinal cord efficiently activated ERKs in the supraspinal neurons and induced axonal regeneration across the transection site, which was confirmed by anterograde labeling with wheat-germ-agglutinin conjugated peroxidase (WGA-HRP). Spinal cord evoked potentials (SCEP) showed that these regenerated axons were electroconductive. Most importantly, CA-MEK virus-treated rats showed significant recovery of hind limb function 2 weeks after operation compared to the control rats treated with no virus or LacZ virus. These results suggest that adenovirus-mediated CA-MEK gene transduction offers a novel strategy for the gene therapy of spinal cord injury.

Surgery or trauma often results in restrictive adhesions around joints or tendons that cause severe functional impairment. The formation of adhesion is essentially a fibrogenetic process; therefore, peptide growth factors, such as transforming growth factor-beta, are assumed to play central roles in its development. The purpose of this study was to test the hypothesis that suppression of transforming growth factor-beta1 activity reduces adhesion formation. Sixty rabbits were prepared and randomly divided into six groups of 10. Intraarticular adhesions were created in the right knee joints by cortical bone shaving and subsequent cast immobilization for 4 weeks. In animals in three of the six groups, transforming growth factor-beta1 activity was suppressed by continuous administration of the neutralizing antibody in three graded doses; animals in the other three groups were used as controls. Four weeks after the surgery, the casts were removed and the adhesions were assessed macroscopically, histologically, biomechanically, and biochemically. Gross observation showed that the neutralizing antibody had suppressed adhesion formation in a dose-dependent manner. This is consistent with biomechanical measurement results demonstrating that the antibody reduced the flexion contractures. Histologically, the adhesion in our model was fibrous tissue and the adhesions in the animals in the antibody groups were thin and loose in comparison with the controls. Biochemical analyses further supported these results, demonstrating that administration of the antibody reduced collagen content in the adhesions with a predominance of type-I collagen. Thus, this study showed that suppression of the actions of transforming growth factor-beta1 reduced adhesion formation. Considering the various possible measures to control the activity of the growth factor, suppression of transforming growth factor-beta may be a novel, potent approach to preventing adhesions.

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.

We have explored the use of adenovirus vector-mediated gene transfer to introduce foreign genes into osteoclasts, terminally differentiated cells responsible for bone resorption. A replication-deficient adenovirus vector that contains a reporter gene encoding beta-galactosidase efficiently infected human osteoclast-like cells (OCLs) derived from human giant cell tumors and mouse OCLs formed in vitro. We then constructed an adenovirus vector carrying human epidermal growth factor receptor (EGFR) cDNA (Ax1CAhEGFR) and introduced the EGFR gene into mouse OCLs. Clear induction of EGF receptor was detected in Ax1CAhEGFR-infected OCLs (EGFR-OCLs) by immunocytochemistry and immunoblotting, and EGF stimulation induced rapid tyrosine phosphorylation of several proteins including EGF receptor itself. Large vacuoles appeared in EGFR-OCLs in response to EGF treatment, and pit-forming activity by EGFR-OCLs was dose-dependently suppressed by recombinant human EGF. In addition, survival of EGFR-OCLs was prolonged by EGF. No expression of EGF receptor or effects of EGF were observed in noninfected OCLs or control vector-infected OCLs. These results suggest that adenoviral vectors are useful for modulating osteoclast function by introducing foreign genes into osteoclasts and that they will be a good means of gene therapy of metabolic bone diseases.

Bone-resorbing multinucleated cells were efficiently formed in primary culture of cells isolated from synovial tissues of patients with rheumatoid arthritis in 2-3 weeks in the presence of 1,25(OH) 2 vitaminD 3 without any additional stromal cells, and that formation was further facilitated by macrophage-colony stimulating factor. Furthermore, we show that osteoclast-like cells are formed in co-culture of peripheral blood mononuclear cells and rheumatoid synovial fibroblasts obtained by continued sub-cultures. The multinucleated cells showed all the phenotypical and functional characteristics of osteoclasts including the expression of tartrate resistant acid phosphatase, vitronectin receptors, receptors for human calcitonin and the ability to resorb bone. These results indicate that synovial macrophages are capable of differentiating into osteoclasts in the presence of rheumatoid synovial fibroblasts which can support differentiation of monocytes/macrophages, implicating that osteoclasts generated within the synovial membrane are probably involved in bone destruction in rheumatoid arthritis.

The midportion of the anterior cruciate ligament (ACL) of rabbits was partially transected, and the effect of hyaluronan (HA) on its healing was determined. A 1% solution of HA (HA group) or physiological phosphate-buffered saline (control group) was administered intraarticularly, at 0.1 ml/kg body weight, once a week from 1 week after the operation. Two, 4, and 6 weeks after the initiation of HA administration, the ACLs were examined grossly, histologically and immunohistochemically. At 2 weeks, the lacerated portions were completely covered with scar-like tissue in both groups. These tissue areas were smaller in the HA group than in the control group. Histologically in the HA group, the regularity of collagen fibers (indicating the maturity of regenerated collagen fibers) had increased compared to findings in the control group, and the number of fibroblastic cells decreased gradually at a significantly faster rate. The number of inflammatory cells and blood vessels decreased gradually in both groups, with these values being lower in the HA group at each time point but not significantly so. Immunohistochemical examination of the repaired tissue revealed strong staining with anti-chondroitin sulfate proteoglycan antibody in the HA group 2 weeks after the first HA administration. The staining gradually became reduced, with the rate of reduction being faster in the HA group than in the control group. The stimulation of chondroitin sulfate proteoglycan production and the faster reduction of it in the HA group suggests that HA facilitated tissue repair and inhibited the formation of scar tissue.

Background Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes progressive joint destruction. A line of evidence suggests that synovial fibroblasts (SFs) play an important role in the pathogenesis of RA. RASFs produce matrix metalloproteinases (MMPs) that degrade articular cartilage. Although interleukin-6 (IL-6) is an inflammatory cytokine and is proved to be involved in the pathogenesis of RA, it is unknown whether IL-6 affects MMPs gene transcription in RASFs. Furthermore, recent advances have revealed that epigenetic mechanisms, including histone modifications, are considered to be important regulators in gene transcription. We hypothesized that epigenetic dysregulation might induce RASF activation. Objectives The aim of this study is to examine whether IL-6 regulates MMPs expression in RASFs and whether histone modifications are associated with the MMPs gene activation in RASFs. Methods We compared MMPs gene expression by quantitative RT-PCR and histone methylation in the MMP promoters by chromatin immunoprecipitation (ChIP) assay after stimulation with IL-6 and/or soluble IL-6 receptor α (sIL-6Rα) in RASFs and control, osteoarthritis (OA) SFs. Chromatin structures in the MMP promoters were evaluated with micrococcal nuclease (MNase) assay in RASFs and OASFs. We investigated the change in the MMPs gene expression after silencing of WDR5 that is required for generating H3K4me3, an active histone marker. IL-6 signal induces Signal Transducer and Activator of Transcription 3 (STAT3) activation. To elucidate the mechanisms of IL-6-induced MMPs gene activation in RASFs, we investigated cell surface expression of the IL-6 receptor (gp130 and membrane-bound IL-6Rα) by flow cytometry, phospho-STAT3 (p-STAT3) expression by immunoblotting, and binding of STAT3 to the MMPs 1, 3, 9, and 13 promoters after IL-6 stimulation by ChIP assay in RASFs and OASFs. Results MMPs 1, 3, 9 and 13 genes were actively transcribed in RASFs. The histone methylation profiles (H3K4me3 and H3K27me3) and the result of MNase assay indicated that chromatin structures were open in the MMPs 1, 3, 9 and 13 promoters in RASFs. The depletion of WDR5 reduced the levels of H3K4me3 as well as the MMPs 1, 3, 9 and 13 gene expression. Interestingly, IL-6 and sIL-6Rα significantly increased the expression of MMPs 1, 3 and 13, but not MMP-9, in RASFs. Although the expression levels of gp130 as well as membrane-bound IL-6Rα were comparable and STAT3 was similarly phosphorylated after IL-6 stimulation in RASFs and OASFs, STAT3 bound to the MMPs 1, 3 and 13 promoters, but not the MMP-9 promoter, after stimulation with IL-6 and sIL-6Rα only in RASFs. It was suggested that binding of STAT3 to the promoters resulted in MMPs 1, 3 and 13 gene activation after IL-6 stimulation in RASFs. Conclusions Our data indicate that altered profiles of histone methylation and binding of STAT3 to the promoters regulate constitutive and IL-6-induced MMPs gene activation in RASFs and possibly arthritogenic properties of RASFs. References Araki Y et al. Arthritis Rheum. Epub. 2015 Dec 29. Disclosure of Interest None declared

Objectives and Design: The difference in cell proliferation and release of soluble factors in response to interleukin 1β (IL-1β) in fibroblasts obtained from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) and from normal skin has been investigated.¶Treatment: The cells were treated with recombinant IL-1β in the presence or absence of pharmacological agents for 24 h or 48 h.¶Methods: Cell proliferation was examined by WST-1 assay, and the amounts of interleukin-6 (IL-6), interleukin-8 (IL-8), macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEGF), matrix metalloproteinase-1 (MMP-1), and prostaglandin E2 (PGE2) were measured by enzyme linked immunosorbent assay (ELISA).¶Results: IL-1β dose-dependently enhanced the proliferation of all fibroblasts. The proliferative response to IL-1β in RA synovial fibroblasts was greater than that in OA synovial and skin fibroblasts. However, there was no difference in spontaneous levels of soluble factors between OA and RA fibroblasts, though medium concentrations of IL-1β-released VEGF, MMP-1, and PGE2, but not cytokines, in RA were slightly higher than those in OA. Ability to release soluble mediators was pronouncedly increased at 3 h to 9 h after stimulating fibroblasts with IL-1β for 1h. The proliferative response to IL-1β in all fibroblasts was inhibited by dexamethasone and the NF-κB inhibitor hymenialdisine but not the cyclooxygenase 2 (COX-2) inhibitor NS-398. But PGE2 prevented proliferation of RA fibroblasts when added to medium up to 3 h after IL-1β stimulation. Dexamethasone also inhibited the release of IL-6, IL-8, and PGE2 induced by IL-1β in both OA and RA fibroblasts. NS-398 exhibited an inhibition of IL-1β-induced IL-6 production as well as PGE2 production. Hymenialdisine inhibited IL-6 production and reduced IL-8 production dependent on synovial cell strains. Methotrexate had no effect on the response to IL-1β in synovial fibroblasts.¶Conclusion: The present results indicate that the activation of NF-κB plays an important role in the proliferative response to IL-1β in human fibroblasts, and suggest that PGE2 acts as a modulator of cell proliferation in inflamed synovial tissue. It appears that the ability to produce soluble factors in RA synovial fibroblasts is not intrinsic. However, the response to IL-1β in RA cells seems to be greater than that in OA cells.¶

The aim of this study was to determine the effect of increasing estrogen and decreasing androgen in males and increasing androgen and decreasing estrogen in females on bone metabolism in patients with gender identity disorder (GID). We measured and examined bone mineral density (BMD) and bone metabolism markers retrospectively in GID patients who were treated in our hospital. In addition, we studied the effects of treatment on those who had osteoporosis. Patients who underwent a change from male to female (MtF) showed inhibition of bone resorption and increased L2–4 BMD whereas those who underwent a change from female to male (FtM) had increased bone resorption and decreased L2–4 BMD. Six months after administration of risedronate to FtM patients with osteoporosis, L2–4 BMD increased and bone resorption markers decreased. These results indicate that estrogen is an important element with regard to bone metabolism in males.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic bone formation in muscle tissues. A common mutation among FOP patients has been identified in ALK2, ALK2(R206H), which encodes a constitutively active bone morphogenetic protein (BMP) receptor. Recently, a unique mutation of ALK2, ALK2(G356D), was identified to be a novel mutation in a Japanese FOP patient who had unique clinical features. Over-expression of ALK2(G356D) induced phosphorylation of Smad1/5/8 and activated Id1-luc and alkaline phosphatase activity in myoblasts. However, the over-expression failed to activate phosphorylation of p38, ERK1/2, and CAGA-luc activity. These ALK2(G356D) activities were weaker than those of ALK2(R206H), and they were suppressed by a specific inhibitor of the BMP-regulated Smad pathway. These findings suggest that ALK2(G356D) induces heterotopic bone formation via activation of a BMP-regulated Smad pathway. The quantitative difference between ALK2(G356D) and ALK2(R206H) activities may have caused the phenotypic differences in these patients.

The role of Rac1 in osteoclast survival and bone-resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts. Introduction: Rac1 is a member of Rho family small G-proteins, and recent studies have revealed that it mediates anti-apoptotic signals in some types of cells. Rac1 is reported to be required for the cytoskeletal organization and bone-resorbing activity of osteoclasts, but their roles in osteoclast survival and function are not fully elucidated. Materials and Methods: We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1DN) or constitutively active Rac1 (Rac1CA) gene, and osteoclast-like cells (OCLs) generated in mouse co-culture system were infected with these viruses. To examine the role of Rac1 in osteoclast survival and function, we performed pit formation assays, survival assays, and Western blotting, including an activated-Rac1 pull-down assay using adenovirus-infected OCLs. To further clarify the mechanism of Rac1 regulation in osteoclast survival, some specific inhibitors and adenovirus vectors of signal transduction molecules were used. To quantify membrane movement before and after macrophage colony-stimulating factor (M-CSF) treatment, OCLs expressing either enhanced green fluorescent protein (EGFP) or Rac1DN were recorded with a time-lapse video microscope. Results: Adenovirus vector-mediated dominant negative Rac1 (Rac1DN) expression significantly reduced pit formation, and promoted their apoptosis. M-CSF rapidly activated Rac1, and the prosurvival effect of M-CSF for OCLs was abrogated by Rac1DN overexpression. Constitutively active Rac1 enhanced OCL survival, which was completely suppressed by phosphatidylinositol 3′-kinase (PI3K) inhibitors, whereas a Mek inhibitor had only partial effect. Rac1DN also partially blocked the activation of Akt induced by the overexpressing catalytic subunit of PI3K. Using time-lapse video microscopy, we found that Rac1DN expression reduced membrane ruffling and the spreading of OCLs in response to M-CSF. Conclusions: Small guanosine triphosphatase (GTPase) Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling of osteoclasts primarily by modulating PI3K/Akt pathways. Rac1 also plays a significant role in the bone resorptive activity of cells, probably by regulating the motility of osteoclasts.

Osteoclasts (OCs) undergo rapid apoptosis without trophic factors, such as macrophage colony‐stimulating factor (M‐CSF). Their apoptosis was associated with a rapid and sustained increase in the pro‐apoptotic BH3‐only Bcl‐2 family member Bim. This was caused by the reduced ubiquitylation and proteasomal degradation of Bim that is mediated by c‐Cbl. Although the number of OCs was increased in the skeletal tissues of bim−/− mice, the mice exhibited mild osteosclerosis due to reduced bone resorption. OCs differentiated from bone marrow cells of bim−/− animals showed a marked prolongation of survival in the absence of M‐CSF, compared with bim+/+ OCs, but the bone‐resorbing activity of bim−/− OCs was significantly reduced. Overexpression of a degradation‐resistant lysine‐free Bim mutant in bim−/− cells abrogated the anti‐apoptotic effect of M‐CSF, while wild‐type Bim did not. These results demonstrate that ubiquitylation‐dependent regulation of Bim levels is critical for controlling apoptosis and activation of OCs.

Objective To determine the role of Ras-mediated signaling pathways in synovial cell activation and bone destruction in arthritic joints. Methods The E11 rheumatoid synovial cell line and primary synovial fibroblast-like cells (SFCs) from patients with rheumatoid arthritis (RA) were gene-transferred by replication-deficient adenovirus vector carrying the dominant-negative mutant of the ras gene (AxRasDN). The effects of RasDN overexpression on cellular proliferation, interleukin-1 (IL-1)–induced activation of mitogen-activated protein kinases (extracellular signal–regulated kinase [ERK], p38, c-Jun N-terminal kinase [JNK]), and IL-6 production by synovial cells were analyzed. The in vivo effects of Ras inhibition on synovial cell activation and arthritic bone destruction were analyzed by injection of AxRasDN into ankle joints of rats with adjuvant arthritis. Results AxRasDN markedly reduced the proliferation of RA SFCs. IL-1, a proinflammatory cytokine involved in RA pathology, induced activation of ERK, p38, and JNK in the cells. Adenovirus vector–mediated RasDN overexpression suppressed ERK activation, but not p38 or JNK activation, in SFCs. IL-6 is also an important proinflammatory cytokine, and RasDN inhibited IL-1–induced production of IL-6 by RA SFCs at both the transcriptional and protein levels. Injection of AxRasDN into ankle joints of rats with adjuvant arthritis ameliorated inflammation and suppressed bone destruction in the affected joints. Conclusion Ras-mediated signaling pathways are involved in the activation of RA SFCs and the destruction of bone in arthritic joints, suggesting that inhibition of Ras signaling can be a novel approach for RA treatment that targets both synovial cell activation and bone destruction in the RA joint.

Osteoclasts are multinucleated cells that resorb bones, and are derived from hematopoietic cells of the monocyte/macrophage lineage. The receptor activator of NF-κB ligand (RANKL, also called ODF/TRANCE/OPGL) stimulates both osteoclast differentiation from osteoclast progenitors and activation of mature osteoclasts. To identify genes responsible for osteoclast differentiation, we used a molecular indexing technique. Here, we report a clone of one of these genes whose transcription is induced by soluble RANKL (sRANKL) in both the RAW264.7 cells of the mouse macrophage cell line and the mouse primary bone marrow cells. The predicted protein was found to be a mouse homologue of Jun dimerization protein 2 (JDP2), a member of the AP-1 family of transcription factors, containing a basic region-leucine zipper motif. Transient transfection experiments revealed that overexpression of JDP2 leads to activation of both tartrate-resistant acid phosphatase (TRAP) and cathepsin K gene promoters in RAW264.7 cells. Infection of mouse primary bone marrow cells with retroviruses expressing JDP2-facilitated sRANKL-mediated formation of TRAP-positive multinuclear osteoclasts. Importantly, antisense oligonucleotide to JDP2 strongly suppressed sRANKL-induced osteoclast formation of RAW264.7 cells. Our findings suggest that JDP2 may play an important role in the RANK-mediated signal transduction system, especially in osteoclast differentiation.

Targeted disruption of either c-Src or TNFR-associated factor 6 (TRAF6) in mice causes osteoclast dysfunction and an osteopetrotic phenotype, suggesting that both molecules play important roles in osteoclastic bone resorption. We previously demonstrated that IL-1 induces actin ring formation and osteoclast activation. In this study, we examined the relationship between IL-1/TRAF6-dependent and c-Src-mediated pathways in the activation of osteoclast-like cells (prefusion cells (pOCs); multinucleated cells) formed in the murine coculture system. In normal pOCs, IL-1 induces actin ring formation and tyrosine phosphorylation of p130Cas, a known substrate of c-Src. However, in Src-deficient pOCs, p130Cas was not tyrosine phosphorylated following IL-1 treatment. In normal pOCs treated with IL-1, anti-TRAF6 Abs coprecipitate p130Cas, protein tyrosine kinase 2, and c-Src. In Src-deficient pOCs, this molecular complex was not detected, suggesting that c-Src is required for formation of the TRAF6, p130Cas, and protein tyrosine kinase 2 complex. Moreover, an immunocytochemical analysis revealed that in osteoclast-like multinucleated cells, IL-1 induced redistribution of TRAF6 to actin ring structures formed at the cell periphery, where TRAF6 also colocalized with c-Src. Taken together, these data suggest that IL-1 signals feed into the tyrosine kinase pathways through a TRAF6-Src molecular complex, which regulates the cytoskeletal reorganization essential for osteoclast activation.

Recent studies have revealed the essential role of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) in osteoclast differentiation and activation. Adenovirus vector could efficiently transduce genes into RAW264.7 cells, which differentiate into osteoclast-like multinucleated cells in the presence of RANKL. The role of NF-kappaB and c-jun N-terminal kinase (JNK) activation in RANKL-induced osteoclast differentiation was investigated using an adenovirus vector carrying the dominant negative 1kappaB kinase 2 gene (AxIKK2DN) or dominant negative MKK7 gene (AxMKK7DN). IKK2DN and MKK7DN overexpression in RAW cells specifically suppressed the NF-kappaB activation and JNK activation in response to RANKL, respectively, without affecting other signaling pathways. Either inhibition of NF-kappaB or JNK pathways dose-dependently inhibited osteoclast formation induced by RANKL. These results suggest that both NF-kappaB and JNK activation are independently required for osteoclast differentiation.

Small proteoglycan decorin is known to suppress the bioactivity of TGF-beta through a competitive binding with the cell surface receptors for the cytokine. Based on this knowledge, we hypothesized that decorin could reduce the formation of fibrous adhesion, because our previous study showed the neutralizing antibody to TGF-beta1 has that effect. An intra-articular adhesion model in the rabbit knee joint was employed in this study, and decorin was administered into the joint cavity continuously during the 4 weeks of the experiment. The results of the dose-response study demonstrated that decorin suppresses formation of fibrous adhesion in a dose-dependent manner. When the administration of decorin was limited to shorter periods, this effect was considerably impaired and the necessity of long-term administration was demonstrated. On the other hand, when administered together with TGF-beta1, decorin still suppressed adhesion but to a lesser extent, and it was suggested that this proteoglycan could have other significant mechanism(s) to suppress adhesion besides the neutralization of TGF-beta. Thus, the present study showed that decorin could inhibit adhesion formation by both TGF-beta dependent and independent mechanisms. Considering that decorin exists ubiquitously in the body, its administration might be a promising approach to suppress adhesion.

Background Rheumatoid Arthritis (RA) is characterised by severe and irreversible bone destruction in affected joints on its last stage. On the other hand, bone absorption by osteoclasts may be a natural physiological phenomenon as bone remodelling in mature manhood. Throughout our life, bone is constantly renewed with the process of old bone removing and new bone supplement. Osteoclasts are derived from the hematopoietic granulocyte/macrophage colony forming units, which also become to monocytes and macrophages, and their progenitors move from bone marrow to bone surface through the circulation or direct migration, and differentiate to functional mature osteoclasts with stimulation of some cytokines and/or direct contact. In RA, or another bone destructive disease, activated osteoclasts certainly have a critical role in bone destruction. But, is that all? How bone is broke down in RA? We have speculation that some cells derived from rheumatoid synovia were activated by inflammatory cytokines abundant in the joint of RA and take a significant role in the destruction of the bone. Objectives To elucidate what mechanism is there, we have observed the pannus-bone junctions in detail. Methods The specimens of the destructive bone derived from the RA patients under arthroplasty with their consent. Tissue sections from the pannus-bone junctions at sites of bone erosion were examined by hematoxylin-eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, and electron microscopy. Synovial tissues were also examined immunohistochemically. Results The specimens of the destructive bone derived from the RA patients showed that there were many not-classical osteoclasts in the area of bone erosions. Cells in the erosive front of RA are divided in four groups, the authentic osteoclasts, mononuclear osteoclasts, fibroblast-like cells and monocytes. The electron microscopy observations revealed that the fibroblast-like cells certainly have some contact with bone matrix. TRAP-positive cells and vitronectin receptor -positive cells were seen in frozen sections of synovial membrane from the patient with RA. Conclusion Our result showed that not only authentic osteoclasts, but the mononuclear cells and fibroblast-like cells were activated and have some role in bone destruction of RA. References Arthritis Rheum. 1984;27:968–75 Ann Rhuem Dis. 1993;52:133–7

The purpose of this study is to observe type I and type III procollagen gene expressions in the healing ligament using in situ hybridization histochemistry. The rabbit medial collateral ligaments were incised and harvested at 3, 7, 14, and 28 days postoperatively. The healing ligament showed increased expression of both procollagen genes through this period compared with the unoperated ligament. The peak expression level was observed at 7 or 14 days for type I and at 7 days for type III, respectively. The strongest expression of both genes was detected in the scar tissue created between the ends of the old ligament. Although type III procollagen gene expression was observed almost only in the newly created scar tissue, type I procollagen gene was expressed not only in the scar tissue, but also at the ends of the previously normal ligament. These results suggest that type I collagen synthesis begins shortly after ligament injury and occurs at the ends of the injured ligament as well as in the scar tissue, and that type III collagen is largely synthesized in the scar tissue around one week after injury but continues being synthesized for at least four weeks after injury.

Adhesion is a serious complication after trauma or surgery. Because adhesion formation is essentially a fibrogenetic process, a series of growth factors are assumed to be involved in its development. If this is true, it may be possible that inhibition of the growth factor activity suppresses adhesion formation. The current study was conducted to verify this hypothesis on fibroblast growth factor-2 using an intraarticular adhesion model in the rabbit knee. Forty Japanese White rabbits were used. They were divided randomly into five groups of eight animals, and in three of them, activity of endogenous fibroblast growth factor-2 was suppressed locally by a neutralizing antibody. The remaining two groups served as controls, and formation of adhesions was evaluated 4 weeks after surgery. The results showed that the administration of the antibody reduced the extent of adhesions macroscopically, whereas histologic observation and collagen content measurement suggested the adhesion tissue was not affected significantly. Corresponding to the macroscopic findings, contraction of the knee was improved in the antibody groups. The findings showed that suppression of fibroblast growth factor-2 activity reduces adhesions. It is expected that control of the cytokine activity may become a novel method for reducing adhesions.

Objective To clarify the mechanism by which osteoclasts are formed in culture of rheumatoid synoviocytes by exploring the involvement of receptor activator of nuclear factor κB ligand (RANKL)/osteoclast differentiation factor (ODF). Methods Osteoclast formation was evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood mononuclear cells (PBMC) in the presence of macrophage colony stimulating factor and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) utilizing separating membrane filters. RANKL/ODF expression was examined by Northern blotting in synovial tissues from 5 rheumatoid arthritis (RA) patients and tissues from patients with giant cell tumor (GCT), osteosarcoma (OS), and osteoarthritis (OA). RANKL/ODF expression and the ability of synovial fibroblasts to support osteoclastogenesis were investigated in coculture with PBMC in the presence or absence of 1,25(OH)2D3, and soluble RANKL/ODF and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) were measured by enzyme-linked immunosorbent assay. The effects of OPG/OCIF on the osteoclastogenesis in the primary culture of rheumatoid synoviocytes and the coculture system were determined. Results Synovial fibroblasts did not induce osteoclastogenesis when separately cocultured with PBMC. Northern blotting revealed that RANKL/ODF was highly expressed in all tissues from RA and GCT patients, but not from OA or OS patients. Cultured rheumatoid synovial fibroblasts efficiently induced osteoclastogenesis in the presence of 1,25(OH)2D3, which was accompanied by up-regulated expression of RANKL/ODF and decreased production of OPG/OCIF. Osteoclastogenesis from synoviocytes was dose-dependently inhibited by OPG/OCIF. Conclusion RANKL/ODF expressed on synovial fibroblasts is involved in rheumatoid bone destruction by inducing osteoclastogenesis and would therefore be a good therapeutic target.

Leukotriene (LT) C4 in the synovial fluid of patient with osteoarthritis deformans (OA) and rheumatoid arthritis (RA) was measured by radioimmunoassay after its extraction with Sep-Pak C18 cartridge. The amounts of immunoreactive LTC4 (i-LTC4) in samples from patients with OA and RA were not significantly different, being 0.198±0.018 pmol/ml (n=11) and 0.179±0. 016 pmol/ml (n=12), respectively. By high performance liquid chromatography and radioimmunoassay, the levels of other sulfidopeptide LTs, such as LTD4 and LTE4, in synovial fluid from patients with RA were found to be significantly higher than those in fluid from patients with OA. The leukocyte number in synovial fluids was not correlated with the i-LTC4 level.The metabolic activities of these synovial fluids were determined by incubating them with 3H-LTC4 and then separating sulfidopeptide LTs by high performance liquid chromatography. LTC4 was found to be converted by γ-glutamyltranspeptidase activities to LTD4 which was further metabolized by dipeptidase of synovial fluid to LTE4. The conversion of LTC4 to LTD4 in synovial fluids of patients with OA and RA were similar, but the dipeptidase activity converting LTD4 to LTE4 was higher in fluid from patients with RA. It is suggested that a high level of LTE4 may play cause exudation of synovial fluid, since LTE4 has a strong effect in increasing vascular permeability.

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.

We isolated osteoblastic cells derived from human periosteum and established them in culture. Their growth depended on the presence of ascorbic acid, and the doubling time was 40 to 60 h. The requirement for ascorbic acid was used to high production of collagen. These cells produced mainly type I collagen and only small amounts of type III collagen determined by reducing sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis. The total collagen yield was about 10 mg from 2 X 10(7) cells. The cells could be continuously cultured in alpha-minimum essential medium supplemented with 10% fetal bovine serum for 18 to 40 population doubling levels, depending on the age of the donated periosteum. These cells have the ability to calcify when incubated with 2 mM alpha-glycerophosphate-Na2. Calcification as viewed by the naked eye appeared from Day 15 after treatment. Treatment with the active formed vitamin D3, 1, 25 dihydroxyvitamin D3 enhanced calcification significantly and stimulated osteocalcin production. By electron microscopy, cells with many projections on their surfaces showed well-developed rough endoplasmic reticulum and actinlike fibers, and larger numbers of lysosomes, mitochondria, and secretion granules. Many matrix vesicles, in which minerals were initially localized, and well-banded collagen fibrils were seen in the intercellular spaces. These observations demonstrate typical osteoblastic morphology. The above results indicate that cultured cells from human periostem are osteoblastic cells that have the capacity to differentiate into osteocytes and to deposit calcified minerals in response to 1,25 dihydroxyvitamin D3.

Summary Hedgehog (Hh)-Patched1 (Ptch1) signaling plays essential roles in various developmental processes, but little is known about its role in postnatal homeostasis. Here, we demonstrate regulation of postnatal bone homeostasis by Hh-Ptch1 signaling. Ptch1 -deficient ( Ptch1 +/− ) mice and patients with nevoid basal cell carcinoma syndrome showed high bone mass in adults. In culture, Ptch1 +/− cells showed accelerated osteoblast differentiation, enhanced responsiveness to the runt-related transcription factor 2 (Runx2), and reduced generation of the repressor form of Gli3 (Gli3rep). Gli3rep inhibited DNA binding by Runx2 in vitro, suggesting a mechanism that could contribute to the bone phenotypes seen in the Ptch1 heterozygotes. Moreover, systemic administration of the Hh signaling inhibitor cyclopamine decreased bone mass in adult mice. These data provide evidence that Hh-Ptch1 signaling plays a crucial role in postnatal bone homeostasis and point to Hh-Ptch1 signaling as a potential molecular target for the treatment of osteoporosis.

The proto-oncogene c-src, which encodes a non-receptor-type tyrosine kinase c-Src, has been shown to be essential for osteoclastic bone resorption by the finding that the targeted disruption of the c-src gene induced osteopetrosis in mice. The csk (C-terminal Src family kinase) gene encodes a cytoplasmic protein-tyrosine kinase that specifically phosphorylates the negative regulatory site of c-Src (Tyr-527), thereby inhibiting its kinase activity. To regulate osteoclast function by modulating the kinase activity of c-Src, we constructed an adenovirus vector that carries this gene. The recombinant adenovirus vector carrying csk cDNA induced Csk expression in mouse osteoclast-like cells formed in vitro and clearly reduced c-Src kinase activity in a dose-dependent manner. The expression of Csk caused cytoskeletal disorganization of osteoclast-like cells and strongly suppressed pit-forming activity of the cells in vitro. In addition, the viral vector carrying csk gene dramatically suppressed interleukin-1 alpha-induced bone resorption in vivo. Conversely, kinase-inactive Csk caused an increase in c-Src kinase activity and bone resorbing activity of the cells both in vitro and in vivo, acting as a dominant negative molecule against intrinsic Csk. These findings indicate that the inhibition of c-Src activity by adenovirus vector-mediated csk expression offers an efficient means for inhibiting pathological bone resorption by suppressing osteoclast function.

To examine the effects of direct electric current on ligament healing in rabbits, a full-thickness defect of the patellar ligament was electrically stimulated for time periods of up to seven weeks. The rabbits were randomly assigned to biomechanical and biochemical studies, and healing was evaluated by these parameters. Electrical stimulation was shown to restore tensile stiffness in a short period of time and to decrease the relative proportion of Type III collagen more rapidly than in the control group. However, electrical stimulation did not change the collagen content of newly formed tissue. Electricity enhances the repair process of the ligament by changing the ratio of collagen types.