NF-κB-mediated effects on behavior and cartilage pathology in a non-invasive loading model of post-traumatic osteoarthritis.

OBJECTIVE: This study aimed to examine the temporal activation of NF-κB and its relationship to the development of pain-related sensitivity and behavioral changes in a non-invasive murine knee loading model of PTOA. METHOD: Following knee injury NF-κB activity was assessed longitudinally via in vivo imaging in FVB. Cg-Tg (HIV-EGFP,luc)8Tsb/J mice. Measures of pain-related sensitivity and behavior were also assessed longitudinally for 16 weeks. Additionally, we antagonized NF-κB signaling via intra-articular delivery of an IκB kinase two antagonist to understand how local NF-κB inhibition might alter disease progression. RESULTS: Following joint injury NF-κB signaling within the knee joint was transiently increased and peaked on day 3 with an estimated 1.35 p/s/cm2/sr (95% CI 0.913.1.792 p/s/cm2/sr) fold increase in signaling when compared to control joints. Furthermore, injury resulted in the long-term development of hindpaw allodynia. Hyperalgesia withdrawal thresholds were reduced at injured knee joints, with the largest reduction occurring 2 days following injury (estimate of between group difference 129.1 g with 95% CI 60.9,197.4 g), static weight bearing on injured limbs was also reduced. Local delivery of an NF-κB inhibitor following joint injury reduced chondrocyte death and influenced the development of pain-related sensitivity but did not reduce long-term cartilage degeneration. CONCLUSION: These findings underscore the development of behavioral changes in this non-invasive loading model of PTOA and their relationships to NF-κB activation and pathology. They also highlight the potential chondroprotective effects of NF-κB inhibition shortly following joint injury despite limitations in preventing the long-term development of joint degeneration in this model of PTOA.

Poly-ADP-ribosylation-mediated degradation of ARTD1 by the NLRP3 inflammasome is a prerequisite for osteoclast maturation.

Evidence implicates ARTD1 in cell differentiation, but its role in skeletal metabolism remains unknown. Osteoclasts (OC), the bone-resorbing cells, differentiate from macrophages under the influence of macrophage colony-stimulating factor (M-CSF) and receptor-activator of NF-κB ligand (RANKL). We found that M-CSF induced ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1) auto-ADP-ribosylation in macrophages, a modification that marked ARTD1 for cleavage, and subsequently, for degradation upon RANKL exposure. We established that ARTD1 proteolysis was NLRP3 inflammasome-dependent, and occurred via the proteasome pathway. Since ARTD1 is cleaved at aspartate(214), we studied the impact of ARTD1 rendered uncleavable by D214N substitution (ARTD1(D214N)) on skeletal homeostasis. ARTD1(D214N), unlike wild-type ARTD1, was resistant to cleavage and degradation during osteoclastogenesis. As a result, ARTD1(D214N) altered histone modification and promoted the abundance of the repressors of osteoclastogenesis by interfering with the expression of B lymphocyte-induced maturation protein 1 (Blimp1), the master regulator of anti-osteoclastogenic transcription factors. Importantly, ARTD1(D214N)-expressing mice exhibited higher bone mass compared with controls, owing to decreased osteoclastogenesis while bone formation was unaffected. Thus, unless it is degraded, ARTD1 represses OC development through transcriptional regulation.

Myeloid lineage skewing due to exacerbated NF-κB signaling facilitates osteopenia in Scurfy mice.

Immune surveillance through Foxp3+ regulatory T cells plays a crucial role in bone homeostasis. Scurfy, the mouse model of autoimmune IPEX syndrome, bears a loss-of-function mutation in Foxp3 that leads to multi-organ inflammation. Herein, we report that scurfy mice exhibit severe bone loss mediated by accelerated osteoclastogenesis. Mechanistically, Foxp3 deficiency results in the upregulation of NF-κB in T helper cells through the loss of repressive Foxp3/NEMO interaction, thereby unleashing NF-κB-mediated over-production of pro-osteoclastogenic cytokines. Flow cytometry analysis shows marked increase in lin-Sca-1+c-kit+ hematopoietic stem cells (LSK HSCs) and granulocyte/macrophage progenitors (GMPs) in bone marrow of scurfy mice with corresponding exacerbated osteoclastogenic potential, implying that osteoclast progenitors are affected at a very primitive stage in this disorder. Scurfy LSK HSCs exhibit greater sensitivity to M-CSF and contain abundant PU.1+ Sf LSK HSCs compared with WT. Accordingly, genetic or pharmacological inhibition of M-CSF or mTOR signaling, but not IL-17 signaling, attenuates osteoclastogenesis and osteopenia in scurfy. Thus, our study suggests that Foxp3 deficiency leads to osteopenia owing to dysregulated NF-κB activity and subsequent cytokine-mediated hyper-proliferation of myeloid precursors, and positions the NF-κB pathway as a potential target for therapeutic intervention for this disorder.

Activation of peroxisome proliferator-activated receptor-gamma pathway inhibits osteoclast differentiation.

The nuclear receptor and transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), regulates the activity of other transcription factors in the adipogenic differentiation and inflammatory response pathways. We examined the possible function of the PPAR-gamma pathway in osteoclast (Ocl) formation from CD34(+) hematopoietic stem cells (CD34(+) HSCs), using a co-culture system comprised of human mesenchymal stem cells (hMSCs) and CD34(+) HSCs, both derived from bone marrow. Ocl formation in this co-culture system is enhanced by the addition of exogenous osteoprotegerin ligand (OPGL), an essential Ocl differentiation factor, and macrophage-colony stimulating factor (M-CSF). The data indicate that soluble OPGL (sOPGL) and M-CSF stimulate Ocl formation in the co-cultures up to 4-fold compared with CD34(+) HSCs alone treated with sOPGL and M-CSF. CD34(+) HSCs, but not hMSCs, express PPAR-gamma, and 15-deoxy-Delta(12, 14)-prostaglandin-J2 (15d-PG-J2), a PPAR-gamma agonist, completely blocked the effects of sOPGL and M-CSF on Ocl formation and activity. The inhibitory effect of 15d-PG-J2 is specific to the Ocl lineage in both human and mouse models of osteoclastogenesis. Accordingly, parallel experiments demonstrate that sOPGL activates the NF-kappaB pathway within mouse Ocl progenitors, and this effect was abolished by 15d-PG-J2. These data establish a link between PPAR-gamma and OPGL signaling within Ocl progenitors, and support a role for PPAR-gamma pathway in the modulation of osteoclastogenesis.

Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase.

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.

Human mesenchymal stem cells promote human osteoclast differentiation from CD34+ bone marrow hematopoietic progenitors.

Interactions between osteoclast progenitors and stromal cells derived from mesenchymal stem cells (MSCs) within the bone marrow are important for osteoclast differentiation. In vitro models of osteoclastogenesis are well established in animal species; however, such assays do not necessarily reflect human osteoclastogenesis. We sought to establish a reproducible coculture model of human osteoclastogenesis using highly purified human marrow-derived MSCs (hMSCs) and CD34+ hematopoietic stem cells (HSCs). After 3 weeks, coculture of hMSCs and HSCs resulted in an increase in hematopoietic cell number with formation of multinucleated osteoclast-like cells (Ocls). Coculture of hMSCs with HSCs, transduced with a retroviral vector that expresses enhanced green fluorescent protein, produced enhanced green fluorescent protein+ Ocls, further demonstrating that Ocls arise from HSCs. These Ocls express calcitonin and vitronectin receptors and tartrate-resistant acid phosphatase and possess the ability to resorb bone. Ocl formation in this assay is cell contact dependent and is independent of added exogenous factors. Conditioned medium from the coculture contained high levels of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), and macrophage-colony stimulating factor. IL-6 and LIF were present at low levels in cultures of hMSCs but undetectable in cultures of HSCs alone. These data suggest that coculture with HSCs induce hMSCs to secrete cytokines involved in Ocl formation. Addition of neutralizing anti-IL-6, IL-11, LIF, or macrophage-colony stimulating factor antibodies to the coculture inhibited Ocl formation. hMSCs seem to support Ocl formation as undifferentiated progenitor cells, because treatment of hMSCs with dexamethasone, ascorbic acid, and beta-glycerophosphate (to induce osteogenic differentiation) actually inhibited osteoclastogenesis in this coculture model. In conclusion, we have developed a simple and reproducible assay using culture-expanded hMSCs and purified HSCs with which to study the mechanisms of human osteoclastogenesis.

Cadherin-6 mediates the heterotypic interactions between the hemopoietic osteoclast cell lineage and stromal cells in a murine model of osteoclast differentiation.

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell-cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell-cell contact caused evident morphological changes accompanied with tight cell-cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow-derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.

E-cadherin expression in human breast cancer cells suppresses the development of osteolytic bone metastases in an experimental metastasis model.

The molecular mechanisms by which human cancer cells spread to bone are largely unexplored. The process likely involves cell adhesion molecules (CAMs) that are responsible for homophilic and heterophilic cell-cell interactions. One relevant CAM may be the calcium-dependent transmembrane glycoprotein E-cadherin. To investigate the involvement of E-cadherin in breast cancer metastasis to bone, we used an in vivo model in which osteolytic bone metastases preferentially occur after injections of cancer cells directly into the arterial circulation through the left ventricle of the hearts of nude mice. We have found that E-cadherin-negative human breast cancer cells MDA-MB-231 (MDA-231) develop radiographically detectable multiple osteolytic bone metastases and cachexia in this model. However, MDA-231 breast cancer cells that were transfected with E-cadherin cDNA showed a dramatically impaired capacity to form osteolytic metastases and induce cachexia. Histological and histomorphometrical analyses of bones of mice bearing mock-transfected MDA-231 revealed aggressive metastatic tumor, whereas metastatic tumor burden was significantly decreased in the bones of mice bearing E-cadherin-expressing MDA-231. Nude mice bearing E-cadherin-transfected MDA-231 breast cancer cells survived longer than mice bearing mock-transfected MDA-231 breast cancer cells. Anchorage-dependent and -independent growth in culture and tumor enlargement in the mammary fat pad of nude mice were unchanged between mock-transfected and E-cadherin-expressing MDA-231, suggesting that these differences in metastatic behavior are not due to an impairment of cell growth and tumor-igenicity. Our results show the suppressive effects of E-cadherin expression on bone metastasis by circulating breast cancer cells and suggest that the modulation of expression of this CAM may reduce the destructive effects of breast cancer cells on bone.

Short-term local injections of transforming growth factor-beta 1 decrease ovariectomy-stimulated osteoclastic resorption in vivo in rats.

Estrogen deficiency in rats is responsible for increased osteoclastic resorption and a subsequent rapid bone loss. TGF-beta, which is known to have acute effects on bone resorption in several in vitro models, has been shown to be secreted by osteoblastic cells in vitro in response to 17 beta-estradiol, but little is known about its in vivo effects on bone resorption. We therefore decided to investigate the short-term effect of TGF-beta 1 on bone resorption in ovariectomized rats. TGF-beta 1 (0.04-20 ng/injection), or vehicle, was injected daily directly into the bone marrow space, through a thin catheter implanted in the distal end of the right femur, during 4 consecutive days, starting 14 days after the ovariectomy. Bone histomorphometry was performed in the secondary spongiosa of the metaphysis of injected femurs and compared with vehicle-injected femurs of sham ovariectomized rats. Ovariectomy was associated with a marked increase in the resorption surface, a 2-fold increase in the number of osteoclasts, and no change in the number of TRAP-positive marrow cells distant from bone surfaces. Bone resorption was significantly lower in the TGF-beta 1-injected bones of ovariectomized rats, as compared with vehicle injected bones: the osteoclast surface and the number of osteoclasts were, respectively, 11.0 +/- 5.1% versus 20.8 +/- 1.3% and 287 +/- 41 versus 505 +/- 53, in bones injected with 0.2 ng of TGF-beta 1 as compared with vehicle-injected bones (mean +/- SE, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of cadherin in the generation of multinucleated osteoclasts from mononuclear precursors in murine marrow.

A critical step in bone resorption is the fusion of mononuclear osteoclast precursors to form multinucleated osteoclasts. However, little is known of the molecular mechanisms that are responsible for this important process. Since the expression of proteins in the cadherin family of homophilic calcium-dependent cell adhesion molecules is involved in the fusion process for certain other cells, we examined their role in osteoclast formation. Immunohistochemical examination of human and mouse bone using monoclonal antibodies to human and mouse E-cadherin clearly demonstrated positive staining in osteoclasts. N- and P-cadherin were not detected. In cultures of murine marrow mononuclear cells in which osteoclasts form by cell fusion, E-cadherin expression determined by Western blotting reached the highest levels as fusion was taking place. Expression of E-cadherin gene fragment was also detected in the marrow cultures by polymerase chain reaction. To study the functional role of E-cadherin expression in osteoclastic differentiation, neutralizing monoclonal antibodies were examined for their effects on osteoclast formation. The antibodies decreased the number of tartrate-resistant acid phosphatase (a marker of murine osteoclast)-positive multinucleated cell (TRAP-positive MNC) by inhibiting the fusion of mononuclear osteoclast precursors, but not proliferation of these cells or their attachment to plastic dish surfaces. This inhibitory effect was reversible. Furthermore, synthetic peptides containing the cell adhesion recognition sequence of cadherins also decreased TRAP-positive MNC formation. The antibodies and peptides inhibited not only osteoclast formation but also bone resorption. Antibodies to other types of cadherins and control rat IgG had no effects in these culture systems. Our findings suggest that E-cadherin expression may be involved in fusion (differentiation) of hemopoietic osteoclast precursors into mature multinucleated osteoclasts.

Osteoclast formation from human cord blood mononuclear cells co-cultured with mice embryonic metatarsals in the presence of M-CSF.

Investigating the potentiality of cord monocytes to differentiate toward osteoclast-like cells (OCL) in vitro, we previously reported that in the presence of 1,25(OH)2 vitamin D3 (1,25-(OH)2D3), multinucleated-cells generated by cord monocyte cultures though displaying morphological features of OCL failed to resorb devitalized bones. We thus hypothesized that full differentiation of cord monocytes toward bone-resorbing cells may require the presence of factors released from and/or direct interactions with living osteogenic cells. In the present study, we tested these hypotheses using two culture systems supporting the development of bone-resorbing cells in the presence of bone matrix. First, cord mononuclear cells were co-cultured with murine fetal metatarsals depleted of osteoclast progenitor cells (stripped metatarsals) in the presence of 1,25-(OH)2D3. We found that cord mononuclear cells failed to differentiate toward OCL as indicated by the absence of the release of 45Ca previously incorporated in fetal bones and by the absence of formation of TRAP-positive (TRAP[+]) multinucleated cells which have invaded mineralized cartilage during the co-culture period. In the same model, we then investigated the effect of some soluble factors known as stimulators of osteoclast differentiation. Whereas exogenous rhIL6 and rhIL3 were ineffective in this assay, rhM-CSF consistently increased both the number of TRAP(+) multinucleated cells inside the mineralized cartilage and the release of 45Ca into the culture media. The effects of rhM-CSF were time-dependent reaching the maximum after 3 weeks of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta enhances calcitonin-induced cyclic AMP production and the number of calcitonin receptors in long-term cultures of human umbilical cord blood monocytes in the presence of 1,25-dihydroxycholecalciferol.

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide, abundant in bone, that regulates both proliferation and differentiation of a wide variety of cells, but its role in osteoclast differentiation remains controversial. We have recently shown that long-term cultures of human cord blood monocytes, in the presence of 1,25 dihydroxycholecalciferol (1,25-(OH)2D3), give rise to cells that express two markers of the osteoclast phenotype, namely, the vitronectin receptor (VNR) and the calcitonin receptor (CTR). TGF-beta enhanced the proportion of cells expressing the VNR. In the present study, we investigated the effect of TGF-beta on the expression of CTR in cord blood monocytes cultured during 3 weeks in the presence of 1,25-(OH)2D3. When added within the first 2 weeks of culture, TGF-beta (500 pg/ml) significantly decreased the cell protein content. TGF-beta alone did not stimulate basal cAMP production. The 10 nM-sCT-stimulated cAMP production was enhanced by increasing TGF-beta concentrations from 50 pg/ml to 1,000 pg/ml: for 500 pg/ml TGF-beta, it was 294 +/- 28% vs. 140 +/- 25% for control cultures (p less than 0.01). The sCT dose-response curves showed a higher cAMP production from 10(-9) M to 10(-7) M of sCT in the presence of 500 pg/ml TGF-beta than in control cultures. The increase was 325 +/- 36% in the presence of TGF-beta and 195 +/- 13% in the absence of TGF-beta, for 10(-7) M sCT (p less than 0.01). This effect of TGF-beta on cAMP production was not observed either when it was added to monocyte cultures the last day or 2 hours before the end of the culture or in MCF7, a human breast cancer cell line that expresses CTR. [125I]-sCT binding studies performed on confluent cells showed similar Kd in control and TGF-beta-treated cells. By contrast, the CTR number was significantly increased in the presence of TGF-beta: 6.1 +/- 2 x 10(4) receptors per cell in control cultures and 28.8 +/- 8.1 x 10(4) receptors per cell in TGF-beta-treated cultures (p less than 0.05). It is thus suggested that TGF-beta increases the number of CTR of these cells that have other features of preosteoclasts. The role of this cytokine on the process of osteoclast differentiation and in bone resorption is thus emphasized.

Human umbilical cord blood monocytes express calcitonin receptors in culture in the presence of 1,25 dihydroxyvitamin D.

Calcitonin (CT) is a potent inhibitor of bone resorption and there are abundant CT receptors on mature osteoclasts. The relationship between osteoclast precursors and monocyte lineage is far from clear. We recently showed that human cord monocytes in culture, by contrast to adult monocytes, develop some features of osteoclast precursors. We therefore assessed the presence of CT receptors on monocytes. We could not demonstrate any CT receptors on adult monocytes. By contrast, we observed in cultured cord monocytes increased cAMP production in presence of CT. This cAMP response was observed after a 2-week culture and only in the presence of 10(-9) M 1,25-dihydroxyvitamin D. After a 3-week culture, CT 10(-9) to 10(-6) M increased cAMP production dose dependently from 10(-9) M; however the curve was shallower than the one observed in a control CT receptor positive tumoral cell line, MCF7. 125I sCT bound specifically to cord monocytes cultured during 3 weeks; apparent dissociation constant (Kd) was 3.3 +/- 2.2 10(-10) M and average receptor number was 5.1 +/- 0.4 10(4)/cell. On autoradiography all the cells, whether mono or multinucleated, were labeled with 125sCT. One or 2-h exposure to salmon CT did not induce cell contraction. In conclusion, CT receptors can be induced on newborn cord monocytes in the presence of 1,25-dihydroxyvitamin D. This observation shows that osteoclasts and fetal monocytes share a common membrane determinant.