Receptor Activator Of NF-kappaB Ligand Production Research Articles

Alpha-7 Nicotinic Receptor Dampens Murine Osteoblastic Response to Inflammation and Age-Related Osteoarthritis

IntroductionOsteoarthritis (OA) is a whole-joint disease characterized by a low-grade inflammation that is involved in both cartilage degradation and subchondral bone remodeling. Since subchondral bone has a cholinergic innervation and that acetylcholine (Ach) might have an anti-inflammatory effect through the α7 nicotinic Ach receptor (α7nAchR), we aimed (i) to determine the expression of non-neuronal cholinergic system and nicotinic receptor subunits by murine and human osteoblasts, (ii) to address the role of α7nAchR in osteoblastic response to inflammation, and (iii) to study the role of α7nAchR in a spontaneous aging OA model.MethodsPrimary cultures of WT and α7nAchR knock-out mice (Chrna7-/-) murine osteoblasts and of subchondral bone human OA osteoblasts were performed. The expressions of the non-neuronal cholinergic system and of the nAchR subunits were assessed by PCR. In vitro, IL1β-stimulated WT, Chrna7-/-, and human osteoblasts were pretreated with nicotine. At 24 h, expressions of interleukin-6 (IL6) and metalloproteinase-3 and -13 (MMP), RANK-ligand (RANKL), and osteoprotegerin (OPG) were quantified by qPCR and ELISA. Spontaneous aging OA was evaluated and compared between male WT and Chrna7-/- mice of 9 and 12 months.ResultsMurine WT osteoblasts express the main components of the cholinergic system and α7 subunit composing α7nAchR. Nicotine partially prevented the IL1β-induced expression and production of IL6, MMP3, and RANKL in WT osteoblasts. The effect for IL6 and MMP was mediated by α7nAchR since nicotine had no effect on Chrna7-/- osteoblasts while the RANKL decrease persisted. Chrna7-/- mice displayed significantly higher cartilage lesions than their WT counterparts at 9 and 12 months, without difference in subchondral bone remodeling. Human OA osteoblasts also expressed the non-neuronal cholinergic system and α7 subunit as well as CHRFAM7A, the dominant negative duplicate of Chrna7. Nicotine pretreatment did not significantly reduce IL6 and MMP3 production in IL-1β-stimulated human osteoarthritic osteoblasts (n = 4), possibly due to CHRFAM7A.ConclusionCholinergic system counteracts murine osteoblastic response to IL-1β through α7nAchR. Since α7nAchR deletion may limit cartilage degradation during murine age-related OA, enhancing cholinergic system could be a new therapeutic target in OA but may depend on CHRFAM7A expression.

Local promotion of B10 function alleviates experimental periodontitis bone loss through antagonizing RANKL-expressing neutrophils.

Persistent host immune responses initiated by oral bacteria protect host against infection but may also elicit the process of sustained periodontal inflammation and subsequent alveolar bone loss. Interleukin-10 (IL-10), an anti-inflammatory cytokine, can downregulate pro-inflammatory cytokine and inhibit neutrophil migration in inflammation. IL-10-expressing regulatory B cells (B10) is termed by negatively regulating immune response through IL-10 and are mainly restricted in CD19+ CD1dhi CD5+ B cells in mice. Our current study was aimed to explore the effect of locally transferred CD19+ CD1dhi CD5+ B cells on inflammation and alveolar bone loss in an experimental periodontitis mouse model. Ligation plus P. gingivalis (Pg) infection was used to induce periodontitis in a mouse model. CD19+ CD1dhi CD5+ B cells were sorted by flow cytometry and transferred into the gingivae immediately on the fifth day after ligation. All the mice were sacrificed on day 14 after ligation. H&E staining showed that inflammatory cell infiltration was significantly reduced by the CD19+ CD1dhi CD5+ B cells. Toluidine blue staining showed that the CD19+ CD1dhi CD5+ B cells alleviated alveolar bone loss in the ligature/Pg-induced periodontitis in mice. Immunohistochemical staining showed Receptor Activator of NF-KappaB Ligand (RANKL), Interleukin-1β(IL-1β) and Interleukin-17 (IL-17) were decreased after the CD19+ CD1dhi CD5+ B cell transfer. Immunofluorescent staining showed that IL-10 was increased while the number of Ly6G+ neutrophil and its RANKL production were decreased in gingival tissue. These results indicated that locally transferred CD19+ CD1dhi CD5+ B cells may alleviate alveolar bone loss through inhibiting pro-inflammatory cytokine expression and RANKL-expressing neutrophils in the periodontitis mouse model.

Effects of antibody to receptor activator of nuclear factor κ-B ligand on inflammation and cartilage degradation in collagen antibody-induced arthritis in mice.

BackgroundRheumatoid arthritis (RA) is an inflammatory disease that leads to destruction of both articular cartilage and bone tissues. In rheumatic joints, synoviocytes and T-lymphocytes as well as bone cells produce the receptor activator of nuclear factor κ-B (RANK) ligand (RANKL), which binds to RANK on the surface of osteoclasts and their precursor cells to induce differentiation and activation of osteoclasts. Hence, inhibition of RANKL may be a promising approach to suppress osteolysis in RA. On the other hand, RANKL production by lymphocytes indicates the possibility that its inhibition would be effective to suppress inflammation in RA. In addition, it has been reported that cathepsin K, a predominant cysteine protease in osteoclasts, is involved in cartilage destruction in RA model mice. Here, we evaluated the effects of an anti-RANKL antibody on inflammation in footpads and degradation of articular cartilage in RA model mice.ResultsWe induced arthritis in mice by injection of anti-type II collagen antibodies and lipopolysaccharide (LPS). Inhibition of RANKL by an anti-RANKL antibody (OYC1, Oriental Yeast, Tokyo, Japan) was confirmed by increased bone volume in the metaphysis of tibias. Swelling in either limb until day 14 was seen in 5 of 6 mice injected with anti-collagen antibodies and LPS without treatment with OYC1, while that was seen in 4 of 5 mice treated with OYC1. The average arthritis scores on day 14 in those groups were 2.17 and 3.00, respectively, indicating that OYC1 did not ameliorate inflammation in the limbs. Histological analyses indicated that OYC1 does not protect articular cartilage from destruction in mice with arthritis.ConclusionsOur present study failed to show the effectiveness of an anti-RANKL antibody to ameliorate inflammation in the limbs or protect articular cartilage from degradation in a collagen antibody-induced arthritis mouse model.Electronic supplementary materialThe online version of this article (doi:10.1186/s12952-014-0018-0) contains supplementary material, which is available to authorized users.

PTX3 Stimulates Osteoclastogenesis by Increasing Osteoblast RANKL Production

Pentraxin-3 (PTX3), also known as tumor necrosis factor-stimulated gene 14 (TSG-14), is produced by immune and vascular cells in response to pro-inflammatory signals and is therefore a multipotent inflammatory mediator. The present study showed that during human osteoblast (OB) differentiation, precursor OBs (pOBs), but not mature OB, highly expressed PTX3. TNFα treatment elevated the PTX3 expression of pOBs. When mice were injected with lipopolysaccharide, which induces an inflammatory osteolytic condition characterized by trabecular bone destruction and high osteoclastogenesis, their bone marrow cells expressed elevated levels of PTX3 protein. Exogenous PTX3 did not directly affect osteoclast (OC) or OB differentiation. However, when pOBs and precursor OCs were co-cultured, exogenous PTX3 significantly increased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (i.e., OC cells) by increasing the pOB mRNA expression and protein secretion of RANK ligand (RANKL). This was accompanied with increased Runt-related transcription factor 2 (Runx2) expression in the pOBs. Knock-down of endogenous PTX3 with small-interfering RNA did not change the osteogenic potential of pOBs but suppressed their production of RANKL and reduced osteoclastogenesis. Finally, TNFα treatment of the co-culture elevated PTX3 expression by the pOBs and increased OC formation. This effect was suppressed by PTX3 knock-down by decreasing RANKL expression. Thus, the PTX3-driven increase in the osteoclastogenic potential of pOBs appears to be mediated by the effect of PTX3 on pOB RANKL production. These findings suggest that PTX3 is an inflammatory mediator that contributes to the deteriorating osteolytic condition of inflamed bone. J. Cell. Physiol. 229: 1744-1752, 2014. © 2014 Wiley Periodicals, Inc.

Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo.

Osteosarcoma (OS) is the most common cancer of bone. Parathyroid hormone (PTH) regulates calcium homeostasis and bone development, while the paracrine/autocrine PTH-related protein (PTHrP) has central roles in endochondral bone formation and bone remodeling. Using a murine OS model, we found that OS cells express PTHrP and the common PTH/PTHrP receptor (PTHR1). To investigate the role of PTHR1 signaling in OS cell behavior, we used shRNA to reduce PTHR1 expression. This only mildly inhibited proliferation in vitro, but markedly reduced invasion through collagen and reduced expression of RANK ligand (RANKL). Administration of PTH(1-34) did not stimulate OS proliferation in vivo but, strikingly, PTHR1 knockdown resulted in a profound growth inhibition and increased differentiation/mineralization of the tumors. Treatment with neutralizing antibody to PTHrP did not recapitulate the knockdown of PTHR1. Consistent with this lack of activity, PTHrP was predominantly intracellular in OS cells. Knockdown of PTHR1 resulted in increased expression of late osteoblast differentiation genes and upregulation of Wnt antagonists. RANKL production was reduced in knockdown tumors, providing for reduced homotypic signaling through the receptor, RANK. Loss of PTHR1 resulted in the coordinated loss of gene signatures associated with the polycomb repressive complex 2 (PRC2). Using Ezh2 inhibitors, we demonstrate that the increased expression of osteoblast maturation markers is in part mediated by the loss of PRC2 activity. Collectively these results demonstrate that PTHR1 signaling is important in maintaining OS proliferation and undifferentiated state. This is in part mediated by intracellular PTHrP and through regulation of the OS epigenome.

AB0390 Serum RANKL Activity is Suppressed by TNF Alpha Inhibition but not with MTX Alone

BackgroundBone is, together with cartilage a central target of rheumatoid arthritis (RA). Chronic synovitis attacks the juxtaarticular bone, where it causes bone erosions. Formation of bone erosions is a consequence...

Hydrogen Sulfide

Vascular calcification is the major cause of cardiovascular morbidity and mortality in patients with type 2 diabetes mellitus, chronic kidney disease, and in aging patients.1 Considerable progress has been made in the past 2 decades in understanding the molecular mechanisms of vascular calcification.2–4 Regardless of the morphology and location, most evidence indicates that vascular calcification involves an organized process recapitulating many cellular and molecular events that govern skeletal bone formation. Although a large body of evidence shows that osteoblastic and osteochonrocytic cells contribute to vascular calcification, it remains unclear how osteoclasts are differentiated from their precursors and how osteoclasts play a role in calcium reabsorption in calcifying arteries. Osteoclasts develop from monocytic precursors of the hematopoietic lineage, and 2 distinct signaling systems are both necessary and sufficient for the early differentiation into multinucleated osteoclasts as demonstrated by osteopetrotic mice models with loss-of-function mutation of either gene.5–7 One is a signaling system that involves macrophage colony-stimulating factor (MCSF) and its receptor c-fms, and the other involves receptor activator of nuclear factor κB (RANK) ligand (RANKL), RANK, and osteoprotegerin (OPG), a soluble decoy receptor for RANKL.8–10 MCSF and RANKL induce osteoclast differentiation, fusion, and maturation on binding to its specific receptors, c-fms and RANK, respectively, on the surface of preosteoclastic monocytes. Increased production of RANKL in atherosclerotic lesions, particularly in endothelial cells and vascular smooth muscle cells (SMCs), has been well documented.11–13 See accompanying article on page 626 Studies of …

Immunomodulatory drugs lenalidomide and pomalidomide inhibit multiple myeloma-induced osteoclast formation and the RANKL/OPG ratio in the myeloma microenvironment targeting the expression of adhesion molecules

Multiple myeloma (MM)-induced osteoclast (OC) formation is mainly due to an imbalance of the receptor activator NF-κB ligand (RANKL)-osteoprotegerin (OPG) ratio in favor of RANKL in the bone microenvironment and to the CCL3 production by MM cells. The purpose of the study was to investigate the effect of the immunomodulatory drugs on RANKL/OPG ratio, the production of pro-osteoclastogenic cytokines, and MM-induced OC formation. We found that invivo concentrations of both lenalidomide (LEN) and pomalidomide (POM) significantly blunted RANKL upregulation normalizing the RANKL/OPG ratio in human osteoprogenitor cells (PreOBs) when co-cultured with MM cells and also inhibited CCL3 production by MM cells. A reduction in CD49d expression, a molecule critically involved in RANKL upregulation in the MM microenvironment, accompanied this effect. Consistently, the pro-osteoclastogenic property of MM cells co-cultured with PreOBs was reduced by both LEN and POM. We further investigated the effect of these drugs on the transcriptional profile of both MM cells and PreOBs by microarray analysis, which showed that adhesion molecules, such as ITGA8 and ICAM2, are significantly downregulated in MM cells. Our data suggest that LEN and POM inhibit MM-induced OC formation through normalization of the RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells.

Abstract 445: Fibroblast-recruited, tumor-infiltrating CD4+ T cells stimulate mammary cancer metastasis through RANKL-RANK signaling

Abstract Inflammatory mechanisms influence tumor development and metastatic progression. Of interest is the role of such mechanisms in metastatic spread of tumors whose etiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. We found that prostate cancer metastasis is associated with lymphocyte infiltration into advanced tumors and elevated expression of the tumor necrosis factor (TNF) family members receptor activator of NF-κB (RANK) ligand (RANKL) and lymphotoxin (LT). But the source of RANKL and its role in metastasis were not established. RANKL and its receptor RANK control proliferation of mammary lobuloalveolar cells during pregnancy through activation of IκB kinase α(IKKα), a protein kinase that is required for self-renewal of mammary cancer progenitors and prostate cancer metastasis. We therefore examined whether RANKL, RANK and IKKα are also involved in mammary/breast cancer metastasis. Indeed, RANK signaling in mammary carcinoma cells that overexpress the ErbB2 (c-Neu) proto-oncogene, which is frequently amplified in metastatic human breast cancers, was important for pulmonary metastasis. Metastatic spread of ErbB2-transformed carcinoma cells was also dependent on CD4+CD25+ T cells, whose major pro-metastatic function appeared to be RANKL production. RANKL-producing T cells were mainly FoxP3+ and found in close proximity to smooth muscle actin (SMA)-positive stromal cells in mouse and human breast cancers. The T cell-dependence of pulmonary metastasis was replaced by administration of exogenous RANKL, a procedure that also stimulated pulmonary metastasis of RANK-positive human breast carcinoma cells. These results are consistent with the adverse prognostic impact of tumor-infiltrating CD4+ or FoxP3+ T cells on human breast cancer, and suggest that targeting of RANKL-RANK signaling can be used in conjunction with other therapies to prevent subsequent metastatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 445. doi:10.1158/1538-7445.AM2011-445

Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL–RANK signalling

Inflammatory mechanisms influence tumorigenesis and metastatic progression even in cancers whose aetiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. For instance, prostate cancer metastasis is associated with the infiltration of lymphocytes into advanced tumours and the upregulation of two tumour-necrosis-factor family members: receptor activator of nuclear factor-κB (RANK) ligand (RANKL) and lymphotoxin. But the source of RANKL and its role in metastasis have not been established. RANKL and its receptor RANK control the proliferation of mammary lobuloalveolar cells during pregnancy through inhibitor of nuclear factor-κB (IκB) kinase-α (IKK-α), a protein kinase that is needed for the self-renewal of mammary cancer progenitors and for prostate cancer metastasis. We therefore examined whether RANKL, RANK and IKK-α are also involved in mammary/breast cancer metastasis. Indeed, RANK signalling in mammary carcinoma cells that overexpress the proto-oncogene Erbb2 (also known as Neu), which is frequently amplified in metastatic human breast cancers, was important for pulmonary metastasis. Metastatic spread of Erbb2-transformed carcinoma cells also required CD4(+)CD25(+) T cells, whose major pro-metastatic function was RANKL production. Most RANKL-producing T cells expressed forkhead box P3 (FOXP3), a transcription factor produced by regulatory T cells, and were located next to smooth muscle actin (SMA)(+) stromal cells in mouse and human breast cancers. The dependence of pulmonary metastasis on T cells was replaceable by exogenous RANKL, which also stimulated pulmonary metastasis of RANK(+) human breast cancer cells. These results are consistent with the adverse impact of tumour-infiltrating CD4(+) or FOXP3(+) T cells on human breast cancer prognosis and suggest that the targeting of RANKL-RANK can be used in conjunction with the therapeutic elimination of primary breast tumours to prevent recurrent metastatic disease.

Dysregulation of the Receptor Activator of NF-κB Ligand and Osteoprotegerin Production Influence the Apoptosis of Multiple Myeloma Patients’ Bone Marrow Stromal Cells Co-Cultured with Myeloma Cells

The interaction of multiple myeloma (MM) cells and bone marrow stromal cells (BMSCs) induces profound changes in the bone marrow environment, influencing osteoclastogenesis and MM cell survival. Differences in receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) production in BMSCs derived from MM patients and control subjects and the apoptosis of BMSCs and MM cells in co-cultures of both cell types were examined. RANKL and OPG expressions were examined by ELISA and semiquantitative RT-PCR. Apoptosis of BMSCs after contact with RPMI8226 and U266 cells was measured by flow cytometry and the level of ALP activity by the spectrophotometric method. OPG production by BMSCs was significantly inhibited after direct contact with RPMI8226 cells. Production of soluble RANKL was enhanced and the increase was more significant in the BMSCs of the MM patients than in those of the controls. In co-cultures of BMSCs and MM cells, significant apoptosis was detected with a concomitant decrease in ALP activity. This apoptosis decreased significantly in the presence of RANK-Fc, an antagonist of RANKL. Disturbances in the RANKL/OPG system are more profound in the BMSCs of MM patients than in those of control subjects after direct contact with RPMI8226 cells. Moreover, direct contact with RPMI8226 and U266 cells induces apoptosis of BMSCs which is mediated by an overproduction of RANKL.

Modulation of OPG, RANK and RANKL by human chondrocytes and their implication during osteoarthritis

Earlier studies suggest the involvement of osteoprotegerin (OPG), RANK and RANK ligand (RANKL) in OA subchondral bone metabolism; however, few studies have looked at their functional consequences on chondrocytes. We compared the expression/production of OPG, RANK and RANKL on human normal and OA chondrocytes, and evaluated, on OA chondrocytes, their modulation by some catabolic factors. Furthermore, the role of OPG and RANKL on the production of catabolic/anabolic factors was assessed. Expression was determined using real-time PCR, production of RANK and RANKL by flow cytometry and that of OPG by ELISA. Modulation of these factors was determined upon treatment with IL-1beta, TNF-alpha and PGE(2). The functional consequences were examined following treatment with soluble RANKL or OPG-Fc (OPG without the heparin-binding domain). OPG, RANK and RANKL were expressed and produced by human chondrocytes. Membranous RANK was produced only by an OA chondrocyte subpopulation (29%) localized throughout the cartilage. The OPG/RANKL ratio was significantly (P = 0.05) reduced on the OA chondrocytes, whereas the RANK/RANKL ratio was significantly (P < 0.03) increased. OPG and membranous RANKL levels were significantly enhanced by IL-1beta, TNF-alpha and PGE(2), whereas membranous RANK was significantly increased only with IL-1beta. Administration of soluble RANKL had no effect on the OA chondrocytes. However, addition of OPG-Fc significantly stimulated MMP-13 (P = 0.05) and protease-activated receptor-2 (PAR-2) (P < 0.04) production. Our findings showed that human chondrocytes express and produce OPG, RANK and RANKL. OA chondrocyte treatment with catabolic factors pointed towards an increased biological effect of OPG. Interestingly, OPG appears to be involved in OA progression by increasing two catabolic factors involved in cartilage pathophysiology.

Oxidized lipids enhance RANKL production by T lymphocytes: Implications for lipid-induced bone loss

Osteoporosis is a systemic disease that is associated with increased morbidity, mortality and health care costs. Whereas osteoclasts and osteoblasts are the main regulators of bone homeostasis, recent studies underscore a key role for the immune system, particularly via activation-induced T lymphocyte production of receptor activator of NFkappaB ligand (RANKL). Well-documented as a mediator of T lymphocyte/dendritic cell interactions, RANKL also stimulates the maturation and activation of bone-resorbing osteoclasts. Given that lipid oxidation products mediate inflammatory and metabolic disorders such as osteoporosis and atherosclerosis, and since oxidized lipids affect several T lymphocyte functions, we hypothesized that RANKL production might also be subject to modulation by oxidized lipids. Here, we show that short term exposure of both unstimulated and activated human T lymphocytes to minimally oxidized low density lipoprotein (LDL), but not native LDL, significantly enhances RANKL production and promotes expression of the lectin-like oxidized LDL receptor-1 (LOX-1). The effect, which is also observed with 8-iso-Prostaglandin E2, an inflammatory isoprostane produced by lipid peroxidation, is mediated via the NFkappaB pathway, and involves increased RANKL mRNA expression. The link between oxidized lipids and T lymphocytes is further reinforced by analysis of hyperlipidemic mice, in which bone loss is associated with increased RANKL mRNA in T lymphocytes and elevated RANKL serum levels. Our results suggest a novel pathway by which T lymphocytes contribute to bone changes, namely, via oxidized lipid enhancement of RANKL production. These findings may help elucidate clinical associations between cardiovascular disease and decreased bone mass, and may also lead to new immune-based approaches to osteoporosis.

α1β1 integrin and interleukin‐7 receptor up‐regulate the expression of RANKL in human T cells and enhance their osteoclastogenic function

Activated T cells, through the production of the receptor activator of NF-kappaB ligand (RANKL) cytokine, have been implicated in the osteoclast development and bone loss that are associated with autoimmune diseases such as rheumatoid arthritis. However, the cellular pathways that regulate the expression of RANKL and the induction of osteoclasts are still unclear. In this study, we show that, in human effector CD4(+) T cells, activation of alpha1beta1 integrin and interleukin (IL)-7 receptor (IL-7R) up-regulates the expression and production of RANKL but has no effect on the production of interferon-gamma, an inhibitor of T-cell-mediated osteoclastogenesis. Thus, both alpha1beta1 integrin and IL-7R enhance the ability of these cells to induce the formation of osteoclasts from human monocytes. Furthermore, we found that simultaneous activation of effector CD4(+) T cells via alpha1beta1 integrin and IL-7R synergistically increases the production of RANKL and enhances their osteoclastogenic function. We also show that, although alpha1beta1 integrin does not protect human effector CD4(+) T cells from IL-2-withdrawal-induced apoptosis, it does enhance the pro-survival effect of IL-7, further emphasizing the importance of the alpha1beta1/IL-7R synergistic effect. Together our results identify a new function of alpha1beta1 integrin in T cells and suggest that activation of effector CD4(+) T cells through alpha1beta1 integrin and IL-7R is an important regulatory pathway in T-cell-dependent osteoclastogenesis. Further understanding of the mechanisms by which IL-7R and alpha1beta1 integrin promote T-cell-mediated osteoclastogenesis will lead to new insights into the regulatory pathways of T-cell-dependent bone resorption associated with autoimmune diseases.

Staphylococcus aureusInduces Expression of Receptor Activator of NF-κB Ligand and Prostaglandin E2in Infected Murine Osteoblasts

Osteomyelitis is an inflammatory disease of the bone that is characterized by the presence of necrotic bone tissue and increased osteoclast activity. Staphylococcus aureus is responsible for approximately 80% of all cases of human osteomyelitis. While the disease is especially difficult to treat, the pathogenesis of S. aureus-induced osteomyelitis is poorly understood. Elucidating the molecular mechanisms by which S. aureus induces osteomyelitis could lead to a better understanding of the disease and its progression and development of new treatments. Osteoblasts can produce several soluble factors that serve to modulate the activity or formation of osteoclasts. Receptor activator of NF-kappaB ligand (RANK-L) and prostaglandin E(2) (PGE(2)) are two such molecules which can promote osteoclastogenesis and stimulate bone resorption. In addition, previous studies in our laboratory have shown that osteoblasts produce inflammatory cytokines, such as interleukin 6, following infection with S. aureus, which could induce COX-2 and in turn PGE(2), further modulating osteoclast recruitment and differentiation. Therefore, we hypothesized that following infection with S. aureus, osteoblasts will express increased levels of RANK-L and PGE(2). The results presented in this study provide evidence for the first time that RANK-L mRNA and protein and PGE(2) expression are upregulated in S. aureus-infected primary osteoblasts. In addition, through the use of the specific COX-2 inhibitor NS 398, we show that when PGE(2) production is inhibited, RANK-L production is decreased. These data suggest a mechanism whereby osteoblasts regulate the production of RANK-L during infection.

Intracellular and surface RANKL are differentially regulated in patients with ankylosing spondylitis

Ankylosing spondylitis (AS) is characterized by ankylosis of axial joints but osteoporosis is also a well-reported feature. T cells have been implicated as a source of receptor activator of NFkappaB ligand (RANKL) in inflammatory bone diseases. Hence, we assessed whether T cells in patients with AS act as a source of RANKL too. Therefore, we investigated the expression of RANKL on T cells from 21 patients with AS by flow cytometry. Bone mineral density (BMD) was evaluated by quantitative computer tomography (QCT) and dual X-ray absorptiometry (DXA) and correlated with serum levels of osteoprotegerin (OPG) and RANKL. BMD was decreased in 45% of all patients when measured with DXA (48% with QCT) and correlated negatively with OPG. Expression of intracellular RANKL was increased on CD4+ (84 vs. 70%) and CD8+ (85.2 vs. 65.3%, P < 0.05) T cells in patients with AS, whereas expression of membrane-bound RANKL was significantly lower (CD4+: 2.2 vs. 8.5% and CD8+: 0.7 vs. 3.2%, P < 0.01). Our results indicate that surface and intracellular RANKL production is differentially regulated on T cells of patients with AS.

P62 Signaling Is Increased in Multiple Myeloma Microenvironment.

The bone microenvironment plays a critical role in promoting both tumor growth and bone destruction in myeloma (MM). Marrow stromal cells produce factors, which stimulate both the growth of MM cells and osteoclastic bone destruction and are key regulators of these processes. Marrow stromal cells produce these factors in increased amounts when they bind MM cells through adhesive interactions mediated via VCAM-1 on stromal cells and β1 integrins on the surface of MM cells. We have examined the role of sequestosome-1 (p62), a recently described member of the NF-κB signaling pathway, in this process, since it sits at the crossroads of multiple signaling pathways potentially involved in both osteoclastogenesis and MM cell growth. In previous studies, we found that stromal cells lacking p62 minimally supported the growth of MM cells or osteoclast (OCL) formation compared to wild-type p62 containing stromal cells. We have further shown that stromal cells lacking p62 produce much lower levels of TNF-α, interleukin (IL)-6, MCP-1 and RANK ligand (RANKL), factors which increase OCL formation, and express lower levels of VCAM-1. However, the mechanisms responsible for this decreased cytokine production and VCAM-1 expression in multiple myeloma patient stromal cells lacking p62 are unknown. Further, it is unknown that targeting PKCζ, a downstream enzyme activity activated by p62, will have similar effects on myeloma patient stromal cells. Our hypotheses are that inhibiting PKCζ expression will block the effects of cytokines produced in the MM-bone microenvironment in response to TNF-α, decrease VCAM-1 expression in stromal cells, and markedly diminish osteolytic bone destruction and MM growth in MM patients. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patient and normals and screened for p62 and PKCζ/λ activation in MM marrow. We measured levels of PKCζ and p62 by Western blot analysis in marrow stromal cells from 13 patients and 11 healthy controls. We found significantly elevated levels of phosho-PKCζ/λ and total PKCζ in MM patients although the levels varied greatly among the patients. We next measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in these marrow stromal cells. Since inhibiting p62 expression should profoundly diminish osteolytic bone destruction and myeloma growth in patients, by blocking production of RANKL, MCP-1, TNF-α and IL-6 in the tumor-bone microenvironment, and the upregulation of VCAM-1 on stromal cells, we assessed the effects of blocking p62 activity in human stromal cells. p62 siRNA (40 nM) was transduced into human stromal cells using a commercial transfection substrate. We confirmed that p62 expression was decreased by at least 50% in these stromal cells by Western blot analysis using an anti-p62 antibody. Stromal cells from the p62siRNA and control siRNA treatment and non-treatment stromal cells were cultured with or without MM1s cells for 3 days in separate experiments. The levels of VCAM-1 in p62siRNA transducer stromal cells were lower than control siRNA and untreated stromal cells. In addition, IL-6 production by MM1.s was increased when cocultured with control stromal cells but not with p62siRNA treated bone marrow stromal cells. These results show that p62 plays an important role in myeloma cell growth induced by cytokines that are upregulated in the marrow microenvironment and myeloma cells in patients with myeloma. Therefore, p62 is an attractive therapeutic target for MM growth and bone destruction.

Estradiol rapidly inhibits osteoclastogenesis and RANKL expression in bone marrow cultures in postmenopausal women: a pilot study

We examined RANKL expression and OCL formation in cultured bone marrow cells from eight postmenopausal women in response to E(2). E(2) treatment inhibited the ability of hematopoietic cells to form OCLs in response to RANKL, and decreased RANKL production. These changes are likely involved in the ability of E(2) to influence the development of osteoporosis. Estrogen (E(2)) deficiency at menopause increases osteoclast (OCL) formation and bone resorption, predisposing women to osteoporosis. We examined receptor activator of NF-kappa B-ligand (RANKL) expression and in vitro OCL formation in cultured bone marrow cells from eight postmenopausal women before and after 3 weeks of E(2) therapy and three untreated premenopausal women. TRAP staining and resorption pit assay determined OCL number and function. Flow cytometry measured the distribution of marrow cell types and expression of RANKL in the macrophage-enriched fraction (R1) and a lymphocyte-enriched fraction (R2). RANKL (3-100 ng/ml) produced a dose-dependent increase in in vitro OC formation and E(2) therapy significantly (p < 0.01) inhibited OCL formation by 33 to 50%. A small proportion of marrow cells bound anti- RANKL Ab (0.2-4.3%). There was no effect of E(2) on the percentage of cells binding the anti-RANKL Ab in the R1 fraction. In the R2 fraction E(2) treatment decreased the percentage of cells binding anti-RANKL Ab by 68 +/- 9% (p < 0.01). Three weeks of E(2) treatment had a dual action. It inhibited the ability of hematopoietic cells to form OCLs in response to RANKL, and decreased the production of RANKL in cells of the bone marrow. The observed changes in the osteoclastic potential of bone marrow cells are likely involved in the ability of E(2) to regulate bone mass and influence the development of osteoporosis.

The new concept of periodontal disease pathogenesis requires new and novel therapeutic strategies

In this issue Bostanci et al. (2007) demonstrate that receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) were oppositely regulated in gingival crevice fluid (GCF) from periodontitis patients. RANKL, RANK and OPG are key molecules that regulate osteoclast recruitment, differentiation and activation. New concepts of the pathogenesis of periodontitis have implicated inflammation triggered by host immune response to periodontal biofilm microorganisms(s) in disease. Host response to bacteria involves activation of T and B cells in the inflammatory infiltrate which bear abundant RANKL that promotes osteoclastic bone resorption. Periodontal tissue destruction can be ameliorated by immunobiological interference with immune cell RANKL expression or function. The new disease concepts provide a foundation to build biological approaches to target RANKL production in periodontal lesions.

Targeting p62 in Marrow Stromal Cells Is Effective at Inhibiting Myeloma Cell Growth.

Release of growth factors from bone by osteoclastic bone destruction and by tumor-stromal cell interactions play critical roles in promoting myeloma cell growth. In particular, expression of the cytokines RANK ligand (RANKL), a potent inducer of osteoclast (OCL) formation, IL-6, tumor necrosis factor-α (TNF-α) and MCP-1 are upregulated in the bone microenvironment in response to myeloma (MM) cells as well as by adhesive interactions between myeloma cells and marrow stromal cells through VCAM-1 on stromal cells. Therefore, agents that can both inhibit OCL formation and block the effects of myeloma stromal cell interactions should have a major impact on both bone destruction and tumor growth. p62 plays a critical role in NF-κB activation induced by TNF-α, RANKL and IL-1 and is involved in multiple signaling pathways that result in enhanced tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62 expression will profoundly diminish osteolytic bone destruction and myeloma growth in patients, by blocking production of RANKL, MCP-1, TNF-α and IL-6 in the tumor-bone microenvironment, and the upregulation of VCAM-1 on stromal cells. Therefore, we used p62 −/− mice to determine the effects of deleting p62 in stromal cells on the growth of myeloma cells. Marrow cells from p62 −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. IL-6 and TNF-α production by p62 −/− stromal cells was decreased compared to WT stromal cells. To determine the effects of the lack of p62 on MM cell growth, GFP-labeled MM.1S cells were co-cultured with p62 −/− stromal cells. Growth of MM.1S cells was decreased by 70% in cocultures of p62 −/− mice, and IL-6 and TNF-α levels were not increased in cocultures of tumor cells with p62 −/− stromal cells. Next, we measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in cocultures of human myeloma cells with marrow stromal cells. Stromal cells from the p62 −/− or wild type were cultured with and without MM.1S cells for 3 days in separate experiments. The levels of VCAM-1 in p62 −/− stromal cells were lower than p62 +/− stromal cells. In addition, VCAM-1 levels on p62 −/− bone marrow stromal cells were decreased compared to p62 +/− stromal cells when cocultured with MM cells. The addition of 25 ng/ml mouse TNF-α to p62 −/− stromal cells cocultured with MM.1S cells resulted in enhanced MM.1S growth and VCAM-1 production to similar levels as seen with p62 +/− stromal cells cocultured with MM.1S cells. We then determined the capacity of p62 −/− stromal cells to increase MCP-1 production, a chemoattract for myeloma cells, when they were cocultured with human myeloma cells for 48 hours. The conditioned media were collected after 48 hours of culture. Wild type stromal cells produced increased levels of MCP-1 when cocultured with MM.1S cells. MCP-1 levels in p62 −/− stromal cell conditioned media were decreased compared to wild type stromal cell cultures, regardless of whether MM.1S cells were present in the culture (MCP-1 in p62 +/− stromal cell culture, 980± 70pg/ml vs. p62 −/− 380± 10 pg/ml). These results show that p62 plays an important role in myeloma cell growth through regulation of production of cytokines that are upregulated in the marrow microenvironment in response to myeloma, and suggest that p62 is a novel target for treating myeloma.