Human RANK Ligand Research Articles

Expression of the RANK/RANKL/OPG system in the human intervertebral disc: implication for the pathogenesis of intervertebral disc degeneration

BackgroundThe expression of the receptor activator of nuclear factor kappa B (RANK) /RANK ligand (RANKL) /osteoprotegerin (OPG) system and its association with the progression of intervertebral disc (IVD) degeneration has recently been reported in a human IVD. However, the effect of the RANK/RANKL/OPG system on the matrix metabolism of human IVD cells, especially on the expression of catabolic factors relevant to IVD degeneration, remains unknown. The purpose of this study was to examine the expression of the RANK/RANKL/OPG system, and then to evaluate the effect of this system on the expression of catabolic factors by human IVD cells.MethodsAnnulus fibrosus (AF) and nucleus pulposus (NP) cells isolated by sequential enzyme digestion from human IVD tissues obtained during spine surgeries were monolayer cultured. The expression of the RANK/RANKL/OPG system was determined using immunohistochemical methods and real-time polymerase chain reaction (PCR). To evaluate the influence of interleukin-1 beta (IL-1β) stimulation on the mRNA expression of RANK, RANKL, and OPG, recombinant human IL-1β (rhIL-1β) was administered in the culture media of IVD cells. To examine the influence of RANKL signaling on the expression of matrix metalloprotease-3 (MMP-3), MMP-13, and IL-1β, the cells were cultured with exogenous recombinant human RANKL (rhRANKL), recombinant human OPG (rhOPG) or anti-human RANKL mouse monoclonal antibody (ahRANKL-mAB) with or without rhIL-1β.ResultsImmunoreactivity to RANK/RANKL/OPG and the mRNA expression of the three genes were obviously identified in both AF and NP cells. rhIL-1β stimulation significantly upregulated the mRNA expression level of RANK/RANKL/OPG. The mRNA expression of catabolic factors was significantly upregulated by stimulation of rhRANKL in the presence of rhIL-1β. On the other hand, the administration of either rhOPG or ahRANKL-mAB significantly suppressed the mRNA expression of catabolic factors that had been upregulated by rhIL-1β stimulation. The suppressive effect of ahRANKL-mAB against rhIL-1β stimulation was also confirmed by the protein expression of MMP-3.ConclusionsThe present study showed that the RANK/RANKL/OPG system may be involved in the progression of IVD degeneration. This study also suggested the potential use of anti-RANKL monoclonal antibody and OPG as therapeutic agents to suppress the progression of IVD degeneration.

Selective inhibition of receptor activator of NF-\u03baB ligand (RANKL) in hematopoietic cells improves outcome after experimental myocardial infarction

The RANK (receptor activator of nuclear factor κB)/RANKL (RANK ligand)/OPG (osteoprotegerin) axis is activated after myocardial infarction (MI), but its pathophysiological role is not well understood. Here, we investigated how global and cell compartment-selective inhibition of RANKL affects cardiac function and remodeling after MI in mice. Global RANKL inhibition was achieved by treatment of human RANKL knock-in (huRANKL-KI) mice with the monoclonal antibody AMG161. huRANKL-KI mice express a chimeric RANKL protein wherein part of the RANKL molecule is humanized. AMG161 inhibits human and chimeric but not murine RANKL. To dissect the pathophysiological role of RANKL derived from hematopoietic and mesenchymal cells, we selectively exchanged the hematopoietic cell compartment by lethal irradiation and across-genotype bone marrow transplantation between wild-type and huRANKL-KI mice, exploiting the specificity of AMG161. After permanent coronary artery ligation, mice were injected with AMG161 or an isotype control antibody over 4 weeks post-MI. MI increased RANKL expression mainly in cardiomyocytes and scar-infiltrating cells 4 weeks after MI. Only inhibition of RANKL derived from hematopoietic cellular sources, but not global or mesenchymal RANKL inhibition, improved post-infarct survival and cardiac function. Mechanistically, hematopoietic RANKL inhibition reduced expression of the pro-inflammatory cytokine IL-1ß in the cardiac cellular infiltrate. In conclusion, inhibition of RANKL derived from hematopoietic cellular sources is beneficial to maintain post-ischemic cardiac function by reduction of pro-inflammatory cytokine production.Key messagesExperimental myocardial infarction (MI) augments cardiac RANKL expression in mice.RANKL expression is increased in cardiomyocytes and scar-infiltrating cells after MI.Global or mesenchymal cell RANKL inhibition has no influence on cardiac function after MI.Inhibition of RANKL derived from hematopoietic cells improves heart function post-MI.Hematopoietic RANKL inhibition reduces pro-inflammatory cytokines in scar-infiltrating cells.

RANK expression as a prognostic and predictive marker in breast cancer

RANK ligand (RANKL) is crucial for the development of mouse mammary glands during pregnancy. RANKL functions as a major paracrine effector of the mitogenic action of progesterone in mammary epithelium via its receptor RANK and has a role in expansion and regenerative potential of mammary stem cells. Pharmacologic inhibition of RANKL attenuates the development of mammary carcinoma and inhibits metastatic progression in multiple mouse models. Primary breast carcinoma samples from the neoadjuvant GeparTrio study were analyzed to correlate the expression of human RANK and RANKL with pathological complete response (pCR), disease-free (DFS), and overall (OS) survival. Pre-treatment FFPE core biopsies (n=601) were analyzed for percentage and intensity of immunohistochemical RANK and RANKL expression. Antibodies against human RANK (N-1H8; Amgen) and human RANKL (M366; Amgen) were used. RANK protein was expressed in 160 (27%) patients. Increased RANK expression was observed in 14.5% of patients and correlated with high tumor grade (p<0.023) and negative hormone receptor (HR) status (p<0.001). Patients with high RANK expression showed a higher pCR rate (23.0% vs. 12.6%, p=0.010), shorter DFS (p=0.038), and OS (p=0.011). However, prognostic and predictive information was not an independent parameter. Only 6% of samples expressed RANKL, which was not correlated with any clinical features. Higher RANK expression in the primary tumor is associated with a higher sensitivity to chemotherapy, but also a higher risk of relapse and death. Our study provides a basis for further exploration of the antitumor activity of clinical antibodies against RANKL.

Abstract P1-08-25: RANK expression is prognostic and predictive in primary breast carcinoma: Analysis of samples from the GeparTrio study

Abstract Purpose: RANK ligand (RANKL), a key factor for bone remodeling and metastasis, is crucial for the development of mouse mammary glands during pregnancy. RANKL functions as a major paracrine effector of the mitogenic action of progesterone in mouse and human mammary epithelium via its receptor RANK and has a role in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells. Pharmacologic inhibition of RANKL attenuates the development of mammary carcinoma and inhibits metastatic progression in multiple mouse models. Many published expression analyses of RANK and RANKL have been performed using immunohistochemistry (IHC) without documented validation of antibody specificity. This study analyzed primary breast carcinoma samples from the GeparTrio study to correlate the expression of human RANK and RANKL (using specific, monoclonal antibodies validated and optimized for IHC) with pathological complete response (pCR: ypT0 ypN0), disease-free (DFS), and overall (OS) survival after anthracycline-taxane-based neoadjuvant chemotherapy. Methods: Pre-treatment FFPE core biopsies (n = 601) from participants of the neoadjuvant GeparTrio study were analyzed for percentage and intensity (H-score) of immunohistochemical RANK and RANKL expression. Antibodies against human RANK (N-1H8; Amgen) and human RANKL (M366; Amgen) were used for IHC staining. The overall expression measured by IHC was calculated using the H scoring method. The specificity of the antibodies was substantiated by concordant signals observed using multiple independent analyses, including IHC, flow cytometry and Western blots of positive and negative control cells and xenograft samples. Results: RANK protein was expressed within the tumor epithelium in 160 (27%) patients. A cutoff of an H-score of &amp;gt;8.5 was identified as optimal for prediction of pCR, DFS, and OS using an automated cut-off finder (http://molpath.charite.de/cutoff). High RANK expression (&amp;gt;8.5 H-Score) was observed in 14.5% of patients and was strongly correlated with high tumor grade (p&amp;lt;0.001) and negative hormone-receptor status (p&amp;lt;0.001). Patients with high RANK expression showed a higher pCR rate (23.0% vs 12.6%, p = 0.010), shorter DFS (p = 0.038) and OS (p = 0.011) compared to patients with low RANK expression. However, prognostic and predictive information was not independent from other baseline parameters (age, histologic type, stage, grade, and hormone/HER2-receptor) in multivariate analyses. Only 6% of samples expressed RANKL within the tumor epithelium, which was not correlated with any clinical features. Conclusion: Immunohistochemical RANK and RANKL protein expression was observed in the epithelial carcinoma in human primary breast cancers. Higher RANK expression in the primary tumor is associated with a higher sensitivity to chemotherapy, but also a higher risk of relapse and death. However, due to its strong correlation with grade and hormone-receptor status, RANK does not provide independent predictive and prognostic information. Denosumab, a clinically available antibody against RANKL, might have direct anti-tumor activity and should be further explored in this group of patients with high RANK expression. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-25.

Abstract P5-03-02: Expression of RANK and RANK ligand (RANKL) in breast carcinoma and distinct breast epithelial cells from BRCA1 mutation carriers

Abstract Background: Breast tumors in BRCA1 mutation carriers likely arise from luminal progenitor (LP) cells, previously shown to exhibit aberrant growth properties. Oophorectomy, and possibly tamoxifen, reduce breast cancer risk in BRCA1 carriers, potentially via inhibition of paracrine mediated signaling to stem/progenitor cells. RANKL is a major paracrine effector of progesterone's mitogenic action in mammary epithelium via its receptor RANK, and has a role in ovarian hormone-dependent activation of stem cells. Here we assessed RANK and RANKL expression in breast tumors and normal breast epithelial subtypes from women with mutations of BRCA1 (mBRCA1) or BRCA2 (mBRCA2). Methods: RANK and RANKL expression in breast cancer or normal breast tissue samples with mBRCA1, mBRCA2 or wildtype (WT) BRCA1/2 were analyzed in formalin fixed paraffin embedded (FFPE) sections by IHC. kConFab and The Royal Melbourne Hospital Tissue Bank provided the samples used in this analysis; these samples were obtained with relevant IRB approval. RANK expression on normal breast epithelial cells was measured by flow cytometry. Antibodies against human RANK (N-1H8, N-2B10; Amgen) and RANKL (M366; Amgen) were used in both assays. Incidence of IHC staining was scored as a positive IHC signal of any intensity. The overall expression was generated using the H scoring method which is calculated as the staining intensity of the tumor (0-3) multiplied by the percentage of positively staining cells. Results: Breast tumors from women with mBRCA1 had a higher incidence of RANK expression (68/162; 42%) compared with mBRCA2 (17/113; 15%) or WT (34/314; 11%) and higher overall H score (21.3) compared with mBRCA2 (8.0) or WT (3.4); RANKL expression did not vary greatly between groups: mBRCA1 (13/135; 10%), mBRCA2 (5/114; 4%), WT (23/212; 11%). In normal breast tissue, LP (Lin−EpCAM+CD49f+) and basal/stem cells (Lin−EpCAM−CD49fhi) expressed RANK on their surface. Similar expression patterns were seen in these epithelial subtypes from each BRCA1/2 genotype. Stromal cells (Lin−EpCAM−CD49f−) had minimal RANK expression. Conclusions: RANK expression intensity and incidence scores are both enriched approximately 4-fold in breast tumors from BRCA1 carriers compared with other genotypes. Also, RANK is normally expressed in breast LP cells as well as the basal/stem cell containing population. Ongoing studies will assess functional regulation of LP or mammary stem cell activity by RANKL and determine if the RANKL/RANK signaling pathway affects the aberrant growth characteristics of these cells from BRCA1 mutation carriers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-02.

P3-01-14: RANK and RANK Ligand (RANKL) Expression in Invasive Breast Carcinoma and Human Breast Cancer Cell Lines.

Abstract Purpose: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of mouse mammary gland during pregnancy. RANKL functions as a major paracrine effector of the mitogenic action of progesterone in mouse mammary epithelium and has a role in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells (MaSC). RANKL inhibition has been shown to reduce mammary tumor formation and pulmonary metastases in mouse models. Many published expression analyses of RANK and RANKL have been performed using immunohistochemistry (IHC) without documented validation of antibody specificity. This study assessed the expression of human RANK and RANKL in human invasive breast carcinoma (IBC) and human breast cancer cell lines using specific, monoclonal antibodies validated and optimized for IHC or flow cytometry. Methods: RANK and RANKL expression was analyzed in a panel of human breast cancer cell lines representing luminal or basal breast subtypes using qPCR, flow cytometry and surface receptor quantitation. Antibodies against human RANK (N-1H8, N-2B10; Amgen) and human RANKL (M366, AMG161; Amgen) were used for flow cytometry, surface receptor quantitation or IHC staining. For human IBC, the intensity of IHC staining was scored on a semiquantitative scale (0=absent, 1=weak, 2=moderate, 3=intense). Incidence was scored as a positive IHC signal (any intensity). In vitro responses of cell lines to RANKL were also tested. Results: The specificity of the antibodies was substantiated by concordant signals observed using multiple independent analyses, including IHC, flow cytometry and Western blots of positive and negative control cells and xenograft samples. Analysis of primary human IBC using IHC demonstrated that 25/114 (22%) IBC samples expressed RANK and 18/97 IBC (19%) expressed RANKL protein within the tumor epithelium. RANK protein was observed in monocytic cells infiltrating the tumor in 87/114 (76%) and within normal mammary epithelium adjacent to tumors in 35/79 (44%) of samples. RANKL was observed in infiltrating monocytic cells within the tumor in 60/115 (52%) and within normal mammary epithelium adjacent to tumors in 15/68 (22%) of samples. Both mRNA and RANK surface protein were detected in multiple breast cancer cell lines, including basal and luminal subtypes. Functional RANK expression on cell lines was confirmed in vitro by the observation of RANKL-dependent increases in mRNAs (e.g. MMP-9, IL-6 or IL-8) or proteins in conditioned media (e.g. IL-6, IL-8), despite the relatively low surface expression of RANK observed (range = 1240–9120 sites/cell). Conclusion: RANK and RANKL expression was observed in the epithelial carcinoma element in human IBC using IHC. RANK and RANKL expression was also observed in normal mammary epithelium and monocytic cells adjacent to breast tumors. Functional RANK expression was observed in human breast cancer cell lines, including both basal and luminal subtypes. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-14.

The RANKL/RANK/OPG Signaling Pathway Mediates Medial Arterial Calcification in Diabetic Charcot Neuroarthropathy

OBJECTIVEThe receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) signaling pathway (RANKL/RANK/OPG signaling) is implicated in the osteolysis associated with diabetic Charcot neuroarthropathy (CN); however, the links with medial arterial calcification (MAC) seen in people with CN are unclear. This study aimed to investigate the role of RANKL/OPG in MAC in patients with CN.RESEARCH DESIGN AND METHODSEnzyme-linked immunosorbent assay and Bio-plex multiarray technology were used to quantify a range of cytokines, including RANKL and OPG in sera from 10 patients with diabetes, 12 patients with CN, and 5 healthy volunteers. Human tibial artery segments were immunohistochemically stained with Alizarin red and human RANKL antibody. Human vascular smooth muscle cells (VSMCs) were also explanted from arterial segments for in vitro studies.RESULTSWe demonstrate colocalization and upregulation of RANKL expression in areas displaying MAC. Systemic levels of RANKL, OPG, and inflammatory cytokines (interleukin-8, granulocyte colony–stimulating factor) were elevated in those with CN compared with diabetic patients and healthy control subjects. Human VSMCs cultured in CN serum showed accelerated osteoblastic differentiation (alkaline phosphatase activity) and mineralization (alizarin red staining) compared with cells treated with diabetic or control serum (P < 0.05). Coincubation with OPG, the decoy receptor for RANKL, attenuated osteogenic differentiation of VSMCs and was independent of a high calcium-phosphate milieu. The accelerated mineralization induced by RANKL and CN serum correlated with nuclear translocation of nuclear factor-κB, a process abrogated by OPG.CONCLUSIONSOur data provide direct evidence that RANKL/RANK/OPG signaling is modulated in patients with CN and plays a role in vascular calcification. This study highlights this pathway as a potential target for intervention.

Functional Dissection of Osteoprotegerin and Its Interaction with Receptor Activator of NF-κB Ligand

The receptor activator of NF-kappaB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nM. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of approximately 3 microM, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction.

Targeting p62ZIP in Marrow Stromal Cells Is Highly Effective at Inhibiting Myeloma Cell Growth and Osteoclast Formation.

Marrow stromal cells play a critical role in osteolytic bone destruction in multiple myeloma, and promote tumor growth. In particular, adhesive interactions between myeloma cells and marrow stromal cells increase RANK ligand (RANKL), a potent inducer of osteoclast formation, IL-6 and TNF-α production by marrow stromal cells. IL-6 enhances the growth and prevents apoptosis of myeloma cells, and TNF increases production of RANKL and IL-6. Recently, a new member of NF-κB signaling pathway, p62ZIP, has been identified. p62ZIP plays a critical role in NF-κB activation induced by TNF-α and RANKL and is involved in multiple signaling pathways that result in enhanced IL-6 production, tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62ZIP expression will profoundly diminish myeloma growth by blocking the effects of IL-6 produced in the tumor-bone microenvironment in response to TNF-α. Therefore, we used p62ZIP−/− mice to determine the effects of deleting p62ZIP in stromal cells on the growth of myeloma cells. Marrow cells from p62ZIP −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. Marrow stromal cells from p62ZIP −/− or wild type mice were cocultured for 48 h with a GFP-labeled human MM.1S myeloma cell line (MM.1S) and murine and human RANKL, IL-6 and TNF-α levels were determined in conditioned media from these cocultures using commercial ELISA assays. Cocultures of MM.1S with wild type marrow stromal cells resulted in much greater upregulation of murine IL-6 than p62−/− stromal cell coculture (IL-6 in p62−/− stromal cell cultures; 114+70 vs. WT 1900+9 pg/ml). In addition, deleting p62ZIP in stromal cells markedly decreased the growth of tumor cells. Coculture with wild type stromal cells induce 1.4-fold greater increase in MM.1S cell growth at 72 h compared to p62−/− stromal cells. Further, addition of neutralizing antibodies to TNF-α and IL-6 to the cocultures of MM.1S cells with WT stromal cells similarly affected the growth of the MM.1S. Since TNF-alpha can increase the expression of adhesion molecules on stromal cells and tumor cells, we measured expression levels of ICAM-1 and VCAM-1 by Western blot. VCAM-1 and ICAM-1 levels on p62ZIP−/− bone marrow stromal cells were not changed compared to WT stromal cells. We then determined the capacity of p62 −/− cells to support OCL formation by normal spleen CFU-blast. OCL formation was decreased about 50 % in cocultures containing p62−/− stromal cells treated with PTH-rp, IL-6 and TNF-α compare with WT stromal cell culture. These results show that p62 plays an important role in myeloma cell growth and OCL formation induced by cytokines that are upregulated in the marrow microenvironment in patients with myeloma.

Functional role for heat shock factors in the transcriptional regulation of human RANK ligand gene expression in stromal/osteoblast cells.

RANK Ligand (RANKL) is a critical osteoclastogenic factor that is expressed on stromal cells and osteoblasts. Most resorption stimuli induce osteoclast formation by modulating RANKL gene expression in marrow stromal/osteoblast cells. However, it is unclear how these stimuli modulate RANKL gene expression in the bone microenvironment. To characterize the transcriptional control of human RANKL gene expression in stromal/osteoblast cells, we PCR-amplified and cloned a 2-kb 5'-flanking sequence of the RANKL gene, using normal human osteoblast derived genomic DNA as a template. Sequence analysis identified the presence of several potential Heat Shock Factor (HSF) responsive elements (HSE) in the human RANKL gene promoter region. Co-expression of HSF-1 or HSF-2 with the RANKL gene promoter-luciferase reporter plasmid in human osteoblastic cells (NOBC) demonstrated a 2-fold and 4.5-fold increase in promoter activity, respectively. RT-PCR analysis for HSF-1 and 2 mRNA expression in human bone marrow-derived stromal cells (SAKA-T) and osteoblast cells detected only HSF-2 expression. As evident from EMSA analysis, in contrast to 1,25(OH)(2)D(3) SAKA-T cells treated with b-FGF demonstrated increased levels of HSF-2 binding to the HSE present in the RANKL gene promoter region. Immunocytochemical staining further confirmed nuclear localization of HSF-2 in both SAKA-T transformed stromal cells and human bone marrow derived primary stromal/preosteoblastic cells in response to b-FGF treatment. Furthermore, b-FGF treatment of SAKA-T cells transfected with the luciferase reporter plasmid containing the hRANKL HSE region (-2 kb to -1275 bp) upstream to a heterologous promoter showed increased levels of transactivation. Western blot analysis further demonstrated enhanced levels of RANKL expression and HSP-27 phosphorylation in SAKA-T cells treated with b-FGF. In addition, overexpression of HSF-2 in SAKA-T cells resulted in a 5-fold increase in the levels of RANKL expression in these cells. These data further suggest that HSF-2 is a downstream target of b-FGF to induce RANKL expression in stromal/osteoblast cells, and that HSF may play an important role in modulating RANKL gene expression in the bone microenvironment.