RANK Signaling Research Articles

Heterologous expression, purification and function of the extracellular domain of human RANK

BackgroundReceptor activator of NF-κB ligand (RANKL)/RANK signaling essentially functions within the skeletal system, particularly participating in osteoclastogenesis and bone resorption. In addition, this signaling pathway has also been shown to influence tumor progression as well as the development and function of the immune system. Therefore, blocking the interaction between RANKL and RANK is a new therapeutic approach to prevent bone-related diseases and cancer.ResultsThe coding sequence encoding the extracellular domain of human RANK (RANK-N) was codon optimized for Pichia pastoris and cloned into the pPIC9K vector, and the recombinant plasmid was then transformed into P. pastoris. The expression of RANK-N protein was confirmed using SDS-PAGE with Coomassie Brilliant Blue stain and western blotting. Recombinant RANK-N protein was purified by a multistep process including ultrafiltration (UF), Sephadex G-50 size-exclusion chromatography and Q-Sepharose Fast Flow ion exchange chromatography, which resulted in a purity >95%. We found that the RANK-N protein can block RANKL-RANK signaling both in vitro and in vivo. Furthermore, using a patient-derived xenograft of human colon cancer, we found that the recombinant RANK-N protein can inhibit the growth of colorectal cancer.ConclusionsThe results show that a simple system to express and purify functional RANK-N protein has been developed. This work has thus laid a foundation for further research and clinical applications of RANK-N protein in treating bone-related diseases or even colorectal cancer.

W9 peptide enhanced osteogenic differentiation of human adipose-derived stem cells

W9 is a peptide that abrogates osteoclast differentiation via blockade of nuclear factor-κB ligand (RANKL)-RANK signaling, which activates bone formation. However, W9 stimulated osteogenesis in osteoblasts and mesenchymal stem cells. The present study demonstrated that the W9 peptide promoted osteogenic differentiation of human adipose-derived stem cells (hAdSCs) even under non-osteogenic differentiation culture conditions. W9-treated hAdSCs exhibited several osteocalcin-expressing cells and great mineralization compared to the BMP2-treated hAdSCs, which suggests that the W9 peptide had potent osteogenic potential in hAdSCs. W9 treatment also markedly enhanced the phosphorylation of p38, JNK, Erk1/2, and Akt, and BMP2 treatment only enhanced the phosphorylation of p38 and Erk1/2 in hAdSCs. hAdSCs did not express the RANKL gene, but W9 treatment upregulated Runx2, Collagen type 1A1 and TGF receptor genes and increased Akt phosphorylation. These results suggest that the W9-induced potent osteogenic induction was attributed to activation of TGF and the PI3 kinase/Akt signaling pathway in hAdSCs.

Receptor Activator of NF-κB Orchestrates Activation of Antiviral Memory CD8 T Cells in the Spleen Marginal Zone

SummaryThe spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169+ MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity.

C/EBPα and PU.1 exhibit different responses to RANK signaling for osteoclastogenesis

The transcription factors C/EBPα and PU.1 are upregulated by RANKL through activation of its receptor RANK during osteoclastogenesis and are critical for osteoclast differentiation. Herein we investigated the mechanisms underlying how C/EBPα and PU.1 regulate osteoclast differentiation in response to RANK signaling. We showed that C/EBPα or PU.1 overexpression could initiate osteoclastogenesis and upregulate the expressions of the osteoclast genes encoding the nuclear factor of activated T-cells, C1, cathepsin K, and tartrate-resistant acid phosphatase independently of RANKL. However, while PU.1 upregulated C/EBPα, C/EBPα could not upregulate PU.1. RANK has a unique cytoplasmic domain, 535IVVY538 motif, which is crucial for osteoclast differentiation. We demonstrated that mutational inactivation of RANK IVVY motif blocked osteoclast differentiation and significantly attenuated C/EBPα, but not PU.1, expression, indicating that RANK-IVVY-induced signaling is dispensable to PU.1 upregulation during osteoclastogenesis. However, C/EBPα or PU.1 overexpression failed to promote osteoclastogenesis in cells expressing mutated RANK IVVY motif. We noted that RANK-IVVY-motif inactivation significantly repressed osteoclast genes as compared with a vector control, suggesting that IVVY motif might also negatively regulate osteoclast inhibitors during osteoclastogenesis. Consistently, IVVY-motif inactivation triggered upregulation of RBP-J, a potent osteoclast inhibitor, during osteoclastogenesis. Notably, C/EBPα or PU.1 overexpression in cells expressing mutated RANK IVVY motif failed to control the deregulated RBP-J expression, resulting in repression of osteoclast genes. Accordingly, RBP-J silencing in the mutant cells rescued osteoclastogenesis with C/EBPα or PU.1 overexpression. In conclusion, we revealed that while PU.1 and C/EBPα are critical for osteoclastogenesis, they respond differently to RANKL-induced activation of RANK IVVY motif.

Circulating RANKL and RANKL/OPG and Breast Cancer Risk by ER and PR Subtype: Results from the EPIC Cohort.

Receptor activator of nuclear factor-kappa B (RANK)-RANK ligand (RANKL) signaling promotes mammary tumor development in experimental models. Circulating concentrations of soluble RANKL (sRANKL) may influence breast cancer risk via activation of RANK signaling; this may be modulated by osteoprotegerin (OPG), the decoy receptor for RANKL. sRANKL and breast cancer risk by hormone receptor subtype has not previously been investigated. A case-control study was nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. This study included 1,976 incident invasive breast cancer cases [estrogen receptor positive (ER+), n = 1,598], matched 1:1 to controls. Women were pre- or postmenopausal at blood collection. Serum sRANKL was quantified using an ELISA, serum OPG using an electrochemiluminescent assay. Risk ratios (RR) and 95% confidence intervals (95% CI) were calculated using conditional logistic regression. Associations between sRANKL and breast cancer risk differed by tumor hormone receptor status (Phet = 0.05). Higher concentrations of sRANKL were positively associated with risk of ER+ breast cancer [5th vs. 1st quintile RR 1.28 (95% CI, 1.01-1.63); Ptrend = 0.20], but not ER- disease. For both ER+ and estrogen and progesterone receptor positive (ER+PR+) breast cancer, results considering the sRANKL/OPG ratio were similar to those for sRANKL; we observed a suggestive inverse association between the ratio and ER-PR- disease [5th vs. 1st quintile RR = 0.60 (0.31-1.14); Ptrend = 0.03]. This study provides the first large-scale prospective data on circulating sRANKL and breast cancer. We observed limited evidence for an association between sRANKL and breast cancer risk. Cancer Prev Res; 10(9); 525-34. ©2017 AACR.

Boldine isolated from Litsea cubeba inhibits bone resorption by suppressing the osteoclast differentiation in collagen-induced arthritis

ObjectiveTo investigate the effect of boldine isolated from Litsea cubeba on collagen-induced arthritis (CIA) rats and explore the molecular mechanism predicted by network pharmacology. Material and methodsCIA rats were orally administered with boldine. The bone destruction of paws was analyzed by histologic examination, tartrate-resistant acid phosphatase (TRACP) staining and micro-computed tomography. Prediction of signal pathway associated with boldine network molecules and CIA genes was applied by the network pharmacology analysis. The expressions of osteoprotegerin (OPG), receptor activator of nuclear factor-κB (RANK) and its ligand (RANKL) in the ankle were detected by immunohistochemistry. In vitro osteoclasts were cultured in the presence of variable doses of boldine and the RANK expressions were evaluated using Real-time polymerase chain reaction and western blot. ResultsBoldine reduced ankle swelling, alleviated pathological damage and significantly prevented bone destruction in CIA rats. Consistent with this, enzyme linked immunosorbent assay revealed boldine decreased serum TRACP5b levels and osteoclast number in the ankle region by TRACP staining from CIA rats. The network pharmacology analysis indicated that RANK signaling in osteoclasts was the most significant canonical pathway associated with boldine network molecules and CIA genes, which was verified by the increased expression of OPG, reduced expression of RANK, RANKL and RANKL/OPG in boldine-treated CIA rats. The in vitro study further confirmed that boldine inhibited osteoclastogenesis by inhibiting the RANKL/RANK signaling pathway. ConclusionTaken together, our study first indicates that boldine from Litsea cubeba suppresses osteoclastogenesis, improves bone destruction by down-regulating the OPG/RANKL/RANK signal pathway and may be a potential therapeutic agent for rheumatoid arthritis.

RANK and EGFR in invasive breast carcinoma

Breast cancer is the most common malignancy, affecting one in eight women in North America and Europe. The human epidermal growth factor receptor (EGFR) protein comprises a major determinant of normal development but also cancer. RANK receptor (Receptor Activator of Nuclear factor-κB) is a tumor necrosis superfamily member and a binding partner for RANKL, which was recently implicated in breast cancer initiation, progression and metastasis. Here we provide preliminary evidence of a possible interplay between RANK and EGFR signaling in breast cancer. TCGA (cancergenome.nih.gov) publicly available data for EGFR and TNFRSF11A (RANK) genes from breast cancer patients and breast cancer cell lines were retrieved and analyzed. RANK mRNA showed a statistically significant positive correlation (p <0.001) with the mRNA and protein expression of EGFR, but not with ERBB2/3/4. Further analyses of survival data of a group of breast cancer patients (n = 248) from TCGA, revealed an EGFRhi/RANKhi subpopulation that showed a statistically significant (p = 0.001) reduced overall survival when compared to EGFRlow/RANKlow group of patients. Finally, EGFR and RANK combinatorial in vitro analyses revealed a significant upregulation of AKT and ERK signaling after EGF stimulation in cell lines and also an increase of breast cancer cell invasiveness.

The ABC7 regimen: a new approach to metastatic breast cancer using seven common drugs to inhibit epithelial-to-mesenchymal transition and augment capecitabine efficacy.

Breast cancer metastatic to bone has a poor prognosis despite recent advances in our understanding of the biology of both bone and breast cancer. This article presents a new approach, the ABC7 regimen (Adjuvant for Breast Cancer treatment using seven repurposed drugs), to metastatic breast cancer. ABC7 aims to defeat aspects of epithelial-to-mesenchymal transition (EMT) that lead to dissemination of breast cancer to bone. As add-on to current standard treatment with capecitabine, ABC7 uses ancillary attributes of seven already-marketed noncancer treatment drugs to stop both the natural EMT process inherent to breast cancer and the added EMT occurring as a response to current treatment modalities. Chemotherapy, radiation, and surgery provoke EMT in cancer generally and in breast cancer specifically. ABC7 uses standard doses of capecitabine as used in treating breast cancer today. In addition, ABC7 uses 1) an older psychiatric drug, quetiapine, to block RANK signaling; 2) pirfenidone, an anti-fibrosis drug to block TGF-beta signaling; 3) rifabutin, an antibiotic to block beta-catenin signaling; 4) metformin, a first-line antidiabetic drug to stimulate AMPK and inhibit mammalian target of rapamycin, (mTOR); 5) propranolol, a beta-blocker to block beta-adrenergic signaling; 6) agomelatine, a melatonergic antidepressant to stimulate M1 and M2 melatonergic receptors; and 7) ribavirin, an antiviral drug to prevent eIF4E phosphorylation. All these block the signaling pathways – RANK, TGF-beta, mTOR, beta-adrenergic receptors, and phosphorylated eIF4E – that have been shown to trigger EMT and enhance breast cancer growth and so are worthwhile targets to inhibit. Agonism at MT1 and MT2 melatonergic receptors has been shown to inhibit both breast cancer EMT and growth. This ensemble was designed to be safe and augment capecitabine efficacy. Given the expected outcome of metastatic breast cancer as it stands today, ABC7 warrants a cautious trial.

Thymic homing of activated CD4+ T cells induces degeneration of the thymic epithelium through excessive RANK signaling

Activated T cells have been shown to be able to recirculate into the thymus from the periphery. The present study was aimed to elucidate the functional consequences of thymic homing of activated T cells upon developing thymocytes and thymic epithelial cells (TEC). In the presence of activated T cells, especially CD4+ T cells, T cell development was found to be inhibited in thymic organ cultures with markedly reduced cellularity. Thymic transplantation demonstrated that the inhibitory effect was most likely due to a defective microenvironment. As the major component of the thymic stroma, the TEC compartment was severely disturbed after prolonged exposure to the activated T cells. In addition to reduced cell proliferation, TEC differentiation was heavily skewed to the mTEC lineage. Furthermore, we demonstrated that RANKL highly expressed by activated CD4+ T cells was primarily responsible for the detrimental effects. Presumably, excessive RANK signaling drove overproduction of mTECs and possibly exhaustion of epithelial progenitors, thereby facilitating the deterioration of the epithelial structures. These findings not only reveal a novel activity of activated T cells re-entering the thymus, but also provide a new perspective for understanding the mechanism underlying thymic involution.

Abstract 430: Myeloid RANK Deletion Accelerate the Development of Atherosclerosis in ApoE Deficient Mice with Chronic Kidney Disease

Objective: Chronic kidney disease (CKD) greatly increases the risk of cardiovascular disease morbidity and mortality. Mounting data has identified the receptor activator of NF-κB (RANK) pathway as an important mediator of the accelerated atherosclerosis and vascular calcification observed in CKD. Here we investigated the role of myeloid RANK in a model of CKD accelerated atherosclerosis and vascular calcification in apolipoprotein E-deficient mice (apoE -/- ). Methods and Results: We used the Lys M promoter to conditionally delete granulocyte and monocyte RANK in atherogenic apoE -/- mice. To induce CKD RANK (flox/flox) LysM (cre/+) Apoe (-/-) (apoE ΔRANK ) and LysM (cre/+) Apoe (-/-) (apoE CTL ) underwent a two-step surgical procedure to achieve 5/6 th nephrectomy. Additional control animals underwent sham surgeries. Blood urea nitrogen (BUN) levels were elevated approximately two-fold in the CKD mice relative to sham-operated mice regardless of genotype. CKD was accompanied by a trend toward lower body weight, as well as slightly elevated circulating phosphate levels at the time of sacrifice. CKD induced a 47% elevation of plasma cholesterol levels in the apoE ΔRANK and apoE CTL mice, with no significant difference in cholesterol levels between the apoE ΔRANK and apoE CTL CKD mice. Interestingly, CKD resulted in elevated triglycerides in apoE ΔRANK mice, but not in apoE CTL mice. CKD mice age 22 and 34 weeks had larger average lesional cross-section areas in the root of the aorta as compared to sham operated mice (p&lt;0.001 and p=0.03). Most importantly, the aortic root lesions in CKD apoE ΔRANK mice were significantly larger than those from apoE CTL CKD mice. In all genotypes and disease conditions we observed very little vascular calcification. Conclusions: These results suggest an atheroprotective role for myeloid RANK signaling in CDK-dependent lesion acceleration.

-643C > T RANKL gene polymorphism is associated with osteoporosis in Tunisian postmenopausal women

Objectives: The dynamic nature of the skeleton is achieved by a remodeling process. Receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) stimulates bone resorption by activating RANK signaling. Therefore it is considered as a candidate gene regulating susceptibility to osteoporosis. In the current study, we have investigated the association between the RANKL gene -693G > C and -643 C > T polymorphisms and bone mineral density (BMD) in a population of postmenopausal Tunisian women.Methods: Polymorphic sites in RANKL gene (rs9533155 -693G > C and rs9533156 -643 C > T polymorphisms) were determined using PCR-RFLP analysis in 566 postmenopausal Tunisian women. All statistical analysis were examined by SPSS software.Results: We have detected a significant difference in lumbar spine and hip BMD for -643C > T genotypes. For -693G > C genotypes, a significant difference was detected only in hip BMD. The distribution of -643C > T genotypes and alleles between three groups (osteoporotic, osteopenic and normal women) revealed a significant association of the TT genotype with development of osteoporosis (p = 0.01; odds ratio 2.15), although for the -693G > C polymorphism, no significant results were found.Conclusion: We have demonstrated the association of the -643C > T polymorphism with BMD variation and osteoporosis risk in postmenopausal Tunisian women.

Poor prognosis of patients with triple-negative breast cancer can be stratified by RANK and RANKL dual expression.

As clinical studies have correlated RANK expression levels with survival in breast cancer, and that RANK signaling is dependent on its cognate ligand RANKL, we hypothesized that dual protein expression further stratifies the poor outcome in TNBC. RANK mRNA and protein expression was evaluated in TNBC using genomic databases, cell lines and in a tissue microarray of curated primary tumor samples derived from 87 patients with TNBC. RANK expression was evaluated either by Mann-Whitney U test on log-normalized gene expression data or by Student's t test on FACS data. Analysis of RANK and RANKL immunostaining was calculated by H-score, and correlations to clinical factors performed using χ 2 or Fisher's exact test. Associations with RFS and OS were assessed using univariate and multivariate Cox proportional hazard models. Survival estimates were generated using the Kaplan-Meier method. In three distinct datasets spanning 684 samples, RANK mRNA expression was higher in primary tumors derived from TNBC patients than from those with other molecular subtypes (P<0.01). Cell surface-localized RANK protein was consistently higher in TNBC cell lines (P=0.037). In clinical samples, TNBC patients that expressed both RANK and RANKL proteins had significantly worse RFS (P=0.0032) and OS (P=0.004) than patients with RANK-positive, RANKL-negative tumors. RANKL was an independent, poor prognostic factor for RFS (P=0.04) and OS (P=0.01) in multivariate analysis in samples that expressed both RANK and RANKL. RANK and RANKL co-expression is associated with poor RFS and OS in patients with TNBC.

Implicating Receptor Activator of NF-κB (RANK)/RANK Ligand Signalling in Microglial Responses to Toll-Like Receptor Stimuli

Inflammation in the perinatal brain caused by maternal or intrauterine fetal infection is now well established as an important contributor to the development of perinatal brain injury. Exposure to inflammatory products can impair perinatal brain development and act as a risk factor for neurological dysfunction, cognitive disorders, cerebral palsy, or preterm birth. Pre-exposure to inflammation significantly exacerbates brain injury caused by hypoxic/ischaemic insult. Tumour necrosis factor (TNF) is a family of cytokines largely involved in inflammation signalling. In our previous study, we identified the importance of TNF-related apoptosis-inducing ligand (TRAIL) signalling in the development of perinatal brain injury. We observed a significant increase in the expression levels of a soluble decoy receptor for TRAIL, osteoprotegerin (OPG). Besides TRAIL, OPG is able to bind the receptor activator of the NF-κB (RANK) ligand (RANKL) and inhibit its signalling. The function of the RANK/RANKL/OPG system in the brain has not come under much scrutiny. The aim of this research study was to elucidate the role of RANK, RANKL, and OPG in microglial responses to the proinflammatory stimuli lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C). Here, we show that RANK signalling is important for regulating the activation of the BV2 microglial cell line. We found that LPS treatment causes a significant decrease in the expression of RANK in the BV2 cell line while significantly increasing the expression of OPG, Toll-like receptor (TLR)3, and the adaptor proteins MyD88 and TRIF. We found that pretreatment of BV2 cells with RANKL for 24 h before the LPS or Poly I:C exposure decreases the expression of inflammatory markers such as inducible nitric oxide synthase and cyclooxygenase. This is accompanied by a decreased expression of the TLR adaptor proteins MyD88 and TRIF, which we observed after RANKL treatment. Similar results were obtained in our experiments with primary mouse microglia. Using recently developed CRISPR/Cas9 technology, we generated a BV2 cell line lacking RANK (RANK^-/- BV2). We showed that most effects of RANKL pretreatment were abolished, thereby proving the specificity of this effect. Taken together, these findings suggest that RANK signalling is important for modulating the inflammatory activation of microglial cells to a moderate level, and that RANK attenuates TLR3/TLR4 signalling.

Afatinib ameliorates osteoclast differentiation and function through downregulation of RANK signaling pathways.

Non-small-cell lung cancer (NSCLC) is the third most common cancer that spreads to the bone, resulting in osteolytic lesions caused by hyperactivation of osteoclasts. Activating mutations in epidermal growth factor receptor-tyrosine kinase (EGF-TK) are frequently associated with NSCLC, and afatinib is a first-line therapeutic drug, irreversibly targeting EGF-TK. However, the effects of afatinib on osteoclast differentiation and activation as well as the underlying mechanism remain unclear. In this study, afatinib significantly suppressed receptor activator of nuclear factor κB (RANK) ligand (RANKL)-induced osteoclast formation in bone marrow macrophages (BMMs). Consistently, afatinib inhibited the expression of osteoclast marker genes, whereas, it upregulated the expression of negative modulator genes. The bone resorbing activity of osteoclasts was also abrogated by afatinib. In addition, afatinib significantly inhibited RANKL-mediated Akt/protein kinase B and c-Jun N-terminal kinase phosphorylation. These results suggest that afatinib substantially suppresses osteoclastogenesis by downregulating RANK signaling pathways, and thus may reduce osteolysis after bone metastasis.

Osteoclast precursors do not express CD68: results from CD68 promoter-driven RANK transgenic mice.

Macrophages and osteoclasts are thought to derive from CD68 lineage marker-positive common myeloid precursors. We used the CD68 promoter to drive an inducible receptor activator of NF-κB (iRANK) construct that selectively activates RANK signaling in myeloid cells in vivo. The cytoplasmic portion of RANK was fused to a mutant FK506 binding domain, which selectively binds the chemical inducer of dimerization AP20187 and initiates signaling. iRANK mRNA was expressed in macrophages isolated from peritoneal cavity, spleen-, and bone marrow-derived myeloid cells. Unexpectedly, AP20187 did not induce osteoclast formation in spleen- and bone marrow-derived myeloid cells. However, AP20187-dependent RANK signaling induced ERK1/2 phosphorylation and mRNA expression of MMP9 and CathepsinK in peritoneal macrophages. Importantly, CD68 was not expressed until day 3 and day 5 in bone marrow and spleen myeloid cells, respectively. Contrary to dogma, osteoclast precursors do not express the lineage marker CD68.

Osteoblastic Lrp4 promotes osteoclastogenesis by regulating ATP release and adenosine-A2AR signaling.

Bone homeostasis depends on the functional balance of osteoblasts (OBs) and osteoclasts (OCs). Lrp4 is a transmembrane protein that is mutated in patients with high bone mass. Loss of Lrp4 in OB-lineage cells increases bone mass by elevating bone formation by OBs and reducing bone resorption by OCs. However, it is unclear how Lrp4 deficiency in OBs impairs osteoclastogenesis. Here, we provide evidence that loss of Lrp4 in the OB lineage stabilizes the prorenin receptor (PRR) and increases PRR/V-ATPase-driven ATP release, thereby enhancing the production of the ATP derivative adenosine. Both pharmacological and genetic inhibition of adenosine-2A receptor (A2AR) in culture and Lrp4 mutant mice diminishes the osteoclastogenic deficit and reduces trabecular bone mass. Furthermore, elevated adenosine-A2AR signaling reduces receptor activator of nuclear factor κB (RANK)-mediated osteoclastogenesis. Collectively, these results identify a mechanism by which osteoblastic Lrp4 controls osteoclastogenesis, reveal a cross talk between A2AR and RANK signaling in osteoclastogenesis, and uncover an unrecognized pathophysiological mechanism of high-bone-mass disorders.

Abstract TP77: New treatment

Background and purpose: We previously showed that stimulation of RANKL (receptor activator of nuclear factor-B ligand)/ RANK (receptor for RANKL) signal with novel RANKL-based partial peptide, MHP1, worked as anti-inflammatory in microglia and macrophages without induction of osteoclast activation, but the effects of systemic injection of MHP1 in ischemic brain is still unclear. Here, we examined the effects of systemically injected MHP1 in ischemic brain. Methods: FITC-conjugated MHP1 was injected via jugular vein 4 hrs after transient middle cerebral artery occlusion (tMCAo) in C57BL6J mice to check whether MHP1 could penetrate into infarct parenchyma. To examine whether MHP1 has therapeutic effects, MHP1 was injected intravenously (IV) followed by successive subcutaneous injection with Alzet pump beginning at 4 or 6 hrs after tMCAo. The effects of MHP1 for osteoclast activation in the radial bone in paralyzed upper arm were examined by TRAP staining. Results: Immunohistochemistry for FITC showed that MHP1 could successfully penetrated into infarct parenchyma 5 min after IV injection of MHP1, but the penetrated MHP1 was not observed 1 hr after injection. These results indicated that MHP1 should be administered continuously to achieve successive penetration into infarct parenchyma. The mice treated with MHP1 showed less neurological severity score and infarct volume at 48 hrs after tMCAo even when the treatment was started at 6 hrs after MCAo. TRAP staining showed that osteoclast activation in paralyzed radial bone was inhibited in MHP1-treated mice, suggesting that MHP1 could prevent osteoclast activation as well as ischemic injury. Conclusion: Systemically injected MHP1 could successfully penetrated into infarct parenchyma and prevent exacerbation of paralysis and infarct size after tMCAo. Although further studies for improvement of its stability are needed, MHP1 might be a novel agent to treat ischemic brain.

Current Understanding of RANK Signaling in Osteoclast Differentiation and Maturation.

Osteoclasts are bone-resorbing cells that are derived from hematopoietic precursor cells and require macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) for their survival, proliferation, differentiation, and activation. The binding of RANKL to its receptor RANK triggers osteoclast precursors to differentiate into osteoclasts. This process depends on RANKL-RANK signaling, which is temporally regulated by various adaptor proteins and kinases. Here we summarize the current understanding of the mechanisms that regulate RANK signaling during osteoclastogenesis. In the early stage, RANK signaling is mediated by recruiting adaptor molecules such as tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of mitogen-activated protein kinases (MAPKs), and the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Activated NF-κB induces the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), which is the key osteoclastogenesis regulator. In the intermediate stage of signaling, the co-stimulatory signal induces Ca2+ oscillation via activated phospholipase Cγ2 (PLCγ2) together with c-Fos/AP-1, wherein Ca2+ signaling facilitates the robust production of NFATc1. In the late stage of osteoclastogenesis, NFATc1 translocates into the nucleus where it induces numerous osteoclast-specific target genes that are responsible for cell fusion and function.

Up-Regulation of RANK Expression via ERK1/2 by Insulin Contributes to the Enhancement of Osteoclast Differentiation.

Despite the importance of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-RANK signaling mechanisms on osteoclast differentiation, little has been studied on how RANK expression is regulated or what regulates its expression during osteoclastogenesis. We show here that insulin signaling increases RANK expression, thus enhancing osteoclast differentiation by RANKL. Insulin stimulation induced RANK gene expression in time- and dose-dependent manners and insulin receptor shRNA completely abolished RANK expression induced by insulin in bone marrow-derived monocyte/macrophage cells (BMMs). Moreover, the addition of insulin in the presence of RANKL promoted RANK expression. The ability of insulin to regulate RANK expression depends on extracellular signal-regulated kinase 1/2 (ERK1/2) since only PD98059, an ERK1/2 inhibitor, specifically inhibited its expression by insulin. However, the RANK expression by RANKL was blocked by all three mitogen-activated protein (MAP) kinases inhibitors. The activation of RANK increased differentiation of BMMs into tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts as well as the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of vacuolar (H+) ATPase (v-ATPase) Vo domain (Atp6v0d2), genes critical for osteoclastic cell-cell fusion. Collectively, these results suggest that insulin induces RANK expression via ERK1/2, which contributes to the enhancement of osteoclast differentiation.

NADPH oxidase gp91phox contributes to RANKL-induced osteoclast differentiation by upregulating NFATc1.

Bone-marrow derived monocyte-macrophages (BMMs) differentiate into osteoclasts by M-CSF along subsequent RANKL stimulation possibly in collaboration with many other unknown cytokines released by pre- or mature osteoblasts. The differentiation process requires receptor activator of nuclear factor kappa-B ligand (RANKL)/RANK signaling and reactive oxygen species (ROS) such as superoxide anion (O2•−). Gp91phox, a plasma membrane subunit of NADPH oxidase (Nox), is constitutively expressed in BMMs and plays a major role in superoxide anion production. In this study, we found that mice deficient in gp91phox (gp91phox−/−) showed defects in osteoclast differentiation. Femurs of these mice produced osteoclasts at about 70% of the levels seen in femurs from wild-type mice, and accordingly exhibited excessive bone density. This abnormal bone growth in the femurs of gp91phox−/− mice resulted from impaired osteoclast differentiation. In addition, gp91phox−/− mice were defective for RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1). However, H2O2 treatment compensated for gp91phox deficiency in BMMs, almost completely rescuing osteoclast differentiation. Treating wild-type BMMs with antioxidants and superoxide inhibitors resulted in a differentiation defect resembling the phenotype of gp91phox−/− BMMs. Therefore, our results demonstrate that gp91phox-derived superoxide is important for promoting efficient osteoclast differentiation by inducing NFATc1 as a downstream signaling mediator of RANK.