An LGR4 agonist activates the GSK­3ß pathway to inhibit RANK­RANKL signaling during osteoclastogenesis in bone marrow­derived macrophages.

The binding between receptor­activated nuclear factor­κB (RANK) and the RANK ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. The leucine­rich repeat­containing G­protein­coupled receptor 4 (LGR4) acts as a negative regulator of RANK­RANKL that suppresses canonical RANK signaling during osteoclast differentiation. Therefore, LGR4 agonists may be useful in inhibiting osteoclastogenesis and effectively treating osteoporosis. In the present study, bone marrow­derived macrophages and a mouse model of RANKL­induced bone loss were used to investigate the effect of mutant RANKL (MT RANKL), which was previously developed based on the crystal structure of the RANKL complex. In the present study, the binding affinity of wild­type (WT) RANKL and MT RANKL to RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT­glycogen synthase kinase­3ß (GSK­3ß)­nuclear factor of activated T cells, cytoplasmic, calcineurin­dependent 1 (NFATc1) signaling cascade was investigated using western blotting and confocal microscopy. In addition, the expression of LGR4 and the colocalization of LGR4 with MT RANKL were analyzed in a mouse model of RANKL­induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4 and increased GSK­3ß phosphorylation independently of AKT, resulting in the inhibition of NFATc1 nuclear translocation. In the mouse model, MT RANKL colocalized with LGR4 and inhibited bone resorption. These results indicated that MT RANKL may inhibit RANKL­induced osteoclastogenesis through an LGR4­dependent pathway and this could be exploited to develop new therapies for osteoporosis.

RANKL immunisation inhibits prostate cancer metastasis by modulating EMT through a RANKL-dependent pathway.

Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa. RANKL was associated with epithelial-mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Therefore, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of ß-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and ß-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa.

A novel modified RANKL variant can prevent osteoporosis by acting as a vaccine and an inhibitor.

BACKGROUND: The discovery of receptor activator of nuclear factor-ĸB ligand (RANKL) as the final effector in the pathogenesis of osteoporosis has led to a better understanding of bone remodeling. When RANKL binds to its receptor (RANK), osteoclastic differentiation and activation are initiated. Herein, we propose a strategy using a novel RANKL variant as a competitive inhibitor for RANKL. The RANKL variant activates LGR4 signaling, which competitively regulates RANK and acts as an immunogen that induces anti-RANKL antibody production. METHODS: We modified the RANK-binding site on RANKL using minimal amino acid changes in the RANKL complex and its counterpart receptor RANK and tried to evaluate the inhibitory effects on osteoclastogenesis. RESULTS: The novel RANKL variant did not bind RANK in osteoclast progenitor cells, but activated LGR4 through the GSK3-ß signaling pathway, thereby suppressing activated T cell cytoplasmic nuclear factor calcineurin-dependent 1 (NFATc1) expression and activity during osteoclastogenesis. Our RANKL variant generated high levels of RANKL-specific antibodies, blocked osteoclastogenesis, and inhibited osteoporosis in ovariectomized mouse models. Generated anti-RANKL antibodies showed a high inhibitory effect on osteoclastogenesis in vivo and in vitro. CONCLUSIONS: We observed that the novel RANKL indeed blocks RANKL via LGR4 signaling and generates anti-RANKL antibodies, demonstrating an innovative strategy in the development of general immunotherapy.

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL.

BACKGROUND: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK-RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. RESULTS: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3ß phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

Effect of CD133 overexpression on bone metastasis in prostate cancer cell line LNCaP.

Prostate cancer (PC) metastasizes to the bone, and a small number of cancer cells, described as cancer stem cells (CSCs), have the ability to differentiate into tumor cells. CSCs are responsible for tumor recurrence and metastases. In the present study, we examined whether ectopic overexpression of CD133, a key molecule maintaining the stability of CSCs in the human PC cell line, LnCaP, caused bone metastasis in a mouse model. Ectopic overexpression of CD133 was induced in LnCaP cells, and CSC-related protein expression was measured. Furthermore, a colony-forming assay was performed to compare results against the blank green fluorescent protein-expressing cells. Furthermore, epithelial to mesenchymal transition-related protein expression, cell migration and wound healing were investigated. To assess the role of CD133 in bone metastasis, CD133-overexpressing LnCaP cells were inoculated into mice via intracardiac injection, and bone metastasis was assessed via histological and immunohistochemical study. In addition, cytokine arrays were used to determine the cytokines involved in bone metastasis. Ectopic overexpression of CD133 in LnCaP cells increased CSC properties such as Oct-4 and Nanog expression and colony-forming ability. Furthermore, epithelial-to-mesenchymal transition (EMT) properties, including decreased E-cadherin and increased vimentin expression, wound gap distance, and cell migration increased. CD133 overexpression led to formation of bone metastatic tumors in mice, consistent with results of hematoxylin and eosin staining. In addition, an increase in expression of the macrophage-migration inhibitory factor was observed at the tumor margin in mice inoculated with CD133+ LNCaP cells. These findings suggest a regulatory role of CD133 in stem cell and EMT properties, and the sustained acquisition of osteolytic features in PC. Therefore, our results may facilitate development of a novel classification system and therapeutic strategies for bone metastasis of PC.

Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ.

Mesenchymal stem cells (MSCs) are of particular interest for the treatment of immune-related diseases owing to their immunosuppressive properties. In this study, we aimed to identify the effect of interferon (IFN)-γ priming on immunomodulation by MSCs and elucidate the possible mechanism underlying their properties for the clinical treatment of allogeneic conflicts. Infusion of MSCs primed with IFN-γ significantly reduced the symptoms of graft-versus-host disease (GVHD) in NOD-SCID mice, thereby increasing survival rate when compared with naïve MSC-infused mice. However, infusion of IFN-γ-primed MSCs in which indoleamine 2,3-dioxygenase (IDO) was downregulated did not elicit this effect. The IDO gene was expressed in MSCs via the IFN-γ-Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) pathway, and the infusion of IDO-over-expressing MSCs increased survival rate in an in vivo GVHD model, similar to infusion of IFN-γ-primed MSCs. These data indicate that IFN-γ production by activated T-cells is correlated with the induction of IDO expression in MSCs via the IFN-γ-JAK-STAT1 pathway, which in turn results in the suppression of T-cell proliferation. Our findings also suggest that cell therapy based on MSCs primed with IFN-γ can be used for the clinical treatment of allogeneic conflicts, including GVHD.

Application of human mesenchymal stem cells cultured in different oxygen concentrations for treatment of graft-versus-host disease in mice.

Human mesenchymal stem cell (MSC) heterogeneity and problems associated with the ex vivo expansion of MSC are linked with the failure of MSC clinical trials. In this study, we compared the effect of MSCs cultured in different oxygen concentrations on GVHD in mice to elucidate whether hypoxia improves the immunosuppressive capacity of MSCs. Hypoxia increased the proliferative activity and the expression of several stemness and chemokine genes, such as KLF4, OCT4, C-MYC, CCL2, and CXCL10. Mice that received MSCs cultured in normoxia or hypoxia showed alleviated symptoms of GVHD and increased survival times. However, there was no significant difference in survival rates between mice that received MSCs cultured in normoxia and hypoxia. These data suggest that hypoxic culture is a useful method for maintaining and obtaining MSCs used for cell therapy and that the therapeutic potential of MSCs cultured in hypoxia warrants further investigation.

Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells.

Culture of mesenchymal stem cells (MSCs) under ambient conditions does not replicate the low oxygen environment of normal physiological or pathological states and can result in cellular impairment during culture. To overcome these limitations, we explored the effect of hypoxia (1 % O2) on the biological characteristics of MSCs over the course of different culture periods. The following biological characteristics were examined in human bone marrow-derived MSCs cultured under hypoxia for 8 weeks: proliferation rate, morphology, cell size, senescence, immunophenotypic characteristics, and the expression levels of stemness-associated factors and cytokine and chemokine genes. MSCs cultured under hypoxia for approximately 2 weeks showed increased proliferation and viability. During long-term culture, hypoxia delayed phenotypic changes in MSCs, such as increased cell volume, altered morphology, and the expression of senescence-associated-ß-gal, without altering their characteristic immunophenotypic characteristics. Furthermore, hypoxia increased the expression of stemness and chemokine-related genes, including OCT4 and CXCR7, and did not decrease the expression of KLF4, C-MYC, CCL2, CXCL9, CXCL10, and CXCR4 compared with levels in cells cultured under normoxia. In conclusion, low oxygen tension improved the biological characteristics of MSCs during ex vivo expansion. These data suggest that hypoxic culture could be a useful method for increasing the efficacy of MSC cell therapies.

Cell culture density affects the proliferation activity of human adipose tissue stem cells.

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions.

RAD001 (everolimus) enhances TRAIL cytotoxicity in human leukemic Jurkat T cells by upregulating DR5.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, is a promising new strategy for the treatment of cancer. However, aberrant PI3K/Akt/mTOR survival signaling may confer TRAIL resistance by altering the balance between pro- and anti-apoptotic proteins. In the present study, we showed that the Akt/mTOR inhibitor RAD001 (everolimus) induced cell death in a dose-dependent manner and enhanced TRAIL-induced apoptosis in human leukemic Jurkat T cells, which show PI3K/Akt/mTOR pathway activation and basal expression levels of death receptor (DR) 5 (TRAIL-R2). Investigation of the effect of RAD001 treatment on the expression of TRAIL receptors (TRAIL-Rs) in Jurkat T cells showed that RAD001 significantly upregulated DR5 by up to 51.22%, but not other TRAIL-Rs such as DR4 (TRAIL-R1), decoy receptor (DcR) 1 (TRAIL-R3), and DcR2 (TRAIL-R4). Pretreatment with DR5:Fc chimera abrogated the RAD001-induced increase of TRAIL cytotoxicity, indicating that the upregulation of DR5 by RAD001 plays a role in enhancing the susceptibility of Jurkat T cells to TRAIL. Our results indicate that combination treatment with RAD001 and TRAIL may be a novel therapeutic strategy in leukemia.

Dual matrilineal geographic distribution of Korean type 2 diabetes mellitus-associated -11,377 G adiponectin allele.

The present study was performed to identify the susceptible single nucleotide polymorphisms (SNPs) for the prediction of Korean type 2 diabetes mellitus (T2DM) and to clarify the matrilineal origin of Korean T2DM­specific SNPs. Fourteen SNPs from the adiponectin (ADIPOQ), hepatocyte nuclear factor 4α, phosphoenolpyruvate carboxykinase 1 and glucokinase genes in the Korean population were analyzed. Only one SNP, ­11,377 C/G on the ADIPOQ gene, was finally determined to be responsible for the incidence of Korean T2DM (P=0.028). The G­T­T­A haplotype at positions ­11,377, +45, +276 and +349 on the ADIPOQ gene was also associated with a high incidence of Korean T2DM (P=0.023). In addition, the susceptibility of Korean individuals to T2DM appears to be affected by their matrilineal origin. Of note, the group of Southern origin, consisting of mitochondrial DNA macrohaplogroups F and R, was predisposed to T2DM, whereas the group of Northern origin, consisting of haplogroups A and Y, was resistant to T2DM. This implied that the differential genetics between the two groups, which were formed from the initial peopling of the proto­Korean population via Southern and Northern routes to the present time, may explain their differing susceptibility to T2DM. In conclusion, from Southern Asia Northward, a matrilineal origin of Korean individuals appears to be responsible for the prevalence of Korean T2DM caused by the ­11,377 G allele.

Selective death of cancer cells by preferential induction of reactive oxygen species in response to (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG) induces apoptosis in cancer cells without adversely affecting normal cells. Understanding the cancer-specific cytotoxic activity of EGCG is very important in defining the mechanism of tumorigenesis and identifying superb chemotherapeutic agents against cancer. We comparatively assayed human telomerase reverse transcriptase (hTERT)-mediated apoptosis by EGCG-induced reactive oxygen species (ROS) in normal cells and cancer cells. EGCG showed differential levels of ROS induction between the cell types; ROS, especially hydrogen peroxide, was highly induced in cancer cells, while it was not in normal cells. In addition, the higher level of ROS down-regulated hTERT via binding of CCCTC binding factor (CTCF) to the core promoter region of hTERT, which repressed hTERT expression. CTCF binding was epigenetically controlled by the demethylation of the previously hypermethylated site for CTCF, which was induced by down-regulation of DNA methyltransferase 1 (DNMT1). In contrast, hTERT down-regulation was not observed in normal cells. These results suggest that preferential death of cancer cells by EGCG could be caused by the cancer-specific induction of ROS and epigenetic modulation of expression of apoptosis-related genes, such as hTERT.

Effects of 635nm light-emitting diode irradiation on angiogenesis in CoCl(2) -exposed HUVECs.

BACKGROUND AND OBJECTIVES: It is recognized that hypoxic/ischemic conditions leading to production of reactive oxygen species (ROS) are an important mediator of angiogenesis in the wound-healing process. Recently, low level light irradiation at 635 nm, which is used in many clinical fields, was found to decrease intracellular ROS levels, and consequently alleviate oxidative stress. The purpose of the present study was to investigate the effects of 635 nm light-emitting diode (LED) irradiation on angiogenesis in human umbilical vein endothelial cells, in an in vitro CoCl(2) -induced severe hypoxia model. STUDY DESIGN/MATERIALS AND METHODS: The effects were assessed on cell viability, tube formation, hypoxia-inducible factor-1, vascular endothelial growth factor (VEGF), VEGF-1 and -2 protein expression, mitogen-activated protein kinase (MAPK) phosphorylation, and ROS dissociation. RESULTS: The results showed that, under hypoxic/ischemic conditions, irradiation with 635 leads to reduced production and increased scavenging of intracellular ROS, which results in alleviation of VEGFR-1 suppression, enhanced VEGF expression and ERK MAPK activation, and subsequent acceleration of angiogenesis with improved cell viability and tube formation. CONCLUSION: Taken together, irradiation with 635 nm was shown to reduce intracellular ROS production, which results in increased angiogenesis. Thus, we suggest that irradiation with 635 nm accelerate angiogenesis under hypoxic/ischemic conditions, and may prove to be a useful alternative tool in wound healing.

TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116.

Trichostatin A (TSA), an inhibitor of histone deacetylase, is a well-known antitumor agent that effectively and selectively induces tumor growth arrest and apoptosis. Recently, it was reported that hTERT is one of the primary targets for TSA-induced apoptosis in cancer cells but the mechanism of which has not yet been elucidated. In the present study, to better understand the epigenetic regulation mechanism responsible for the repression of hTERT by TSA, we examined expression of hTERT in the HCT116 colon cancer cell line after treatment with TSA and performed site-specific CpG methylation analysis of the hTERT promoter. We found that TSA-induced the demethylation of site-specific CpGs on the promoter of hTERT, which was caused by down-regulation of DNA methyltransferase 1 (DNMT1). Among the demethylated region, the 31st-33rd CpGs contained a binding site for CTCF, an inhibitor of hTERT transcription. ChIP analysis revealed that TSA-induced demethylation of the 31st-33rd CpGs promoted CTCF binding on hTERT promoter, leading to repression of hTERT. Taken together, down-regulation of DNMT1 by TSA caused demethylation of a CTCF binding site on the hTERT promoter, the result of which was repression of hTERT via recruitment of CTCF to the promoter.

Inhibition of airway allergic disease by co-administration of flagellin with allergen.

Bacterial flagellin, which activates Toll-like receptor 5 and cytosolic pattern recognition receptor Ipaf, has a strong immunomodulatory activity. In the present study, we examined whether intranasal co-administration of flagellin with allergen could modulate established airway hyperresponsiveness and Th2 response using an ovalbumin (OVA)-sensitized mouse model. Balb/c mice sensitized with OVA were treated with OVA-flagellin (FlaB) mixture three times at 1-week intervals. Seven days after the final OVA-FlaB administration, the mice were challenged with OVA inhalation, and airway responses and OVA-specific immune responses were evaluated. The OVA-FlaB treatment significantly suppressed OVA-induced airway hyperresponsiveness, airway eosinophilic inflammation, and OVA-specific Th2 cytokine productions in splenocytes. These results indicate that flagellin co-administered with allergen can modulate airway inflammatory response through inhibition of Th2 responses, and flagellin can be considered as a component for allergen-specific immunotherapy.