Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes.

N-Myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family of differentiation-related genes, has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34(+) progenitor cells, and myelomonocytic leukemic cells. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (RANKL). U937 cells stably expressing NDRG2 are unable to differentiate into multinucleated osteoclast-like cells and display reduced tartrate-resistant acid phosphatase (TRAP) activity and resorption pit formation. Furthermore, NDRG2 expression significantly suppresses the expression of genes that are crucial for the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, and OSCAR. The activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with the down-regulation of the expression of the transcription factor PU.1. Taken together, our results suggest that the expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis.

Protective Effects of High-Fat Diet against Murine Colitis in Association with Leptin Signaling and Gut Microbiome.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal-tract inflammation with dysregulated immune responses, which are partly attributable to dysbiosis. Given that diet plays a critical role in IBD pathogenesis and progression, we elucidated the effects of a high-fat diet (HFD) feeding on IBD development in relation to immune dysfunction and the gut microbiota. Five-week-old male C57BL/6J mice were fed either a normal diet (ND) or HFD for 14 weeks. The animals were further divided into ND, ND+ dextran sulfate sodium (DSS), HFD, and HFD+DSS treatment groups. The HFD+DSS mice exhibited lower body weight loss, lower disease activity index, longer colon length, and increased tight-junction protein expression and goblet-cell proportions compared with the ND+DSS mice. The T helper (h)1 and Th17 cell populations and pro-inflammatory cytokines involved in colitis pathogenesis were significantly more reduced in the HFD+DSS mice than in the ND+DSS mice. The HFD+DSS mice showed significantly increased serum leptin concentrations, colonic leptin receptor expression, enhanced anti-apoptotic AKT expression, and reduced pro-apoptotic MAPK and Bax expression compared with the ND+DSS mice, suggesting the involvement of the leptin-mediated pathway in intestinal epithelial cell apoptosis. The alterations in the gut-microbiota composition in the HFD+DSS group were the opposite of those in the ND+DSS group and rather similar to those of the ND group, indicating that the protective effects of HFD feeding against DSS-induced colitis are associated with changes in gut-microbiota composition. Overall, HFD feeding ameliorates DSS-induced colitis and colonic mucosal damage by reinforcing colonic barrier function and regulating immune responses in association with changes in gut-microbiota composition.

Analysis of the Expression and Regulation of PD-1 Protein on the Surface of Myeloid-Derived Suppressor Cells (MDSCs).

Myeloid-derived suppressor cells (MDSCs) that are able to suppress T cell function are a heterogeneous cell population frequently observed in cancer, infection, and autoimmune disease. Immune checkpoint molecules, such as programmed death 1 (PD-1) expressed on T cells and its ligand (PD-L1) expressed on tumor cells or antigen-presenting cells, have received extensive attention in the past decade due to the dramatic effects of their inhibitors in patients with various types of cancer. In the present study, we investigated the expression of PD-1 on MDSCs in bone marrow, spleen, and tumor tissue derived from breast tumor-bearing mice. Our studies demonstrate that PD-1 expression is markedly increased in tumor-infiltrating MDSCs compared to expression in bone marrow and spleens and that it can be induced by LPS that is able to mediate NF-κB signaling. Moreover, expression of PD-L1 and CD80 on PD-1⁺ MDSCs was higher than on PD-1⁻ MDSCs and proliferation of MDSCs in a tumor microenvironment was more strongly induced in PD-1⁺ MDSCs than in PD-1⁻ MDSCs. Although we could not characterize the inducer of PD-1 expression derived from cancer cells, our findings indicate that the study on the mechanism of PD-1 induction in MDSCs is important and necessary for the control of MDSC activity; our results suggest that PD-1⁺ MDSCs in a tumor microenvironment may induce tumor development and relapse through the modulation of their proliferation and suppressive molecules.

Z-ajoene from Crushed Garlic Alleviates Cancer-Induced Skeletal Muscle Atrophy.

Skeletal muscle atrophy is one of the major symptoms of cancer cachexia. Garlic (Allium sativum), one of the world's most commonly used and versatile herbs, has been employed for the prevention and treatment of diverse diseases for centuries. In the present study, we found that ajoene, a sulfur compound found in crushed garlic, exhibits protective effects against muscle atrophy. Using CT26 tumor-bearing BALB/c mice, we demonstrate in vivo that ajoene extract alleviated muscle degradation by decreasing not only myokines secretion but also janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) and SMADs/forkhead box (FoxO) signaling pathways, thereby suppressing muscle-specific E3 ligases. In mouse skeletal myoblasts, Z-ajoene enhanced myogenesis as evidenced by increased expression of myogenic markers via p38 mitogen-activated protein kinase (MAPK) activation. In mature myotubes, Z-ajoene protected against muscle protein degradation induced by conditioned media from CT26 colon carcinoma cells, by suppressing expression of muscle specific E3 ligases and nuclear transcription factor kappa B (NF-κB) phosphorylation which contribute to muscle atrophy. Moreover, Z-ajoene treatment improved myofiber formation via stimulation of muscle protein synthesis. These findings suggest that ajoene extract and Z-ajoene can attenuate skeletal muscle atrophy induced by cancer cachexia through suppressing inflammatory responses and the muscle wasting as well as by promoting muscle protein synthesis.

Kazinol U inhibits melanogenesis through the inhibition of tyrosinase-related proteins via AMP kinase activation.

BACKGROUND AND PURPOSE: Kazinol U is a prenylated flavan isolated from an extract of Broussonetia kazinoki Sieb (Moraceae). Kazinol U has shown cytoprotective effects against cytokine-induced apoptotic cell death and induces AMP kinase (AMPK) activation through LKB1 activation. However, kazinol U has not been tested as a regulator of melanogenesis, although bark extract of B. kazinoki has been used as a cosmetic ingredient for skin conditioning. EXPERIMENTAL APPROACH: We cultured mouse, human melanoma cells and normal human melanocytes to demonstrate anti-melanogenic effects of kazinol U. A tyrosinase activity assay, Western blot, RT-qPCR and a luciferase reporter gene assay were performed to determine the anti-melanogenic mechanisms of kazinol U. We confirmed its effect on melanogenesis in vivo using zebrafish. KEY RESULTS: Kazinol U inhibited the expression and activity of tyrosinase, the rate-limiting enzyme in melanogenesis, and reduced tyrosinase expression and activity in response to cAMP-inducing agents. Kazinol U reduced the expression of other melanogenic enzymes, such as tyrosinase-related protein (Tyrp) 1 and Tyrp2, and down-regulated microphthalmia-associated transcription factor (MITF), the master regulator of the tyrosinase gene family. Moreover, kazinol U induced phosphorylation of AMPK and MAPK proteins, which are MITF inhibitors. It also exhibited anti-melanogenic effects in zebrafish, a recently developed in vivo model. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that kazinol U reduces melanogenesis via its inhibitory effect on MITF and its downstream target genes, tyrosinase, Tyrp1 and Tyrp2. This work may provide a basis for the application of kazinol U for the treatment of hyperpigmentation skin disorders.

Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages.

The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet-fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage-associated genes IL-4 receptor α (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs.

Essential role of interferon regulatory factor 4 (IRF4) in immune cell development.

The family of interferon regulatory factors, which includes nine mammalian members (IRF1-IRF9), acts as transcription factors for interferons and thus exerts regulatory functions in the immune system and in oncogenesis. Among these members, IRF4 expression is restricted to immune cells such as T and B lymphocytes, macrophages, and dendritic cells where it is a key factor in the regulation of differentiation and is required during the immune response for lymphocyte activation and the generation of immunoglobulin-secreting plasma cells. Consequently, dysregulation of IRF4 is associated with many lymphoid malignancies. Recent studies have demonstrated that depending on the context and stage of hematopoietic cell differentiation in which its expression is dysregulated, IRF4 may act as either an oncogene or a tumor-suppressor-like factor. In addition, it has been shown that IRF4 plays a pivotal role in the development and function of several autoimmune-associated cells. Various genetic and functional studies have also pointed to IRF4 as a master regulator for autoimmunity. In this review, the roles of IRF4 in the immune response are briefly summarized and discussed, with particular focus on its essential and distinct functions in immune cell development.

NDRG2 Expression Decreases Tumor-Induced Osteoclast Differentiation by Down-regulating ICAM1 in Breast Cancer Cells.

Bone matrix is properly maintained by osteoclasts and osteoblasts. In the tumor microenvironment, osteoclasts are increasingly differentiated by the various ligands and cytokines secreted from the metastasized cancer cells at the bone metastasis niche. The activated osteoclasts generate osteolytic lesions. For this reason, studies focusing on the differentiation of osteoclasts are important to reduce bone destruction by tumor metastasis. The N-myc downstream-regulated gene 2 (NDRG2) has been known to contribute to the suppression of tumor growth and metastasis, but the precise role of NDRG2 in osteoclast differentiation induced by cancer cells has not been elucidated. In this study, we demonstrate that NDRG2 expression in breast cancer cells has an inhibitory effect on osteoclast differentiation. RAW 264.7 cells, which are monocytic preosteoclast cells, treated with the conditioned media (CM) of murine breast cancer cells (4T1) expressing NDRG2 are less differentiated into the multinucleated osteoclast-like cells than those treated with the CM of 4T1-WT or 4T1-mock cells. Interestingly, 4T1 cells stably expressing NDRG2 showed a decreased mRNA and protein level of intercellular adhesion molecule 1 (ICAM1), which is known to enhance osteoclast maturation. Osteoclast differentiation was also reduced by ICAM1 knockdown in 4T1 cells. In addition, blocking the interaction between soluble ICAM1 and ICAM1 receptors significantly decreased osteoclastogenesis of RAW 264.7 cells in the tumor environment. Collectively, these results suggest that the reduction of ICAM1 expression by NDRG2 in breast cancer cells decreases osteoclast differentiation, and demonstrate that excessive bone resorption could be inhibited via ICAM1 down-regulation by NDRG2 expression.

Interferon regulatory factor 4 (IRF4) controls myeloid-derived suppressor cell (MDSC) differentiation and function.

Myeloid-derived suppressor cells (MDSCs) are immature cells that do not differentiate into mature myeloid cells. Two major populations of PMN-MDSCs (Ly6GhighLy6ClowGr1highCD11b+) and MO-MDSCs (Ly6G-Ly6ChighGr-1intCD11b+) have an immune suppressive function. Interferon regulatory factor 4 (IRF4) has a role in the negative regulation of TLR signaling and is associated with lymphoid cell development. However, the roles of IRF4 in myeloid cell differentiation are unclear. In this study, we found that IRF4 expression was remarkably suppressed during the development of MDSCs in the tumor microenvironment. Both the mRNA and protein levels of IRF4 in MDSCs were gradually reduced, depending on the development of tumors in the 4T1 model. siRNA-mediated knockdown of IRF4 in bone marrow cells promoted the differentiation of PMN-MDSCs. Similarly, IRF4 inhibition in bone marrow cells using simvastatin, which has been known to inhibit IRF4 expression, increased PMN-MDSC numbers. In contrast, IRF4 overexpression in bone marrow cells inhibited the total numbers of MDSCs, especially PMN-MDSCs. Notably, treatment with IL-4, an upstream regulator of IRF4, induced IRF4 expression in the bone marrow cells, and consequently, IL-4-induced IRF4 expression resulted in a decrease in PMN-MDSC numbers. Finally, we confirmed that IRF4 expression in MDSCs can modulate their activity to inhibit T cell proliferation through IL-10 production and ROS generation, and myeloid-specific deletion of IRF4 leads to the increase of MDSC differentiation. Our present findings indicate that IRF4 reduction induced by tumor formation can increase the number of MDSCs, and increases in the IRF4 expression in MDSCs may infringe on the immune-suppressive function of MDSCs.

NDRG2 Promotes GATA-1 Expression through Regulation of the JAK2/STAT Pathway in PMA-stimulated U937 Cells.

BACKGROUND: N-myc downstream-regulated gene 2 (NDRG2), a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMA-stimulated U937 cells. METHODS: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression. RESULTS: NDRG2 overexpression in U937 cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1 were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells. CONCLUSION: The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.