Inhibition of Lipopolysaccharide-Induced Inflammatory Bone Loss by Saikosaponin D is Associated with Regulation of the RANKL/RANK Pathway.

BACKGROUND: Osteolytic diseases such as osteoporosis are featured with accelerated osteoclast differentiation and strong bone resorption. Considering the complications and other limitations of current drug treatments, it is necessary to develop a safer and more reliable drug to deal with osteoclast-related diseases. Saikosaponin D (SSD) is the active extract of Bupleurum, which has anti-inflammation, anti-tumor and liver protection functions. However, the role of SSD in regulating the differentiation and function of osteoclasts is not clear. PURPOSE: To explore whether SSD could prevent osteoclast differentiation and bone resorption induced by M-CSF and RANKL, and further evaluate the potential therapeutic properties of SSD in LPS-induced inflammatory bone loss mouse models. METHODS: BMMs were cultured in complete medium stimulated by RANKL with different concentrations of SSD. TRAP staining, bone resorption determination, qRT-PCR, immunofluorescence and Western blotting were performed. A mouse model of LPS-induced calvarial bone loss was established and treated with different doses of SSD. The excised calvaria bones were used for TRAP staining, micro-CT scan and histological analysis. RESULTS: SSD inhibited the formation and bone resorption of osteoclasts induced by RANKL in vitro. SSD suppressed LPS-induced inflammatory bone loss in vivo. CONCLUSION: SSD inhibited osteoclastogenesis and LPS-induced osteolysis in mice both which served as a new potential agent for the treatment of osteoclast-related conditions.

Electrosprayed minocycline hydrochloride-loaded microsphere/SAIB hybrid depot for periodontitis treatment.

Minocycline hydrochloride (MINO) has been one of the most frequently used antibiotics in the treatment of periodontitis due to its antibacterial activity and osteogenesis effects; however, high levels of MINO administered during the treatment halt the formation of new bone. Therefore, the purpose of the present study was to prepare a MINO-microsphere/sucrose acetate isobutyrate (SAIB) hybrid depot to reduce the burst release of MINO and ensure antibacterial and osteogenesis effects of MINO in the treatment of periodontitis. Uniform microspheres, approximately 5 µm size, with a slightly rough surface and different MINO loading (10, 12, and 14%) were prepared, and the microspheres were added into SAIB, after which the burst release significantly decreased from 66.18 to 2.92%, from 71.82 to 3.82%, and from 73.35 to 4.45%, respectively, and the release from all the MINO-microspheres/SAIB hybrid depots lasted for 77 days. In addition, cytotoxicity test showed that the MINO-microsphere with 12% drug loading promoted the proliferation of osteoblasts the most and was subsequently used in vivo experiments. Moreover, in the model of ligatured-induced periodontitis in SD rats, the MINO-microsphere/SAIB hybrid depot not only significantly increased the alveolar bone height and bone volume but also reduced the inflammation of the periodontal tissue. Additionally, it also inhibited the expression of the receptor activator of nuclear factor-kappa B ligand (RANKL) and promoted the expression of osteoprotegerin (OPG).. These results indicated that the MINO-microsphere/SAIB hybrid depot might be promising in the treatment of periodontitis.

Controversies in the use of new bone-modifying therapies in multiple myeloma.

Bone-modifying therapies are essential in the treatment of patients with multiple myeloma. Zoledronic acid is preferred over other bisphosphonates due to its superiority in reducing the incidence of skeletal-related events and improving survival. The anti-receptor activator of nuclear factor-κΒ ligand (RANKL)-targeted agent denosumab has shown its non-inferiority compared to bisphosphonates in preventing skeletal-related events among newly diagnosed patients with myeloma bone disease. Denosumab may confer a survival benefit in patients eligible for autologous transplantation. Denosumab may present a safer profile for patients with renal impairment. Discontinuation of bone-directed therapies can be considered for patients with deep responses and after an adequate time period on treatment; however, a rebound effect may become evident especially in the case of denosumab. Three-monthly infusions of zoledronic acid or at-home denosumab administration should be considered during the coronavirus disease 2019 (COVID-19) pandemic. Measures to prevent hypocalcaemia, renal toxicity and osteonecrosis of the jaw are important for all bone-modifying agents.

Blockade of RANKL/RANK and NF-ĸB signalling pathways as novel therapeutic strategies for allergic asthma: A comparative study in a mouse model of allergic airway inflammation.

The main aims of this study were: (1) to investigate whether a blockade of the interaction between the receptor activator of nuclear factor-κB (NF-ĸB) ligand (RANKL) and its receptor RANK may have potential as a novel therapeutic strategy for allergic asthma; (2) to compare the efficacies of the blockade of RANKL/RANK interaction as well as the blockade of NF-κB inhibitor kinase (IKK) and of NF-κB translocation to the nucleus, also in comparison with glucocorticosteroid treatment, in terms of the development of a mouse model of allergic airway inflammation (AAI) and accompanying immune response. The blockade of each of the targets fully prevented the development of AAI. All the tested therapeutic strategies seemed to have a certain advantage over glucocorticosteroids with regard to counteracting the development of AAI. Prevention of the activation and clonal expansion of CD4+ effector T (Teff) cells in the mediastinal lymph nodes (MLNs) constitutes a fundamental event underlying the anti-asthmatic effect induced by the blockade of IKK, NF-κB translocation or of RANKL/RANK interaction. The results indicate that attenuation of the CD11b+CD103-CD11chigh dendritic cell response in the MLNs is an initial but not the main mechanism responsible for this effect. In turn, the direct anti-proliferative action on CD4+ Teff cells seems to constitute the chief mechanism responsible for the anti-asthmatic effect of all the tested therapeutic strategies. A clinical implication is that local inhibition of RANKL/RANK interaction achieved via inhalatory administration of a RANKL antagonist can be considered as a novel therapeutic strategy in treatment of allergic asthma.

MiR-143 Inhibits Osteoclastogenesis by Targeting RANK and NF-κB and MAPK Signaling Pathways.

OBJECTIVE: To explore the effect of miRNA-143 on osteoclast formation and provide new ideas for the treatment of osteoporosis. METHODS: Mice macrophage lines RAW264.7 cells after transfection were divided into four groups: control group, RANKL group, RANKL combined with miR-143 mimics group and RANKL combined with miR-NC group. TARCP staining was used to observe the effect of miR-143 on osteoclast formation. The expression of RANK, TRAF6 and NFATc-1 in the upstream of RANKL pathway was detected by real-time quantitative PCR (RT qPCR) and Western blotting (WB). The binding of miR-143 to TNFRSF11A was detected by double Luciferase Reporter Analysis. The effect of miR-143 on the expression of NF-κB (p65, I-κB-α) signal pathway in osteoclasts was detected. The effects of I-BET151 on the expression of osteoclast-specific genes TRACP, MMP 9, CtsK and c-Src were detected. RESULTS: The positive level of osteoclasts in RANKL group and RANKL combined with miR-NC group was significantly higher than that of RANKL combined with miR-143 mimics group and control group (P < 0.05). The expression levels of RANK, TRAF6, NFATc-1, TRACP, MMP-9, CtsK and c-Src in RANKL group and RANKL combined with miR-NC group were significantly higher than those of RANKL combined with miR-143 mimics group and control group (P < 0.05). The expression levels of I-κB-α were significantly lower than that of RANKL combined with miR-143 mimics group and control group (P<0.05). CONCLUSION: MiR-143 can inhibit the expression of RANK, TRAF6 and downstream NFATc-1 in the RANKL pathway, thereby inhibiting the RANK/RANKL pathway. MiR-143 can inhibit the signal pathway of NF-κB (p65, I-κB-α). MiR-143 inhibits the expression of osteoclast-specific genes TRACP, MMP 9, CtsK and c-Src. That is to say, miR-143 inhibits osteoclast formation by targeting RANK, NF- κB and MAPK signaling pathways.

l-tetrahydropalmatine suppresses osteoclastogenesis in vivo and in vitro via blocking RANK-TRAF6 interactions and inhibiting NF-κB and MAPK pathways.

Bone homeostasis is delicately orchestrated by osteoblasts and osteoclasts. Various pathological bone loss situations result from the overactivated osteoclastogenesis. Receptor activator of nuclear factor κB ligand (RANKL)-activated NF-κB and MAPK pathways is vital for osteoclastogenesis. Here, we for the first time explored the effects of l-tetrahydropalmatine (l-THP), an active alkaloid derived from corydalis, on the formation and function of osteoclasts in vitro and in vivo. In RAW264.7 cells and bone marrow monocytes cells (BMMCs), l-THP inhibited osteoclastic differentiation at the early stage, down-regulated transcription level of osteoclastogenesis-related genes and impaired osteoclasts functions. Mechanically, Western blot showed that l-THP inhibited the phosphorylation of P50, P65, IκB, ERK, JNK and P38, and the electrophoretic mobility shift assay (EMSA) revealed that DNA binding activity of NF-κB was suppressed, ultimately inhibiting the expression of nuclear factor of activated T cells (NFATc1). Besides, Co-immunoprecipitation indicated that l-THP blocked the interactions of RANK and TNF receptor associated factor 6 (TRAF6) at an upstream site. In vivo, l-THP significantly inhibited ovariectomy-induced bone loss and osteoclastogenesis in mice. Collectively, our study demonstrated that l-THP suppressed osteoclastogenesis by blocking RANK-TRAF6 interactions and inhibiting NF-κB and MAPK pathways. l-THP is a promising agent for treating osteoclastogenesis-related diseases such as post-menopausal osteoporosis.

Combining RANK/RANKL and ERBB-2 targeting as a novel strategy in ERBB-2-positive breast carcinomas.

BACKGROUND: ERBB-2 is overexpressed in about 20% of breast cancers (BCs), indicating poor prognosis. The receptor activator of nuclear factor-κB (RANK) pathway is implicated in ERBB-2 (+) BC. The purpose of this study was to elucidate the underlying molecular mechanism of this interaction and the beneficial impact of dual targeting of RANK and ERBB-2 pathways. METHODS: We used SKBR3, MCF7, MDA-MB-453, and BT-474 human BC cell lines. We examined RANK and RANKL expression using RT-PCR, Western blot, and immunofluorescence. The evaluation of RANK expression in a cohort of BC patients was performed using immunohistochemistry. The interaction between RANK and ERBB family members was detected using proximity ligation assay (PLA), which enables the visualization of interacting proteins. We used inhibitors of both pathways [trastuzumab (T), pertuzumab (P), denosumab (D)]. NF-κB pathway activation was studied using Western blot. Cell growth and viability was evaluated using XTT, flow cytometry, and clonogenic assay. For cell migration evaluation, scratch assay was performed. Data were analyzed by one-way ANOVA. RESULTS: Cell lines express RANK and RANKL. RANK immunostaining was also detected in human BC tissue samples. RANK receptor dimerizes with ERBB family members. RANK/ERBB-2 dimer number seems to be associated with ERBB-2 expression (SKBR3, 5.4; BT-474, 8.2; MCF7, 0.7; MDA-MB-453, 0.3). RANK/ERBB-2 dimers were decreased in the presence of the inhibitors D, T, and P, while they were increased after RANKL (R) treatment in SKBR3 (m, 5.4; D, 1.2; T, 1.9; DT, 0.6; TP, 1; DTP, 0.4; R, 11.8) and BT-474 (m, 8.2; D, 3.1; T, 4.3; DT, 0.7; TP, 3.4; DTP, 3.2; R, 11.6). Combination targeting of SKBR3 further decreased NF-κB pathway activation compared to single targeting. In SKBR3, RANKL and ERBB-2 blockage resulted in reduced cell proliferation, increased apoptosis, and lower metastatic potential compared to mock cells (m) and reversed values in RANKL presence. The combination treatment of SKBR3 with D, T, and P had an advantage in functional traits compared to single targeting. Denosumab suppressed NF-κB signaling and diminished proliferation rate in MDA-MB-453 cells. MCF7 did not correspond to inhibitors. CONCLUSIONS: The results indicate a novel physical and molecular association between ERBB-2 and RANK pathways that affects ERBB-2 (+) BC growth. We also present data suggesting that the combination of anti-ERBB-2 agents and RANKL inhibitors have a potential direct anti-tumor effect and should be further tested in certain BC patients.

Adjuvant bisphosphonates or RANK-ligand inhibitors for patients with breast cancer and bone metastases: A systematic review and network meta-analysis.

Bone-modifying agents like bisphosphonates and receptor activator of nuclear factor kappaß ligand (RANK-L) inhibitors are used as supportive treatments in breast cancer patients with bone metastases to prevent skeletal-related events (SREs). Due to missing head-to-head comparisons, a network meta-analysis was performed to provide a hierarchy of these therapeutic options. Through a systematic literature search, 21 randomized controlled trials (RCTs) that fulfilled the inclusion criteria were identified. To prevent SREs, the ranking through P-scores showed denosumab (RR: 0.62; 95%CI: 0.50-0.76), zoledronic acid (RR: 0.72; 95%CI: 0.61-0.84) and pamidronate (RR: 0.76; 95%CI: 0.67-0.85) to be significantly superior to placebo. Due to insufficient or heterogeneous data, overall survival, quality of life, pain response and adverse events were not able to be analyzed within the network. Although data were sparse on adverse events, the risk of significant adverse events appeared low. The results of this review can therefore be used to formulate clinical studies more precisely in order to standardise and focus on patient-relevant outcomes.

RANK/RANKL signaling inhibition may improve the effectiveness of checkpoint blockade in cancer treatment.

Binding between the receptor activator of nuclear factor-kB (RANK) and its ligand (RANKL) triggers recruitment of TNF receptor associated factor (TRAF) adaptor proteins and activation of downstream pathways. RANK/RANKL signaling is controlled by a decoy receptor called osteoprotegerin (OPG) which interacts with RANKL. Additional networks regulating RANK/RANKL signaling are active in a context specific manner. RANK/RANKL signaling is essential for the differentiation of bone-resorbing osteoclasts, and is deregulated in pathological processes such as postmenopausal osteoporosis or cancer induced bone destruction. Cells expressing RANK and RANKL are commonly found in the tumor microenvironment. The RANKL/RANK pathway is often overexpressed in tumors of the breast, prostate, endometrium, cervix, stomach, oesophagus and bladder, thyroid and correlated with poor prognosis. RANK signaling plays an important role in the innate and adaptive immune response as it generates regulatory T (Treg) cells and increases production of cytokines. RANK expression induces chemoresistance in vitro through the activation of multiple signal transduction pathways. RANKL blockade improves the efficacy of anti-CTLA-4 monoclonal antibodies against solid tumors and experimental metastases. As RANK inhibition enhances the immune response there is an increasing interest in combining it with immune therapy in an attempt to sensitize immune resistant tumors to immune therapies. Several studies are ongoing to assess this concept. The role of RANK/RANKL inhibition should be further pursued as an immunomodulatory strategy in combination with other treatment modalities.

Development of PEI-RANK siRNA Complex Loaded PLGA Nanocapsules for the Treatment of Osteoporosis.

Osteoporosis, which is characterized by low bone mineral density and susceptibility to fracture, is caused by increased osteoclastic activity. Receptor activator of nuclear factor kappa B ligand (RANKL)/RANK signaling plays an important role in osteoclast differentiation and activation. The current treatment strategies for osteoporosis do not directly address this underlying cause and generates undesired side effects. This led to emergence of controlled delivery systems to increase drug bioavailability and efficacy specifically at the bone tissue. With better understanding of molecular pathology of bone, the use of small interfering RNA (siRNA) to inhibit translation of abnormal gene expression in cells is becoming a promising approach. In this study, we report a siRNA delivery system consisting of PEI:RANK siRNA complex entrapped in nanosized poly(lactic acid-co-glycolic acid) (PLGA) capsules intended to be used in the treatment of osteoporosis. The nanosize will enable the nanoparticles to be administered by intravenous injection. The RANK siRNA was complexed with polyethylenimine (PEI) and loaded into biodegradable PLGA nanocapsules (NCs). The PEI:RANK siRNA loaded nanocapsules significantly reduced (47%) RANK mRNA levels. The differentiation of osteoclast precursors to mature osteoclasts was significantly suppressed (∼54%). The reduction in the osteoclastic activity of the differentiated osteoclasts (55%) was found to be statistically significant. The siRNA delivery system developed in the study is planned to be tested i.v. in mouse and has the potential to be used as a novel alternative approach for the systemic treatment of osteoporosis.

Roles of the RANKL-RANK axis in antitumour immunity - implications for therapy.

Recognizing that the transformative effects of immunotherapy are currently limited to a minority of patients with cancer, research efforts are increasingly focused on expanding and enhancing clinical responses by combining immunotherapies; the repurposing of existing drugs is an attractive approach, given their well-characterized safety and pharmacokinetic profiles. Receptor activator of nuclear factor-κB (RANK) and the RANK ligand (RANKL) were initially described in the context of T cell-dendritic cell interactions; however, the discovery of an obligate role of RANK signalling in osteoclastogenesis led to the development of the anti-RANKL antibody denosumab for antiresorptive indications, including bone metastases. Randomized clinical trials and post-marketing surveillance studies have established the acceptable safety profile of denosumab. More recently, several case reports involving patients with advanced-stage melanoma have described remarkable responses following concurrent treatment with denosumab and immune-checkpoint inhibitors. Randomized trials assessing similar combinations in patients with melanoma or renal cell carcinoma are now underway. Herein, we discuss the hallmark clinical trials of denosumab in light of possible immunological effects of this agent. We highlight the role of immune cells as sources of RANK and RANKL in the tumour microenvironment and review data on RANKL inhibition in mouse models of cancer. Finally, we describe hypothetical immune-related mechanisms of action, which could be assessed in clinical trials of immune-checkpoint inhibitors and denosumab in patients with cancer.

Adiponectin inhibits osteoclastogenesis by suppressing NF-κB and p38 signaling pathways.

Adiponectin (APN) has been shown to play a key role in regulating bone mineral density (BMD). Nevertheless, the effects of APN on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and mechanism of regulation are not entirely clear. The study, therefore, aimed to evaluate the effect of APN on osteoclastogenesis. Our results showed that APN inhibits osteoclastogenesis and resorption function in vitro by suppressing nuclear factor-κB (NF-κB) and p38 signaling pathways, which is essential for osteoclast formation. Moreover, APN blocked the formation of F-actin rings and attenuated osteoclast-mediated bone resorptive function. Therefore, we concluded that APN may provide a potential treatment for osteoclast-related diseases, such as osteoporosis.

[Multiple Vertebral Fractures after Denosumab Discontinuation: How to Avoid Them?] / Fractures vertébrales multiples à l'arrêt du denosumab: comment les éviter?

Multiple Vertebral Fractures after Denosumab Discontinuation: How to Avoid Them? Abstract. Denosumab is a monoclonal antibody raised against the RANK ligand that inhibits the maturation and activity of osteoclasts. It decreases bone resorption, increases bone density and reduces fracture risk. However, after its discontinuation, a significant rebound effect appears that lasts about two years. It results in increased markers of bone remodeling, a loss of bone density that may be greater than gain, and an increased risk of multiple vertebral fractures. These fractures occur at a frequency of 1 to 10 %. Due to this high risk, denosumab should be a second-line treatment limited to very specific indications. At denosumab discontinuation, in order to limit the rebound effect, the current recommendation is to prescribe a strong bisphosphonate (alendronate, zoledronate) and regularly monitor the bone resorption markers.

Current Status and Future Prospects of Small-molecule Protein-protein Interaction (PPI) Inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL).

The overexpression of Tumor Necrosis Factor (TNF) is directly related to the development of several autoimmune diseases, such as rheumatoid and psoriatic arthritis, inflammatory bowel disease, Crohn's disease, refractory asthma, and multiple sclerosis. Receptor Activator of Nuclear Factor Kappa- B Ligand (RANKL) belongs to the TNF family and is the primary mediator of osteoclast-induced bone resorption through interaction with its receptor RANK. The function of RANKL is physiologically inhibited by the action of osteoprotegerin (OPG), which is a decoy receptor that binds to RANKL and prevents the process of osteoclastogenesis. Malfunction among RANK/RANKL/OPG can also result in bone loss diseases, including postmenopausal osteoporosis, rheumatoid arthritis, bone metastasis and multiple myeloma. To disrupt the unwanted functions of TNF and RANKL, current attempts focus on blocking TNF and RANKL binding to their receptors. In this review, we present the research efforts toward the development of low-molecular-weight pharmaceuticals that directly block the detrimental actions of TNF and RANKL.

Synthesis of some novel orcinol based coumarin triazole hybrids with capabilities to inhibit RANKL-induced osteoclastogenesis through NF-κB signaling pathway.

A total of twenty-two novel coumarin triazole hybrids (4a-4k and 6a-6k) were synthesized from orcinol in good to excellent yields of 70-94%. The structures of all the synthesized compounds were elucidated by spectroscopic techniques such as 1H NMR, 13C NMR, and HRMS. The anti-inflammatory potential of synthesized compounds was investigated against the proinflammatory cytokine, TNF-α on U937 cell line and compounds 4d, 4j, and 6j were found to exhibit promising anti-inflammatory activity. These three compounds were further screened against TNF-α on LPS-stimulated RAW 264.7 cells, which confirm their anti-inflammatory potential. Furthermore, the above said active compounds were tested for their inhibitory effect on RANKL-induced osteoclastogenesis in RAW 264.7 cells by using tartrate resistant acid phosphatase (TRAP) staining assay at 10 µM. Molecular mechanism studies demonstrated that compound 4d exhibited dose dependent inhibition of RANKL-induced osteoclastogenesis by suppression of the NF-kB pathway. Thus, compound 4d is a promising candidate for further optimization to develop as a potent anti-osteoporotic agent.

RANKL and RANK: From Mammalian Physiology to Cancer Treatment.

The tumor necrosis factor (TNF) receptor RANK (TNFRSF11A) and its ligand RANKL (TNFSF11) regulate osteoclast development and bone metabolism. They also control stem cell expansion and proliferation of mammary epithelial cells via the sex hormone progesterone. As such, RANKL and RANK have been implicated in the onset of hormone-induced breast cancer. Recently, RANK/RANKL were identified as crucial regulators for BRCA1 mutation-driven breast cancer. Current prevention strategies for BRCA1 mutation carriers are associated with wide-ranging risks; therefore, the search for alternative, non-invasive strategies is of paramount importance. We summarize here the functions of the RANKL/RANK pathway in mammalian physiology and focus on its recently uncovered role in breast cancer. We propose that anti-RANKL therapy should be pursued as a preventative strategy for breast cancer.

RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer.

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.

Porphyrin Derivatives Inhibit the Interaction between Receptor Activator of NF-κB and Its Ligand.

Receptor activator of NF-κB (RANK), a member of the TNF-receptor superfamily, plays an important role in bone resorption and stimulates immune and epithelial cell activation. Denosumab, a human monoclonal antibody that blocks the RANK ligand (RANKL), is approved for the treatment of osteoporosis and bone metastasis. However, a small molecule that inhibits the RANK-RANKL interaction would be beneficial to decrease cost and to facilitate treatments with orally available therapeutic agents. Herein we report the discovery of the first nonpeptidic inhibitors of RANK-RANKL interactions. In screening a chemical library by competitive ELISA, the porphyrin verteporfin was identified as a hit. Derivatives were screened, and the chlorin-macrocycle-containing pheophorbide A and purpurin 18 were found to bind recombinant RANKL, to inhibit RANK-RANKL interactions in the ELISA, and to suppress the RANKL-dependent activation of model cells and the differentiation of RANK-expressing precursors into osteoclasts. This discovery of a family of small molecules that inhibit RANK activation presents an initial basis for further development of nonpeptidic therapeutic agents targeting the interaction between RANK and RANKL.

N-(2-Hydroxyphenyl)acetamide: a Novel Suppressor of RANK/RANKL Pathway in Collagen-Induced Arthritis Model in Rats.

RANKL and RANK are potential contributors of inflammatory cascade in human and animal model of arthritis. The current study aims to investigate the effect of N-(2-hydroxyphenyl)acetamide (NA-2) on regulation of RANKL pathway in collagen-induced arthritis (CIA) model in rats. CIA was induced using bovine type II collagen in female Wistar rats. The clinical parameters, level of pro-inflammatory and oxidative stress markers were measured to determine the progression of the disease. The mRNA level of RANKL and RANK and downstream mediators of inflammation i.e. c-fos, c-jun, NF-κB and Akt were analysed in spleen tissue using real-time PCR. Immunohistochemical analysis of iNOS, pAkt and c-Fos was also done in spleen tissue. Treatment with NA-2 and indomethacin showed increase in body weight and significant reduction in paw volume and arthritic score (p < 0.0001). Marked reduction in the level of oxidative stress markers, NO, PO and GSH (p < 0.0001), and pro-inflammatory markers, IL-1ß (p < 0.0001) and TNF-α (p < 0.01), was also observed. Likewise, NA-2 and indomethacin treatment also significantly suppressed the mRNA expression of RANKL, RANK, c-fos, c-jun, NF-κB (p < 0.0001) and Akt (p < 0.01) and protein expression of iNOS, pAkt and c-Fos (p < 0.0001) compared to the arthritic control group. Our findings suggest that NA-2 is an antiarthritic agent acting in a pleiotropic manner in CIA rats by not only reducing the clinical signs of arthritis, inflammatory cytokines and free radical production but also attenuating the RANK/RANKL signaling pathway.

Novel factors modulating human ß-cell proliferation.

ß-Cell dysfunction in type 1 and type 2 diabetes is accompanied by a progressive loss of ß-cells, and an understanding of the cellular mechanism(s) that regulate ß-cell mass will enable approaches to enhance hormone secretion. It is becoming increasingly recognized that enhancement of human ß-cell proliferation is one potential approach to restore ß-cell mass to prevent and/or cure type 1 and type 2 diabetes. While several reports describe the factor(s) that enhance ß-cell replication in animal models or cell lines, promoting effective human ß-cell proliferation continues to be a challenge in the field. In this review, we discuss recent studies reporting successful human ß-cell proliferation including WS6, an IkB kinase and EBP1 inhibitor; harmine and 5-IT, both DYRK1A inhibitors; GNF7156 and GNF4877, GSK-3ß and DYRK1A inhibitors; osteoprotegrin and Denosmab, receptor activator of NF-kB (RANK) inhibitors; and SerpinB1, a protease inhibitor. These studies provide important examples of proteins and pathways that may prove useful for designing therapeutic strategies to counter the different forms of human diabetes.