Nuclear Factor Of Activated T Cells Cytoplasmic 1 Research Articles

Napyradiomycin B4 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Alveolar Bone Destruction in Experimental Periodontitis.

The unique structure and beneficial biological properties of marine natural products have drawn interest in drug development. Here, we examined the therapeutic potential of napyradiomycin B4 isolated from marine-derived Streptomyces species for osteoclast-related skeletal diseases. Bone marrow-derived macrophages were treated with napyradiomycin B4 in an osteoclast-inducing medium, and osteoclast formation, osteoclast-specific gene expression, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) localization were evaluated using tartrate-resistant acid phosphatase staining, real-time PCR, and immunostaining, respectively. Phosphorylation levels of signaling proteins were assessed by immunoblot analysis to understand the molecular action of napyradiomycin B4. The in vivo efficacy of napyradiomycin B4 was examined under experimental periodontitis, and alveolar bone destruction was evaluated by microcomputed tomography (micro-CT) and histological analyses. Among the eight napyradiomycin derivatives screened, napyradiomycin B4 considerably inhibited osteoclastogenesis. Napyradiomycin B4 significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation and disrupted the expression of NFATc1 and its target genes. Mitogen-activated extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation levels were reduced by napyradiomycin B4 in response to RANKL. Under in vivo experimental periodontitis, napyradiomycin B4 significantly attenuated osteoclast formation and decreased the distance between the cementoenamel junction and alveolar bone crest. Our findings demonstrate the antiosteoclastogenic activity of napyradiomycin B4 by inhibiting the RANKL-induced MEK-ERK signaling pathway and its protective effect on alveolar bone destruction.

Ethanol extract of Cyathulae Radix inhibits osteoclast differentiation and bone loss

Cyathulae Radix, a traditional Chinese medicine and a common vegetable, boasts a history spanning millennia. It enhances bone density, boosts metabolism, and effectively alleviates osteoporosis-induced pain. Despite its historical use, the molecular mechanisms behind Cyathulae Radix's impact on osteoporosis remain unexplored. In this study, we investigated the effects and mechanisms of Cyathulae Radix ethanol extract (CEE) in inhibiting osteoporosis and osteoclastogenesis. Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks. Micro-computed tomography (micro-CT) assessed histomorphometric parameters, bone tissue staining observed distal femur histomorphology, and three-point bending tests evaluated tibia mechanical properties. Enzyme-linked immunosorbent assay (ELISA) measured serum estradiol (E2), receptor activator for nuclear factor B ligand (RANKL), and osteoprotegerin (OPG) levels. Osteoclastogenesis-related markers were analyzed via Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase (TRAP) staining, qRT-PCR, and WB assay. Compared with the ovariectomy (OVX) group, CEE treatment enhanced trabecular bone density, maximal load-bearing capacity, and various histomorphometric parameters. Serum E2 and OPG levels significantly increased, while Receptor activator of nuclear factor-κB (RANK) decreased in the CEE group. CEE downregulated matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), and TRAP gene and protein expression. In bone marrow macrophages (BMMs), CEE reduced mature osteoclasts, bone resorption pit areas, and MMP-9, CTSK, and TRAP expression during osteoclast differentiation. Compared with DMSO treatment, CEE markedly inhibited RANK, TNF receptor associated factor 6 (TRAF6), Proto-oncogene c-Fos (c-Fos), Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expressions, and Extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), NF-kappa B-p65 (p65) phosphorylation in osteoclasts. In conclusion, CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis, potentially through modulating the Estrogen Receptor (ER)/RANK/NFATc1 signaling pathway.

The Role of Rosavin in the Pathophysiology of Bone Metabolism.

Rosavin, a phenylpropanoid in Rhodiola rosea's rhizome, and an adaptogen, is known for enhancing the body's response to environmental stress. It significantly affects cellular metabolism in health and many diseases, particularly influencing bone tissue metabolism. In vitro, rosavin inhibits osteoclastogenesis, disrupts F-actin ring formation, and reduces the expression of osteoclastogenesis-related genes such as cathepsin K, calcitonin receptor (CTR), tumor necrosis factor receptor-associated factor 6 (TRAF6), tartrate-resistant acid phosphatase (TRAP), and matrix metallopeptidase 9 (MMP-9). It also impedes the nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), c-Fos, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways and blocks phosphorylation processes crucial for bone resorption. Moreover, rosavin promotes osteogenesis and osteoblast differentiation and increases mouse runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) expression. In vivo studies show its effectiveness in enhancing bone mineral density (BMD) in postmenopausal osteoporosis (PMOP) mice, restraining osteoclast maturation, and increasing the active osteoblast percentage in bone tissue. It modulates mRNA expressions by increasing eukaryotic translation elongation factor 2 (EEF2) and decreasing histone deacetylase 1 (HDAC1), thereby activating osteoprotective epigenetic mechanisms, and alters many serum markers, including decreasing cross-linked C-telopeptide of type I collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRACP5b), receptor activator for nuclear factor κ B ligand (RANKL), macrophage-colony-stimulating factor (M-CSF), and TRAP, while increasing alkaline phosphatase (ALP) and OCN. Additionally, when combined with zinc and probiotics, it reduces pro-osteoporotic matrix metallopeptidase 3 (MMP-3), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), and enhances anti-osteoporotic interleukin 10 (IL-10) and tissue inhibitor of metalloproteinase 3 (TIMP3) expressions. This paper aims to systematically review rosavin's impact on bone tissue metabolism, exploring its potential in osteoporosis prevention and treatment, and suggesting future research directions.

Sucrosephenylpropanoid esters and isoflavonoids isolated from Belamcanda chinensis roots and their potential anti-osteoclastogenic activity

Repeated chromatography of the CH2Cl2 and EtOAc soluble fractions from the methanol extract of Belamcanda chinensis root yielded six new sucrosephenylpropanoid esters (1–6) and twenty-one known compounds (7–27). The structures of 1–6 were elucidated using diverse nuclear magnetic resonance (NMR) techniques and high-resolution mass spectrometry (HRMS) data analysis, together with chemical methods. All the twenty-seven isolated compounds were evaluated for their anti-osteoclastogenic activities. Preliminary screening results revealed that compounds 1 and 19 exhibited strong effects against RANKL-induced osteoclast formation in RAW264.7 cells. In addition, the treatment of mouse bone marrow macrophages (BMMs) with compounds 1 and 19 significantly decreased RANKL-induced TRAP-positive multinucleated osteoclast formation in a concentration-dependent manner without affecting cell viability. Further bioassay investigation showed that compounds 1 and 19 inhibited the expression of some osteoclast-specific marker genes and the transcription factor nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. To the best of our knowledge, this is the first investigation of anti-osteoclastogenic activity for compounds isolated from B. chinensis.

2,5-Dihydroxyacetophenone attenuates RANKL-mediated osteoclastogenesis by affecting the NFATc1 signaling pathway in vitro

Excessive bone resorption activity of osteoclasts is a common characteristic of osteolytic conditions such as osteoporosis and inflammatory bone diseases. Natural compounds with antiosteoclastogenic function seem to be beneficial for the treatment of osteolytic diseases. In this study, we evaluated the effects of 2,5-dihydroxyacetophenone (DHAP), a phenolic compound in Ganoderma bambusicola, on osteoclastogenesis induced in vitro by the receptor activator of nuclear factor-κB ligand (RANKL). DHAP inhibited the differentiation, actin ring formation, and bone resorption activity of osteoclasts. In particular, DHAP inhibited the transcriptional activity of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) during osteoclastogenesis. This inhibition resulted in reduced expression levels of cathepsin k (Ctsk), tartrate-resistant acid phosphatase (Trap), and NFATc1 (Nfatc1), thereby reducing the differentiation of osteoclasts. However, DHAP did not affect reactive oxygen species production or activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) signaling. Our findings suggest that DHAP inhibits RANKL-induced osteoclastogenesis by inhibiting the NFATc1 signaling pathway.

Oleanolic acid promotes skeletal muscle fiber type transformation by activating TGR5-mediated CaN signaling pathway

In recent years, the impact of bile acids and their representative G protein-coupled bile acid receptor 1 Takeda-G-protein-receptor-5 (TGR5) signaling pathway on muscle function and metabolic health has gained considerable interest. Increasing the content of slow muscle fibers has been recognized as an effective strategy to improve metabolic health. Oleanolic acid (OA) is a naturally occurring triterpenoid compound derived from plants, which can activate TGR5. The aim of this study was to investigate the effect of OA and TGR5 on muscle fiber types and further explore the underlying TGR5-dependent mechanisms. In this study, mice were divided into three groups and dietary supplementation with 0, 50, or 100 mg/kg OA. In addition, C2C12 cells were treated with OA at concentrations of 0, 5, 10, and 20 µM. Our studies revealed that OA promoted the conversion of fast to slow muscle fibers. In addition, it was found that OA activated the TGR5-mediated calcineurin (CaN)/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway. Further mechanistic investigations demonstrated that inhibiting TGR5 and CaN abolished the effects of OA on muscle fiber types transformation. In conclusion, this study found that OA promotes the transformation of fast muscle fibers to slow muscle fibers through the TGR5-mediated CaN/NFATc1 signaling pathway.

Abstract P398: Reduced Expression Of Nfatc1 Protects Against Salt-sensitive Hypertension In Mice

Background: While nuclear factor of activated T cells-cytoplasmic 1 (NFATc1) is a transcription factor that canonically plays a role in the immune response, it is also widely expressed in non-immune cells. A recent trans-ethnic meta-analysis of genome-wide association studies (GWAS) found that the NFATC1 gene in humans was associated with chronic kidney disease. Additionally, renal Nfact1 mRNA expression was decreased during high-salt exposure in a salt-sensitive murine model, suggesting a link between NFATc1 and salt-sensitive hypertension. Methods: Here, young adult heterozygous Nfatc1 knockout mice (Het) and wild-type (WT) male mice on a salt-sensitive 129S6 background were studied. Systolic blood pressure (SBP) measurements were obtained by either tail cuff manometry with at least 2 weeks of training or implanted radiotelemetry with at least 2 weeks of recovery following surgery. SBP was recorded on standard chow (NSD), on a matched high-salt diet (HSD, 6% total NaCl), or on standard chow with 1000 ng/kg/min angiotensin II (AngII) delivered by osmotic minipump. Pressure natriuresis was examined by correlating 24 h SBP from telemetry with 24 h urine sodium collected in metabolic cages before and for 3 days after switching from NSD to HSD. Results: On NSD, there was no difference in SBP between WT and Het mice (127.8±9.9 vs 125.8±8.7 mmHg, n = 9/group). However, while WT mice displayed an expected elevation in SBP after 3 weeks on HSD, Het mice showed no change in SBP, resulting in a statistically significant difference between genotypes (143.4±8.9 vs 125.7±10.6 mmHg, p = 0.0024, n = 9/group). Pressure natriuresis curves were not significantly different between genotypes. SBP was not significantly different on AngII (167.6±22.2 vs 161.8±10.9 mmHg, n = 2/3). Conclusion: Our preliminary data shows that decreased Nfatc1 expression protects against salt-sensitive hypertension. However, this effect is not mediated through a change in pressure natriuresis, suggesting possible involvement of a vascular mechanism. NFATc1 is a potential therapeutic target in salt-sensitive hypertension.

Anandamide modulates WNT-5A/BCL-2, IP3/NFATc1, and HMGB1/NF-κB trajectories to protect against mercuric chloride-induced acute kidney injury.

Endocannabinoid anandamide (AEA) has a physiological role in regulating renal blood flow, whereas its analogs ameliorated renal ischemia/reperfusion injury. Nonetheless, the role of AEA against mercuric chloride (HgCl2)-induced renal toxicity has not been unraveled. Rats were allocated into control, HgCl2, and HgCl2/AEA treated groups. The administration of AEA quelled the HgCl2-mediated increase in inositol trisphosphate (IP3) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). The endocannabinoid also signified its anti-inflammatory potential by turning off the inflammatory cascade evidenced by the suppression of high mobility group box protein-1 (HMGB1), receptor of glycated end products (RAGE), nuclear factor-κB p65 (NF-κB), and unexpectedly PPAR-γ. Additionally, the aptitude of AEA to inhibit malondialdehyde and boost glutathione points to its antioxidant capacity. Moreover, AEA by enhancing the depleted renal WNT-5A and reducing cystatin-C and KIM-1 (two kidney function parameters) partly verified its anti-apoptotic capacity, confirmed by inhibiting caspase-3 and increasing B-cell lymphoma-2 (BCL-2). The beneficial effect of AEA was mirrored by the improved architecture and kidney function evidenced by the reduction in cystatin-C, KIM-1, creatinine, BUN, and caspase1-induced activated IL-18. In conclusion, our results verify the reno-protective potential of AEA against HgCl2-induced kidney injury by its anti-inflammatory, antioxidant, and anti-apoptotic capacities by modulating WNT-5A/BCL-2, IP3/NFATC1, HMGB-1/RAGE/NF-κB, caspase-1/IL-18, and caspase-3/BCL-2 cues.

Inhibition of CD4 + T cells by fanchinoline via miR506-3p/NFATc1 in Sjögren's syndrome.

The hyperproliferation and hyperactivation of CD4 + T cells in salivary gland tissues are hallmarks of Sjögren's syndrome (SS). Fangchinoline (Fan) is extracted from the root of Stephania tetrandra Moore, which is used for treating rheumatic diseases in many studies. This study aimed to identify the mechanism underlying the inhibition of CD4 + T cells by Fan in the SS model NOD/ShiLtj mice. In vivo, Fan alleviated the dry mouth and lymphocyte infiltration in the salivary gland tissues of the NOD/ShiLtj mice and inhibited the number of CD4 + T cells in the infiltrating focus. In vitro, Fan's inhibitory effect on the proliferation of mouse primary CD4 + T cells was verified by CFSE and EdU tests. Furthermore, qRT-PCR and WB analysis confirmed that Fan could inhibit the expression of NFATc1 (Nuclear factor of activated T-cells, cytoplasmic 1) by upregulating miR-506-3p. Dual luciferase reporter gene assay suggested that miR-506-3p interacted with NFATc1. CFSE and EdU tests showed that Fan could inhibit the proliferation of CD4 + T cells through miR-506-3p/NFATc1. The key role of NFATc1 in the activation of CD4 + T cells and the high expression of NFATc1 in samples from SS patients suggested that NFATc1 might become a therapeutic target for SS. In vivo, 11R-VIVIT (NFATc1 inhibitor) alleviated SS-like symptoms. This study not only explained the new mechanism of Fan inhibiting proliferation of CD4 + T cells and alleviating SS-like symptoms but also provided NFATc1 as a potential target for the subsequent research and treatment of SS.

SIRT3-and FAK-mediated acetylation-phosphorylation crosstalk of NFATc1 regulates Nε-carboxymethyl-lysine-induced vascular calcification in diabetes mellitus

Background and aimsArterial calcification is the predictor of cardiovascular risk in diabetic patients. Nε-carboxymethyl-lysine (CML), a toxic metabolite, is associated with accelerated vascular calcification in diabetes mellitus (DM). However, the mechanism remains elusive. This study aims to explore the key regulators involved in CML-induced vascular calcification in DM. MethodsWe used Western blot and immuno-staining to test the expression and localization of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) in human samples, a diabetic apolipoprotein E-deficient (ApoE−/−) mouse model, and a vascular smooth muscle cells (VSMC) model. Further, we confirmed the regulator of NFATc1 phosphorylation and acetylation induced by CML. The role of NFATc1 in VSMCs calcification and osteogenic differentiation was explored in vivo and in vitro. ResultsIn diabetic patients, CML and NFATc1 levels increased in the severe calcified anterior tibial arteries. CML significantly promoted NFATc1 expression and nuclear translocation in VSMCs and mouse aorta. Knockdown of NFATc1 significantly inhibited CML-induced calcification. CML promoted NFATc1 acetylation at K549 by downregulating sirtuin 3 (SIRT3), which antagonized the focal adhesion kinase (FAK) induced NFATc1 phosphorylation at the Y270 site. FAK and SIRT3 affected the nuclear translocation of NFATc1 by regulating the acetylation-phosphorylation crosstalk. NFATc1 dephosphorylation mutant Y270F and deacetylation mutant K549R had opposite effects on VSMC calcification. SIRT3 overexpression and FAK inhibitor could reverse CML-promoted VSMC calcification. ConclusionsCML enhances vascular calcification in DM through NFATc1. In this process, CML increases NFATc1 acetylation by downregulating SIRT3 to antagonize FAK-induced NFATc1 phosphorylation.

Cardiopreventive effect of Klotho protein on oxidative stress, inflammation and apoptosis during cardiac ischemia/reperfusion injury

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre, Poland. Background The injury of the myocardium during ischemia/reperfusion (IRI) involves metabolic, morphological and contractile disorders [1]. Klotho is a membrane or soluble anti-aging protein. Recent studies have proven the correlation of Klotho deficiency and the occurrence and development of cardiovascular diseases [2]. Purpose This study aimed to evaluate the effect of Klotho protein supply on oxidative stress, inflammation and apoptosis in hearts subjected to IRI. Methods Isolated Wistar rat hearts perfused with the Langendorff method were subjected to ischemia, followed by reperfusion, in the presence or absence of Klotho protein. Hemodynamic parameters, and the levels of nitrate/nitrite (NOX)-, glutathione peroxidase (GPx), superoxide dismutase (SOD), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-Κb), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), interleukin-6 (IL-6), matrix metalloproteinase 2 (MMP-2), cytochrome c (cyt c) and caspase-9 were evaluated. Results Infusion supply of Klotho during IRI resulted in a recovery of contractile function (p=0.031) and heart rate (p=0.02). Klotho protein contributed to increase in the production of GPx (p=0.045) and SOD (p=0.006). The levels of (NOX)- (p=0.022), NF-Κb (p=0.006), NFATc1 (p=0.027), IL-6 (p=0.002), MMP-2 (p=0.008), cyt c (p=0.0002) and caspase-9 (p=0.009) were decreased in the IRI+Klotho group, as compared to the IRI group. Conclusions Klotho protein preserved heart mechanical function, enhanced antioxidant capacity, and reduced inflammation and apoptosis in the injured heart. Thus, Klotho can be recognized as a novel cardiopreventive/cardioprotective agent in ischemic damage.

Abstract 4725: The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia

Abstract Introduction: The introduction of Bruton’s tyrosine kinase (BTK) inhibitors (BTKi) to target B cell receptor (BCR) pathways in chronic lymphocytic leukemia (CLL) has significantly improved clinical responses. However, the effectiveness of BTKi is limited by the development of adaptive drug resistance. Therefore, there is an urgent need to identify critical biologic features that determine BTKi efficacy in order to develop more effective therapies. Epigenetic aberrations play an important role in tumor initiation, progression, and drug resistance. However, it is currently unknown if BCR signaling is also dependent on epigenetic mechanisms to support oncogenic gene expression and survival in CLL B cells. Methods and Results: To gain new insights into the role of epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in other cohorts at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies show that suppressing BCR signaling by BTKi treatment leads to an alteration of a pro-survival epigenetic signature (defined by histone modifications and chromatin accessibility landscapes) in CLL and that the genes regulated by these chromatin changes are enriched in pathways associated with malignant B cell survival (i.e., BCR signaling and apoptosis pathways). Importantly, disruption of this pro-survival epigenetic signature is necessary for effective BTKi treatment. We further find that the transcription factor (TF) Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) couples the BCR signaling to control this pro-survival epigenetic program that supports CLL B cell survival. In specific, we show that nuclear depletion of NFATc1 is required for BTKi induced epigenetic alteration and effective BTKi treatment. Depletion of NFATc1 disrupts the pro-survival epigenetic signature and inhibits CLL B cells survival, which improves the efficacy of BTKi treatment in CLL. Conclusion: Here using leukemic cells from CLL patients with BTKi treatment, we demonstrate that epigenetic regulators are exploited by the oncogenic signaling pathway to support malignant cell survival. Specifically, NFATc1 is utilized by the BCR signaling pathway to control an epigenetic signature that supports CLL cell survival. This BCR regulated epigenetic machinery can be targeted to maintain and enhance BTKi treatment efficacy in CLL. Citation Format: Zhiquan Wang, Huihuang Yan, Justin C. Boysen, Esteban Braggio, Sameer A. Parikh, Neil E. Kay. The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4725.

Histone Deacetylase 6 Inhibitor CKD-WID Suppressed Monosodium Urate-Induced Osteoclast Formation by Blocking Calcineurin-NFAT Pathway in RAW 264.7 Cells

Histone deacetylase (HDAC) has been found to play a crucial role in the regulation of osteoclast differentiation and formation. This study was designed to identify the effect of the HDAC6 inhibitor CKD-WID on the receptor for the activation of nuclear factor-κB ligand (RANKL)-mediated osteoclast formation in the presence of monosodium urate (MSU) in RAW 264.7 murine macrophage cells. The expression of osteoclast-specific target genes, calcineurin, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was evaluated in RAW 264.7 murine macrophages treated with MSU, RANKL, or CKD-WID by real-time quantitative polymerase chain reaction and Western blot assay. The effect of CKD-WID on osteoclast formation was measured by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring formation staining, and assays for bone resorption activity. RANKL in the presence of MSU significantly induced HDAC6 gene and protein expression in RAW 264.7 cells. CKD-WID markedly suppressed the expression of osteoclast-related markers such as c-Fos, TRAP, cathepsin K, and carbonic anhydrase II induced by co-stimulation with RANKL and MSU in RAW 264.7 cells. Transcription factor NFATc1 mRNA expression and nuclear NFATc1 protein expression induced by co-stimulation with RANKL and MSU were significantly inhibited by CKD-WID treatment. CKD-WID also decreased the number of TRAP-positive multinuclear cells and F-actin ring-positive cells and attenuated bone resorption activity. Co-stimulation with RANKL and MSU increased calcineurin gene and protein expression, which was significantly blocked by CKD-WID treatment. The HDAC6 inhibitor CKD-WID suppressed MSU-induced osteoclast formation through blocking the calcineurin-NFAT pathway in RAW 264.7 cells. This suggests that HDAC6 is considered a therapeutic target in uric acid-mediated osteoclastogenesis.

NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation.

The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overexpressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription.

Genetic studies in the Pakistani population reveal novel associations with ventricular septal defects (VSDs)

BackgroundWith prevalence up to 4%, Ventricular Septal Defect (VSD) is one of the leading causes of neonatal deaths. VSD is a common complex genetic disorder that has been associated with many genetic determinants. Variants from genes for the transcription factors including T-Box TBX5 and NFATc1 (nuclear factor of activated T cells, cytoplasmic 1), Vascular endothelial growth factor (VEGF), ISLET1 (encoded by the ISL1 gene) and enzyme MTHFR, a methylene tetrahydrofolate reductase were selected. Genetic risk score (GRS) is a widely accepted approach used to convert the genetic data into prediction and assessment tool for disease susceptibility.MethodsA total of 200 participants were recruited for the current study, 100 VSD patients and 100 controls. Genotyping of the ISL1: rs1017, NFATc1: rs7240256, VEGF: rs36208048, TBX5: rs11067075, and MTHFR: rs1801133 variants was performed using tetra primer ARMS PCR and PCR-RFLP. For the statistical analysis, the software SPSS version 23 was used. Genotypic frequencies of cases and controls were calculated using chi-square (χ²) whereas allelic frequencies were calculated by using the SNPStats tool. The association of GRS quartiles with VSD was examined using binary logistic regression. Adjusted p-value 0.01 was used as significance threshold for all analyses.ResultsThe ISL1 (OD: 0.242, CI: 0.158–0.37, p-value: 2.15 × 10− 4 :), NFATc1 (OD: 2.53, CI: 1.64–3.89, p-value: 2.11 × 10− 5), TBX5 (OD: 2.24, CI: 1.47–3.41, p-value:1.6 × 10− 4) and MTHFR (OD: 10.46, CI: 5.68–19.26, p-value: 2.09 × 10− 9:) variants were found to be in association with VSD. In contrast, the VEGF (OD: 0.952, CI: 0.56–1.62, p-value: 0.8921) variant did not show significance association with the VSD. For cases, the mean GRS score was 3.78 ± 1.285 while in controls it was 2.95 ± 1.290 (p-value: 0.479, CI: 0.474–1.190). Comparison of GRS between cases and control showed that mean GRS of cases was 1.90 ± 0.480 while in controls it was 1.68 ± 0.490 (p-value: 0.001, CI: 0.086–0.354). Higher quartiles were more prevalent in cases whereas lower quartiles were more prevalent in controls.ConclusionGRS of these five loci was strongly associated with VSD. Moreover, genetic risk score can provide better information for the association between variants and disease as compared to a single SNP. We also illustrated that the cumulative power of GRS is greater over the single SNP effect. This is a pilot scale study with a relatively small sample size whose findings should be replicated in a larger sample size for the unique local Pakistani population.

Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption

ABSTRACT Regulation of osteoclastogenesis and bone-resorbing activity can be an efficacious strategy for treating bone loss diseases because excessive osteoclastic bone resorption leads to the development of such diseases. Here, we investigated the role of (-)-tubaic acid, a thermal degradation product of rotenone, in osteoclast formation and function in an attempt to identify alternative natural compounds. (-)-Tubaic acid significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation at both the early and late stages, suggesting that (-)-tubaic acid affects the commitment and differentiation of osteoclast progenitors as well as the cell-cell fusion of mononuclear osteoclasts. (-)-Tubaic acid attenuated the activation of extracellular signal-regulated kinase (ERK) and expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and its target genes in response to RANKL. Furthermore, a pit-formation assay revealed that (-)-tubaic acid significantly impaired the bone-resorbing activity of osteoclasts. Our results demonstrated that (-)-tubaic acid exhibits anti-osteoclastogenic and anti-resorptive effects, indicating its therapeutic potential in the management of osteoclast-related bone diseases.

Dental Implant Osseointegration Inhibition by Nicotine through Increasing nAChR, NFATc1 Expression, Osteoclast Numbers, and Decreasing Osteoblast Numbers.

The success of dental implants is determined by the osteointegration process. Many studies state that smoking cigarettes can inhibit osseointegration, but the inhibition mechanism is still unclear.The aim of this study was to identify and analyze the effect of nicotine on the inhibition of dental implant osseointegration through the expression of nicotinic acetylcholine receptor (nAChR), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), osteoclast, and osteoblast numbers. This study is an experimental study of 16 New Zealand rabbits, randomized across two groups. Group 1 (eight rabbits) was a control group, and group 2 (eight rabbits) was a treatment group. The treatment group was given 2.5 mg/kg body weight/day of nicotine by injection 1 week before placement of the implant until the end of research. Observations were made in the first and the eighth week by measuring the number of osteoblast and osteoclast by immunohistology test and the expression of nAChR and NFATc1 by immunohistochemistry test. Data was analyzed using a one-way analysis of variance and Student's t-test. A p-value of < 0.05 was considered statistically significant. Significant differences were found between the control and treatment groups (p < 0.05). Results showed that nicotine increases the expression of nAChR and decreases the number of osteoblasts and the expression of BMP2 and osteocalcin. Nicotine inhibits the osseointegration of dental implants by increasing nAChR, NFATc1, osteoclast numbers, and decreasing osteoblast numbers.

Evaluation of culture conditions for osteoclastogenesis in RAW264.7 cells.

Osteoclasts are the only multinucleated cells in vivo responsible for bone resorption and are vital for regulating bone remodeling and maintaining bone mass. The RAW264.7 cell line is widely used to study osteoclastic differentiation and biological molecular mechanism. However, protocols for inducing osteoclast formation in RAW264.7 cells vary considerably between laboratories, hindering the replication of results. Therefore, we tested the influence of culture conditions on osteoclast differentiation, including cell density and receptor activator of nuclear factor kappa-B ligand (RANKL) concentrations with or without macrophage colony-stimulating factors (M-CSF). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the morphology of osteoclasts. qPCR was used to detect gene expression of osteoclast-specific gene marker cathepsin K (CTSK), osteoclast transcription factors c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The bone resorption function was evaluated by a scanning electron microscope (SEM). RANKL treatment increased multinucleated osteoclasts formation and increased CTSK, c-Fos and NFATc1 gene expression. Compared with RANKL treatment, M-CSF significantly decreased multinucleated osteoclasts formation, reduced CTSK gene expression and had little effect on c-Fos and NFATc1 gene expression. Concerning bone resorption activity, RANKL treatment increased bone resorption pits on bovine bone slices. Significantly higher levels of osteoclastogenesis were observed with RAW264.7-cell density of 2×104 cells/well in 24-well plates. Our results suggest that the addition of 50 ng/ml M-CSF has no positive effect on osteoclastogenesis. RANKL treatment and cell density contribute to osteoclast formation, and the optimal conditions are beneficial when exploring osteoclast function and mechanism.

Lacticaseibacillus rhamnosus MG4706 Suppresses Periodontitis in Osteoclasts, Inflammation-Inducing Cells, and Ligature-Induced Rats.

Periodontitis is a chronic inflammatory disease characterized by tooth loss due to inflammation and the loss of alveolar bone. Periodontitis is closely related to various systemic diseases and is emerging as a global health problem. In this study, we investigated the anti-inflammatory effect of lactic acid bacteria (LAB) in vitro on Porphyromonas gingivalis (P. gingivalis) LPS-activated RAW264.7 and human gingival fibroblasts-1 (HGF-1) cells and the anti-osteoclastogenic effect of LAB on RANKL-induced RAW264.7 cells. All LAB strains (Lacticaseibacillus rhamnosus MG4706, MG4709, and MG4711) inhibited nitric oxide (NO)/inducible nitric oxide synthase (iNOS) in P. gingivalis LPS-activated RAW264.7 cells and pro-inflammatory cytokines (IL-1β and IL-6) and matrix metalloproteinase (MMP-8 and MMP-9) in HGF-1 cells. In addition, LAB treatment inhibited osteoclastogenesis by reducing tartrate-resistant acid phosphatase (TRAP) activity and cathepsin K (CtsK) through the downregulation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and c-fos gene expression in RANKL-induced RAW264.7 cells. Administration of MG4706 alleviated alveolar bone loss indices and reduced the gene expression of IL-1β, IL-6, MMP-8,&amp;nbsp;MMP-9, and RANKL/OPG ratio in gingival tissue. In conclusion, L. rhamnosus MG4706 has the potential to alleviate periodontitis.

Hypoxia-inducible factor 1α enhances RANKL-induced osteoclast differentiation by upregulating the MAPK pathway

Hypoxia (low-oxygen tension) and excessive osteoclast activation are common conditions in many bone loss diseases, such as osteoporosis, rheumatoid arthritis (RA), and pathologic fractures. Hypoxia-inducible factor 1 alpha (HIF1α) regulates cellular responses to hypoxic conditions. However, it is not yet known how HIF1α directly affects osteoclast differentiation and activation. This study sought to. explore the effects of HIF1α on osteoclast differentiation and it's molecular mechanisms. L-mimosine, a prolyl hydroxylase (PHDs) domain inhibitor, was used to stabilize HIF1α in normoxia. In the presence of receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL), RAW264.7 cells were cultured and stimulated by treatment with L-mimosine at several doses to maintain various levels of intracellular HIF1α. The multi-nucleated cells were assessed by a tartrate-resistant acid phosphatase (TRAP) and F-actin ring staining assays. The osteoclast-specific genes, such as Cathepsin K, β3-Integrin, TRAP, c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and matrix metallo-proteinase 9 (MMP9), were analyzed by real time-polymerase chain reaction (RT-PCR). The expression of relevant proteins was analyzed by Western blot. L-mimosine increased the content of intracellular HIF1α in a dose-dependent manner, which in turn promoted RANKL-induced osteoclast formation and relevant protein expression by upregulating the mitogen-activated protein kinase (MAPK) pathways. Our findings suggest that HIF1α directly increases the osteoclast differentiation of RANKL-mediated RAW264.7 cells in vitro by upregulating the MAPK pathways.