Nuclear Translocation Of NFATc1 Research Articles

Lactate-induced metabolic remodeling and myofiber type transitions via activation of the Ca2+-NFATC1 signaling pathway.

Lactate can serve as both an energy substrate and a signaling molecule, exerting diverse effects on skeletal muscle physiology. Due to the apparently positive effects, it would be interesting to consider it as a sports supplement. However, the mechanism behind these effects are yet to be comprehensively understood. In this study, we observed that lactate administration could improve the ability of antifatigue, and we further found that lactate upregulated the expression of myosin heavy chain (MYHC I) and MYHC IIa, while downregulating the expression of MYHC IIb. Besides, transcriptomics and metabolomics revealed significant changes in the metabolic profile of gastrocnemius muscle following lactate administration. Furthermore, lactate enhanced the activities of metabolic enzymes, including HK, LDHB, IDH, SDM, and MDH, and promoted the expression of lactate transport-related proteins MCT1 and CD147, thereby improving the transport and utilization of lactate in both vivo and vitro. More importantly, lactate administration increased cellular Ca2+ concentration and facilitated nuclear translocation of nuclear factor of activated T cells (NFATC1) in myotubes, whereas inhibition of NFATC1 significantly attenuated the effects of lactate treatment on NFATC1 nuclear translocation and MyHC expression. Our results elucidate the ability of lactate to induce metabolic remodeling in skeletal muscle and promote myofiber-type transitions by activating the Ca2+-NFATC1 signaling pathway. This study is useful in exploring the potential of lactate as a nutritional supplement for skeletal muscle adaptation and contributing to a mechanistic understanding of the central role of lactate in exercise physiology.

Abstract 6686: The mechanism of gut microbiome-mediated immune activation on the clinical efficacy of PD-1 blocking treatment in solid tumors

Abstract Gut microbiota is associated with antitumor efficacy of immune checkpoint blockade therapies, yet the mechanism is not largely understood. Here, we show that a novel bacterial strain of Ruminococcaceae isolated from the feces of patients who respond to PD-1 (programmed cell death 1) blockade efficiently primes/activates tumor-specific CD8+ T cells through effective stimulation/maturation of CD103+ dendritic cells (DCs) during migration from the gut to the tumor microenvironment (TME). The administration of this bacteria strain with PD-1 blockade significantly inhibited tumor growth, even when adding them to fecal transplantation of non-responder patients in animal models. Mechanistically, the bacterial strain promoted the IRF8-driven differentiation of CD103+ DCs by stimulating multiple Toll-like receptors and the accumulation of these DCs in tumors and tumor-draining lymph nodes. These CD103+ DCs prolonged engagement with cognate antigen-specific CD8+ T cells, which induced the activation of CD8+ T cells specific for a wide variety of tumor antigens and fostered their PD-1 expression through enhanced T-cell activation signals with NFATc1 nuclear translocation, thereby leading to abundant PD-1+CD8+ T cells, a key effector cell population in PD-1 blockade, with a broader T-cell receptor repertoire in the TME. Our findings with novel single bacterium isolated from feces of responder patients demonstrate the mode of action by gut microbiota to augment antitumor immunity. Citation Format: Nina Yi-Tzu Lin, Shota Fukuoka, Daisuke Motooka, Yuko Shigeno, Takuma Irie, Dieter M. Tourlousse, Kazutoshi Murotomi, Yuji Sekiguchi, Hideyuki Tamaki, Mariko Shindo, Takahiro Tsuji, Hiroaki Wake, Koichi Goto, Toshihiko Doi, Kohei Shitara, Keisuke Watanabe, Yuka Maeda, Shota Nakamura, Yoshimi Benno, Hiroyoshi Nishikawa, Shohei Koyama. The mechanism of gut microbiome-mediated immune activation on the clinical efficacy of PD-1 blocking treatment in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6686.

Identifying Marine-Derived Tanzawaic Acid Derivatives as Novel Inhibitors against Osteoclastogenesis and Osteoporosis via Downregulation of NF-κB and NFATc1 Activation.

To discover novel osteoclast-targeting antiosteoporosis leads from natural products, we identified 40 tanzawaic acid derivatives, including 22 new ones (1-8, 14-19, 27-32, 37, and 38), from the South China Sea mangrove-derived fungus Penicillium steckii SCSIO 41025. Penicisteck acid F (2), one of the new derivatives showing the most potent NF-κB inhibitory activity, remarkably inhibited osteoclast generation in vitro. Mechanistically, 2 reduced RANKL-induced IκBα degradation, NF-κB p65 nuclear translocation, the activation and nuclear translocation of NFATc1, and the relevant mRNA expression. NF-κB p65 could be a potential molecular target for 2, which has been further determined by the cellular thermal shift assay, surface plasmon resonance, and the gene knock-down assay. Moreover, 2 could also alleviate osteoporosis in ovariectomized mice by reducing the quantities of osteoclasts. Our finding offered a novel potential inhibitor of osteoclastogenesis and osteoporosis for further development of potent antiosteoporosis agents.

An LGR4 agonist activates the GSK‑3β pathway to inhibit RANK‑RANKL signaling during osteoclastogenesis in bone marrow‑derived macrophages.

The binding between receptor‑activated nuclear factor‑κB (RANK) and the RANK ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. The leucine‑rich repeat‑containing G‑protein‑coupled receptor 4 (LGR4) acts as a negative regulator of RANK‑RANKL 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 marrow‑derived macrophages and a mouse model of RANKL‑induced 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 wild‑type (WT) RANKL and MT RANKL to RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT‑glycogen synthase kinase‑3β (GSK‑3β)‑nuclear factor of activated T cells, cytoplasmic, calcineurin‑dependent 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 RANKL‑induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4 and increased GSK‑3β 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 RANKL‑induced osteoclastogenesis through an LGR4‑dependent pathway and this could be exploited to develop new therapies for osteoporosis.

Abstract 15636: NFATC1 Modulates Atrial Excitability and Arrhythmogenesis

Transcription factors have emerged as important loci for atrial fibrillation (AF) susceptibility. We identified a novel mutation (M527L) in a conserved site of the transcription factor NFATC1 in a multigenerational family with an autosomal dominant young onset AF (<40 years old) phenotype. M527L reduces NFATC1 nuclear translocation (nuclear/total cellular ratio: M527L 0.50±0.02; WT 0.64±0.04, p<1E-5; n=10/each), DNA binding (M527L 0.69±0.02; WT 0.83±0.01, p=0.02; n=3/ each), and transcriptional activity (M527L 0.0003±0.00005; WT 0.0009±0.0002, p=0.01; n=5/each) in expression systems, underscoring the functional consequences of this mutation. To study the broader role of NFATC1 in atrial excitability we employed a CRISPR/Cas9 strategy to create an nfatc1 null (KO) zebrafish. KO fish exhibited reduced survival, with ~80% of juvenile fish succumbing to sudden death (log rank test p=0.0001), with no observable change in body or heart gross morphology. Explanted juvenile atria displayed spontaneous tachyarrhythmias or irregular rhythm (29 /40 KO atria vs 11/28 WT atria; Chi-Squared, p=0.0013) accompanied by irregular Ca 2+ handling (10/11 KO atria vs 5/16 WT atria). Furthermore, we found an abbreviated action potential duration in KO atrium (80±1 ms in KO (n=607) vs 91±1 ms in WT (n=441), p=0.0001), but not ventricle (186±3 ms in KO (n=109) vs 191±2 ms in WT (n=160), p=0.8). Taken together, our data suggests that NFATC1 is a modulator of atrial excitability and plays a role in AF susceptibility. Experiments are ongoing to further define the mechanisms underlying arrhythmia susceptibility and to identify the gene networks downstream of NFATC1 in the atria.

Tacrolimus Improves Therapeutic Efficacy of Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Diabetic Retinopathy by Suppressing DRP1-Mediated Mitochondrial Fission.

Diabetic retinopathy (DR) is a leading cause of blindness in diabetic patients. Umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) are emerging as a promising new drug for degenerative disease associated with diabetes. Recent studies have shown that high glucose-increased excessive calcium levels are a major risk factor for mitochondrial reactive oxygen species (mtROS) accumulation and apoptosis. This study aimed to investigate the role of high glucose-induced NFATC1 signaling in mitochondrial oxidative stress-stimulated apoptosis and the effect of tacrolimus on the therapeutic efficacy of subconjunctival transplantation of UCB-MSCs in a DR rat model. High glucose increased mtROS and cleaved caspase-9 expression in UCB-MSCs. High glucose conditions increased O-GlcNAcylated protein expression and nuclear translocation of NFATC1. Tacrolimus pretreatment recovered high glucose-induced mtROS levels and apoptosis. In the DR rat model, subconjunctival transplantation of tacrolimus-pretreated MSCs improved retinal vessel formation, retinal function, and uveitis. In high glucose conditions, tacrolimus pretreatment reduced protein and mRNA expression levels of DRP1 and inhibited mitochondrial fission. In conclusion, we demonstrated that high glucose-induced O-GlcNAcylation activates NFATC1 signaling, which is important for DRP1-mediated mitochondrial fission and mitochondrial apoptosis. Finally, we proposed NFATC1 suppression by tacrolimus as a promising therapeutic strategy to improve the therapeutic efficacy of UCB-MSC transplantation for DR treatment.

Microglial LRRK2-mediated NFATc1 attenuates α-synuclein immunotoxicity in association with CX3CR1-induced migration and the lysosome-initiated degradation.

Synucleinopathies refer to a range of neurodegenerative diseases caused by abnormal α-synuclein (α-Syn) deposition, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Their pathogenesis is strongly linked to microglial dysfunction and neuroinflammation, which involves the leucine-rich-repeat kinase 2 (LRRK2)-regulated nuclear factor of activated T-cells (NFAT). Of the NFAT family, NFATc1 has been found to be increasingly translocated into the nucleus in α-syn stimulation. However, the specific role of NFATc1-mediated intracellular signaling in PD remains elusive in regulating microglial functions. In the current study, we crossbred LRRK2 or NFATc1 conditional knockout mice with Lyz2Cre mice to generate mice with microglia-specific deletion of LRRK2 or NFATc1, and by stereotactic injection of fibrillary α-Syn, we generated PD models in these mice. We found that LRRK2 deficiency enhanced microglial phagocytosis in the mice after α-Syn exposure and that genetic inhibition of NFATc1 markedly diminished phagocytosis and α-Syn elimination. We further demonstrated that LRRK2 negatively regulated NFATc1 in α-Syn-treated microglia, in which microglial LRRK2-deficiency facilitated NFATc1 nuclear translocation, CX3CR1 upregulation, and microglia migration. Additionally, NFATc1 translocation upregulated the expression of Rab7 and promoted the formation of late lysosomes, resulting in α-Syn degradation. In contrast, the microglial NFATc1 deficiency impaired CX3CR1 upregulation and the formation of Rab7-mediated late lysosomes. These findings highlight the critical role of NFATc1 in modulating microglial migration and phagocytosis, in which the LRRK2-NFATc1 signaling pathway regulates the expression of microglial CX3CR1 and endocytic degradative Rab7 to attenuate α-synuclein immunotoxicity.

4'‑O‑methylbavachalcone inhibits succinate induced cardiomyocyte hypertrophy via the NFATc4 pathway.

Pathological cardiac hypertrophy is an independent risk factor for complications such as arrhythmia, myocardial infarction, sudden mortality and heart failure. Succinate, an intermediate product of the Krebs cycle, is released into the bloodstream by cells; its levels increase with exacerbations of hypertension, myocardial and other tissue damage and metabolic disease. Succinate may also be involved in several metabolic pathways and mediates numerous pathological effects through its receptor, succinate receptor 1 (SUCNR1; previously known as GPR91). Succinate-induced activation of SUCNR1 has been reported to be related to cardiac hypertrophy, making SUCNR1 a potential target for treating cardiac hypertrophy. Traditional Chinese medicine (TCM) and its active ingredients have served important roles in improving cardiac functions and treating heart failure. The present study investigated whether 4'-O-methylbavachadone (MeBavaC), an active ingredient of the herbal remedy Fructus Psoraleae, which is often used in TCM and has protective effect on myocardial injury and hypertrophy induced by adriamycin, ischemia-reperfusion and sepsis, could ameliorate succinate-induced cardiomyocyte hypertrophy by inhibiting the NFATc4 pathway. Using immunofluorescence staining, reverse transcription-quantitative PCR, western blotting and molecular docking analysis, it was determined that succinate activated the calcineurin/NFATc4 and ERK1/2 pathways to promote cardiomyocyte hypertrophy. MeBavaC inhibited cardiomyocyte hypertrophy, nuclear translocation of NFATc4 and ERK1/2 signaling activation in succinate-induced cardiomyocytes. Molecular docking analysis revealed that MeBavaC interacts with SUCNR1 to form a relatively stable binding and inhibits the succinate-SUCNR1 interaction. The results demonstrated that MeBavaC suppressed cardiomyocyte hypertrophy by blocking SUCNR1 receptor activity and inhibiting NFATc4 and ERK1/2 signaling, which will contribute to the preclinical development of this compound.

Icariin inhibits thioacetamide-induced osteoclast differentiation through RANKL-p38/ERK-NFAT pathway

This study aims to investigate the therapeutic effect of icariin(ICA) on thioacetamide(TAA)-induced femoral osteolysis in rats. RAW264.7 cells were treated with TAA and ICA. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation, and tartrate-resistant acid phosphatase(TRAP) staining to examine the formation of osteoclasts. The expression of TRAP, cathepsin K, c-FOS, and NFATc1 in RAW264.7 cells was determined by Western blot and immunofluorescence method. Thirty-two SD rats were randomized into the control group, TAA group(intraperitoneal injection of TAA at 300 mg·kg~(-1)), ICA group(gavage of ICA at 600 mg·kg~(-1)) and TAA + ICA group(intraperitoneal injection of TAA at 300 mg·kg~(-1) and gavage of ICA at 600 mg·kg~(-1)). Administration was performed every other day for 6 weeks. Body weight and length of femur were recorded at execution. Pathological injury and osteoclast differentiation of femur were observed based on hematoxylin-eosin(HE) staining and TRAP staining, and the changes of bone metabolism-related indexes alkaline phosphatase(ALP), calcium(Ca), phosphorus(P), magnesium(Mg), and cross-linked N-telopeptide of type Ⅰ collagen(NTX-Ⅰ) in serum were detected. Three-point bending test and micro-CT were applied to evaluate the quality of femur, and Western blot to detect the levels of osteoclast-related proteins TRAP, cathepsin K, RANK, RANKL, p38, p-p38, ERK, p-ERK, JNK, p-JNK, c-Fos, and NFATc1. The results showed ICA could inhibit TAA-induced production of TRAP-positive cells, the expression of osteoclast-related proteins, and nuclear translocation of NFATc1. ICA alleviated the weight loss, reduction of femur length, and growth inhibition induced by TAA in SD rats. ICA ameliorated the decline of femur elastic modulus caused by TAA and significantly restored trabecular bone mineral density(BMD), trabecular pattern factor(Tb.Pf), trabecular number(Tb.N), trabecular thickness(Tb.Th), and structure model index(SMI), thus improving bone structure. Western blot results showed ICA suppressed femoral osteoclast differentiation induced by TAA through RANKL-p38/ERK-NFATc1 signaling pathway. ICA inhibits osteoclast differentiation and prevents TAA-induced osteolysis by down-regulating RANKL-p38/ERK-NFAT signaling pathway.

Parathyroid Hormone Promotes Human Umbilical Vein Endothelial Cell Migration and Proliferation Through Orai1-Mediated Calcium Signaling.

Parathyroid hormone is the main endocrine regulator of extracellular calcium and phosphorus levels. Secondary hyperparathyroidism–induced endothelial dysfunction may be related to calcium homeostasis disorders. Here, we investigated the effects of parathyroid hormone on human umbilical vein endothelial cells (HUVECs) and characterized the involvement of store-operated Ca2+ entry (SOCE) and the nuclear factor of activated T cells (NFAT) signaling pathway. We used immunoblot experiments to find that parathyroid hormone significantly enhanced the expression of the Orai1 channel, a type of channel mediating SOCE, SOCE activity, and Orai1-mediated proliferation of HUVECs but did not increase Orai2 and Orai3. RNA-seq was utilized to identify 1,655 differentially expressed genes (823 upregulated and 832 downregulated) in parathyroid hormone–treated HUVECs as well as enhanced focal adhesion signaling and expression levels of two key genes, namely, COL1A1 and NFATC1. Increased protein and mRNA expression levels of COL1A1 and NFATC1 were confirmed by immunoblotting and quantitative RT-PCR, respectively. Cytosol and nuclei fractionation experiments and immunofluorescence methods were used to show that parathyroid hormone treatment increased NFATC1 nuclear translocation, which was inhibited by a calcineurin inhibitor (CsA), a selective calmodulin antagonist (W7), an Orai channel inhibitor (BTP2), or Orai1 small interfering RNA (siRNA) transfection. Parathyroid hormone also increased COL1A1 expression, cell migration, and proliferation of HUVECs. The PTH-induced increase in HUVEC migration and proliferation were inhibited by CsA, W7, BTP2, or COL1A1 siRNA transfection. These findings indicated that PTH increased Orai1 expression and Orai1-mediated SOCE, causing the nuclear translocation of NFATC1 to increase COL1A1 expression and COL1A1-mediated HUVEC migration and proliferation. These results suggest potential key therapeutic targets of Orai1 and the downstream calmodulin/calcineurin/NFATC1/COL1A1 signaling pathway in parathyroid hormone–induced endothelial dysfunction and shed light on underlying mechanisms that may be altered to prevent or treat secondary hyperparathyroidism–associated cardiovascular disease.

Tumor-derived miR-378a-3p-containing extracellular vesicles promote osteolysis by activating the Dyrk1a/Nfatc1/Angptl2 axis for bone metastasis

Most prostate cancer (PCa)-related deaths are caused by progression to bone metastasis. Recently, the importance of extracellular vesicles (EVs) in pre-metastatic niche formation has been reported. However, whether and how tumor-derived EVs interact with bone marrow macrophages (BMMs) to release EV-delivered microRNAs to promote osteolysis and induce pre-metastatic niche formation for PCa bone metastasis remain unclear. Our in vitro and in vivo functional and mechanistic assays revealed that EV-mediated release of miR-378a-3p from tumor cells was upregulated in bone-metastatic PCa, maintaining low intracellular miR-378a-3p concentration to promote proliferation and MAOA-mediated epithelial-to-mesenchymal transition. Moreover, miR-378a-3p enrichment in tumor-derived EVs was induced by hnRNPA2B1 (a transfer chaperone) overexpression. After tumor-derived EVs were taken in by BMMs, enriched miR-378a-3p promoted osteolytic progression by inhibiting Dyrk1a to improve Nfatc1 (an osteolysis-related transcription factor) nuclear translocation, to activate the expression of downstream target gene Angptl2. As a feedback, increased Angptl2 secretion into the tumor environment promoted PCa progression. In conclusion, tumor-derived miR-378a-3p-containing EVs play a significant role in PCa bone metastasis by activating the Dyrk1a/Nfatc1/Angptl2 axis in BMMs to induce osteolytic progression, making miR-378a-3p a potential predictor of metastatic PCa. Reducing the release of miR-378a-3p-containing EVs or inhibiting the recruitment of miR-378a-3p into EVs can be a therapeutic strategy against PCa metastasis.

RANK promotes colorectal cancer migration and invasion by activating the Ca2+-calcineurin/NFATC1-ACP5 axis

The tumor necrosis factor (TNF) receptor superfamily member 11a (TNFRSF11a, also known as RANK) was demonstrated to play an important role in tumor metastasis. However, the specific function of RANK in colorectal cancer (CRC) metastasis and the underlying mechanism are unknown. In this study, we found that RANK expression was markedly upregulated in CRC tissues compared with that in matched noncancerous tissues. Increased RANK expression correlated positively with metastasis, higher TNM stage, and worse prognosis in patients with CRC. Overexpression of RANK promoted CRC cell metastasis in vitro and in vivo, while knockdown of RANK decreased cell migration and invasion. Mechanistically, RANK overexpression significantly upregulated the expression of tartrate-resistant acid phosphatase 5 (TRAP/ACP5) in CRC cells. Silencing of ACP5 in RANK-overexpressing CRC cells attenuated RANK-induced migration and invasion, whereas overexpression of ACP5 increased the migration and invasion of RANK-silencing cells. The ACP5 expression was transcriptionally regulated by calcineurin/nuclear factor of activated T cells c1 (NFATC1) axis. The inhibition of calcineurin/NFATC1 significantly decreased ACP5 expression, and attenuated RANK-induced cell migration and invasion. Furthermore, RANK induced phospholipase C-gamma (PLCγ)-mediated inositol-1,4,5-trisphosphate receptor (IP3R) axis and stromal interaction molecule 1 (STIM1) to evoke calcium (Ca2+) oscillation. The RANK-mediated intracellular Ca2+ mobilization stimulated calcineurin to dephosphorylate NFATC1 and induce NFATC1 nuclear translocation. Both blockage of PLCγ-IP3R axis and STIM1 rescued RANK-induced NFATC1 nuclear translocation, ACP5 expression, and cell metastasis. Our study revealed the functional expression of RANK in human CRC cells and demonstrated that RANK induced the Ca2+-calcineurin/NFATC1-ACP5 axis in the regulation of CRC metastasis, that might be amenable to therapeutic targeting.

DGG-100629 inhibits lung cancer growth by suppressing the NFATc1/DDIAS/STAT3 pathway

DNA damage-induced apoptosis suppressor (DDIAS) promotes the progression of lung cancer and hepatocellular carcinoma through the regulation of multiple pathways. We screened a chemical library for anticancer agent(s) capable of inhibiting DDIAS transcription. DGG-100629 was found to suppress lung cancer cell growth through the inhibition of DDIAS expression. DGG-100629 induced c-Jun NH(2)-terminal kinase (JNK) activation and inhibited NFATc1 nuclear translocation. Treatment with SP600125 (a JNK inhibitor) or knockdown of JNK1 restored DDIAS expression and reversed DGG-100629-induced cell death. In addition, DGG-100629 suppressed the signal transducer and activator of transcription (STAT3) signaling pathway. DDIAS or STAT3 overexpression restored lung cancer cell growth in the presence of DGG-100629. In a xenograft assay, DGG-100629 inhibited tumor growth by reducing the level of phosphorylated STAT3 and the expression of STAT3 target genes. Moreover, DGG-100629 inhibited the growth of lung cancer patient-derived gefitinib-resistant cells expressing NFATc1 and DDIAS. Our findings emphasize the potential of DDIAS blockade as a therapeutic approach and suggest a novel strategy for the treatment of gefitinib-resistant lung cancer.

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-3β 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.

Maternal high-fat diet promotes the expansion of abdominal aortic aneurysm in adult offspring by enhancing osteoclast-like macrophage differentiation through down-regulation of IRF8 expression

Abstract Background and objective Maternal high-fat diet (HFD) has been shown to modulate vascular function and remodeling in adult offspring. Here, we investigated the impact of maternal HFD on abdominal aortic aneurysm (AAA) formation. Methods and results Eight-week-old female wild-type mice (C57BL/6) were fed a HFD or normal diet (ND) one week prior to mating, and the diet was continued throughout gestation and lactation. In eight-week-old male offspring, AAA was induced with the application of 0.5 M calcium chloride (CaCl2) on the infrarenal aorta. Offspring of HFD-fed dams (O-HFD) showed a significant increase in maximum outer diameter of AAA at 1, 4 and 8 weeks after surgery compared with offspring of ND-fed dams (O-ND). The lengths of outer circumference assessed by histological analysis were increased in O-HFD (p<0.05). Likewise, female O-HFD showed a greater length of outer circumference than female O-ND (p<0.05). While the number of F4/80-positive cells at 1 wk after surgery was comparable in the O-HFD and O-ND, the percentage of MMP-9/F4/80 double-positive cells was significantly increased in O-HFD. Consistently, fluorescent image of abdominal aorta taken by IVIS at 1 wk after surgery revealed a 2-fold increase in MMP activity. Intriguingly, F4/80-positive cells in O-HFD showed a 2.5-fold increase in co-staining with tartrate-resistant acid phosphate (TRAP), typical marker of osteoclast-like macrophages which abundantly secrete proteases than classically activated macrophages, while the percentage of TNF-α/F4/80 double-positive cells was comparable in the two groups. Pharmacological inhibition of osteoclastogenesis by zoledronic acid (ZA) (100μg/kg) completely abolished the exaggerated AAA development in O-HFD to an extent similar to that in O-ND, while AAA development in O-ND mice did not change after ZA treatment. Furthermore, in vitro TNF-α-induced osteoclast differentiation of bone marrow-derived macrophages (BMDMs) showed a significantly higher number of TRAP-positive cells in O-HFD, accompanied by a significant increase in osteoclast-related genes expression. Western blotting analysis revealed that the expression of NFATc1, master regulator of osteoclastogenesis, was significantly higher in O-HFD than that in O-ND, and immunofluorescent imaging showed that nuclear translocation of NFATc1 upon TNF-α stimulation was significantly enhanced in O-HFD. We further examined the expression of IFN regulatory factor 8 (IRF8) which suppresses osteoclastogenesis by inhibiting the function and expression of NFATc1. IRF8 mRNA and nuclear protein expression levels were significantly lower in O-HFD than those in O-ND. Conclusion Our findings demonstrate that maternal HFD accelerates CaCl2-induced AAA expansion, accompanied by the exaggerated accumulation of osteoclast-like macrophages and augmented activity of MMPs. Inhibition of macrophages skewing toward osteoclast-like cells could be a potential therapeutic target for preventing AAA development. Funding Acknowledgement Type of funding source: None

Kirenol inhibits RANKL-induced osteoclastogenesis and prevents ovariectomized-induced osteoporosis via suppressing the Ca2+-NFATc1 and Cav-1 signaling pathways

BackgroundOsteoporosis is a threat to aged people who have excessive osteoclast activation and bone resorption, subsequently causing fracture and even disability. Inhibiting osteoclast differentiation and absorptive functions has become an efficient approach to treat osteoporosis, but osteoclast-targeting inhibitors available clinically remain rare. Kirenol (Kir), a bioactive diterpenoid derived from an antirheumatic Chinese herbal medicine Herba Siegesbeckiae, can treat collagen-induced arthritis in vivo and promote osteoblast differentiation in vitro, while the effects of Kir on osteoclasts are still unclear. PurposeWe explore the role of Kir on RANKL-induced osteoclastogenesis in vitro and bone loss in vivo. MethodsThe in vitro effects of Kir on osteoclast differentiation, bone resorption and the underlying mechanisms were evaluated with bone marrow-derived macrophages (BMMs). In vivo experiments were performed using an ovariectomy (OVX)-induced osteoporosis model. ResultsWe found that Kir remarkably inhibited osteoclast generation and bone resorption in vitro. Mechanistically, Kir significantly inhibited F-actinring formation and repressed RANKL-induced NF-κB p65 activation and p-p38, p-ERK and c-Fos expression. Moreover, Kir inhibited both the expression and nuclear translocation of NFATc1. Ca2+ oscillation and caveolin-1 (Cav-1) were also reduced by Kir during osteoclastogenesis in vitro. Consistent with these findings, 2–10 mg/kg Kir attenuated OVX-induced osteoporosis in vivo as evidenced by decreased osteoclast numbers and downregulated Cav-1 and NFATc1 expression. ConclusionsKir suppresses osteoclastogenesis and the Cav-1/NFATc1 signaling pathway both in vitro and in vivo and protects against OVX-induced osteoporosis. Our findings reveal Kir as a potential safe oral treatment for osteoporosis.

Monocyte Subsets With High Osteoclastogenic Potential and Their Epigenetic Regulation Orchestrated by IRF8.

Osteoclasts (OCs) are bone-resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type (WT) mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C- monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8-deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR).

Stachydrine hydrochloride suppresses phenylephrine-induced pathological cardiac hypertrophy by inhibiting the calcineurin/nuclear factor of activated T-cell signalling pathway

The calcineurin (CaN)/nuclear factor of activated T-cell (NFAT) signalling pathway plays an important role in pathological cardiac hypertrophy. Here, we investigated the potential effects of stachydrine hydrochloride, a bioactive constituent extracted from the Chinese herb Leonurus japonicus Houtt. (Yimucao), on pathological cardiac hypertrophy during chronic α1-adrenergic receptor (α1-AR) activation and the underlying mechanisms. First, by transcriptome analysis, we determined that pathological hypertrophy models could be prepared after phenylephrine stimulation. In primary cultured neonatal rat ventricular myocytes, stachydrine hydrochloride reduced phenylephrine-induced cardiomyocyte surface area and the mRNA expression of cardiac hypertrophy biomarkers (atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and β-myosin heavy chain/α-myosin heavy chain (β-MHC/α-MHC)). In addition, phenylephrine stimulation potently induced activation of the CaN/NFAT pathway. Interestingly, stachydrine hydrochloride inhibited CaN activation and reduced NFATc3 nuclear translocation in phenylephrine-stimulated neonatal rat ventricular myocytes. In mice treated with phenylephrine, stachydrine hydrochloride treatment decreased cardiac hypertrophy and regulated heart function. Collectively, our data show that stachydrine hydrochloride decreases cardiac hypertrophy in phenylephrine-stimulated hearts by inhibiting the CaN/NFAT pathway, which might contribute to alleviation of pathological cardiac hypertrophy and cardiac dysfunction by stachydrine hydrochloride after phenylephrine stimulation This also indicated that governing of CaN/NFAT pathway might serve as a preventive or therapeutic strategy for pathological cardiac hypertrophy.

LncRNA Nron regulates osteoclastogenesis during orthodontic bone resorption

Activation of osteoclasts during orthodontic tooth treatment is a prerequisite for alveolar bone resorption and tooth movement. However, the key regulatory molecules involved in osteoclastogenesis during this process remain unclear. Long noncoding RNAs (lncRNAs) are a newly identified class of functional RNAs that regulate cellular processes, such as gene expression and translation regulation. Recently, lncRNAs have been reported to be involved in osteogenesis and bone formation. However, as the most abundant noncoding RNAs in vivo, the potential regulatory role of lncRNAs in osteoclast formation and bone resorption urgently needs to be clarified. We recently found that the lncRNA Nron (long noncoding RNA repressor of the nuclear factor of activated T cells) is highly expressed in osteoclast precursors. Nron is downregulated during osteoclastogenesis and bone ageing. To further determine whether Nron regulates osteoclast activity during orthodontic treatment, osteoclastic Nron transgenic (Nron cTG) and osteoclastic knockout (Nron CKO) mouse models were generated. When Nron was overexpressed, the orthodontic tooth movement rate was reduced. In addition, the number of osteoclasts decreased, and the activity of osteoclasts was inhibited. Mechanistically, Nron controlled the maturation of osteoclasts by regulating NFATc1 nuclear translocation. In contrast, by deleting Nron specifically in osteoclasts, tooth movement speed increased in Nron CKO mice. These results indicate that lncRNAs could be potential targets to regulate osteoclastogenesis and orthodontic tooth movement speed in the clinic in the future.

The RCAN1.4-calcineurin/NFAT signaling pathway is essential for hypoxic adaption of intervertebral discs

Calcipressin-1, also known as regulator of calcineurin 1 (RCAN1), can specifically bind calcineurin at or near the calcineurin A catalytic domain and downregulate calcineurin activity. However, whether RCAN1 affects the hypoxic intervertebral disc (IVD) phenotype through the calcineurin/NFAT signaling pathway remains unclear. First, we confirmed the characteristics of the degenerative nucleus pulposus (NP) by H&E, safranin O/fast green and Alcian blue staining, and detected increased RCAN1 levels in the degenerative NP by immunohistochemistry. Then, we demonstrated that the protein level of RCAN1.4 was higher than that of RCAN1.1 and progressively elevated from the control group to the Pfirrmann grade V group. In vitro, both hypoxia (1% O2) and overexpression of HIF-1α reduced the protein level of RCAN1.4 in rat NP cells in a dose- and time-dependent manner. We further found that miRNA-124, through a nondegradative pathway (without the proteasome or lysosome), suppressed the expression of RCAN1.4. As expected, calcineurin in NP cells was activated and primarily promoted nuclear translocation of NFATc1 under hypoxia or RCAN1.4 siRNA transfection. Furthermore, SOX9, type II collagen and MMP13 were elevated under hypoxia, RCAN1.4 siRNA transfection or NFATc1 overexpression. Using chromatin immunoprecipitation (ChIP) and a luciferase reporter assay (with mutation), we clarified that NFATc1 increasingly bound the SOX9 promotor region (bp −367~−357). Interaction of HIF-1α and NFATc1 promoted MMP13 transcription. Finally, we found that FK506 reversed hypoxia-induced activation of the calcineurin/NFAT signaling pathway in NP cells and an ex vivo model. Together, these findings show that the RCAN1.4-calcineurin/NFAT signaling pathway has a vital role in the hypoxic phenotype of NP cells. RCAN1.4 might be a therapeutic target for degenerative disc diseases.