Oligomeric proanthocyanidins ameliorates osteoclastogenesis through reducing OPG/RANKL ratio in chicken's embryos.

Skeletal disorders can seriously threaten the health and the performance of poultry, such as tibial dyschondroplasia (TD) and osteoporosis (OP). Oligomeric proanthocyanidins (OPC) are naturally occurring polyphenolic flavonoid compounds that can be used as potential substances to improve the bone health and the growth performance of poultry. Eighty 7-day-old green-eggshell yellow feather layer chickens were randomly divided into 4 groups: basal diet and basal diet supplementation with 25, 50, and 100 mg/kg OPC. The results have indicated that the growth performance and bone parameters of chickens were significantly improved supplementation with OPC in vivo, including the bone volume (BV), the bone mineral density (BMD) and the activities of antioxidative enzymes, but ratio of osteoprotegerin (OPG)/receptor activator of NF-κB (RANK) ligand (RANKL) was decreased. Furthermore, primary bone marrow mesenchymal stem cells (BMSCs) and bone marrow monocytes/macrophages (BMMs) were successfully isolated from femur and tibia of chickens, and co-cultured to differentiate into osteoclasts in vitro. The osteogenic differentiation derived from BMSCs was promoted treatment with high concentrations of OPC (10, 20, and 40 µmol/L) groups in vitro, but emerging the inhibition of osteoclastogenesis by increasing the ratio of OPG/RANKL. In contrary, the osteogenic differentiation was also promoted treatment with low concentrations of OPC (2.5, 5, and 10 µmol/L) groups, but osteoclastogenesis was enhanced by decreasing the ratio of OPG/RANKL in vitro. In addition, OPG inhibits the differentiation and activity of osteoclasts by increasing the autophagy in vitro. Dietary supplementation of OPC can improve the growth performance of bone and alter the balance of osteoblasts and osteoclasts, thereby improving the bone health of chickens.

VPS41-mediated incomplete autophagy aggravates cadmium-induced apoptosis in mouse hepatocytes.

Exposure to cadmium (Cd), an environmental heavy metal contaminant, is a serious threat to global health that increases the burden of liver diseases. Autophagy and apoptosis are important in Cd-induced liver injury. However, the regulatory mechanisms involved in the progression of Cd-induced liver damage are poorly understood. Herein, we investigated the role of vacuolar protein sorting 41 (VPS41) in Cd-induced autophagy and apoptosis in hepatocytes. We used targeted VPS41 regulation to elucidate the mechanism of Cd-induced hepatotoxicity. Our data showed that Cd triggered incomplete autophagy by downregulating VPS41, aggravating Cd-induced hepatocyte apoptosis. Mechanistically, Cd-induced VPS41 downregulation interfered with the mTORC1-TFEB/TFE3 axis, leading to an imbalance in autophagy initiation and termination and abnormal activation of autophagy. Moreover, Cd-induced downregulation of VPS41 inhibited autophagosome-lysosome fusion, leading to blocked autophagic flux. This triggers incomplete autophagy, which causes excessive P62 accumulation, accelerating Caspase-9 (CASP9) cleavage. Incomplete autophagy blocks clearance of cleaved CASP9 (CL-CASP9) via the autophagic pathway, promoting apoptosis. Notably, VPS41 overexpression alleviated Cd-induced incomplete autophagy and apoptosis, independent of the homotypic fusion and protein sorting complex. This study provides a new mechanistic understanding of the relationship between autophagy and apoptosis, suggesting that VPS41 is a new therapeutic target for Cd-induced liver damage.

Vitamin D Alleviates Cadmium-Induced Inhibition of Chicken Bone Marrow Stromal Cells' Osteogenic Differentiation In Vitro.

Vitamin D is a lipid soluble vitamin that is mostly used to treat bone metabolism-related diseases. In this study, the effect of Cd toxicity in vitro on osteogenic differentiation derived from BMSCs and the alleviating effect of lα, 25-(OH)2D3 were investigated. Cell index in real time was monitored using a Real-time cell analyzer (RTCA) system. The activity of alkaline phosphatase (ALP), and the calcified nodules and the distribution of Runx2 protein were detected using ALP staining, alizarin red staining, and immunofluorescence, respectively. Furthermore, the mitochondrial membrane potential and the apoptotic rate of BMSCs, the mRNA levels of RUNX2 and type Ⅰ collagen alpha2 (COL1A2) genes, and the protein expression of Col1 and Runx2 were detected using flow cytometry, qRT-PCR and western blot, respectively. The proliferation of BMSCs and osteogenic differentiation were enhanced after treatment with different concentrations of lα, 25-(OH)2D3 compared with the control group. However, 5 µmol/L Cd inhibited the proliferation of BMSCs. In addition, 10 nmol/L lα,25-(OH)2D3 attenuated the toxicity and the apoptosis of BMSCs treated by Cd, and also promoted the osteogenic differentiation including the activity of ALP, and the protein expression of Col1 and Runx2. lα, 25-(OH)2D3 can alleviate cadmium-induced osteogenic toxicity in White Leghorn chickens in vitro.

Cadmium accelerates autophagy of osteocytes by inhibiting the PI3K/AKT/mTOR signaling pathway.

Cadmium (Cd) can damage bone cells and cause osteoporosis. Osteocytes are the most numerous bone cells and also important target cells for Cd-induced osteotoxic damage. Autophagy plays important role in the progression of osteoporosis. However, osteocyte autophagy in Cd-induced bone injury is not well characterized. Thus, we established a Cd-induced bone injury model in BALB/c mice and a cellular damage model in MLO-Y4 cells. Aqueous Cd exposure for 16 months showed an increase in plasma alkaline phosphatase (ALP) activity and increase in urine calcium (Ca) and phosphorus (P) concentrations in vivo. Moreover, expression level of autophagy-related microtubule-associated protein 1A/1B-light chain 3 II (LC3II) and autophagy-related 5 (ATG5) proteins were induced, and the expression of sequestosome-1 (p62) was reduced, along with Cd-induced trabecular bone damage. In addition, Cd inhibited the phosphorylation of mammalian target of rapamycin (mTOR), protein kinase B (AKT), and phosphatidylinositol 3-kinase (PI3K). In vitro, 80 µM Cd concentrations exposure upregulated LC3II protein expression, and downregulated of p62 protein expression. Similarly, we found that treatment with 80 µM Cd resulted in a reduction in the phosphorylation levels of mTOR, AKT, and PI3K. Further experiments revealed that addition of rapamycin, an autophagy inducer, enhanced autophagy and alleviated the Cd-induced damage to MLO-Y4 cells. The findings of our study reveal for the first time that Cd causes damage to both bone and osteocytes, as well as induces autophagy in osteocytes and inhibits PI3K/AKT/mTOR signaling, which could be a protective mechanism against Cd-induced bone injury.

Polystyrene exacerbates cadmium-induced mitochondrial damage to lung by blocking autophagy in mice.

Cadmium (Cd) is an environmental heavy metal, and its accumulation is harmful to animal and human health. The cytotoxicity of Cd includes oxidative stress, apoptosis, and mitochondrial histopathological changes. Furthermore, polystyrene (PS) is a kind of microplastic piece derived from biotic and abiotic weathering courses, and has toxicity in various aspects. However, the potential mechanism of action of Cd co-treated with PS is still poorly unclear. The objective of this study was to investigate the effects of PS on Cd-induced histopathological injury of mitochondria in the lung of mice. In this study, the results have showed that Cd could induce the activity of oxidative enzymes of the lung cells in mice, increasing the content of partial microelement and the phosphorylation of inflammatory factor NF-κB p65. Cd further destroys the integrity of mitochondria by increasing the expression of apoptotic protein and blocking the autophagy. In addition, PS solely group aggravated the lung damage in mice, especially mitochondrial toxicity, and played a synergistic effect with Cd in lung injury. However, how PS can augment mitochondrial damage and synergism with Cd in lung of mice requiring further exploration. Therefore, PS was able to exacerbate Cd-induced mitochondrial damage to the lung in mice by blocking autophagy, and was associated with the apoptosis.

The Mechanism of Osteoprotegerin-Induced Osteoclast Pyroptosis In Vitro.

Osteoprotegerin (OPG) is a new member of the tumor necrosis factor (TNF) receptor superfamily, which can inhibit the differentiation and activity of osteoclasts by binding to nuclear factor kappa B receptor activator (RANK) competitively with nuclear factor kappa B receptor activator ligand (RANKL). The previous experiments found that OPG can induce apoptosis of mature osteoclasts in vitro, which can inhibit the activity of mature osteoclasts, thereby exerting its role in protecting bone tissue. In addition, pyroptosis is a new type of cell death that is different from apoptosis. It is unclear whether OPG can induce mature osteoclast pyroptosis and thereby play its role in protecting bone tissue. In this study, the results showed that compared with the control group, the survival rate of osteoclasts in the OPG group was significantly reduced, and the contents of IL-1ß, IL-18, and LDH in the supernatant both increased. Many osteoclast plasma membranes were observed to rupture in bright fields, and OPG induced loss of their morphology. Flow cytometry was used to analyze the pyroptosis rate; OPG significantly increased the osteoclast pyroptosis rate. To further reveal the mechanism of OPG-induced osteoclast pyroptosis, we examined the expression level of pyroptosis-related genes and proteins, and the results found that OPG increased the expression of NLRP3, ASC, caspase-1, and GSDMD-N compared with the control group. In summary, OPG can induce osteoclast pyroptosis, and its mechanism is related to the expression levels of ASC, NLRP3, caspase 1 and GSDMD, which were included in the classical pathway of pyroptosis.

Gut microbiome dysregulation drives bone damage in broiler tibial dyschondroplasia by disrupting glucose homeostasis.

Tibial dyschondroplasia (TD) with multiple incentives is a metabolic skeletal disease that occurs in fast-growing broilers. Perturbations in the gut microbiota (GM) have been shown to affect bone homoeostasis, but the mechanisms by which GM modulates bone metabolism in TD broilers remain unknown. Here, using a broiler model of TD, we noted elevated blood glucose (GLU) levels in TD broilers, accompanied by alterations in the pancreatic structure and secretory function and damaged intestinal barrier function. Importantly, faecal microbiota transplantation (FMT) of gut microbes from normal donors rehabilitated the GM and decreased the elevated GLU levels in TD broilers. A high GLU level is a predisposing factor to bone disease, suggesting that GM dysbiosis-mediated hyperglycaemia might be involved in bone regulation. 16S rRNA gene sequencing and short-chain fatty acid analysis revealed that the significantly increased level of the metabolite butyric acid derived from the genera Blautia and Coprococcus regulated GLU levels in TD broilers by binding to GPR109A in the pancreas. Tibial studies showed reduced expression of vascular regulatory factors (including PI3K, AKT and VEFGA) based on transcriptomics analysis and reduced vascular distribution, contributing to nonvascularization of cartilage in the proximal tibial growth plate of TD broilers with elevated GLU levels. Additionally, treatment with the total flavonoids from Rhizoma drynariae further validated the improvement in bone homoeostasis in TD broilers by regulating GLU levels through the regulation of GM to subsequently improve intestinal and pancreatic function. These findings clarify the critical role of GM-mediated changes in GLU levels via the gut-pancreas axis in bone homoeostasis in TD chickens.

Morinda officinalis Polysaccharides Ameliorates Bone Growth by Attenuating Oxidative Stress and Regulating the Gut Microbiota in Thiram-Induced Tibial Dyschondroplasia Chickens.

Tibial dyschondroplasia (TD) occurs in chickens and other fast-growing birds, affecting their cartilage growth and leading to reduced meat quality in broilers. Morinda officinalis polysaccharide (MOP) is one of the chief active components of Morinda officinalis, which promotes bone formation, inhibiting bone loss and having anti-oxidant and anti-inflammatory properties. A total of 120 AA chickens were randomly divided into the CON group (basal diet), TD group (100 mg/kg thiram + basal diet), and MOP group (100 mg/kg thiram + basal diet + water with 500 mg/kg MOP). The experiment lasted 21 days. The results showed that MOP could alleviates broiler lameness caused by TD, restore the morphological structure of tibial growth plate (TGP), increase tibial weight (p < 0.05), balance the disorder of calcium and phosphorus metabolism, and promote bone formation by increasing the expression of BMP-2, Smad4, and Runx2 genes In addition, MOP supplementation stimulated the secretion of plasma antioxidant enzymes (T-SOD and GSH-Px) by regulating the expression of SOD and GPX-1 genes, thereby enhancing the antioxidant capacity of TD broilers. Interestingly, we observed MOP can also improve gut microbiota by increasing the beneficial bacteria count and decreasing the harmful bacteria count. These findings indicated that MOP can regulate bone formation through the BMP/Smads signaling pathway, attenuating oxidative stress and regulating the gut microbiota of TD broilers, so as to achieve the effect of treating TD. This suggests that MOP might be a potential novel drug in the treatment of TD in chickens.

Effects of Probiotics and Gut Microbiota on Bone Metabolism in Chickens: A Review.

Broiler leg diseases are a common abnormal bone metabolism issue that leads to poor leg health in growing poultry. Bone metabolism is a complicated regulatory process controlled by genetic, nutritional, feeding management, environmental, or other influencing factors. The gut microbiota constitutes the largest micro-ecosystem in animals and is closely related to many metabolic disorders, including bone disease, by affecting the absorption of nutrients and the barrier function of the gastrointestinal tract and regulating the immune system and even the brain-gut-bone axis. Recently, probiotic-based dietary supplementation has emerged as an emerging strategy to improve bone health in chickens by regulating bone metabolism based on the gut-bone axis. This review aims to summarize the regulatory mechanisms of probiotics in the gut microbiota on bone metabolism and to provide new insights for the prevention and treatment of bone diseases in broiler chickens.

A Review of Signaling Transduction Mechanisms in Osteoclastogenesis Regulation by Autophagy, Inflammation, and Immunity.

Osteoclastogenesis is an ongoing rigorous course that includes osteoclast precursors fusion and bone resorption executed by degradative enzymes. Osteoclastogenesis is controlled by endogenous signaling and/or regulators or affected by exogenous conditions and can also be controlled both internally and externally. More evidence indicates that autophagy, inflammation, and immunity are closely related to osteoclastogenesis and involve multiple intracellular organelles (e.g., lysosomes and autophagosomes) and certain inflammatory or immunological factors. Based on the literature on osteoclastogenesis induced by different regulatory aspects, emerging basic cross-studies have reported the emerging disquisitive orientation for osteoclast differentiation and function. In this review, we summarize the partial potential therapeutic targets for osteoclast differentiation and function, including the signaling pathways and various cellular processes.

Integrated Fecal Microbiome and Metabolomics Reveals a Novel Potential Biomarker for Predicting Tibial Dyschondroplasia in Chickens.

Tibial dyschondroplasia (TD) is a metabolic tibial-tarsal disorder occurring in fast-growing poultry, and its diagnosis is mainly based on an invasive method. Here, we profiled the fecal gut microbiome and metabolome of broilers with and without TD to identify potential non-invasive and non-stress biomarkers of TD. First, TD broilers with the most pronounced clinical signs during the experiment were screened and faecal samples were collected for integrated microbiome and metabolomics analysis. Moreover, the diagnostic potential of identified biomarkers was further validated throughout the experiment. It was noted that the microbial and metabolic signatures of TD broilers differed from those of normal broilers. TD broilers were characterized by enriched bacterial OTUs of the genus Klebsiella, and depleted genera [Ruminococcus], Dorea, Ruminococcus, Oscillospira, Ochrobactrum, and Sediminibacterium. In addition, a total of 189 fecal differential metabolites were identified, mainly enriched in the purine, vitamin and amino acid metabolism, which were closely associated with differential microbiota and tibia-related indicators. Furthermore, three fecal metabolites were screened, including 4-hydroxybenzaldehyde, which distinguished TD from normal broilers with extremely high specificity and was superior to serum bone markers. These results indicated that gut microbiota equilibrium might influence the pathogenesis of TD by modulating host metabolism, and the identified fecal metabolite 4-hydroxybenzaldehyde might be a potential and non-invasive biomarker for predicting TD in chickens.

Puerarin alleviates cadmium-induced oxidative damage to bone by reducing autophagy in rats.

Autophagy is a regulatory mechanism involved in cadmium (Cd)-induced bone toxicity and is suppressed by various stimuli, including oxidative stress. Puerarin is an isoflavonoid compound isolated from Pueraria, a plant used in traditional Chinese medicine. The underlying mechanisms of action of puerarin remain unclear. The objective of this study was to explore the mitigating effects of puerarin on cadmium-induced oxidative damage in the bones of rats. Cadmium exposure increased oxidative damage in rat bones; this was markedly decreased by puerarin treatment, as demonstrated by changes in the activity of antioxidative enzymes. Cadmium-induced blockage of the expression of key bone regulatory proteins, autophagy-related markers, and signaling molecules was also alleviated by puerarin treatment. Additionally, cadmium reduced expression of the autophagic protein Rab7 and of late endosomal/lysosomal adaptor and MAPK and mTOR activator 1 (LAMTOR1); the decrease in these proteins was not restored by puerarin treatment. We speculate that puerarin relieves the inhibition of fusion of autophagosomes with lysosomes that is induced by cadmium; however, this specific effect of puerarin and downstream effects on bone regulatory mechanisms require further investigation. In conclusion, puerarin alleviates cadmium-induced oxidative damage in the bones of rats by attenuating autophagy, which is likely associated with the antioxidant activity of puerarin.

Probiotics Treatment of Leg Diseases in Broiler Chickens: a Review.

Normal development and growth of bones are critical for poultry. With the rapid growth experienced by broiler chickens, higher incidences of leg weakness and lameness are common problems in adolescent meat-type poultry that present huge economic and welfare issues. Leg disorders such as angular bone deformities and tibial dyschondroplasia have become common in broilers and are associated with poor growth, high mortality rates, increased carcass condemnation, and downgrading at slaughter. Probiotics have shown promise for a variety of health purposes, including preventing diarrhea, elevating carcass quality, and promoting growth of the poultry. In addition, recent studies have indicated that probiotics can maintain the homeostasis of the gut microbiota and improve the health of the gastrointestinal tract, which confers a potentially beneficial effect on bone health. This review mainly describes the occurrence of broiler leg disease and the role of probiotics in bone health through regulating the gut microbiota and improving intestinal function, thus providing a relevant theoretical basis for probiotics to hinder the development of skeletal disorders in broiler chickens.

Galectin-3 Contributes to the Inhibitory Effect of lα,25-(OH)2D3 on Osteoclastogenesis.

The active form of vitamin D, 1α,25-(OH)2D3, not only promotes intestinal calcium absorption, but also regulates the formation of osteoclasts (OCs) and their capacity for bone mineral dissolution. Gal-3 is a newly discovered bone metabolic regulator involved in the proliferation, differentiation, and apoptosis of various cells. However, the role of galectin-3 (gal-3) in OC formation and the regulatory effects of 1α,25-(OH)2D3 have yet to be explored. To confirm whether gal-3 contributes to the regulatory effects of 1α,25-(OH)2D3 on osteoclastogenesis, osteoclast precursors (OCPs) were induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). TRAP staining and bone resorption analyses were used to verify the formation and activation of OCs. qPCR, Western blotting, co-immunoprecipitation, and immunofluorescence assays were used to detect gene and protein expression. The regulatory effects of gal-3 in OC formation after treatment with 1α,25-(OH)2D3 were evaluated using gal-3 siRNA. The results showed that 1α,25-(OH)2D3 significantly increased gal-3 expression and inhibited OC formation and bone resorption. Expression levels of OC-related genes and proteins, matrix metalloproteinase 9 (MMP-9), nuclear factor of activated T cells 1 (NFATc1), and cathepsin K (Ctsk) were also inhibited by 1α,25-(OH)2D3. Gal-3 knockdown attenuated the inhibitory effects of 1α,25-(OH)2D3 on OC formation, activation, and gene and protein expression. In addition, gal-3 was co-localized with the vitamin D receptor (VDR). These data suggest that gal-3 contributes to the osteoclastogenesis inhibitory effect of lα,25-(OH)2D3, which is involved in bone and calcium homeostasis.

The Role of Galectin-3 in 1α,25(OH)2D3-Regulated Osteoclast Formation from White Leghorn Chickens In Vitro.

Bones play an important role in maintaining the level of calcium in blood. They provide support for soft tissues and hematopoiesis and undergo continuous renewal throughout life. In addition, vitamin D is involved in regulating bone and calcium homeostasis. Galectin-3 (Gal-3) is a ß-galactoside-binding protein that can regulate bone cell differentiation and function. Here, we aimed to study the regulatory effects of Gal-3 on vitamin-D-regulated osteoclastogenesis and bone resorption in chicken. Gal-3 expression in bone marrow stromal cells (BMSCs) from 18-day-old chicken embryos was inhibited or overexpressed. BMSCs were then co-cultured with bone marrow monocytes/macrophages (BMMs) with or without addition of 1α,25(OH)2D3. The results showed that 1α,25(OH)2D3 upregulated the expression of Gal-3 mRNA and receptor activator of nuclear-factor κB ligand (RANKL) expression in BMSCs and promoted osteoclastogenesis, as shown by the upregulated expression of osteoclast (OC) markers (CtsK, CAII, MMP-9, and TRAP) and increased bone resorption, a method for measuring the bone resorption area in vitro. Knockdown of Gal-3 by small-interfering RNA (siRNA) in BMSCs downregulated the expression of RANKL mRNA and attenuated the effects of 1α,25(OH)2D3 on osteoclastogenesis and bone resorption. Conversely, overexpression of Gal-3 in BMSCs enhanced the effects of osteoclastogenesis and bone resorption by increasing the expression of RANKL mRNA. These results demonstrated that Gal-3 mediates the differentiation and bone resorption of osteoclasts regulated by 1α,25(OH)2D3.

Beclin 1 positively regulates osteoprotegerin-induced inhibition of osteoclastogenesis by increasing autophagy in vitro.

Osteoclastogenesis is induced by receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), and can be suppressed by osteoprotegerin (OPG). Beclin1 has a dual role in osteoclastogenesis. However, the role of Beclin1-mediated autophagy during OPG-induced inhibition of osteoclastogenesis remains unclear. Here, we found that Beclin1 and matrix metalloproteinase 9 (MMP-9) expression were increased during osteoclastogenesis. OPG (20, 40, and 80 ng/mL) decreased Src and MMP-9 expression, but augmented Beclin1 expression and fluorescence intensity. Similarly, treatment with the autophagy activator rapamycin increased Beclin1 expression during OPG-induced inhibition of osteoclastogenesis. Further, Beclin1 knockdown restored osteoclast numbers by reducing autophagy during OPG-induced inhibition of osteoclastogenesis. These results indicate that Beclin1 has a positive role during OPG-induced inhibition of osteoclastogenesis by regulating autophagy, which might provide a potential basis for osteoclastogenesis.

Cadmium Toxicity on Chondrocytes and the Palliative Effects of 1α, 25-Dihydroxy Vitamin D3 in White Leghorns Chicken's Embryo.

Cadmium (Cd) can causes osteoporosis and joint swelling. However, the mechanism of Cd toxicity in chondrocytes and how to alleviate Cd poisoning to chondrocytes are still unclear. Herein, we evaluated the toxicity of Cd to chicken chondrocytes, and whether vitamin D can relieve the toxicity of Cd to chondrocytes. Primary chondrocytes were collected from knee-joint cartilage of 15-day-old chicken embryos. They were treated with (0, 1, 2, and 4) µM Cd alone, 10-8 M 1α,25-(OH)2D3 alone, or 2 µM Cd combined with 10-8 M 1α,25-(OH)2D3. We found that Cd significantly inhibited Sox9 and ACAN mRNA expression, which are markers for chondrocyte differentiation, downregulated the mitochondrial membrane potential, upregulated the Bax/B-cell lymphoma 2 ratio. Furthermore, Cd significantly promoted matrix metalloproteinase (MMP)-9 expression, thus accelerating the degradation of extracellular matrix. And Cd also inhibited the expression of main macromolecular protein of extracellular matrix, Collagen type IIα1 (COL2A1) and acid mucopolysaccharide. However, 1α,25-(OH)2D3 pretreatment significantly alleviated the toxicity effects of Cd on the differentiation, apoptosis and extracellular matrix gene expression in primary chondrocytes. Conclusively, Cd exposure could inhibited chicken embryo chondrocytes differentiation, extracellular matrix gene expression, and induced chondrocyte apoptosis. However, these toxic effects of Cd are alleviated by the pretreatment of chondrocytes with 1α,25-(OH)2D3.

Overexpression of c-Fos reverses osteoprotegerin-mediated suppression of osteoclastogenesis by increasing the Beclin1-induced autophagy.

Osteoclastogenesis requires the involvement of transcription factors and degrading enzymes, and is regulated by upstream and downstream signalling. However, c-Fos how regulates osteoclastogenesis through autophagy remain unclear. This study aimed to explore the role of c-Fos during osteoprotegerin (OPG)-mediated suppression of osteoclastogenesis. We found that the number of osteoclasts and the expression of c-Fos, MMP-9, CAⅡ, Src and p62 were decreased after treated with OPG, including attenuation the PI3K/Akt and the TAK1/S6 signalling pathways, but the expression of Beclin1 and LC3Ⅱ were increased. Knockdown of Beclin1 could reverse the expression of c-Fos and MMP-9 by activating the PI3K/Akt signalling pathway, but inhibiting the autophagy and the TAK1/S6 signalling pathway. In addition, inhibition of autophagy using the PI3K inhibitor LY294002 did not rescues OPG-mediated suppression of osteoclastogenesis, but caused reduction of the expression of c-Fos and CAⅡ by attenuating the autophagy, as well as the PI3K/Akt and the TAK1/S6 signalling pathways. Furthermore, continuous activation of c-Fos could reverse OPG-mediated suppression of osteoclastogenesis by activating the autophagy and the PI3K/Akt and the TAK1/S6 signalling pathways. Thus, overexpression of c-Fos could reverse OPG-mediated suppression of osteoclastogenesis via activation of Beclin1-induced autophagy, indicating c-Fos might serve as a new candidate for bone-related basic studies.

p53 positively regulates osteoprotegerin-mediated inhibition of osteoclastogenesis by downregulating TSC2-induced autophagy in vitro.

Osteoclasts are terminally multinucleated cells that are regulated by nuclear factor-activated T cells c1 (NFATc1), and are responsible for bone resorption while the tartrate resistant acid phosphatase (TRAP) enzymes releases into bone resorption lacunae. Furthermore, tumor suppressor p53 is a negative regulator during osteoclastogenesis. Osteoprotegerin (OPG) inhibits osteoclastogenesis and bone resorption by activating autophagy, however, whether p53 is involved in OPG-mediated inhibition of osteoclastogenesis remains unclear. In the current study, OPG could enhance the expression of p53 and tuberin sclerosis complex 2 (TSC2). Moreover, the expression of p53 is regulated by autophagy during OPG-mediated inhibition of osteoclastogenesis. Inhibition of p53 by treated with pifithrin-α (PFTα) causing augments of osteoclastogenesis and bone resorption, also reversed OPG-mediated inhibition of osteoclastogenesis by reducing the expression of TSC2. In addition, knockdown of TSC2 using siRNA could rescue OPG-mediated inhibition of osteoclastogenesis by reducing autophagy, which is manifested by the decrease of the expression of Beclin1 and the phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase beta 1 (S6K1, also known as p70S6K). Collectively, p53 plays a critical role during OPG-mediated inhibition of osteoclastogenesis via regulating the TSC2-induced autophagy in vitro.

AMP-activated protein kinase (AMPK) regulates autophagy, inflammation and immunity and contributes to osteoclast differentiation and functionabs.

Osteoclasts are multinucleated giant cells, responsible for bone resorption. Osteoclast differentiation and function requires a series of cytokines to remove the old bone, which coordinates with the induction of bone remodelling by osteoblast-mediated bone formation. Studies have demonstrated that AMP-activated protein kinase (AMPK) play a negative regulatory role in osteoclast differentiation and function. Research involving AMPK, a nutrient and energy sensor, has primarily focused on osteoclast differentiation and function; thus, its role in autophagy, inflammation and immunity remains poorly understood. Autophagy is a conservative homoeostatic mechanism of eukaryotic cells, and response to osteoclast differentiation and function; however, how it interacts with inflammation remains unclear. Additionally, based on the regulatory function of different AMPK subunits for osteoclast differentiation and function, its activation is regulated by upstream factors to perform bone metabolism. This review summarises the critical role of AMPK-mediated autophagy, inflammation and immunity by upstream and downstream signalling during receptor activator of nuclear factor kappa-B ligand-induced osteoclast differentiation and function. This pathway may provide therapeutic targets for bone-related diseases, as well as function as a biomarker for bone homoeostasis.