Chuan Liu

HDAC2 regulates FoxO1 during RANKL-induced osteoclastogenesis.

The bone-resorbing osteoclast (OC) is essential for bone homeostasis, yet deregulation of OCs contributes to diseases such as osteoporosis, osteopetrosis, and rheumatoid arthritis. Here we show that histone deacetylase 2 (HDAC2) is a key positive regulator during receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Bone marrow macrophages (BMMs) showed increased HDAC2 expression during osteoclastogenesis. HDAC2 overexpression enhanced, whereas HDAC2 deletion suppressed osteoclastogenesis and bone resorption using lentivirus infection. Mechanistically, upon RANKL activation, HDAC2 activated Akt; Akt directly phosphorylates and abrogates Forkhead box protein O1 (FoxO1), which is a negative regulator during osteoclastogenesis through reducing reactive oxygen species. HDAC2 deletion in BMMs resulted in decreased Akt activation and increased FoxO1 activity during osteoclastogenesis. In conclusion, HDAC2 activates Akt thus suppresses FoxO1 transcription results in enhanced osteoclastogenesis. Our data imply the potential value of HDAC2 as a new target in regulating osteoclast differentiation and function.

Staphylococcal protein A promotes osteoclastogenesis through MAPK signaling during bone infection

Bone infection is a common and serious complication in the orthopedics field, which often leads to excessive bone destruction and non‐union. Osteoclast is the only type of cells which have the function of bone resorption. Its over activation is closely related to excessive bone loss. Staphylococcus aureus (S. aureus) is a major pathogen causing bone infection, which can produce a large number of strong pathogenic substances staphylococcal protein A (SPA). However, few studies were reported about the effects of SPA on osteoclastogenesis. In our study, we observed that S. aureus activated osteoclasts and promoted bone loss in bone infection specimens. Then, we investigated the effects of SPA on RANKL‐induced osteoclastogenesis in vitro, the results revealed that SPA promoted osteoclastic differentiation and fusion, and enhanced osteoclastic bone resorption. In addition, we also showed that SPA upregulated the expression of NFATc1 and c‐FOS through the activation of MAPK signaling to promote osteoclastogenesis. Our findings might help us better understand the pathogenic role of S. aureus in bone infection and develop new therapeutic strategies for infectious bone diseases.

Estrogen Deficiency-Mediated M2 Macrophage Osteoclastogenesis Contributes to M1/M2 Ratio Alteration in Ovariectomized Osteoporotic Mice: M1/M2 RATIO ALTERATION CONTRIBUTES TO OSTEOPOROSIS

In this study, for the first time we discovered that the M1/M2 macrophage phenotype ratio is increased in bone marrow of ovariectomized (OVX) osteoporotic C57BL/6 mice. Considering estrogen is the main variable, we assumed that estrogen participated in this alteration. To determine whether and how estrogen contributes to the change of the M1/M2 ratio, we first isolated bone marrow macrophages (BMMs) from mice femur and stimulated the cells with lipopolysaccharide (LPS)/interferon γ (IFN‐γ) for M1 polarization and interleukin 4 (IL‐4)/IL‐13 for M2 polarization. M1 and M2 macrophages were then exposed to RANKL stimulation, we found that M2 macrophage but not M1 macrophage differentiated into functional osteoclast leading to increased M1/M2 ratio. Intriguingly, 17β‐estradiol (E2) pretreatment prevented osteoclastogenesis from M2 macrophages. By constructing shRNA lentivirus interfering the expression of different estrogen receptors in M2 macrophages, we found that estrogen protects M2 macrophage from receptor activator of nuclear factor κB ligand (RANKL) stimulation selectively through estrogen receptor α (ERα) and the downstream blockage of NF‐κB p65 nuclear translocation. Animal studies showed that ERα selective agonist 4,4′,4″‐(4‐propyl‐[1H]‐pyrazole‐1,3,5‐triyl) trisphenol (PPT) was able to replicate the therapeutic effects of E2 in treating osteoporotic OVX mice. Together, our findings reveal that estrogen deficiency–mediated M2 macrophage osteoclastogenesis leads to increased M1/M2 ratio in OVX mice. Reducing the M1/M2 ratio is a potential therapeutic target in treating postmenopausal osteoporosis.

Long noncoding RNA expression profiles in chondrogenic and hypertrophic differentiation of mouse mesenchymal stem cells

Long noncoding RNAs (lncRNAs) are important regulators for a variety of biological processes. Chondrogenic differentiation of mesenchymal stem cells (MSCs) is a crucial stage in chondrogenesis while chondrocyte hypertrophy is related to endochondral ossification and osteoarthritis. However, the effects of lncRNAs on chondrogenic and hypertrophic differentiation of mouse MSCs are unclear. To explore the potential mechanisms of lncRNAs during chondrogenesis and chondrocyte hypertrophy, microarray was performed to investigate the expression profiles of lncRNA and mRNA in MSCs, pre-chondrocytes, and hypertrophic chondrocytes. Then, we validated microarray data by RT-PCR and screened three lncRNAs from upregulating groups during chondrogenesis and chondrocyte hypertrophy respectively. After downregulating any of the above lncRNAs, we found that the expression of chondrogenesis-related genes such as Sox9 and Col2a1 and hypertrophy-related genes including Runx2 and Col10a1 was inhibited, respectively. Furthermore, the target genes of above lncRNAs were predicted by bioinformatics approaches. Gene ontology and Kyoto encyclopedia of genes and genome biological pathway analysis were also made to speculate the functions of above lncRNAs. In conclusion, the study first revealed the expression profile of lncRNAs in chondrogenic and hypertrophic differentiations of mouse MSCs and presented a new prospect for the underlying mechanisms of chondrogenesis and endochondral ossification.

Staphylococcal lipoteichoic acid promotes osteogenic differentiation of mouse mesenchymal stem cells by increasing autophagic activity

This study sought to explore the effect of staphylococcal lipoteichoic acid (LTA) on autophagy in mouse mesenchymal stem cells (MSCs), and then influence osteogenesis through the change of autophagy. C3H10T1/2xa0cells were induced by osteogenic medium with the treatment of LTA at different concentrations (1, 5, 10xa0μg/mL); 3-methyladenine (3-MA) were used as the autophagy inhibitor, and rapamycin (rapamycin, Rap) were used to activate autophagy; the effects on osteogenesis were detected by alkaline phosphatase staining, alizarin red staining, real-time quantitative PCR, and western blotting; autophagic activity was investigated by the expression of LC3-Ⅱand p62 proteins. Compared with control group, the expression of osteogenesis markers was significantly up-regulated with the LTA treatment on the mRNA and protein level; the positive rate of alkaline phosphatase was enhanced in the LTA groups; and the formation of calcium nodules was increased simultaneously. The expression of LC3-Ⅱ protein was increased in LTA groups, while the expression of p62 protein was decreased. Inhibition of autophagy significantly reduced the effect of LTA on osteogenesis of MSCs; the promotion of LTA on osteogenic differentiation was further enhanced when adding rapamycin to activate autophagic activity. It provides new insight of prevention and treatment for bone infection.

LncRNA-AK131850 Sponges MiR-93-5p in Newborn and Mature Osteoclasts to Enhance the Secretion of Vascular Endothelial Growth Factor a Promoting Vasculogenesis of Endothelial Progenitor Cells

Background/Aims: In the process of bone development and remodeling, the vasculature is regarded as the communicative network between the bone and neighboring tissues. Recently, it has been reported that the processes of angiogenesis and osteogenesis are coupled temporally and spatially. However, few studies reported the relationship and relevant mechanism between osteoclastogenesis and vasculogenesis. Methods: Arraystar Mouse lncRNA microarray V3.0 was firstly used to analyze the differentially expressed lncRNA genes in osteoclast different stages during osteoclastogenesis. Cell counting kit 8 (CCK-8) analysis, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, migration and tube formation assays were used to detect impact of osteoclast different stages on the proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs), respectively. Finally, transfection of AK131850 shRNA, miR-93-5p mimic and miR-93-5p inhibitor, qRT-PCR, western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence in situ hybridization (FISH) and luciferase reporter assay were carried out to dissect molecular mechanisms. Results: In this study, we found that newborn OCs (N-OC) and mature OCs (M-OC) during osteoclastogenesis significantly promoted proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs). Through lncRNA microarray and GO&pathway analysis, we found that AK131850 and co-expressed gene, vascular endothelial growth factor a (VEGFa), were significantly up-regulated in N-OC and M-OC. After inhibition of AK131850 the promoting effect of N-OC and M-OC on EPCs was reversed. Furthermore, we found that AK131850 directly competed miR-93-5p in N-OC and M-OC through sponge, thereby increasing VEGFa transcription, expression and secretion through derepressing of miR-93-5p on VEGFa. Conclusion: Our results provided the first finding that lncRNA-AK131850 sponged miR-93-5p in N-OC and M-OC during osteoclastogenesis to enhance the secretion of VEGFa, thus promoting vasculogenesis of EPCs.

Graphene-Based MicroRNA Transfection Blocks Preosteoclast Fusion to Increase Bone Formation and Vascularization

Abstract The objective of this study is to design a graphene‐based miRNA transfection drug delivery system for antiresorptive therapy. An efficient nonviral gene delivery system is developed using polyethylenimine (PEI) functionalized graphene oxide (GO) complex loaded with miR‐7b overexpression plasmid. GO‐PEI complex exhibits excellent transfection efficiency within the acceptable range of cytotoxicity. The overexpression of miR‐7b after GO‐PEI‐miR‐7b transfection significantly abrogates osteoclast (OC) fusion and bone resorption activity by hampering the expression of an essential fusogenic molecule dendritic cell‐specific transmembrane protein. However, osteoclastogenesis occurs without cell–cell fusion and preosteoclast (POC) is preserved. Through preservation of POC, GO‐PEI‐miR‐7b transfection promotes mesenchymal stem cell osteogenesis and endothelial progenitor cells angiogenesis in the coculture system. Platelet‐derived growth factor‐BB secreted by POC is increased by GO‐PEI‐miR‐7b both in vitro and in vivo. In treating osteoporotic ovariectomized mice, GO‐PEI‐miR‐7b significantly enhances bone mineral density, bone volume as well as bone vascularization through increasing CD31hiEmcnhi cell number. This study provides a cell–cell fusion targeted miRNA transfection drug delivery strategy in treating bone disorders with excessive osteoclastic bone resorption.

Hypertrophic differentiation of mesenchymal stem cells is suppressed by xanthotoxin via the p38‑MAPK/HDAC4 pathway

Chondrocyte hypertrophy is a physiological process in endochondral ossification. However, the hypertrophic-like alterations of chondrocytes at the articular surface may result in osteoarthritis (OA). In addition, the generation of fibrocartilage with a decreased biological function in tissue engineered cartilage, has been attributed to chondrocyte hypertrophy. Therefore, suppressing chondrocyte hypertrophy in OA and the associated regeneration of non-active cartilage is of primary concern. The present study examined the effects of xanthotoxin (XAT), which is classified as a furanocoumarin, on chondrocyte hypertrophic differentiation of mesenchymal stem cells. Following XAT treatment, the expression levels of genes associated with chondrocyte hypertrophy were detected via immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction. The results revealed that XAT inhibited the expression of various chondrocyte hypertrophic markers, including runt related transcription factor 2 (Runx2), matrix metalloproteinase 13 and collagen type X α1 chain. Further exploration indicated that XAT reduced the activation of p38-mitogen activated protein kinase and then increased the expression of histone deacetylase 4 to suppress Runx2. The findings indicated that XAT maintained the chondrocyte phenotype in regenerated cartilage and therefore may exhibit promise as a potential drug for the treatment of OA in the future.

Mangiferin enhances endochondral ossification-based bone repair in massive bone defect by inducing autophagy through activating AMP-activated protein kinase signaling pathway

Endochondral ossification is crucial for bone formation in both adult bone repair process and embryo long‐bone development. In endochondral ossification, bone marrow‐derived mesenchymal stem cells (BMSCs) first differentiate to chondrocytes, then BMSC‐derived chondrocytes endure a hypertrophic process to generate new bone. Endochondral ossification‐based bone repair is a promising strategy to cure massive bone defect, which is a major clinical issue in orthopedics. However, challenges still remain for this novel strategy. One challenge is to ensure the sufficient hypertrophic differentiation. Another is to maintain the survival of the above hypertrophic chondrocytes under the hypoxic environment of massive bone defect. To solve this issue, mangiferin (MAG) was introduced to endochondral ossification‐based bone repair. In this report, we proved MAG to be a novel autophagy inducer, which promoted BMSC‐derived hypertrophic chondrocyte survival against hypoxia‐induced injury through inducing autophagy. Furthermore, MAG enhances hypertrophic differentiation of BMSC‐derived chondrocytes via upregu‐lating key hypertrophic markers. Mechanistically, MAG induced autophagy in BMSC‐derived chondrocytes by promoting AMPKa phosphorylation. Additionally, MAG balanced the expression of sex‐determining region Y‐box 9 and runt‐related transcription factor 2 to facilitate hypertrophic differentiation. These results indicated that MAG was a potential drug to improve the efficacy of endochondral ossification‐based bone repair in massive bone defects.—Bai, Y., Liu, C., Fu, L., Gong, X., Dou, C., Cao, Z., Quan, H., Li, J., Kang, F., Dai, J., Zhao, C., Dong, S. Mangiferin enhances endochondral ossification‐based bone repair in massive bone defect by inducing autophagy through activating AMP‐activated protein kinase signaling pathway. FASEB J. 32, 4573–4584 (2018). www.fasebj.org

Lumichrome inhibits osteoclastogenesis and bone resorption through suppressing RANKL-induced NFAT activation and calcium signaling: LIU et al.

The dynamic balance between bone resorption and bone formation is crucial to maintain bone mass. Osteoclasts are key cells that perform bone resorption while osteoblasts and osteocytes function in bone formation. Osteoporosis, a bone metabolism disease characterized by bone loss and degradation of bone microstructure, occurs when osteoclastic bone resorption outstrips osteoblastic bone synthesis. The interaction between receptor activator of nuclear factor κB ligand (RANKL) and RANK on the surface of bone marrow macrophages promotes osteoclast differentiation and activation. In this study, we found that lumichrome, a photodegradation product of riboflavin, inhibits RANKL‐induced osteoclastogenesis and bone resorption as determined by tartrate‐resistant acid phosphatase staining, immunofluorescence, reverse transcription‐polymerase chain reaction, and western blot. Our results showed that lumichrome represses the expression of osteoclast marker genes, including cathepsin K (Ctsk) and Nfatc1. In addition, lumichrome suppressed RANKL‐induced calcium oscillations, NFATc1, NF‐κB, and MAPK signaling activation. Moreover, lumichrome promoted osteoblast differentiation at an early stage, as demonstrated by upregulated expression of osteoblast marker genes Alp, Runx2, and Col1a1. We also found that lumichrome reduces bone loss in ovariectomized mice by inhibiting osteoclastogenesis. In summary, our data suggest the potential of lumichrome as a therapeutic drug for osteolytic diseases.