Fei Kang

MicroRNAs regulate osteogenesis and chondrogenesis.

MicroRNAs (miRNAs) are a class of small molecules and non-coding single strand RNAs that regulate gene expression at the post-transcriptional level by binding to specific sequences within target genes. miRNAs have been recognized as important regulatory factors in organism development and disease expression. Some miRNAs regulate the proliferation and differentiation of osteoblasts, osteoclasts and chondrocytes, eventually influencing metabolism and bone formation. miRNAs are expected to provide potential gene therapy targets for the clinical treatment of metabolic bone diseases and bone injuries. Here, we review the recent research progress on the regulation of miRNAs in bone biology, with a particular focus on the miRNA-mediated control mechanisms of bone and cartilage formation.

Effect of microRNA‐145 on IL‐1β‐induced cartilage degradation in human chondrocytes

MicroRNA‐145 has been shown to regulate chondrocyte homeostasis. It seems that miR‐145 is implicated in cartilage dysfunction in Osteoarthritis (OA). However, the functional role of miR‐145 in interleukin‐1 beta (IL‐1β)‐induced extracellular matrix (ECM) degradation of OA cartilage has never been clarified. Here, we show that miR‐145 expression increased in OA chondrocytes and in response to IL‐1β stimulation. We confirm that mothers against decapentaplegic homolog 3 (Smad3), a key factor in maintaining chondrocyte homeostasis, is directly regulated by miR‐145. Modulation of miR‐145 affects the expression of Smad3 causing a change of its downstream target gene expression as well as IL‐1β‐induced ECM degradation in OA chondrocytes. This indicates that miR‐145 contributes to impaired ECM in OA cartilage probably in part via targeting Smad3.

MiR-7b directly targets DC-STAMP causing suppression of NFATc1 and c-Fos signaling during osteoclast fusion and differentiation

DC-STAMP is a key regulating molecule of osteoclastogenesis and osteoclast precursor (OCP) fusion. Emerging lines of evidence showed that microRNAs play crucial roles in bone metabolism and osteoclast differentiation, but no microRNA has yet been reported to be directly related to OCPs fusion. Through a microarray, we found that the expression of miR-7b in RAW264.7 cells was significantly decreased after induction with M-CSF and RANKL. The overexpression of miR-7b in RAW264.7 cells attenuated the number of TRAP-positive cells number and the formation of multinucleated cells, whereas the inhibition of miR-7b enhanced osteoclastogenesis. Through a dual luciferase reporter assay, we confirmed that miR-7b directly targets DC-STAMP. Other fusogenic molecules, such as CD47, ATP6v0d2, and OC-STAMP, were detected to be down-regulated in accordance with the inhibition of DC-STAMP. Because DC-STAMP also participates in osteoclast differentiation through the ITAM-ITIM network, multiple osteoclast-specific genes in the ITAM-ITIM network were detected to identify how DC-STAMP is involved in this process. The results showed that molecules associated with the ITAM-ITIM network, such as NFATc1 and OSCAR, which are crucial in osteoclastogenesis, were consistently altered due to DC-STAMP inhibition. These findings suggest that miR-7b inhibits osteoclastogenesis and cell-cell fusion by directly targeting DC-STAMP. In addition, the inhibition of DC-STAMP and its downstream signals changed the expression of other fusogenic genes and key regulating genes, such as Nfatc1, c-fos, Akt, Irf8, Mapk1, and Traf6. In conclusion, our findings indicate that miR-7b may be a potential therapeutic target for the treatment of osteoclast-related bone disorders.

Changing expression profiles of lncRNAs, mRNAs, circRNAs and miRNAs during osteoclastogenesis

Bone is a dynamic organ continuously undergoing shaping, repairing and remodeling. The homeostasis of bone is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts (OCs) are specialized multinucleated cells derived from hematopoietic stem cells (HSCs) or monocytes/macrophage progenitor cells. There are different stages during osteoclastogenesis, and one of the most important steps to form functional osteoclasts is realized by cell-cell fusion. In our study, microarray was performed to detect the expression profiles of lncRNA, mRNA, circRNA and miRNA at different stages during osteoclastogenesis of RAW264.7 cells. Often changed RNAs were selected and clustered among the four groups with Venn analysis. The results revealed that expressions of 518 lncRNAs, 207 mRNAs, 24 circRNAs and 37 miRNAs were often altered at each stage during OC differentiation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis were performed to predict the functions of differentially expressed lncRNAs and co-expressed potential targeting genes. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were constructed based on the correlation analysis between the differentially expressed RNAs. The present study provided a systematic perspective on the potential function of non-coding RNAs (ncRNAs) during osteoclastogenesis.

Dual Effect of Cyanidin on RANKL-Induced Differentiation and Fusion of Osteoclasts.

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts are multinucleated cells derived from hematopoietic stem cells (HSCs) or monocyte/macrophage progenitor cells and formed by osteoclasts precursors (OCPs) fusion. Cyanidin is an anthocyanin widely distributed in food diet with novel antioxidant activity. However, the effect of cyanidin on osteoclasts is still unknown. We investigated the effect of cyanidin on RANKL‐induced osteoclasts differentiation and cell fusion. The results showed that cyanidin had a dual effect on RANKL‐induced osteoclastogenesis. Lower dosage of cyanidin (< 1µg/ml) has a promoting effect on osteoclastogenesis while higher dosage of cyanidin (> 10µg/ml) has an inhibitory effect. Fusogenic genes like CD9, ATP6v0d2, DC‐STAMP, OC‐STAMP, and osteoclasts related genes like NFATc1, mitf, and c‐fos were all regulated by cyanidin consistent to its dual effect. Further exploration showed that low concentration of cyanidin could increase osteoclasts fusion whereas higher dosage of cyanidin lead to the increase of LXR‐β expression and activation which is suppressive to osteoclasts differentiaton. All these results showed that cyanidin exhibits therapeutic potential in prevention of osteoclasts related bone disorders. J. Cell. Physiol. 231: 558–567, 2016.

Cerium Oxide Nanoparticle Modified Scaffold Interface Enhances Vascularization of Bone Grafts by Activating Calcium Channel of Mesenchymal Stem Cells

Insufficient blood perfusion is one of the critical problems that hamper the clinical application of tissue engineering bone (TEB). Current methods for improving blood vessel distribution in TEB mainly rely on delivering exogenous angiogenic factors to promote the proliferation, migration, differentiation, and vessel formation of endothelial cells (ECs) and/or endothelial progenitor cells (EPCs). However, obstacles including limited activity preservation, difficulty in controlled release, and high cost obstructed the practical application of this strategy. In this study, TEB scaffold were modified with cerium oxide nanoparticles (CNPs) and the effects of CNPs existed at the scaffold surface on the growth and paracrine behavior of mesenchymal stem cells (MSCs) were investigated. The CNPs could improve the proliferation and inhibit the apoptosis of MSCs. Meanwhile, the interaction between the cell membrane and the nanoparticle surface could activate the calcium channel of MSCs leading to the rise of intracellular free Ca(2+) level, which subsequently augments the stability of HIF-1α. These chain reactions finally resulted in high expression of angiogenic factor VEGF. The improved paracrine of VEGF could thereby promote the proliferation, differentiation, and tube formation ability of EPCs. Most importantly, in vivo ectopic bone formation experiment demonstrated this method could significantly improve the blood vessel distribution inside of TEB.

rFN/Cad-11-Modified Collagen Type II Biomimetic Interface Promotes the Adhesion and Chondrogenic Differentiation of Mesenchymal Stem Cells

Properties of the cell-material interface are determining factors in the successful function of cells for cartilage tissue engineering. Currently, cell adhesion is commonly promoted through the use of polypeptides; however, due to their lack of complementary or modulatory domains, polypeptides must be modified to improve their ability to promote adhesion. In this study, we utilized the principle of matrix-based biomimetic modification and a recombinant protein, which spans fragments 7-10 of fibronectin module III (heterophilic motif) and extracellular domains 1-2 of cadherin-11 (rFN/Cad-11) (homophilic motif), to modify the interface of collagen type II (Col II) sponges. We showed that the designed material was able to stimulate cell proliferation and promote better chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) in vitro than both the FN modified surfaces and the negative control. Further, the Col II/rFN/Cad-11-MSCs composite stimulated cartilage formation in vivo; the chondrogenic effect of Col II alone was much less significant. These results suggested that the rFN/Cad-11-modified collagen type II biomimetic interface has dual biological functions of promoting adhesion and stimulating chondrogenic differentiation. This substance, thus, may serve as an ideal scaffold material for cartilage tissue engineering, enhancing repair of injured cartilage in vivo.

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.

Xanthotoxin prevents bone loss in ovariectomized mice through the inhibition of RANKL-induced osteoclastogenesis

SummaryXanthotoxin (XAT) is extracted from the seeds of Ammi majus. Here, we reported that XAT has an inhibitory effect on osteoclastogenesis in vitro through the suppression of both receptor activator of nuclear factor-κB ligand (RANKL)-induced ROS generation and Ca2+ oscillations. In vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, and restores bone strength in ovariectomized mice.IntroductionExcessive osteoclast formation and the resultant increase in bone resorption activity are key pathogenic factors of osteoporosis. In the present study, we have investigated the effects of XAT, a natural furanocoumarin, on the RANKL-mediated osteoclastogenesis in vitro and on ovariectomy-mediated bone loss in vivo.MethodsCytotoxicity of XAT was evaluated using bone marrow macrophages (BMMs). Osteoclast differentiation, formation, and fusion were assessed using the tartrate-resistant acid phosphatase (TRAP) stain, the actin cytoskeleton and focal adhesion (FAK) stain, and the fusion assay, respectively. Osteoclastic bone resorption was evaluated using the pit formation assay. Reactive oxygen species (ROS) generation and removal were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). Ca2+ oscillations and their downstream signaling targets were then detected. The ovariectomized (OVX) mouse model was adopted for our in vivo studies.ResultsIn vitro assays revealed that XAT inhibited the differentiation, formation, fusion, and bone resorption activity of osteoclasts. The inhibitory effect of XAT on osteoclastogenesis was associated with decreased intracellular ROS generation. XAT treatment also suppressed RANKL-induced Ca2+ oscillations and the activation of the resultant downstream calcium-CaMKK/PYK2 signaling. Through these two mechanisms, XAT downregulated the key osteoclastogenic factors nuclear factor of activated T cells c1 (NFATc1) and c-FOS. Our in vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, rescues bone microarchitecture, and restores bone strength in OVX mice.ConclusionOur findings indicate that XAT is protective against ovariectomy-mediated bone loss through the inhibition of RANKL-mediated osteoclastogenesis. Therefore, XAT may be considered to be a new therapeutic candidate for treating osteoporosis.

Cordycepin Prevents Bone Loss through Inhibiting Osteoclastogenesis by Scavenging ROS Generation

Cordycepin was previously reported to have anti-tumor, anti-inflammatory and anti-oxidant activity. However, the potential role of cordycepin in bone metabolism and cell biology of osteoclasts remains unclear. In our study, we focused on the in vitro effects of cordycepin on osteoclastogenesis and its in vivo effects in ovariectomized (OVX) mice. Osteoclast differentiation, formation and fusion were evaluated by Tartrate-resistant acid phosphatase (TRAP) stain, focal adhesion stain and fusion assay, respectively. Osteoclastic bone resorption was evaluated by pit formation assay. Reactive oxygen species (ROS) generation and removal were detected by the ROS assay. OVX mice were orally administered with 10 mg/kg of cordycepin daily for four weeks. In vitro results revealed that cordycepin inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation, formation, fusion and bone resorption activity. We further proved that cordycepin treatments scavenged the generation of ROS, upregulated interferon regulatory factor 8 (IRF-8) and suppressed the activity of nuclear factor of activated T cells c1 (NFATc1) during osteoclastogenesis. In vivo results indicated cordycepin prevents bone loss, rescues bone microarchitecture, and restores bone mineralization in OVX mice. Our observations strongly suggested that cordycepin is an efficient osteoclast inhibitor and hold potential therapeutic value in preventing bone loss among postmenopausal osteoporosis patients.