MicroRNA‐92b‐5p modulates melatonin‐mediated osteogenic differentiation of bone marrow mesenchymal stem cells by targeting ICAM‐1

Osteoporosis is closely associated with the dysfunction of bone metabolism, which is caused by the imbalance between new bone formation and bone resorption. Osteogenic differentiation plays a vital role in maintaining the balance of bone microenvironment. The present study investigated whether melatonin participated in the osteogenic commitment of bone marrow mesenchymal stem cells (BMSCs) and further explored its underlying mechanisms. Our data showed that melatonin exhibited the capacity of regulating osteogenic differentiation of BMSCs, which was blocked by its membrane receptor inhibitor luzindole. Further study demonstrated that the expression of miR‐92b‐5p was up‐regulated in BMSCs after administration of melatonin, and transfection of miR‐92b‐5p accelerated osteogenesis of BMSCs. In contrast, silence of miR‐92b‐5p inhibited the osteogenesis of BMSCs. The increase in osteoblast differentiation of BMSCs caused by melatonin was attenuated by miR‐92b‐5p AMO as well. Luciferase reporter assay, real‐time qPCR analysis and western blot analysis confirmed that miR‐92b‐5p was involved in osteogenesis by directly targeting intracellular adhesion molecule‐1 (ICAM‐1). Melatonin improved the expression of miR‐92b‐5p, which could regulate the differentiation of BMSCs into osteoblasts by targeting ICAM‐1. This study provided novel methods for treating osteoporosis.     

Expression of Concern: MiRNA‐218 regulates osteoclast differentiation and inflammation response in periodontitis rats through Mmp9

Periodontitis is a multiple infection and inflammatory disease featured by connective tissue homeostasis loss, periodontal inflammation, and alveolar bone resorption. MicroRNAs (miRNAs) are involved in the mediation of a large scale of pathological processes. Here, we show that miRNA‐218 provides protective effect on periodontitis via regulation of matrix metalloproteinase‐9 (Mmp9). This pathway is aberrant in periodontium from rats with periodontitis and human periodontal ligament progenitor cells stimulated by lipopolysaccharide, with downregulation of miR‐218 and higher levels of Mmp9 compared with periodontium from healthy rats and cells without stimulation. Overexpression of miR‐218 can suppress the degradation of Collagen Types I and IV and dentin sialoprotein (DSP), attenuate osteoclast formation, and inhibit the secretion of proinflammatory cytokines. On the other hand, overexpression of Mmp9 promotes the degradation of Collagen Types I and IV and DSP as well as RANKL‐induced osteoclast formation and elevates inflammatory factors levels. Furthermore, the inhibitory effect of miR‐218 was prevented by rescuing the Mmp9 expression. In addition, we also have showed that miR‐218 was able to attenuate bone resorption and inflammation in a periodontitis rat model. Collectively, our findings therefore suggest that miR‐218 acts as a protective role in periodontitis through the regulation of Mmp9.     

Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.     

Osteoclast inhibitory peptide-1 (OIP-1) inhibits measles virus nucleocapsid protein stimulated osteoclast formation/activity

Paget's disease (PD) of bone is characterized by increased activity of large abnormal osteoclasts (OCLs) which contain paramyxoviral nuclear and cytoplasmic inclusions. MVNP gene expression has been shown to induce pagetic phenotype in OCLs. We previously characterized the osteoclast inhibitory peptide-1 (OIP-1/hSca) which inhibits OCL formation/bone resorption. OIP-1 is a glycophosphatidylinositol (GPI)-linked membrane protein containing a 79 amino acid extra cellular peptide and a 32 amino acid carboxy terminal GPI-linked peptide (c-peptide) which is critical for OCL inhibition. In this study, we demonstrate that OIP-1 c-peptide significantly decreased (43%) osteoclast differentiation of peripheral blood mononuclear cells from patients with PD. Also, OIP-1 treatment to normal human bone marrow mononuclear cells transduced with the MVNP inhibited (41%) osteoclast precursor (CFU-GM) growth in methyl-cellulose cultures. We further tested if OIP-1 overexpression in the OCL lineage in transgenic mice inhibits MVNP stimulated OCL formation. MVNP transduction and RANKL stimulation of OIP-1 mouse bone marrow cells showed a significant decrease (43%) in OCL formation and inhibition (38%) of bone resorption area compared to wild-type mice. Western blot analysis identified that OIP-1 decreased (3.5-fold) MVNP induced TRAF2 expression during OCL differentiation. MVNP or OIP-1 expression did not affect TRAF6 levels. Furthermore, OIP-1 expression resulted in a significant inhibition of MVNP stimulated ASK1, Rac1, c-Fos, p-JNK, and NFATc1 expression during OCL differentiation. These results suggest that OIP-1 inhibits MVNP induced pagetic OCL formation/activity through suppression of RANK signaling. Thus, OIP-1 may have therapeutic utility against excess bone resorption in patients with PD.     

MiR‐9 promotes multiple myeloma progression by regulating TRIM56/NF‐κB pathway

miR‐9 has been reported to play a pivotal role in multiple human cancers by acting as an oncogene or tumor suppressor. In this study, we explored the possible role and molecular mechanism of miR‐9 in multiple myeloma (MM). The miR‐9 expression was examined by quantitative real‐time polymerase chain reaction assay. Transfection with miR‐9‐mimics, miR‐9‐inhibitor, pcDNA‐TRIM56, or si‐TRIM56 into cells was used to change the expression levels of miR‐9 and TRIM56. Western blot analysis was used to detect the expression of TRIM56, p65, p‐p65, IκBα, and p‐IκBα. The potential target of miR‐9 was confirmed by luciferase reporter assay. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium (MTT) assay, colony formation assay, and flow cytometry were used to assess the abilities of cell proliferation and apoptosis. miR‐9 was upregulated in MM patients and cell lines, and miR‐9 overexpression promoted proliferation and repressed apoptosis in MM cell lines. TRIM56 was confirmed as a target of miR‐9. Moreover, TRIM56 reversed miR‐9‐mediated pro‐proliferation and anti‐apoptosis effect on MM cell lines. Furthermore, nuclear factor‐κB (NF‐κB) pathway was involved in miR‐9/TRIM56‐mediated regulation on MM cell lines. miR‐9 promoted the development and progression of MM by regulating TRIM56/NF‐κB pathway, thereby providing a potential microRNA‐based target for MM therapy.     

Inhibition of microRNA‐222 up‐regulates TIMP3 to promotes osteogenic differentiation of MSCs from fracture rats with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is the most common diabetes and has numerous complications. Recent studies demonstrated that T2DM compromises bone fracture healing in which miR‐222 might be involved. Furthermore, tissue inhibitor of metalloproteinase 3 (TIMP‐3) that is the target of miR‐222 accelerates fracture healing. Therefore, we assume that miR‐222 could inhibit TIMP‐3 expression. Eight‐week‐old rats were operated femoral fracture or sham, following the injection of streptozotocin (STZ) to induce diabetes one week later in fractured rats, and then, new generated tissues were collected for measuring the expression of miR‐222 and TIMP‐3. Rat mesenchymal stem cells (MSCs) were isolated and treated with miR‐222 mimic or inhibitor to analyse osteogenic differentiation. MiR‐222 was increased in fractured rats and further induced in diabetic rats. In contrast, TIMP‐3 was reduced in fractured and further down‐regulated in diabetic rats. Luciferase report assay indicated miR‐222 directly binds and mediated TIMP‐3. Furthermore, osteogenic differentiation was suppressed by miR‐222 mimic and promoted by miR‐222 inhibitor. miR‐222 is a key regulator that is promoted in STZ‐induced diabetic rats, and it binds to TIMP3 to reduce TIMP‐3 expression and suppressed MSCs’ differentiation.     

Muscle atrophy‐related myotube‐derived exosomal microRNA in neuronal dysfunction: Targeting both coding and long noncoding RNAs

In mammals, microRNAs can be actively secreted from cells to blood. miR‐29b‐3p has been shown to play a pivotal role in muscle atrophy, but its role in intercellular communication is largely unknown. Here, we showed that miR‐29b‐3p was upregulated in normal and premature aging mouse muscle and plasma. miR‐29b‐3p was also upregulated in the blood of aging individuals, and circulating levels of miR‐29b‐3p were negatively correlated with relative appendicular skeletal muscle. Consistently, miR‐29b‐3p was observed in exosomes isolated from long‐term differentiated atrophic C2C12 cells. When C2C12‐derived miR‐29b‐3p‐containing exosomes were uptaken by neuronal SH‐SY5Y cells, increased miR‐29b‐3p levels in recipient cells were observed. Moreover, miR‐29b‐3p overexpression led to downregulation of neuronal‐related genes and inhibition of neuronal differentiation. Interestingly, we identified HIF1α‐AS2 as a novel c‐FOS targeting lncRNA that is induced by miR‐29b‐3p through down‐modulation of c‐FOS and is required for miR‐29b‐3p‐mediated neuronal differentiation inhibition. Our results suggest that atrophy‐associated circulating miR‐29b‐3p may mediate distal communication between muscle cells and neurons.     

Induced miR‐99a expression represses Mtor cooperatively with miR‐150 to promote regulatory T‐cell differentiation

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4⁺ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T‐cell‐expressed miRNAs in naive mouse CD4⁺ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR‐100, miR‐99a and miR‐10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR‐99a cooperated with miR‐150 to repress the expression of the Th17‐promoting factor mTOR. The comparably low expression of miR‐99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR‐150 could only repress Mtor in the presence of miR‐99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.     

miR‐542‐3p prevents ovariectomy‐induced osteoporosis in rats via targeting SFRP1

Secreted frizzled‐related protein‐1 (SFRP1) is a negative regulatory molecule of the WNT signaling pathway and serves as a therapeutic target for bone formation in osteoporosis. In this study, we first established an ovariectomized (OVX) rat model to simulate postmenopausal osteoporosis and found significant changes in miR‐542‐3p and sFRP1 expression by RNA sequencing and qRT‐PCR. In addition, there was a significant negative correlation between miR‐542‐3p and sFRP1 mRNA levels in postmenopausal women with osteoporosis. We found that miR‐542‐3p inhibited the expression of sFRP1 mRNA by luciferase reporter assay. When the miR‐542‐3p binding site in sFRP1 3'UTR was deleted, it did not affect its expression. Western blot results showed that miR‐542‐3p inhibited the expression of SFRP1 protein. The expression of SFRP1 was significantly increased in osteoblast‐induced mesenchymal stem cells (MSC), whereas the expression of miR‐542‐3p was significantly decreased. And miR‐542‐3p transfected MSCs showed a significant increase in osteoblast‐specific marker expression, indicating that miR‐542‐3p is necessary for MSC differentiation. Inhibition of miR‐542‐3p reduced bone formation, confirmed miR‐542‐3p play a role in bone formation in vivo. In general, these data suggest that miR‐542‐3p play an important role in bone formation via inhibiting SFRP1 expression and inducing osteoblast differentiation.     

MiR‐29b‐3p promotes chondrocyte apoptosis and facilitates the occurrence and development of osteoarthritis by targeting PGRN

This study was aimed to explore the role of miR‐29b‐3p and PGRN in chondrocyte apoptosis and the initiation and progress of osteoarthritis (OA). Both miR‐29b‐3p and PGRN were up‐regulated in cartilage tissue from patients with OA. Transfection of miR‐29b‐3p mimic into rat primary chondrocytes and SW1353 chondrosarcoma cells significantly suppressed PGRN expression and release, induced apoptosis, inhibited proliferation and scratch wound closure. By contrast, transfection of miR‐29b‐3p inhibitor exhibited the opposite effects. Moreover, the expression and secretion of cartilaginous degeneration‐related molecules were also altered by miR‐29b‐3p. Luciferase reporter gene assay showed rat GRN mRNA is directly targeted and repressed by miR‐29b‐3p. The fact that recombinant PGRN or shPGRN‐mediated PGRN interference abolished miR‐29b‐3p mimic‐induced cell apoptosis and growth inhibition suggested miR‐29b‐3p affect the cellular functions of chondrocyte through regulating PGRN expression. In vivo, joint cavity injection of miR‐29b‐3p antagomir prior to surgical induction of OA significantly suppressed the upregulation of miR‐29b‐3p, whereas further promoted the increased expression of PGRN. Articular chondrocytes apoptosis and cartilage loss in the knee joint of surgically induced OA rats were also ameliorated by the injection of miR‐29b‐3p antagomir, demonstrated by TUNEL and safranin O‐fast green staining. This work showed miR‐29b‐3p facilitates chondrocyte apoptosis and OA by targeting PGRN, and miR‐29b‐3p or PGRN may be the potential target for OA treatments.     

Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells

Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts (OCLs) along the stem cell-rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9-dependent manner; however, RANKL did not induce mobilization in young female PTPepsilon-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.     

miRNA‐221 and miRNA‐222 synergistically function to promote vascular calcification

Vascular calcification shares many similarities with skeletal mineralisation and involves the phenotypic trans‐differentiation of vascular smooth muscle cells (VSMCs) to osteoblastic cells within a calcified environment. Various microRNAs (miRs) are known to regulate cell differentiation; however, their role in mediating VSMC calcification is not fully understood. miR‐microarray analysis revealed the significant down‐regulation of a range of miRs following nine days in culture, including miR‐199b, miR‐29a, miR‐221, miR‐222 and miR‐31 (p < 0.05). Subsequent studies investigated the specific role of the miR‐221/222 family in VSMC calcification. Real‐time quantitative polymerase chain reaction data confirmed the down‐regulation of miR‐221 (32.4%; p < 0.01) and miR‐222 (15.7%; p < 0.05). VSMCs were transfected with mimics of miR‐221 and miR‐222, individually and in combination. Increased calcium deposition was observed in the combined treatment (two‐fold; p < 0.05) but not in individual treatments. Runx2 and Msx2 expression was increased during calcification, but no difference in expression was observed following transfection with miR mimics. Interestingly, miR‐221 and miR‐222 mimics induced significant changes in ectonucleotide phosphodiesterase 1 (Enpp1) and Pit‐1 expression, suggesting that these miRs may modulate VSMC calcification through cellular inorganic phosphate and pyrophosphate levels. © 2013 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.