miR-19b enhances osteogenic differentiation of mesenchymal stem cells and promotes fracture healing through the WWP1/Smurf2-mediated KLF5/β-catenin signaling pathway

Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been found to enhance fracture healing. In addition, microRNAs contributing to the healing of various bone fractures have attracted widespread attention in recent years, but knowledge of the mechanisms by which they act is still very limited. In this study, we clarified the function of altered microRNA-19b (miR-19b) expression in BMSCs in fracture healing. We modulated miR-19b expression via mimics/inhibitors in BMSCs and via agomirs in mice to explore the effects of these changes on osteogenic factors, bone cell mineralization and the healing status of modeled fractures. Through gain- and loss-of function assays, the binding affinity between miR-19b and WWP1/Smurf2 was identified and characterized to explain the underlying mechanism involving the KLF5/β-catenin signaling pathway. miR-19b promoted the differentiation of human BMSCs into osteoblasts by targeting WWP1 and Smurf2. Overexpression of WWP1 or Smurf2 degraded the target protein KLF5 in BMSCs through ubiquitination to inhibit fracture healing. KLF5 knockdown delayed fracture healing by modulating the Wnt/β-catenin signaling pathway. Furthermore, miR-19b enhanced fracture healing via the KLF5/β-catenin signaling pathway by targeting WWP1 or Smurf2. Moreover, miR-19b was found to be enriched in BMSC-derived exosomes, and treatment with exosomes promoted fracture healing in vivo. Collectively, these results indicate that mesenchymal stem cell-derived exosomal miR-19b represses the expression of WWP1 or Smurf2 and elevates KLF5 expression through the Wnt/β-catenin signaling pathway, thereby facilitating fracture healing.Subject terms: Diseases, Biotechnology     

Euodia sutchuenensis Dode extract stimulates osteoblast differentiation via Wnt/β-catenin pathway activation

The Wnt/β-catenin pathway has a role in osteoblast differentiation and bone formation. We screened 100 plant extracts and identified an extract from Euodia sutchuenensis Dode (ESD) leaf and young branch as an effective activator of the Wnt/β-catenin pathway. ESD extract increased β-catenin levels and β-catenin nuclear accumulation in murine primary osteoblasts. The ESD extract also increased mRNA levels of osteoblast markers, including RUNX2, BMP2 and COL1A1, and enhanced alkaline phosphatase (ALP) activity in murine primary osteoblasts. Both ESD extract-induced β-catenin increment and ALP activation were abolished by β-catenin knockdown, confirming that the Wnt/β-catenin pathway functions in osteoblast differentiation. ESD extract enhanced terminal osteoblast differentiation as shown by staining with Alizarin Red S and significantly increased murine calvarial bone thickness. This study shows that ESD extract stimulates osteoblast differentiation via the Wnt/β-catenin pathway and enhances murine calvarial bone formation ex vivo.     

Emodin prevents intima thickness via Wnt4/Dvl-1/β-catenin signaling pathway mediated by miR-126 in balloon-injured carotid artery rats

Neointimal proliferation after vascular injury is a key mechanism of restenosis, a major cause of percutaneous transluminal angioplasty failure and artery bypass occlusion. Emodin, an anthraquinone with multiple physiological activities, has been reported to inhibit proliferation of vascular smooth muscle cells (VSMCs) that might cause intimal arterial thickening. Thus, in this study, we established a rat model of balloon-injured carotid artery and investigated the therapeutic effect of emodin and its underlying mechanism. Intimal thickness was analyzed by hematoxylin and eosin staining. Expression of Wnt4, dvl-1, β-catenin and collagen was determined by immunohistochemistry and/or western blotting. The proliferation of VSMC was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and electron microscopy. MicroRNA levels were quantified by real-time quantitative PCR. Emodin relieved injury-induced artery intimal thickness. Results of western blots and immunohistochemistry showed that emodin suppressed expression of signaling molecules Wnt4/Dvl-1/β-catenin as well as collagen protein in the injured artery. In addition, emodin enhanced expression of an artery injury-related microRNA, miR-126. In vitro, MTT assay showed that emodin suppressed angiotensin II (AngII)-induced proliferation of VSMCs. Emodin reversed AngII-induced activation of Wnt4/Dvl-1/β-catenin signaling by increasing expression of miR-126 that was strongly supported by transfection of mimic or inhibitor for miR-126. Emodin prevents intimal thickening via Wnt4/Dvl-1/β-catenin signaling pathway mediated by miR-126 in balloon-injured carotid artery of rats.     

Euphorbiasteroid Abrogates EGFR and Wnt/β-Catenin Signaling in Non-Small-Cell Lung Cancer Cells to Impart Anticancer Activity

EGFR and Wnt/β-catenin signaling pathways play a prominent role in tumor progression in various human cancers including non-small-cell lung carcinoma (NSCLC). Transactivation and crosstalk between the EGFR and Wnt/β-catenin pathways may contribute to the aggressiveness of cancers. Targeting these oncogenic pathways with small molecules is an attractive approach to counteract various types of cancers. In this study, we demonstrate the effect of euphorbiasteroid (EPBS) on the EGFR and Wnt/β-catenin pathways in NSCLC cells. EPBS induced preferential cytotoxicity toward A549 (wildtype EGFR-expressing) cells over PC-9 (mutant EGFR-expressing) cells. EPBS suppressed the expression of EGFR, Wnt3a, β-catenin, and FZD-1, and the reduction in β-catenin levels was found to be mediated through the activation of GSK-3β. EPBS reduced the phosphorylation of GSK-3β^S9 with a parallel increase in β-TrCP and phosphorylation of GSK-3β^Y216. Lithium chloride treatment increased the phosphorylation of GSK-3β^S9 and nuclear localization of β-catenin, whereas EPBS reverted these effects. Forced expression or depletion of EGFR in NSCLC cells increased or decreased the levels of Wnt3a, β-catenin, and FZD-1, respectively. Overall, EPBS abrogates EGFR and Wnt/β-catenin pathways to impart its anticancer activity in NSCLC cells.     

MiR-1224-5p modulates osteogenesis by coordinating osteoblast/osteoclast differentiation via the Rap1 signaling target ADCY2

MicroRNAs (miRNAs) broadly regulate normal biological functions of bone and the progression of fracture healing and osteoporosis. Recently, it has been reported that miR-1224-5p in fracture plasma is a potential therapy for osteogenesis. To investigate the roles of miR-1224-5p and the Rap1 signaling pathway in fracture healing and osteoporosis development and progression, we used BMMs, BMSCs, and skull osteoblast precursor cells for in vitro osteogenesis and osteoclastogenesis studies. Osteoblastogenesis and osteoclastogenesis were detected by ALP, ARS, and TRAP staining and bone slice resorption pit assays. The miR-1224-5p target gene was assessed by siRNA-mediated target gene knockdown and luciferase reporter assays. To explore the Rap1 pathway, we performed high-throughput sequencing, western blotting, RT-PCR, chromatin immunoprecipitation assays and immunohistochemical staining. In vivo, bone healing was judged by the cortical femoral defect, cranial bone defect and femoral fracture models. Progression of osteoporosis was evaluated by an ovariectomy model and an aged osteoporosis model. We discovered that the expression of miR-1224-5p was positively correlated with fracture healing progression. Moreover, in vitro, overexpression of miR-1224-5p slowed Rankl-induced osteoclast differentiation and promoted osteoblast differentiation via the Rap1-signaling pathway by targeting ADCY2. In addition, in vivo overexpression of miR-1224-5p significantly promoted fracture healing and ameliorated the progression of osteoporosis caused by estrogen deficiency or aging. Furthermore, knockdown of miRNA-1224-5p inhibited bone regeneration in mice and accelerated the progression of osteoporosis in elderly mice. Taken together, these results identify miR-1224-5p as a key bone osteogenic regulator, which may be a potential therapeutic target for osteoporosis and fracture nonunion.Subject terms: Translational research, Cell signalling     

The Wnt/β-catenin signaling pathway regulates the development of airway remodeling in patients with asthma

Airway remodeling is a key characteristic of chronic asthma, particularly in patients with a fixed airflow limitation. The mechanisms underlying airway remodeling are poorly understood, and no therapeutic option is available. The Wnt/β-catenin signaling pathway is involved in various physiological and pathological processes, including fibrosis and smooth muscle hypertrophy. In this study, we investigated the roles of Wnt/β-catenin signaling in airway remodeling in patients with asthma. Wnt7a mRNA expression was prominent in induced sputum from patients with asthma compared with that from healthy controls. Next, we induced a chronic asthma mouse model with airway remodeling features, including subepithelial fibrosis and airway smooth muscle hyperplasia. Higher expression of Wnt family proteins and β-catenin was detected in the lung tissue of mice with chronic asthma compared to control mice. Blocking β-catenin expression with a specific siRNA attenuated airway inflammation and airway remodeling. Decreased subepithelial fibrosis and collagen accumulation in the β-catenin siRNA-treated mice was accompanied by reduced expression of transforming growth factor-β. We further showed that suppressing β-catenin in the chronic asthma model inhibited smooth muscle hyperplasia by downregulating the tenascin C/platelet-derived growth factor receptor pathway. Taken together, these findings demonstrate that the Wnt/β-catenin signaling pathway is highly expressed and regulates the development of airway remodeling in chronic asthma.     

Anti-Hair Loss Effect of Adenosine Is Exerted by cAMP Mediated Wnt/β-Catenin Pathway Stimulation via Modulation of Gsk3β Activity in Cultured Human Dermal Papilla Cells

In the present study, we investigated the molecular mechanisms of adenosine for its hair growth promoting effect. Adenosine stimulated the Wnt/β-catenin pathway by modulating the activity of Gsk3β in cultured human dermal papilla cells. It also activated adenosine receptor signaling, increasing intracellular cAMP level, and subsequently stimulating the cAMP mediated cellular energy metabolism. The phosphorylation of CREB, mTOR, and GSK3β was increased. Furthermore, the expression of β-catenin target genes such as Axin2, Lef1, and growth factors (bFGF, FGF7, IGF-1) was also enhanced. The inhibitor study data conducted in Wnt reporter cells and in cultured human dermal papilla cells demonstrated that adenosine stimulates Wnt/β-catenin signaling through the activation of the adenosine receptor and Gsk3β plays a critical role in transmitting the signals from the adenosine receptor to β-catenin, possibly via the Gαs/cAMP/PKA/mTOR signaling cascade.     

Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS,promoted gastric cancer progression via increased β-catenin and CEMIP

ATF3 has been reported to be dysregulated in various cancers and involved in various steps of tumorigenesis. However, the mechanisms underlying the abnormal expression of ATF3 and its biological function in gastric cancer (GC) have not been well investigated. Here, we report ATF3 as one of the key regulators of GC development and progression. Patients with low ATF3 expression had shorter survival and a poorer prognosis. In vitro and in vivo assays investigating ATF3 alterations revealed a complex integrated phenotype that affects cell growth and migration. Strikingly, high-throughput sequencing and microarray analysis of cells with ATF3 silencing or of ATF3-low GC tissues indicated alterations in the Wnt signaling pathway, focal adhesions and adherens junctions. Mechanistically, the expression of β-catenin and cell migration inducing hyaluronidase 1 (CEMIP) was significantly upregulated in GC cells with downregulated ATF3, which was synergistically repressed by the β-catenin/TCF3 signaling axis and noncoding RNA miR-17-5p and HOXA11-AS. In addition, we found that WDR5 expression was promoted by TCF3 and is involved in miR-17-5p and HOXA11-AS activation in GC cells. Taken together, our findings revealed the mechanism of ATF3 downregulation and its biological role in regulating the expression of Wnt signaling-related genes during GC progression, suggesting new informative biomarkers of malignancy and therapeutic directions for GC patients.Subject terms: Gastric cancer, Experimental models of disease     

Anti-Osteoporosis Effect of Perilla frutescens Leaf Hexane Fraction through Regulating Osteoclast and Osteoblast Differentiation

Osteoporosis is the result of an imbalance in the bone-remodeling process via an increase in osteoclastic activity and a decrease in osteoblastic activity. Our previous studies have shown that Perilla frutescens seed meal has anti-osteoclastogenic activity. However, the role of perilla leaf hexane fraction (PLH) in osteoporosis has not yet been investigated and reported. In this study, we aimed to investigate the effects of PLH in osteoclast differentiation and osteogenic potential using cell-based experiments in vitro. From HPLC analysis, we found that PLH contained high luteolin and baicalein. PLH was shown to inhibit RANKL-induced ROS production and tartrate-resistant acid phosphatase (TRAP)-positive multi-nucleated osteoclasts. Moreover, PLH significantly downregulated the RANKL-induced MAPK and NF-κB signaling pathways, leading to the attenuation of NFATc1 and MMP-9 expression. In contrast, PLH enhanced osteoblast function by regulating alkaline phosphatase (ALP) and restoring TNF-α-suppressed osteoblast proliferation and osteogenic potential. Thus, luteolin and baicalein-rich PLH inhibits osteoclast differentiation but promotes the function of osteoblasts. Collectively, our data provide new evidence that suggests that PLH may be a valuable anti-osteoporosis agent.     

980 nm Photobiomodulation Promotes Osteo/Odontogenic Differentiation of the Stem Cells from Human Exfoliated Deciduous Teeth Via the Cross talk Between BMP/Smad and Wnt/β-Catenin Signaling Pathways

Increasing evidence suggests stem cells from human exfoliated deciduous teeth (SHEDs) serve as desirable sources of dentin regeneration. Photobiomodulation (PBM) has shown great potential in enhancing the proliferation and osteogenesis of human bone marrow mesenchymal stem cells (hBMMSCs). However, the specific role of PBM in odontogenic differentiation of SHEDs is little know, and we further investigated potential mechanism of PBM osteo/odontogenisis. A 980 nm diode laser with different energy densities of (0.5, 5, 10 J cm⁻²) in a 100-mW continuous wave was used for irradiation every 24 h. Osteo/odontogenic differentiation of SHEDs was achieved by performing alkaline phosphatase (ALP) and alizarin red staining (ARS) and osteo/odontogenic markers were also evaluated by qRT-PCR and western blotting. Additionally, western blot and immunohistochemical staining were performed to evaluate the levels of BMP/Smad and Wnt/β-catenin signaling-related proteins. We found that PBM at 5 J cm⁻¹ increased mineral deposition and upregulated the expression of related osteo/odontogenic markers along with the elevated expression of β-catenin and phosphorylation level of Smad1/5/9. Furthermore, Wnt signaling inhibition using DKK1 and BMP signaling inhibition using noggin inhibited PBM-induced osteo/odontogenic marker expression when used individually or jointly. In conclusion, PBM induces the osteo/odontogenic differentiation of SHEDs through cross talk between BMP/Smad and Wnt/β-catenin signaling pathways.     

5-Bromo-3,4-dihydroxybenzaldehyde Promotes Hair Growth through Activation of Wnt/β-Catenin and Autophagy Pathways and Inhibition of TGF-β Pathways in Dermal Papilla Cells

Various studies addressing the increasing problem of hair loss, using natural products with few side effects, have been conducted. 5-bromo-3,4-dihydroxybenzaldehyde (BDB) exhibited anti-inflammatory effects in mouse models of atopic dermatitis and inhibited UVB-induced oxidative stress in keratinocytes. Here, we investigated its stimulating effect and the underlying mechanism of action on hair growth using rat vibrissa follicles and dermal papilla cells (DPCs), required for the regulation of hair cycle and length. BDB increased the length of hair fibers in rat vibrissa follicles and the proliferation of DPCs, along with causing changes in the levels of cell cycle-related proteins. We investigated whether BDB could trigger anagen-activating signaling pathways, such as the Wnt/β-catenin pathway and autophagy in DPCs. BDB induces activation of the Wnt/β-catenin pathway through the phosphorylation of GSG3β and β-catenin. BDB increased the levels of autophagic vacuoles and autophagy regulatory proteins Atg7, Atg5, Atg16L, and LC3B. We also investigated whether BDB inhibits the TGF-β pathway, which promotes transition to the catagen phase. BDB inhibited the phosphorylation of Smad2 induced by TGF-β1. Thus, BDB can promote hair growth by modulating anagen signaling by activating Wnt/β-catenin and autophagy pathways and inhibiting the TGF-β pathway in DPCs.     

NF-κB-dependent miR-31/155 biogenesis is essential for TNF-α-induced impairment of endothelial progenitor cell function

Endothelial progenitor cell (EPC) dysfunction impairs vascular function and remodeling in inflammation-associated diseases, including preeclampsia. However, the underlying mechanism of this inflammation-induced dysfunction remains unclear. In the present study, we found increases in TNF-α and miR-31/155 levels and reduced numbers of circulating EPCs in patients with preeclampsia. Patient-derived mononuclear cells (MNCs) cultured in autologous serum had decreased endothelial nitric oxide synthase (eNOS) expression, nitric oxide production, and differentiation into EPCs with angiogenic potential, and these effects were inhibited by a TNF-α-neutralizing antibody and miR-31/155 inhibitors. Moreover, TNF-α treatment of normal MNCs increased miR-31/155 biogenesis, decreased eNOS expression, reduced EPC differentiation, and impaired angiogenic potential. The TNF-α-induced impairment of EPC differentiation and function was rescued by NF-κB p65 knockdown or miR-31/155 inhibitors. In addition, treatment of MNCs with synthetic miR-31/155 or an eNOS inhibitor mimicked the inhibitory effects of TNF-α on eNOS expression and EPC functions. Moreover, transplantation of EPCs that had been differentiated from TNF-α-treated MNCs decreased neovascularization and blood perfusion in ischemic mouse hindlimbs compared with those of normally differentiated EPCs. These findings suggest that NF-κB activation is required for TNF-α-induced impairment of EPC mobilization, differentiation, and function via miR-31/155 biogenesis and eNOS downregulation. Our data provide a new role for NF-κB-dependent miR-31/155 in EPC dysfunction under the pathogenic conditions of inflammation-associated vascular diseases, including preeclampsia.Subject terms: Differentiation, Cell biology     

Msx2 mediates the inhibitory action of TNF-�� on osteoblast differentiation

TNF-α, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-α signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-α-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-α down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2^-/- calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-α induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-α. TNF-α activated the NF-κB and the JNK pathways. Inhibition of NF-κB or JNK activation reduced the inhibitory effect of TNF-α on ALP expression, whereas TNF-α-induced Msx2 expression was only suppressed by the inhibition of the NF-κB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-α on BMP2-regulated osteoblast differentiation and that the TNF-α-activated NF-κB pathway is responsible for Msx2 induction.