microRNA-96 promotes osteoblast differentiation and bone formation in ankylosing spondylitis mice through activating the Wnt signaling pathway by binding to SOST

Ankylosing spondylitis (AS) refers to a type of arthritis manifested with chronic inflammation of spine joints. microRNAs (MiRNAs) have been identified as new therapeutic targets for inflammatory diseases. In this study, we evaluated the influence of microRNA-96 (miR-96) on osteoblast differentiation together with bone formation in a murine model of AS. The speculated relationship that miR-96 could bind to sclerostin (SOST) was verified by dual luciferase reporter assay. After successful model establishment, the mice with AS and osteoblasts isolated from mice with AS were treated with mimics or inhibitors of miR-96, or DKK-1 (a Wnt signaling inhibitor). The effects of gain- or loss-of-function of miR-96 on the inflammatory cytokine release (IL-6, IL-10, and TNF-α), alkaline phosphatase (ALP) activity, calcium nodule formation, along with the viability of osteoblasts were determined. It was observed that miR-96 might target and regulate SOST. Besides, miR-96 was expressed at a high level in AS mice while SOST expressed at a low level. TOP/FOP-Flash luciferase reporter assay confirmed that miR-96 activated the Wnt signaling pathway. Moreover, AS mice overexpressing miR-96 exhibited increased contents of IL-6, IL-10 and TNF-α, ALP activity, calcium nodule numbers, and viability of osteoblasts. In contrast, inhibition of miR-96 resulted in suppression of the osteoblast differentiation and bone formation. In conclusion, the study implicates that overexpressing miR-96 could improve osteoblast differentiation and bone formation in AS mice via Wnt signaling pathway activation, highlighting a potential new target for AS treatment.     

miR-409-5p negatively regulates Wnt/Beta catenin signaling pathway by targeting Lrp-8

Signaling pathways like Wnt play a vital part in all aspects of skeletal development which include osteoblastogenesis and osteoclastogenesis. Inactivation of Wnt signaling pathway leads to bone-related disorders, whereas activation of Wnt signaling pathway can cure bone pathologies like osteoporosis. Certain microRNA(s) have been identified that commune with Wnt signaling molecules to regulate osteogenesis. In this study we reported the identification of miR-409-5p as a suppressor of osteogenesis by targeting Lrp-8 which is a positive effector of Wnt signaling. Our study showed that overexpressing miR-409-5p inhibits osteoblast differentiation whereas obstructing miR-409-5p expression by anti-miR-409 promotes osteoblast functions and matrix mineralization. Using tools like targetscan and 3′-UTR luciferase reporter assay, Lrp-8 was confirmed as a straight target of miR-409-5p. By over expressing miR-409-5p, a repression of canonical Wnt/β catenin signaling was observed. These observations were strengthened by the fact that silencing of miR-409-5p in ovariectomized estrogen deficient Balb/c mice restored the loss of trabecular bone microarchitecture and suppressed bone resorption. Thus, targeting miR-409-5p may be helpful in increasing bone density in conditions like post menopausal osteoporosis.     

The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

To better understand the role of the canonical Wnt signaling pathway in cartilage development, we adenovirally expressed a constitutively active (ca) or a dominant negative (dn) form of lymphoid enhancer factor-1 (LEF-1), the main nuclear effector of the pathway, in undifferentiated mesenchymal cells, chondrogenic cells, and primary chondrocytes, and examined the expression of markers for chondrogenic differentiation and hypertrophy. caLEF-1 and LiCl, an activator of the canonical pathway, promoted both chondrogenic differentiation and hypertrophy, whereas dnLEF-1 and the gene silencing of beta-catenin suppressed LiCl-promoted effects. To investigate whether these effects were dependent on Sox9, a master regulator of cartilage development, we stimulated Sox9-deficient ES cells with the pathway. caLEF-1 and LiCl promoted both chondrogenic differentiation and hypertrophy in wild-type, but not in Sox9-deficient, cells. The response of Sox9-deficient cells was restored by the adenoviral expression of Sox9. Thus, the canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner.     

Inorganic phosphate as a signaling molecule in osteoblast differentiation

The spatial and temporal coordination of the many events required for osteogenic cells to create a mineralized matrix are only partially understood. The complexity of this process, and the nature of the final product, demand that these cells have mechanisms to carefully monitor events in the extracellular environment and have the ability to respond through cellular and molecular changes. The generation of inorganic phosphate during the process of differentiation may be one such signal. In addition to the requirement of inorganic phosphate as a component of hydroxyapatite mineral, Ca(10)(PO(4))(6)(OH)(2), a number of studies have also suggested it is required in the events preceding mineralization. However, contrasting results, physiological relevance, and the lack of a clear mechanism(s) have created some debate as to the significance of elevated phosphate in the differentiation process. More recently, a number of studies have begun to shed light on possible cellular and molecular consequences of elevated intracellular inorganic phosphate. These results suggest a model in which the generation of inorganic phosphate during osteoblast differentiation may in and of itself represent a signal capable of facilitating the temporal coordination of expression and regulation of multiple factors necessary for mineralization. The regulation of protein function and gene expression by elevated inorganic phosphate during osteoblast differentiation may represent a mechanism by which mineralizing cells monitor and respond to the changing extracellular environment.     

miR-27 promotes osteoblast differentiation by modulating Wnt signaling

Canonical Wnt signaling is particularly important for differentiation of human mesenchymal stem cells into osteoblast. MicroRNAs (miRNAs) also play an essential role in regulating cell differentiation. However, the role of miRNAs in osteoblast differentiation remains poorly understood. Here we found that the expression of miR-27 was increased during hFOB1.19 cells differentiation. Moreover, ectopic expression of miR-27 promoted hFOB1.19 cells differentiation, whereas its repression was sufficient to inhibit cell differentiation. Western blot analysis showed that the expression level of miR-27 was positively correlated with that of β-catenin, a key protein in Wnt signaling. Further, we verified that miR-27 directly targeted and inhibited adenomatous polyposis coli (APC) gene expression, and activated Wnt signaling through accumulation of β-catenin. This study suggests miR-27 is an important mediator of osteoblast differentiation, thus offering a new target for the development of preventive or therapeutic agents against osteogenic disorders.     

PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis

The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and β-glycerophosphate, and samples were collected from 0–26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using ¹²⁵I-PTHrP. The numbers of receptors per μg DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1 cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro. © 1996 Wiley-Liss, Inc.     

A blockade in Wnt signaling is activated following the differentiation of F9 teratocarcinoma cells

Aberrant activation of the Wnt signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the effect of Wnt signaling on the differentiation of epithelial cells, we used mouse teratocarcinoma F9 cells as a model. The F9 cells can be differentiated into visceral endoderm (VE) resembling absorptive columnar epithelial cells. We performed comparative gene expression analysis on retinoic acid-differentiated and undifferentiated F9 cells and confirmed that markers of VE and intestinal epithelium were induced upon differentiation. The induction of these markers by retinoic acid was reduced in the presence of Wnt, although Wnt alone did not change their expression. This suggests that Wnt signaling inhibited the differentiation of F9 cells by altering gene expression. This inhibition was also reflected in the morphology of the F9 cells as their apical-basal polarity was disrupted by inclusion of Wnt during differentiation. These results support a model in which Wnt modulates the expression of genes required for normal terminal differentiation of the stem cells. However, it follows that progenitor cells must escape from Wnt signaling to attain the differentiated state. Accordingly, we found that differentiated F9 cells no longer responded to Wnt and that a blockade in Wnt signaling occurred upstream of Axin. Consistent with this, Wnt negative regulators, such as Dickkopf-1 and Disabled-2, were induced upon the differentiation of F9 cells. We propose that a similar system to produce Wnt inhibitors regulates homeostasis of certain stem cell compartments in vivo.     

A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

One of the most important issues in stem cell research is to understand the regulatory mechanisms responsible for their differentiation. An extensive understanding of mechanism underlying the process of differentiation is crucial in order to prompt stem cells to perform a particular function after differentiation. To elucidate the molecular mechanisms responsible for the hematopoietic differentiation of embryonic stem cells (ESCs), we investigated murine ES cells for the presence of hematopoietic lineage markers as well as Wnt signaling pathway during treatments with different cytokines alone or in combination with another. Here we report that Wnt/beta-catenin signaling is down-regulated in hematopoietic differentiation of murine ES cells. We also found that differentiation induced by the interleukin-3, interleukin-6, and erythropoietin combinations resulted in high expression of CD3e, CD11b, CD45R/B220, Ly-6G, and TER-119 in differentiated ES cells. A high expression of beta-catenin was observed in two undifferentiated ES cell lines. Gene and protein expression analysis revealed that the members downstream of Wnt in this signaling pathway including beta-catenin, GSK-3beta, Axin, and TCF4 were significantly down-regulated as ES cells differentiated into hematopoietic progenitors. Our results show that the Wnt/beta-catenin signaling pathway plays a role in the hematopoietic differentiation of murine ESCs and also may support beta-catenin as a crucial factor in the maintenance of ES cells in their undifferentiated state.     

Dickkopf-1 antagonizes Wnt signaling independent of beta-catenin in human mesothelioma

Dickkopf-1 (Dkk-1) is a secreted protein that acts as a potent inhibitor of the Wnt signal transduction pathway. It is thought that the antagonistic effect of Dkk-1 is specific to the canonical (Wnt/beta-catenin) pathway. In this study, we demonstrate that restoration of Dkk-1 expression suppresses cell growth and induces apoptotic cell death in beta-catenin-deficient mesothelioma cell lines H28 and MS-1. Furthermore, we found that a small-molecule inhibitor of JNK inhibited the apoptosis induced by Dkk-1 overexpression in these cells. Together, our data suggest that Dkk-1 may be able to antagonize Wnt signaling and exert its tumor suppressive effects through beta-catenin-independent non-canonical pathways (i.e., the Wnt/JNK pathway).     

Wnt信号通路与前体脂肪细胞

Wnt蛋白是一类分泌型蛋白生长因子,通过自分泌和旁分泌作用调节多种细胞的发生和发育.新近研究表明,Wnt信号通路在前体脂肪细胞的增殖分化中发挥着重要作用.Wnt蛋白的配基通过与细胞膜上的特异性受体Frizzled1/2/5及辅助受体LRP5/6结合,激活经典或非经典的Wnt信号通路,影响下游靶基因产物的磷酸化作用,进而抑制C/EBPα、PPARγ等脂肪细胞关键转录因子,使细胞保持未分化状态,从而抑制脂肪的形成.本文就Wnt信号通路的研究史和主要分支、作用方式及其抑制脂肪细胞的机制方面进行了综述,并对今后的研究方向和应用作了展望.     

Wnt3‐frizzled 1 chimera as a model to study canonical Wnt signaling

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low‐density lipoprotein receptor‐related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF‐luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF‐luciferase activation. DKK‐1 protein blocks upregulation of the TCF‐luciferase reporter by the Wnt3–Fz1 chimera, suggesting involvement of LRP in Wnt3–Fz1 signaling. Expression of a Wnt3–Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3–Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt–Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases. J. Cell. Biochem. 109: 876–884, 2010. © 2009 Wiley‐Liss, Inc.     

ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice

Osteogenic differentiation refers to the process of bone formation and remodeling, which is controlled by complex molecular mechanisms. Activin A receptor type I (ACVR1) is reported to be associated with osteogenic differentiation. However, the underlying molecular mechanism remains elusive. Therefore, this study evaluates the function of ACVR1 in osteogenic differentiation through the Wnt signaling pathway. The expression of osteocalcin (Oc) and osterix together with osteogenic differentiation and mineralization was examined in ACVR1-knockout (KO) mouse. Furthermore, the Wnt signaling pathway was inhibited in bone marrow stromal cells (BMSCs) of mice to explore the role of the Wnt signaling pathway in osteogenic differentiation by means of alkaline phosphatase (ALP) activity detection and evaluation of mineralized nodules and calcium content. Subsequently, the effect of ACVR1 on the Wnt signaling pathway was assessed by determining the expression of ACVR1, β-catenin, glycogen synthase kinase 3 β (GSK3β), dickkopf-related protein 1 (DKK1), and frizzled class receptor 1 (FZD1). Both their effects on osteogenic differentiation were further evaluated by determination of Oc, osterix, and Runx2 expression. AVCR1 KO mice exhibited increased Oc and osterix expression and promoted bone resorption and formation. ACVR1-knockout was observed to activate the Wnt signaling pathway with an increase of β-catenin and reductions in GSK3β, DKK1, and FZD1. With the inhibited Wnt signaling pathway expression of Oc, osterix, and Runx2 was decreased, and ALP activity, mineralized nodule, and calcium content in cellular matrix were decreased as well, indicating that inactivation of the Wnt signaling pathway reduced the differentiation of BMSCs into osteoclasts. These findings indicate that ACVR1-knockout promotes osteogenic differentiation by activating the Wnt signaling pathway in mice.     

Wnt信号通路在毛细胞分化和再生过程中的作用

Wnt信号通路在生物发育和维持内环境稳态过程中起着重要作用。Wnt配体通过与Frizzle受体结合参与体轴的形成、细胞分化和细胞命运决定等生命活动。在小鼠内耳发育过程中,Wnt信号通路扮演了重要角色：在内耳发育早期阶段,参与听基板的特化和听泡的形成;在内耳发育后期阶段,调控毛细胞分化及毛细胞纤毛束的定向。本文综述了Wnt信号通路在内耳毛细胞发育分化及再生过程中的研究进展,以期为从事相关领域的科研人员提供参考。     

Xenopus galectin-VIa shows highly specific expression in cement glands and is regulated by canonical Wnt signaling

Anterior-posterior neural patterning of Xenopus embryo is determined during gastrulation and then followed by differentiation of neural structures including brain and eye. The cement gland is a mucus-secreting neural organ located in the anterior end of the neural plate. This study analyzed expression patterns of Xenopus galectin-VIa (Xgalectin-VIa) by whole-mount in situ hybridization, and found highly restricted expression of this gene in the cement gland region. These patterns were similar to those of XAG-1 and XCG, known cement gland-specific genes. In addition, Xgalectin-VIa was expressed in the dorsal edge of eye vesicles, the otic vesicle, and in part of the hatching gland at the tadpole stage. Although the spatial expression pattern was similar, the temporal expression of Xgalectin-VIa differed from that of XAG-1 and XCG. RT-PCR analysis showed only weak Xgalectin-VIa expression in early neurula embryos, whereas both XAG-1 and CGS were strongly expressed at that stage. We also showed that Xgalectin-VIa expression is repressed by enhancement of Wnt signaling and increased by its inhibition. Furthermore, Xgalectin-VIa expression was activated by neural-gene inducer Xotx2, as is the case for XAG-1 and CGS. Together, these results indicated that Xgalectin-VIa possesses different features from other cement gland genes and is a novel and useful marker of the cement gland in developing embryos.