Achyranthoside D attenuates chondrocyte loss and inflammation in osteoarthritis via targeted regulation of Wnt3a.

BACKGROUND: Achyranthes bidentata Blume (A. bidentata) is a common Chinese herb used to treat osteoarthritis (OA). Achyranthoside D (Ach-D) is a glucuronide saponin isolated from A. bidentata. PURPOSE: To assess the mechanisms of action of Ach-D and its effects on OA. METHODS: The effects of Ach-D were evaluated in rats underwent anterior cruciate ligament transection (ACLT) with medial meniscectomy (MMx) and in interleukin (IL)-1ß-induced chondrocytes. Histological changes in rat cartilage tissues were detected using Safranin O-Fast green and haematoxylin-eosin staining. Immunohistochemical staining, qRT-PCR, ELISA, immunoblotting, and immunofluorescence were conducted to examine cartilage degeneration-related and inflammation-related factor expression. CCK-8, LDH assay, and EdU staining were performed to detect chondrocyte death. RESULTS: Ach-D dose-dependently reduced the Osteoarthritis Research Society International (OARSI) scores, alleviated cartilage injury, and decreased the serum concentrations of CTX-II and COMP in ACLT-MMx models. Ach-D increased the expression levels of collagen II and aggrecan and decreased the levels of cartilage degeneration-related proteins, ADAMTS-5, MMP13, and MMP3, in rat cartilage tissues. Additionally, nod-like receptor protein 3 (NLRP3)-related inflammation was reduced by Ach-D, as shown by the significantly inhibited expression levels of NLRP3, ASC, GSDMD, IL-6, TNF-α, IL-1ß, and IL-18 in rat cartilage tissues. In primary rat chondrocytes, Ach-D protected against IL-1ß-induced viability loss and LDH release. Wnt3a is the target protein of Ach-D. Mechanistically, Ach-D alleviated OA by inhibiting Wnt signalling. CONCLUSION: ACH-D may reduce inflammation and cartilage degeneration by inhibiting the Wnt signalling pathway, thereby reducing OA.

Paeonolide as a Novel Regulator of Core-Binding Factor Subunit Alpha-1 in Bone-Forming Cells.

Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 µM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 µM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 µM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 µM) increased the bone morphogenetic protein (BMP)-Smad1/5/8 and Wnt-ß-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 µM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.

HBO Promotes the Differentiation of Neural Stem Cells via Interactions Between the Wnt3/ß-Catenin and BMP2 Signaling Pathways.

Hyperbaric oxygen (HBO) therapy may promote neurological recovery from hypoxic-ischemic encephalopathy (HIE). However, the therapeutic effects of HBO and its associated mechanisms remain unknown. The canonical Wnt/ß-catenin signaling pathways and bone morphogenetic protein (BMP) play important roles in mammalian nervous system development. The present study examined whether HBO stimulates the differentiation of neural stem cells (NSCs) and its effect on Wnt3/ß-catenin and BMP2 signaling pathways. We showed HBO treatment (2 ATA, 60 min) promoted differentiation of NSCs into neurons and oligodendrocytes in vitro. In addition, rat hypoxic-ischemic brain damage (HIBD) tissue extracts also promoted the differentiation of NSCs into neurons and oligodendrocytes, with the advantage of reducing the number of astrocytes. These effects were most pronounced when these two were combined together. In addition, the expression of Wnt3a, BMP2, and ß-catenin nuclear proteins were increased after HBO treatment. However, blockade of Wnt/ß-catenin or BMP signaling inhibited NSC differentiation and reduced the expression of Wnt3a, BMP2, and ß-catenin nuclear proteins. In conclusion, HBO promotes differentiation of NSCs into neurons and oligodendrocytes and reduced the number of astrocytes in vitro possibly through regulation of Wnt3/ß-catenin and BMP2 signaling pathways. HBO may serve as a potential therapeutic strategy for treating HIE.

Guhong Injection promotes fracture healing by activating Wnt/beta-catenin signaling pathway in vivo and in vitro.

Guhong Injection (GHI), composed of aceglutamide and safflower aqueous extract, has been applied to the clinical treatment of orthopedic diseases, but the relevant mechanism by which GHI exerts effects on bone remodeling has not been reported. In the present study, we investigated the effects of various concentrations of GHI (2.5, 5 and 10 ml/kg) in accelerating rat tibia healing progress by observing haematoxylin and eosin (HE) stained sections, detecting the activity of bone metabolism biochemical markers such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), osteocalcin (OC) and C-terminal crosslinking telopeptide of type Ⅰ collagen (CTX-1) in rat serum, as well as measuring the expressions of collagen I (COL-1) and collagen II (COL-2) in rat tibia. Also, we investigated the effects of different concentrations of GHI (30, 60 and 90 µl/ml) on the proliferation and differentiation of osteoblasts (OBs) through proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP) and type I collagen (COL-1). At the same time, the expression of important factors of Wnt/beta-catenin signaling pathway including Wnt-3a, beta-catenin, disheveled-1 (Dvl-1), glycogen synthase kinases-3beta (GSK-3beta), lymphoid enhancing factor-1 (LEF-1) and axis inhibition protein-2 (Axin-2) after GHI intervention was detected by quantitative real-time PCR (q-PCR), immunohistochemistry and Western blotting. In vivo, rats of tibia fracture model treated with intraperitoneal injection (ip) of GHI had more mature fibroblasts, as well as shorter period formation of new bone. The levels of BMP-2, TGF-beta and OC in rat serum were significantly up-regulated, while the level of CTX was down-regulated. After 4 weeks of drug treatment, the level of COL-1 in the rat tibia increased, but there was no significant change in the level of COL-2. In vitro, after drug intervention, the number of OBs increased significantly, the activities of PCNA, ALP and COL-1 were enhanced. Treatment with GHI increased the mRNA and protein expression of Wnt-3a, beta-catenin, Dvl-1 and LEF-1, and decreased the expression of mRNA of Axin-2 and GSK-3beta. All results demonstrate that GHI accelerates the proliferation of OBs and shortens the recovery time of bone structure, and the Wnt/beta-catenin signaling pathway is involved in the regulation process.

The effects of photobiomodulation therapy on mouse pre-osteoblast cell line MC3T3-E1 proliferation and apoptosis via miR-503/Wnt3a pathway.

Photobiomodulation therapy (PBMT) has been demonstrated as regulating osteoblast proliferation. MicroRNAs (miRNAs) are involved in various pathophysiologic processes in osteoblast, but the role of miRNAs in the PBMT-based promotion of osteoblast proliferation remains unclear. This study aimed to investigate the effects of PBMT treatment (3.75 J/cm2) on mouse pre-osteoblast cell line MC3T3-E1 proliferation and apoptosis via the miR-503/Wnt3a pathway; meanwhile, detect the expressions of miR-503 and Wnt3a after PBMT treatment and the role of miR-503 in regulating Wnt signaling molecules Wnt3a, ß-catenin, Runx2, apoptotic proteins caspase-3, and Bcl-2 in vitro. The PBMT parameters were as follows: 808 nm continuous wavelength, 0.401 W output power, 0.042 W/cm2 power density, 9.6 cm2 spot size, 36 J energy, 3.75 J/cm2 energy density, 90 s irradiation for three times per 12 h, 14.5 cm distance of the laser source and the angle of divergence of the laser beam was 7°. In our present study, the target relationship was predicted and verified by bioinformatics analysis and luciferase reporter assays. Gene mRNA and protein expressions were examined by qPCR and western blot analysis. The MTT method was used to evaluate the effect of miR-503 on MC3T3-E1 cells proliferation. And cell apoptosis was examined by flow cytometry. The results showed that PBMT treatment reduced the expression of miR-503 and increased the level of Wnt3a (p < 0.01). Bioinformatics analysis and luciferase reporter assays revealed that Wnt3a was a target of miR-503, and Wnt3a was regulated by miR-503. Furthermore, miR-503 was found to functionally inhibit proliferation and promote apoptosis (p < 0.01). And during this process, Wnt3a, ß-catenin, Runx2, and Bcl-2 expressions were significantly inhibited (p < 0.01); however, caspase-3 level was upregulated (p < 0.01). These results suggest that miR-503 plays a role in osteoblast proliferation and apoptosis in response to PBMT, which is potentially amenable to therapeutic manipulation for clinical application.

MiR 376c inhibits osteoblastogenesis by targeting Wnt3 and ARF-GEF-1 -facilitated augmentation of beta-catenin transactivation.

Wnt signaling pathway plays important role in all aspects of skeletal development which include chondrogenesis, osteoblastogenesis, and osteoclastogenesis. Induction of the Wnt-3 signaling pathway promotes bone formation while inactivation of the pathway leads to bone related disorders like osteoporosis. Wnt signaling thus has become a desired target to treat osteogenic disorders. MicroRNAs (miRNAs) represent an important category of elements that interact with Wnt signaling molecules to regulate osteogenesis. Here, we show that miR-376c, a well-characterized tumor suppressor which inhibits cell proliferation and invasion in osteosarcoma by targeting to transforming growth factor-alpha, suppresses osteoblast proliferation, and differentiation. Over-expression of miR-376c inhibited osteoblast differentiation, whereas inhibition of miR-376c function by antimiR-376c promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Target prediction analysis tools and experimental validation by luciferase 3' UTR reporter assay along with qRT-PCR identified Wnt-3 and ARF-GEF-1 as direct targets of miR-376c. It was seen that over-expression of miR-376c leads to repression of canonical Wnt/ß-catenin signaling. Our overall results suggest that miR-376c targets Wnt-3 and ARF-GEF-1 suppresses ARF-6 activation which prevents the release of ß-catenin and its transactivation thereby inhibiting osteoblast differentiation. Although miR-376c is known to be a tumor repressor; we have identified a second complementary function of miR-376c where it inhibits Wnt-3-mediated osteogenesis and promotes bone loss.

Thalamic WNT3 Secretion Spatiotemporally Regulates the Neocortical Ribosome Signature and mRNA Translation to Specify Neocortical Cell Subtypes.

Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex. SIGNIFICANCE STATEMENT: The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by ingrowing axons from the thalamus regulates the combinatorial composition of ribosomal proteins in developing neocortex, which we term the "neocortical ribosome signature." Thalamic WNT3 further regulates the specificity of mRNA translation and development of neurons and oligodendrocytes in the neocortex. This study advances our overall understanding of WNT signaling and the spatiotemporal regulation of mRNA translation in highly complex developing systems.

Wnt3 and Wnt3a are required for induction of the mid-diencephalic organizer in the caudal forebrain.

BACKGROUND: A fundamental requirement for development of diverse brain regions is the function of local organizers at morphological boundaries. These organizers are restricted groups of cells that secrete signaling molecules, which in turn regulate the fate of the adjacent neural tissue. The thalamus is located in the caudal diencephalon and is the central relay station between the sense organs and higher brain areas. The mid-diencephalic organizer (MDO) orchestrates the development of the thalamus by releasing secreted signaling molecules such as Shh. RESULTS: Here we show that canonical Wnt signaling in the caudal forebrain is required for the formation of the Shh-secreting MD organizer in zebrafish. Wnt signaling induces the MDO in a narrow time window of 4 hours - between 10 and 14 hours post fertilization. Loss of Wnt3 and Wnt3a prevents induction of the MDO, a phenotype also observed upon blockage of canonical Wnt signaling per se. Pharmaceutical activation of the canonical Wnt pathways in Wnt3/Wnt3a compound morphant embryos is able to restore the lack of the MDO. After blockage of Wnt signaling or knock-down of Wnt3/Wnt3a we find an increase of apoptotic cells specifically within the organizer primordium. Consistently, blockage of apoptosis restores the thalamus organizer MDO in Wnt deficient embryos. CONCLUSION: We have identified canonical Wnt signaling as a novel pathway, that is required for proper formation of the MDO and consequently for the development of the major relay station of the brain - the thalamus. We propose that Wnt ligands are necessary to maintain the primordial tissue of the organizer during somitogenesis by suppressing Tp53-mediated apoptosis.