Loss of Klotho contributes to cartilage damage by derepression of canonical Wnt/β-catenin signaling in osteoarthritis mice.
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ABSTRACT:
SUBMITTER: Gu Y
PROVIDER: S-EPMC6949099 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/β-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.
| S-EPMC5604944 | biostudies-literature
Project description:In past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. FRZB, a soluble antagonist of Wnt signaling, has been studied in osteoarthritis (OA) animal models and OA patients as a modulator of Wnt signaling. We screened for FDA-approved drugs that induce FRZB expression and suppress Wnt/β-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/β-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of β-catenin was attenuated by verapamil in OA chondrocytes. Lack of the verapamil effects in LiCl-treated and FRZB-downregulated OA chondrocytes also suggested that verpamil suppressed Wnt signaling by inducing FRZB. Verapamil enhanced gene expressions of chondrogenic markers of ACAN encoding aggrecan, COL2A1 encoding collagen type II α1, and SOX9, and suppressed Wnt-responsive AXIN2 and MMP3 in human OA chondrocytes. Verapamil ameliorated Wnt3A-induced proteoglycan loss in chondrogenically differentiated ATDC5 cells. Verapamil inhibited hypertrophic differentiation of chondrocytes in the explant culture of mouse tibiae. Intraarticular injection of verapamil inhibited OA progression as well as nuclear localizations of β-catenin in a rat OA model. We propose that verapamil holds promise as a potent therapeutic agent for OA by upregulating FRZB and subsequently downregulating Wnt/β-catenin signaling.
| S-EPMC3962439 | biostudies-literature
Project description:BackgroundGEM (GTP-binding protein overexpressed in skeletal muscle) is one of the atypical small GTPase subfamily members recently identified as a regulator of cell differentiation. Abnormal chondrogenesis coupled with an imbalance in the turnover of cartilaginous matrix formation is highly relevant to the onset and progression of osteoarthritis (OA). However, how GEM regulates chondrogenic differentiation remains unexplored.MethodsCartilage tissues were obtained from OA patients and graded according to the ORASI and ICRS grading systems. The expression alteration of GEM was detected in the Grade 4 cartilage compared to Grade 0 and verified in OA mimic culture systems. Next, to investigate the specific function of GEM during these processes, we generated a Gem knockdown (Gem-Kd) system by transfecting siRNA targeting Gem into ATDC5 cells. Acan, Col2a1, Sox9, and Wnt target genes of Gem-Kd ATDC5 cells were detected during induction. The transcriptomic sequencing analysis was performed to investigate the mechanism of GEM regulation. Wnt signaling pathways were verified by real-time PCR and immunoblot analysis. Finally, a rescue model generated by treating Gem-KD ATDC5 cells with a Wnt signaling agonist was established to validate the mechanism identified by RNA sequencing analysis.ResultsA decreased expression of GEM in OA patients' cartilage tissues and OA mimic chondrocytes was observed. While during chondrogenesis differentiation and cartilage matrix formation, the expression of GEM was increased. Gem silencing suppressed chondrogenic differentiation and the expressions of Acan, Col2a1, and Sox9. RNA sequencing analysis revealed that Wnt signaling was downregulated in Gem-Kd cells. Decreased expression of Wnt signaling associated genes and the total β-CATENIN in the nucleus and cytoplasm were observed. The exogenous Wnt activation exhibited reversed effect on Gem loss-of-function cells.ConclusionThese findings collectively validated that GEM functions as a novel regulator mediating chondrogenic differentiation and cartilage matrix formation through Wnt/β-catenin signaling.
| S-EPMC10548561 | biostudies-literature
Project description:BACKGROUND:Both loss- and gain-of-function of Wnt/?-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/?-catenin signaling in the superficial zone (SFZ) of articular cartilage. METHODS:Wnt/?-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice for loss- and gain-of-function, respectively, of Wnt/?-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/?-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/?-catenin signaling in SFZ cells. RESULTS:Wnt/?-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific ?-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific ?-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by ?-catenin knockout, while it was upregulated by ?-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/?-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4. CONCLUSIONS:We demonstrated that Wnt/?-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.
| S-EPMC6880374 | biostudies-literature
Project description:Aging is characterized by the accumulation of DNA damage and a decrease in stem cell functionality, yet molecular mechanisms that limit the maintenance of stem cells in response to DNA damage remain to be delineated. Here we show in mouse models that DNA damage leads to a transient over-activation of Wnt signaling in hematopoietic stem cells (HSCs), and that high activity of canonical Wnt/?-catenin signaling sensitizes HSCs to DNA damage induced by X-irradiation which results in preferential maintenance of HSCs with low levels of Wnt signaling. The study shows that genetic or chemical activation of canonical Wnt signaling enhances radiosensitivity of HSCs while inhibition of Wnt signaling decreases it. Together, these results indicate that levels of Wnt signaling activity mediate heterogeneity in the sensitivity of HSCs to DNA damage induced depletion. These findings could be relevant for molecular alterations and selection of stem cells in the context of DNA damage accumulation during aging and cancer formation.
| S-EPMC6987068 | biostudies-literature
Project description:Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing ?-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic ?-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.
| S-EPMC5815871 | biostudies-literature
Project description:ObjectiveHepcidin deficiency is known to cause body iron accumulation and bone microarchitecture defects, but the exact underlying mechanisms of hepcidin deficiency-induced bone loss remain unclear. Our objective was to understand the molecular mechanism of hepcidin deficiency-induced bone loss.MethodsThe bone phenotypes of wild type (WT) and hepcidin knockout (Hepcidin-KO) mice were measured by microcomputed tomography. The osteoclastic marker of the bone was measured by tartrate-resistant acid phosphatase staining. The osteoblastic marker of the bone was measured by immunostaining of osteocalcin. Primary osteoblastic and osteoclastic differentiation was performed using bone marrow cells. The mature osteoclast was determined by tartrate-resistant acid phosphatase staining, pit formation assay and relative gene expression. The mature osteoblast was determined by alkaline phosphatase activity, alkaline phosphatase staining, Alizarin Red staining and relative gene expression. The protein expression of β-catenin, TCF4/TCF7L2 and Forkhead box O3a (FOXO3a) was measured by Western blot and their combination by co-immunoprecipitation. In vivo study was performed by tail vein administration of FOXO3a-RNAi using an adeno-associated virus in Hepcidin-KO mice.ResultsWe found that Hepcidin-KO mice exhibited iron accumulation and bone loss compared with WT mice. The osteoclastic differentiation of bone marrow-derived macrophages from Hepcidin-KO mice was not significantly different from that of bone marrow-derived macrophages from WT mice. However, the osteoblastic differentiation of bone marrow-derived mesenchymal stem cells from Hepcidin-KO mice was obviously decreased compared with that of bone marrow-derived mesenchymal stem cells from WT mice. Furthermore, it was confirmed in this study that upon hepcidin deficiency, β-catenin, TCF4/TCF7L2 and FOXO3a expression in bone tissues was not altered, but β-catenin combination with TCF4/TCF7L2 was strongly inhibited by β-catenin combination with FOXO3a, indicating that the canonical Wnt/β-catenin pathway was affected. Tail vein administration of FOXO3a-RNAi using an adeno-associated virus in Hepcidin-KO mice resulted in bone mass recovery.ConclusionThese findings suggested that hepcidin deficiency might cause bone loss by interfering with the canonical Wnt/β-catenin pathway via FOXO3a, and FOXO3a inhibition would be a possible approach to treat hepcidin deficiency-induced bone loss.The translational potential of this articleHepcidin deficiency, as well as iron accumulation, has been considered as a risk factor for osteoporosis. For this kind of osteoporosis, inhibition of FOXO3a either by neutralized antibody or AAV-mediated RNAi, represents an effective and promising method.
| S-EPMC7267010 | biostudies-literature
Project description:Canonical WNT/?-catenin signaling is involved in most of the mechanisms that lead to the formation and development of cancer cells. It plays a central role in three cyclic processes, which are the cell division cycle, the immune cycle, and circadian rhythms. When the canonical WNT pathway is upregulated as in cancers, the increase in ?-catenin in the nucleus leads to activation of the expression of numerous genes, in particular CYCLIN D1 and cMYC, where the former influences the G1 phase of the cell division cycle, and the latter, the S phase. Every stage of the immune cycle is disrupted by the canonical WNT signaling. In numerous cancers, the dysfunction of the canonical WNT pathway is accompanied by alterations of the circadian genes (CLOCK, BMAL1, PER). Induction of these cyclic phenomena leads to the genesis of thermodynamic mechanisms that operate far from equilibrium, and that have been called "dissipative structures." Moreover, upregulation of the canonical WNT/?-catenin signaling is important in the myofibroblasts of the cancer stroma. Their differentiation is controlled by the canonical WNT /TGF-?1 signaling. Myofibroblasts present ultraslow contractile properties due to the presence of the non-muscle myosin IIA. Myofibroblats also play a role in the inflammatory processes, often found in cancers and fibrosis processes. Finally, upregulated canonical WNT deviates mitochondrial oxidative phosphorylation toward the Warburg glycolysis metabolism, which is characteristic of cancers. Among all these cancer-generating mechanisms, the upregulated canonical WNT pathway would appear to offer the best hope as a therapeutic target, particularly in the field of immunotherapy.
| S-EPMC6876670 | biostudies-literature
Project description:Wnt/?-catenin signalling is central to development and its regulation is essential in preventing cancer. Using phosphorylation of Dishevelled as readout of pathway activation, we identified Drosophila Wnk kinase as a new regulator of canonical Wnt/?-catenin signalling. WNK kinases are known for regulating ion co-transporters associated with hypertension disorders. We demonstrate that wnk loss-of-function phenotypes resemble canonical Wnt pathway mutants, while Wnk overexpression causes gain-of-function canonical Wnt-signalling phenotypes. Importantly, knockdown of human WNK1 and WNK2 also results in decreased Wnt signalling in mammalian cell culture, suggesting that Wnk kinases have a conserved function in ensuring peak levels of canonical Wnt signalling.
| S-EPMC3736130 | biostudies-literature
Project description:The development of skeletal muscle from immature precursors is partially driven by canonical WNT/?-catenin signaling. Rhabdomyosarcomas (RMS) are immature skeletal muscle-like, highly lethal cancers with a variably pronounced blockade of muscle differentiation. To investigate whether canonical ?-catenin signaling in RMS is involved in differentiation and aggressiveness of RMS, we analyzed the effects of WNT3A and of a siRNA-mediated or pharmacologically induced ?-catenin knock-down on proliferation, apoptosis and differentiation of embryonal and alveolar RMS cell lines. While the canonical WNT pathway was maintained in all cell lines as shown by WNT3A induced AXIN expression, more distal steps including transcriptional activation of its key target genes were consistently impaired. In addition, activation or inhibition of canonical WNT/?-catenin only moderately affected proliferation, apoptosis or myodifferentiation of the RMS tumor cells and a conditional knockout of ?-catenin in RMS of Ptch del/+ mice did not alter RMS incidence or multiplicity. Together our data indicates a subordinary role of the canonical WNT/?-catenin signaling for RMS proliferation, apoptosis or differentiation and thus aggressiveness of this malignant childhood tumor.
| S-EPMC6290061 | biostudies-literature