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Find video protocols related to scientific articles indexed in Pubmed.
LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation.
Biochem. Biophys. Res. Commun.
PUBLISHED: 07-17-2014
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Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/?-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a ?-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/?-catenin signaling.
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Congenital myasthenic syndrome in Japan: Ethnically unique mutations in muscle nicotinic acetylcholine receptor subunits.
Neuromuscul. Disord.
PUBLISHED: 05-29-2014
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Congenital myasthenic syndromes (CMS) are caused by mutations in genes expressed at the neuromuscular junction. Most CMS patients have been reported in Western and Middle Eastern countries, and only four patients with COLQ mutations have been reported in Japan. We here report six mutations in acetylcholine receptor (AChR) subunit genes in five Japanese patients. Five mutations are novel, and one mutation is shared with a European American patient but with a different haplotype. Among the observed mutations, p.Thr284Pro (p.Thr264Pro according to the legacy annotation) in the epsilon subunit causes a slow-channel CMS. Five other mutations in the delta and epsilon subunits are splice site, frameshift, null, or missense mutations causing endplate AChR deficiency. We also found a heteroallelic p.Met465Thr in the beta subunit in another patient. p.Met465Thr, however, was likely to be polymorphism, because single channel recordings showed mild shortening of channel openings without affecting cell surface expression of AChR, and the minor allelic frequency of p.Met465Thr was 5.1% in the Japanese population. Lack of shared mutant alleles between the Japanese and the other patients suggests that most mutations described here are ethnically unique or de novo in each family.
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Verapamil protects against cartilage degradation in osteoarthritis by inhibiting Wnt/?-catenin signaling.
PLoS ONE
PUBLISHED: 01-01-2014
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In past years, the canonical Wnt/?-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/?-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/?-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of ?-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 ?1, 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 ?-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/?-catenin signaling.
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LRP4 third ?-propeller domain mutations cause novel congenital myasthenia by compromising agrin-mediated MuSK signaling in a position-specific manner.
Hum. Mol. Genet.
PUBLISHED: 11-13-2013
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Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Using Sanger and exome sequencing in a CMS patient, we identified two heteroallelic mutations, p.Glu1233Lys and p.Arg1277His, in LRP4 coding for the postsynaptic low-density lipoprotein receptor-related protein 4. LRP4, expressed on the surface of the postsynaptic membrane of the neuromuscular junction, is a receptor for neurally secreted agrin, and LRP4 bound by agrin activates MuSK. Activated MuSK in concert with Dok-7 stimulates rapsyn to concentrate and anchor AChR on the postsynaptic membrane and interacts with other proteins implicated in the assembly and maintenance of the neuromuscular junction. LRP4 also functions as an inhibitor of Wnt/beta-catenin signaling. The identified mutations in LRP4 are located at the edge of its 3rd beta-propeller domain and decrease binding affinity of LRP4 for both MuSK and agrin. Mutations in the LRP4 3rd beta-propeller domain were previously reported to impair Wnt signaling and cause bone diseases including Cenani-Lenz syndactyly syndrome and sclerosteosis-2. By analyzing naturally occurring and artificially introduced mutations in the LRP4 3rd beta-propeller domain, we show that the edge of the domain regulates the MuSK signaling whereas its central cavity governs Wnt signaling. We conclude that LRP4 is a new CMS disease gene and that the 3rd beta propeller domain of LRP4 mediates the two signaling pathways in a position-specific manner.
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RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-?-catenin signaling.
Science
PUBLISHED: 02-14-2013
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Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-?-catenin network, where it acts as a regulatory subunit of CK1?: In a Wnt-dependent manner, DDX3 binds CK1? and directly stimulates its kinase activity, and promotes phosphorylation of the scaffold protein dishevelled. DDX3 is required for Wnt-?-catenin signaling in mammalian cells and during Xenopus and Caenorhabditis elegans development. The results also suggest that the kinase-stimulatory function extends to other DDX and CK1 members, opening fresh perspectives for one of the longest-studied protein kinase families.
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Meclozine Facilitates Proliferation and Differentiation of Chondrocytes by Attenuating Abnormally Activated FGFR3 Signaling in Achondroplasia.
PLoS ONE
PUBLISHED: 01-01-2013
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Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.
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An ATF2-based luciferase reporter to monitor non-canonical Wnt signaling in Xenopus embryos.
Dev. Dyn.
PUBLISHED: 05-10-2011
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Non-canonical/planar cell polarity (PCP) Wnt signaling plays important roles in embryonic development and tissue homeostasis, and is implicated in human disease. Monitoring Wnt/PCP signaling relies mostly on semi-quantitative bioassays or biochemical analysis. Here we describe a luciferase reporter assay based on an ATF2 response element, which faithfully monitors non-canonical Wnt signaling in Xenopus embryos. The assay is simple, quantitative, and robust. It can be used to detect non-canonical Wnt signaling changes following gain and loss of function of pathway components, including Wnt, Frizzled, Ror2, Disheveled, Rac1, MKK7, and JNK. Wnt/PCP signaling has recently been implicated in left-right asymmetry and our reporter assay suggests that in gastrula embryos there is a right-ward bias in Wnt/PCP signaling. We also mapped Wnt/PCP signaling in the early Xenopus embryo and find that it peaks in the dorso-vegetal region, paralleling Wnt/?-catenin signaling.
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Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis.
Dev. Cell
PUBLISHED: 03-15-2011
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The R-Spondin (Rspo) family of secreted Wnt modulators is involved in development and disease and holds therapeutic promise as stem cell growth factors. Despite growing biological importance, their mechanism of action is poorly understood. Here, we show that Rspo3 binds syndecan 4 (Sdc4) and that together they activate Wnt/PCP signaling. In Xenopus embryos, Sdc4 and Rspo3 are essential for two Wnt/PCP-driven processes-gastrulation movements and head cartilage morphogenesis. Rspo3/PCP signaling during gastrulation requires Wnt5a and is transduced via Fz7, Dvl, and JNK. Rspo3 functions by inducing Sdc4-dependent, clathrin-mediated endocytosis. We show that this internalization is essential for PCP signal transduction, suggesting that endocytosis of Wnt-receptor complexes is a key mechanism by which R-spondins promote Wnt signaling.
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Wnt/Frizzled signaling requires dPRR, the Drosophila homolog of the prorenin receptor.
Curr. Biol.
PUBLISHED: 05-05-2010
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Wnt/Wg signaling pathways are of key importance during development and disease. Canonical and noncanonical Wnt/Frizzled (Fz) pathways share a limited number of signaling components that are part of the membrane proximal signaling complex. In Drosophila, Fz and Dishevelled (Dsh) are the only two components known to be involved in both Wnt/beta-catenin and planar cell polarity (PCP) signaling. PCP signaling is required for the planar polarization of epithelial cells, which occurs, for instance, during hair orientation and gastrulation in vertebrates. Both pathways have been studied intensively in the past years. However, it still remains unresolved whether additional components are required at the receptor complex. Here we identify the Drosophila homolog of the mammalian prorenin receptor (dPRR) as a conserved modulator of canonical Wnt/beta-cat and Fz/PCP signaling. We show that dPRR depletion affects Wg target genes in cultured cells and in vivo. PRR is required for epithelial planar polarity in Drosophila and for convergent extension movements in Xenopus gastrulae. Furthermore, dPRR binds to Fz and Fz2 receptors. In summary, our data suggest that dPRR has an evolutionarily conserved role at the receptor level for activation of canonical and noncanonical Wnt/Fz signaling pathways.
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Requirement of prorenin receptor and vacuolar H+-ATPase-mediated acidification for Wnt signaling.
Science
PUBLISHED: 01-23-2010
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Wnt/beta-catenin signaling is important in stem cell biology, embryonic development, and disease, including cancer. However, the mechanism of Wnt signal transmission, notably how the receptors are activated, remains incompletely understood. We found that the prorenin receptor (PRR) is a component of the Wnt receptor complex. PRR functions in a renin-independent manner as an adaptor between Wnt receptors and the vacuolar H+-adenosine triphosphatase (V-ATPase) complex. Moreover, PRR and V-ATPase were required to mediate Wnt signaling during antero-posterior patterning of Xenopus early central nervous system development. The results reveal an unsuspected role for the prorenin receptor, V-ATPase activity, and acidification during Wnt/beta-catenin signaling.
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