Bisei Ohkawara

Requirement of Prorenin Receptor and Vacuolar H + -ATPase-Mediated Acidification for Wnt Signaling

Of Wnt, Prorenin Receptor, and V-ATPase The Wnt protein binds to receptors at the cell surface and regulates signaling pathways that control a wide range of critical biological processes from stem cell differentiation to generation of cancer. In a screen for components required for Wnt signaling, Cruciat et al. (p. 459) discovered an unexpected partner, the prorenin receptor (PRR). PRR bound to the Wnt receptor proteins Fz8 and LRP6. The PRR protein could interact with the receptors and promote Wnt signaling without its cytoplasmic domain through which it initiates signaling in response to the prorenin protein. Its role in Wnt signaling appears to be rather different. The PRR binds to the vacuolar H+–adenosine triphosphatase (V-ATPase), a proton pump that can influence endocytosis by acidification of vesicles. The V-ATPase was also necessary for phosphorylation of LRP6 and Wnt. Thus, PRR may link the V-ATPase to the Wnt receptor protein LRP6, allowing acidification in the vicinity of the activated receptor, which appears to be necessary for phosphorylation of LRP6 and subsequent signaling. A dual-function protein acts as an adaptor in a major developmental signaling pathway. Wnt/β-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/β-catenin signaling.

RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-β-catenin signaling

Three Tales of Wnt Signaling The Wnt signaling pathway has important roles in regulating many biological processes during development and is also implicated in the behavior of some cancer cells (see the Perspective by Berndt and Moon). Cruciat et al. (p. 1436, published online 14 February) describe the mechanism of action of a protein found in a screen for proteins that influence Wnt signaling. DDX3, a DEAD-box RNA helicase, is required for proper Wnt signaling in Xenopus and Caenorhabditis elegans. It appears to act not through its action as an RNA helicase or through adenosine triphosphate binding, but rather by interacting with the protein kinase, casein kinase 1, and promoting its activation. Huang et al. (p. 1441, published online 31 January) investigated the function of receptor-interacting protein kinase 4 (RIPK4), the product a gene whose mutation causes severe developmental defects in mice and humans. Over-expression of the protein in cultured human cells activated transcription of genes regulated by the Wnt signaling pathway, and loss of RIPK4 function inhibited Wnt signaling in Xenopus embryos. At the molecular level, RIPK4 interacted with the Wnt co-receptor LRP6 and the Wnt signaling adaptor protein DVL2 and promoted phosphorylation of DVL2. Habib et al. (p. 1445) used Wnt-immobilized beads to understand how external cues direct asymmetrical stem cell divisions. Spatially restricted Wnt signals oriented the plane of mitotic division and lead to pluripotency gene expression in the Wnt-proximal daughter cell while the more distal daughter cell acquired hallmarks of differentiation. Thus, asymmetric gene expression patterns can arise as a consequence of orientation by a short-range signal. A multifunctional protein is required for activation of casein kinase 1 in response to Wnt–β-catenin signaling. [Also see Perspective by Berndt and Moon] 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–β-catenin network, where it acts as a regulatory subunit of CK1ε: In a Wnt-dependent manner, DDX3 binds CK1ε and directly stimulates its kinase activity, and promotes phosphorylation of the scaffold protein dishevelled. DDX3 is required for Wnt–β-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.

Anti-MuSK autoantibodies block binding of collagen Q to MuSK

Objective: Muscle-specific receptor tyrosine kinase (MuSK) antibody-positive myasthenia gravis (MG) accounts for 5%–15% of autoimmune MG. MuSK mediates the agrin-signaling pathway and also anchors the collagenic tail subunit (ColQ) of acetylcholinesterase (AChE). The exact molecular target of MuSK–immunoglobulin G (IgG), however, remains elusive. As acetylcholine receptor (AChR) deficiency is typically mild and as cholinesterase inhibitors are generally ineffective, we asked if MuSK-IgG interferes with binding of ColQ to MuSK. Methods: We used 3 assays: in vitro overlay of the human ColQ-tailed AChE to muscle sections of Colq−/− mice; in vitro plate-binding assay to quantitate binding of MuSK to ColQ and to LRP4; and passive transfer of MuSK-IgG to mice. Results: The in vitro overlay assay revealed that MuSK-IgG blocks binding of ColQ to the neuromuscular junction. The in vitro plate-binding assay showed that MuSK-IgG exerts a dose-dependent block of MuSK binding to ColQ by but not to LRP4. Passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the size and density of AChR and MuSK. Conclusions: As lack of ColQ compromises agrin-mediated AChR clustering in Colq−/− mice, a similar mechanism may lead to AChR deficiency in MuSK-MG patients. Our experiments also predict partial AChE deficiency in MuSK-MG patients, but AChE is not reduced in biopsied NMJs. In humans, binding of ColQ to MuSK may be dispensable for clustering ColQ, but is required for facilitating AChR clustering. Further studies will be required to elucidate the basis of this paradox.

NARF, an nemo-like kinase (NLK)-associated ring finger protein regulates the ubiquitylation and degradation of T cell factor/lymphoid enhancer factor (TCF/LEF).

β-Catenin is a key player in the Wnt signaling pathway, and interacts with cofactor T cell factor/lymphoid enhancer factor (TCF/LEF) to generate a transcription activator complex that activates Wnt-induced genes. We previously reported that Nemo-like kinase (NLK) negatively regulates Wnt signaling via phosphorylation of TCF/LEF. To further evaluate the physiological roles of NLK, we performed yeast two-hybrid screening to identify NLK-interacting proteins. From this screen, we isolated a novel RING finger protein that we term NARF (NLK associated RING finger protein). Here, we show that NARF induces the ubiquitylation of TCF/LEF in vitro and in vivo, and functions as an E3 ubiquitin-ligase that specifically cooperates with the E2 conjugating enzyme E2-25K. We found that NLK augmented NARF binding and ubiquitylation of TCF/LEF, and this required NLK kinase activity. The ubiquitylated TCF/LEF was subsequently degraded by the proteasome. Furthermore, NARF inhibited formation of the secondary axis induced by the ectopic expression of β-catenin in Xenopus embryos. Collectively, our findings raise the possibility that NARF functions as a novel ubiquitin-ligase to suppress the Wnt-β-catenin signaling.

LRP4 third β-propeller domain mutations cause novel congenital myasthenia by compromising agrin-mediated MuSK signaling in a position-specific manner

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.

Verapamil Protects against Cartilage Degradation in Osteoarthritis by Inhibiting Wnt/β-Catenin Signaling

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.

Collagen Q and anti-MuSK autoantibody competitively suppress agrin/LRP4/MuSK signaling

MuSK antibody-positive myasthenia gravis (MuSK-MG) accounts for 5 to 15% of autoimmune MG. MuSK and LRP4 are coreceptors for agrin in the signaling pathway that causes clustering of acetylcholine receptor (AChR). MuSK also anchors the acetylcholinesterase (AChE)/collagen Q (ColQ) complex to the synaptic basal lamina. We previously reported that anti-MuSK antibodies (MuSK-IgG) block binding of ColQ to MuSK and cause partial endplate AChE deficiency in mice. We here analyzed the physiological significance of binding of ColQ to MuSK and block of this binding by MuSK-IgG. In vitro plate-binding assay showed that MuSK-IgG blocked MuSK-LRP4 interaction in the presence of agrin. Passive transfer of MuSK-IgG to Colq-knockout mice attenuated AChR clustering, indicating that lack of ColQ is not the key event causing defective clustering of AChR in MuSK-MG. In three MuSK-MG patients, the MuSK antibodies recognized the first and fourth immunoglobulin-like domains (Ig1 and Ig4) of MuSK. In two other MuSK-MG patients, they recognized only the Ig4 domain. LRP4 and ColQ also bound to the Ig1 and Ig4 domains of MuSK. Unexpectedly, the AChE/ColQ complex blocked MuSK-LRP4 interaction and suppressed agrin/LRP4/MuSK signaling. Quantitative analysis showed that MuSK-IgG suppressed agrin/LRP4/MuSK signaling to a greater extent than ColQ.

Meclozine Facilitates Proliferation and Differentiation of Chondrocytes by Attenuating Abnormally Activated FGFR3 Signaling in Achondroplasia

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.

Meclozine Promotes Longitudinal Skeletal Growth in Transgenic Mice with Achondroplasia Carrying a Gain-of-Function Mutation in the FGFR3 Gene

Achondroplasia (ACH) is one of the most common skeletal dysplasias causing short stature owing to a gain-of-function mutation in the FGFR3 gene, which encodes the fibroblast growth factor receptor 3. We found that meclozine, an over-the-counter drug for motion sickness, inhibited elevated FGFR3 signaling in chondrocytic cells. To examine the feasibility of meclozine administration in clinical settings, we investigated the effects of meclozine on ACH model mice carrying the heterozygous Fgfr3(ach) transgene. We quantified the effect of meclozine in bone explant cultures employing limb rudiments isolated from developing embryonic tibiae from Fgfr3(ach) mice. We found that meclozine significantly increased the full-length and cartilaginous primordia of embryonic tibiae isolated from Fgfr3(ach) mice. We next analyzed the skeletal phenotypes of growing Fgfr3(ach) mice and wild-type mice with or without meclozine treatment. In Fgfr3(ach) mice, meclozine significantly increased the body length after 2 weeks of administration. At skeletal maturity, the bone lengths including the cranium, radius, ulna, femur, tibia, and vertebrae were significantly longer in meclozine-treated Fgfr3(ach) mice than in untreated Fgfr3(ach) mice. Interestingly, meclozine also increased bone growth in wild-type mice. The plasma concentration of meclozine during treatment was within the range that has been used in clinical settings for motion sickness. Increased longitudinal bone growth in Fgfr3(ach) mice by oral administration of meclozine in a growth period suggests potential clinical feasibility of meclozine for the improvement of short stature in ACH.

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms

Abstract Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.