RESUMEN
Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets.
Asunto(s)
Tipificación del Cuerpo , Cabeza/embriología , Proteínas de la Membrana/metabolismo , Metaloproteasas/metabolismo , Vía de Señalización Wnt , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Secuencia de Aminoácidos , Animales , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Metaloproteasas/genética , Datos de Secuencia Molecular , Organizadores Embrionarios/metabolismo , Alineación de Secuencia , Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMEN
The membrane-tethered protease Tiki antagonizes Wnt3a signaling by cleaving and inactivating Wnt3a in Wnt-producing cells. Tiki also functions in Wnt-receiving cells to antagonize Wnt signaling by an unknown mechanism. Here, we demonstrate that Tiki inhibition of Wnt signaling at the cell surface requires Frizzled (FZD) receptors. Tiki associates with the Wnt-FZD complex and cleaves the N-terminus of Wnt3a or Wnt5a, preventing the Wnt-FZD complex from recruiting and activating the coreceptor LRP6 or ROR1/2 without affecting Wnt-FZD complex stability. Intriguingly, we demonstrate that the N-terminus of Wnt3a is required for Wnt3a binding to LRP6 and activating ß-catenin signaling, while the N-terminus of Wnt5a is dispensable for recruiting and phosphorylating ROR1/2. Both Tiki enzymatic activity and its association with the Wnt-FZD complex contribute to its inhibitory function on Wnt5a. Our study uncovers the mechanism by which Tiki antagonizes Wnt signaling at the cell surface and reveals a negative role of FZDs in Wnt signaling by acting as Tiki cofactors. Our findings also reveal an unexpected role of the Wnt3a N-terminus in the engagement of the coreceptor LRP6.
Asunto(s)
Receptores Frizzled , Vía de Señalización Wnt , Receptores Frizzled/metabolismo , Proteína Wnt3A/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Membrana Celular/metabolismo , beta Catenina/metabolismoRESUMEN
Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.
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Enfermedad de la Arteria Coronaria , Péptido Hidrolasas , Proteína ADAMTS7 , Animales , Biomarcadores , Endopeptidasas , Células Endoteliales/metabolismo , Ratones , Péptido Hidrolasas/metabolismo , Proteoma/química , Cola (estructura animal)/metabolismoRESUMEN
[Figure: see text].
Asunto(s)
Enfermedad de la Arteria Coronaria/metabolismo , Proteína ADAMTS7/química , Proteína ADAMTS7/genética , Proteína ADAMTS7/metabolismo , Animales , Dominio Catalítico , Células Cultivadas , Enfermedad de la Arteria Coronaria/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Mutación , Polimorfismo de Nucleótido SimpleRESUMEN
The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn(2+)/Co(2+) but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core ß-sheet within an α+ß-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the ß-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.
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Regulación Enzimológica de la Expresión Génica , Metaloendopeptidasas/química , Proteínas Wnt/química , Secuencias de Aminoácidos , Animales , Dominio Catalítico , Cisteína/química , Disulfuros/química , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Luciferasas/metabolismo , Proteínas de la Membrana/química , Metaloproteasas/química , Mutagénesis Sitio-Dirigida , Péptidos/química , Feromonas Humanas/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Transducción de Señal , Proteína Wnt3A/química , XenopusRESUMEN
One major goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. Although many studies have identified candidate driver genes based on recurrence of mutations in individual genes, subsets of genes with nonrecurrent mutations may also be defined as putative drivers if they affect a single biological pathway. In this fashion, we previously identified Wnt signaling as significantly mutated through large-scale massively parallel DNA sequencing of chronic lymphocytic leukemia (CLL). Here, we use a novel method of biomolecule delivery, vertical silicon nanowires, to efficiently introduce small interfering RNAs into CLL cells, and interrogate the effects of 8 of 15 mutated Wnt pathway members identified across 91 CLLs. In HEK293T cells, mutations in 2 genes did not generate functional changes, 3 led to dysregulated pathway activation, and 3 led to further activation or loss of repression of pathway activation. Silencing 4 of 8 mutated genes in CLL samples harboring the mutated alleles resulted in reduced viability compared with leukemia samples with wild-type alleles. We demonstrate that somatic mutations in CLL can generate dependence on this pathway for survival. These findings support the notion that nonrecurrent mutations at different nodes of the Wnt pathway can contribute to leukemogenesis.
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Leucemia Linfocítica Crónica de Células B/genética , Mutación , Transducción de Señal/genética , Vía de Señalización Wnt/genética , beta Catenina/metabolismo , Adulto , Línea Celular Tumoral , Supervivencia Celular/genética , Células Cultivadas , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células HEK293 , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Anorectal malformation (ARM) is a common birth defect but the developmental history and the underlying molecular mechanism are poorly understood. Using murine genetic models, we report here that a signaling molecule Dickkopf-1 (Dkk1) is a critical regulator. The anorectal and genitourinary tracts are major derivatives of caudal hindgut, or the cloaca.Dkk1 is highly expressed in the dorsal peri-cloacal mesenchymal (dPCM) progenitors. We show that the deletion of Dkk1 causes the imperforate anus with rectourinary fistula. Mutant genital tubercles exhibit a preputial hypospadias phenotype and premature urethral canalization.Dkk1 mutants have an ectopic expansion of the dPCM tissue, which correlates with an aberrant increase of cell proliferation and survival. This ectopic tissue is detectable before the earliest sign of the anus formation, suggesting that it is most likely the primary or early cause of the defect. Deletion of Dkk1 results in an elevation of the Wnt/ß-catenin activity. Signaling molecules Shh, Fgf8 and Bmp4 are also upregulated. Furthermore, genetic hyperactivation of Wnt/ß-catenin signal pathway in the cloacal mesenchyme partially recapitulates Dkk1 mutant phenotypes. Together, these findings underscore the importance ofDKK1 in regulating behavior of dPCM progenitors, and suggest that formation of anus and urethral depends on Dkk1-mediated dynamic inhibition of the canonical Wnt/ß-catenin signal pathway.
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Canal Anal/embriología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mesodermo/embriología , Recto/embriología , Sistema Urogenital/embriología , Canal Anal/anomalías , Animales , Malformaciones Anorrectales , Ano Imperforado/embriología , Ano Imperforado/genética , Proteína Morfogenética Ósea 4/biosíntesis , Diferenciación Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Activación Enzimática/genética , Factor 8 de Crecimiento de Fibroblastos/biosíntesis , Proteínas Hedgehog/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Recto/anomalías , Células Madre , Regulación hacia Arriba , Anomalías Urogenitales/embriología , Anomalías Urogenitales/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired ß strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.
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Cisteína/química , Disulfuros/química , Proteína Wnt3A/química , Proteína Wnt3A/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Cisteína/genética , Cisteína/metabolismo , Disulfuros/metabolismo , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Transducción de Señal , Proteína Wnt3A/genéticaRESUMEN
Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in ß-catenin expression. ß-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6(-/-)), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.
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Megacariocitos/citología , Trombopoyesis/fisiología , Vía de Señalización Wnt/fisiología , Animales , Plaquetas/citología , Línea Celular , Células Cultivadas/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Hígado/embriología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Ratones , Ratones Noqueados , Proteínas Recombinantes/farmacología , Trombopoyesis/genética , Proteínas Wnt/farmacología , Proteína Wnt3A/farmacología , beta Catenina/biosíntesis , beta Catenina/genéticaRESUMEN
Population-based association studies have identified many genetic risk loci for coronary artery disease (CAD), but it is often unclear how genes within these loci are linked to CAD. Here, we perform interaction proteomics for 11 CAD-risk genes to map their protein-protein interactions (PPIs) in human vascular cells and elucidate their roles in CAD. The resulting PPI networks contain interactions that are outside of known biology in the vasculature and are enriched for genes involved in immunity-related and arterial-wall-specific mechanisms. Several PPI networks derived from smooth muscle cells are significantly enriched for genetic variants associated with CAD and related vascular phenotypes. Furthermore, the networks identify 61 genes that are found in genetic loci associated with risk of CAD, prioritizing them as the causal candidates within these loci. These findings indicate that the PPI networks we have generated are a rich resource for guiding future research into the molecular pathogenesis of CAD.
Asunto(s)
Enfermedad de la Arteria Coronaria , Humanos , Enfermedad de la Arteria Coronaria/genética , Mapas de Interacción de Proteínas , Redes Reguladoras de Genes , Sitios Genéticos , ProteómicaRESUMEN
Corin is a transmembrane tethered enzyme best known for processing the hormone atrial natriuretic peptide (ANP) in cardiomyocytes to control electrolyte balance and blood pressure. Loss of function mutations in Corin prevent ANP processing and lead to hypertension. Curiously, Corin loss of function variants also result in lighter coat color pigmentation in multiple species. Corin pigmentation effects are dependent on a functional Agouti locus encoding the agouti-signaling protein (ASIP) based on a genetic interaction. However, the nature of this conserved role of Corin has not been defined. Here we report that ASIP is a direct proteolytic substrate of the Corin enzyme.
RESUMEN
Coronary artery disease (CAD) remains the leading cause of death despite scientific advances. Elucidating shared CAD/pneumonia pathways may reveal novel insights regarding CAD pathways. We performed genome-wide pleiotropy analyses of CAD and pneumonia, examined the causal effects of the expression of genes near independently replicated SNPs and interacting genes with CAD and pneumonia, and tested interactions between disruptive coding mutations of each pleiotropic gene and smoking status on CAD and pneumonia risks. Identified pleiotropic SNPs were annotated to ADAMTS7 and IL6R. Increased ADAMTS7 expression across tissues consistently showed decreased risk for CAD and increased risk for pneumonia; increased IL6R expression showed increased risk for CAD and decreased risk for pneumonia. We similarly observed opposing CAD/pneumonia effects for NLRP3. Reduced ADAMTS7 expression conferred a reduced CAD risk without increased pneumonia risk only among never-smokers. Genetic immune-inflammatory axes of CAD linked to respiratory infections implicate ADAMTS7 and IL6R, and related genes.
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Enfermedad de la Arteria Coronaria , Pleiotropía Genética , Neumonía , Proteína ADAMTS7/genética , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/inmunología , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Neumonía/genética , Neumonía/inmunología , Polimorfismo de Nucleótido Simple , Receptores de Interleucina-6/genéticaRESUMEN
AIMS: Genetic studies have implicated the ARHGEF26 locus in the risk of coronary artery disease (CAD). However, the causal pathways by which DNA variants at the ARHGEF26 locus confer risk for CAD are incompletely understood. We sought to elucidate the mechanism responsible for the enhanced risk of CAD associated with the ARHGEF26 locus. METHODS AND RESULTS: In a conditional analysis of the ARHGEF26 locus, we show that the sentinel CAD-risk signal is significantly associated with various non-lipid vascular phenotypes. In human endothelial cell (EC), ARHGEF26 promotes the angiogenic capacity, and interacts with known angiogenic factors and pathways. Quantitative mass spectrometry showed that one CAD-risk coding variant, rs12493885 (p.Val29Leu), resulted in a gain-of-function ARHGEF26 that enhances proangiogenic signalling and displays enhanced interactions with several proteins partially related to the angiogenic pathway. ARHGEF26 is required for endothelial angiogenesis by promoting macropinocytosis of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) on cell membrane and is crucial to Vascular Endothelial Growth Factor (VEGF)-dependent murine vessel sprouting ex vivo. In vivo, global or tissue-specific deletion of ARHGEF26 in EC, but not in vascular smooth muscle cells, significantly reduced atherosclerosis in mice, with enhanced plaque stability. CONCLUSIONS: Our results demonstrate that ARHGEF26 is involved in angiogenesis signaling, and that DNA variants within ARHGEF26 that are associated with CAD risk could affect angiogenic processes by potentiating VEGF-dependent angiogenesis.
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Factores de Intercambio de Guanina Nucleótido , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Animales , Humanos , Ratones , Neovascularización Patológica , Neovascularización Fisiológica/fisiología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Intercambio de Guanina Nucleótido/genéticaRESUMEN
BACKGROUND: Corin is a protease expressed in cardiomyocytes that plays a key role in salt handling and intravascular volume homeostasis via activation of natriuretic peptides. It is unknown if Corin loss-of-function (LOF) is causally associated with risk of coronary artery disease (CAD). METHODS: We analyzed all coding CORIN variants in an Italian case-control study of CAD. We functionally tested all 64 rare missense mutations in Western Blot and Mass Spectroscopy assays for proatrial natriuretic peptide cleavage. An expanded rare variant association analysis for Corin LOF mutations was conducted in whole exome sequencing data from 37 799 CAD cases and 212 184 controls. RESULTS: We observed LOF variants in CORIN in 8 of 1803 (0.4%) CAD cases versus 0 of 1725 controls (P, 0.007). Of 64 rare missense variants profiled, 21 (33%) demonstrated <30% of wild-type activity and were deemed damaging in the 2 functional assays for Corin activity. In a rare variant association study that aggregated rare LOF and functionally validated damaging missense variants from the Italian study, we observed no association with CAD-21 of 1803 CAD cases versus 12 of 1725 controls with adjusted odds ratio of 1.61 ([95% CI, 0.79-3.29]; P=0.17). In the expanded sequencing dataset, there was no relationship between rare LOF variants with CAD was also observed (odds ratio, 1.15 [95% CI, 0.89-1.49]; P=0.30). Consistent with the genetic analysis, we observed no relationship between circulating Corin concentrations with incident CAD events among 4744 participants of a prospective cohort study-sex-stratified hazard ratio per SD increment of 0.96 ([95% CI, 0.87-1.07], P=0.48). CONCLUSIONS: Functional testing of missense mutations improved the accuracy of rare variant association analysis. Despite compelling pathophysiology and a preliminary observation suggesting association, we observed no relationship between rare damaging variants in CORIN or circulating Corin concentrations with risk of CAD.
Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Genómica , Mutación Missense , Análisis de Secuencia de ADN , Serina Endopeptidasas/genética , Adulto , Enfermedad de la Arteria Coronaria/epidemiología , Femenino , Humanos , Italia/epidemiología , Masculino , Factores de RiesgoRESUMEN
Corin (atrial natriuretic peptide-converting enzyme, EC 3.4.21) is a transmembrane serine protease expressed in cardiomyocytes. Corin exerts its cardioprotective effects via the proteolytic cleavage and activation of pro-atrial natriuretic peptide (pro-ANP) to ANP. We recently described an ANP reporter cell line stably expressing the ANP receptor, a cGMP-dependent cation channel used as a real-time cGMP biosensor, and the Ca2+-sensitive photoprotein aequorin. Here, we describe the generation of a novel reporter cell line expressing the calcium biosensor GCaMP6 instead of aequorin. In contrast to the luminescence-based assay, ANP stimulation of our novel GCaMP6 reporter cell resulted in stable, long-lasting fluorescence signals. Using this novel reporter system, we were able to detect pro-ANP to ANP conversion by purified, soluble wildtype corin (solCorin), but not the active site mutant solCorin(S985A), resulting in left-shifted concentration-response curves. Furthermore, cellular pro-ANPase activity could be detected on HEK 293 cells after transient expression of wildtype corin. In contrast, corin activity was not detected after transfection with the inactive corin(S985A) variant. In supernatants from cardiomyocyte-derived HL-1 cells pro-ANP to ANP conversion could also be detected, while in HL-1 corin knockout cells no conversion was observed. These findings underline the role of corin as the pro-ANP convertase. Our novel fluorescence-based ANP reporter cell line is well-suited for the sensitive detection of corin activity, and may be used for the identification and characterization of novel corin modulators.
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Factor Natriurético Atrial/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/farmacología , Calcio/metabolismo , Línea Celular , GMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Células HEK293 , Humanos , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Receptores del Factor Natriurético Atrial/genética , Serina Endopeptidasas/genéticaRESUMEN
The Wnt/beta-catenin signaling pathway has emerged as a key regulator in bone development and bone homeostasis. Loss-of-function mutations in the Wnt co-receptor LRP5 result in osteoporosis and "activating" mutations in LRP5 result in high bone mass. Dickkopf-1 (DKK1) is a secreted Wnt inhibitor that binds LRP5 and LRP6 during embryonic development, therefore it is expected that a decrease in DKK1 will result in an increase in Wnt activity and a high bone mass phenotype. Dkk1-/- knockout mice are embryonic lethal, but mice with hypomorphic Dkk1d (doubleridge) alleles that express low amounts of Dkk1 are viable. In this study we generated an allelic series by crossing Dkk1+/- and Dkk1+/d mice resulting in the following genotypes with decreasing Dkk1 expression levels: +/+, +/d, +/- and d/-. Using muCT imaging we scanned dissected left femora and calvariae from 8-week-old mice (n=60). We analyzed the distal femur to represent trabecular bone and the femur diaphysis for cortical endochondral bone. A region of the parietal bones was used to analyze intramembranous bone of the calvaria. We found that trabecular bone volume is increased in Dkk1 mutant mice in a manner that is inversely proportional to the level of Dkk1 expression. Trabeculae number and thickness were significantly higher in the low Dkk1 expressing genotypes from both female and male mice. Similar results were found in cortical bone with an increase in cortical thickness and cross sectional area of the femur diaphysis that correlated with lower Dkk1 expression. No consistent differences were found in the calvaria measurements. Our results indicate that the progressive Dkk1 reduction increases trabecular and cortical bone mass and that even a 25% reduction in Dkk1 expression could produce significant increases in trabecular bone volume fraction. Thus DKK1 is a negative regulator of normal bone homeostasis in vivo. Our study suggests that manipulation of DKK1 function or expression may have therapeutic significance for the treatment of low bone mass disorders.
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Densidad Ósea/fisiología , Huesos/anatomía & histología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Animales , Huesos/metabolismo , Femenino , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Mutantes , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tomografía Computarizada por Rayos XRESUMEN
Secreted Wnt morphogens are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/ß hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here, we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the "default" neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.
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Esterasas/genética , Cabeza/embriología , Neurogénesis/fisiología , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Tipificación del Cuerpo/genética , Ectodermo/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Silenciador del Gen , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Morfolinos , Placa Neural/metabolismo , Oxidación-Reducción , Ácido Palmítico/química , Unión Proteica , Conformación Proteica , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/química , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Certain missense mutations affecting LRP5 cause high bone mass (HBM) in humans. Based on in vitro evidence, HBM LRP5 receptors are thought to exert their effects by providing resistance to binding/inhibition of secreted LRP5 inhibitors such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). We previously reported the creation of two Lrp5 HBM knock-in mouse models, in which the human p.A214V or p.G171V missense mutations were knocked into the endogenous Lrp5 locus. To determine whether HBM knock-in mice are resistant to SOST- or DKK1-induced osteopenia, we bred Lrp5 HBM mice with transgenic mice that overexpress human SOST in osteocytes ((8kb) Dmp1-SOST) or mouse DKK1 in osteoblasts and osteocytes ((2.3kb) Col1a1-Dkk1). We observed that the (8kb) Dmp1-SOST transgene significantly lowered whole-body bone mineral density (BMD), bone mineral content (BMC), femoral and vertebral trabecular bone volume fraction (BV/TV), and periosteal bone-formation rate (BFR) in wild-type mice but not in mice with Lrp5 p.G171V and p.A214V alleles. The (2.3kb) Col1a1-Dkk1 transgene significantly lowered whole-body BMD, BMC, and vertebral BV/TV in wild-type mice and affected p.A214V mice more than p.G171V mice. These in vivo data support in vitro studies regarding the mechanism of HBM-causing mutations, and imply that HBM LRP5 receptors differ in their relative sensitivity to inhibition by SOST and DKK1.
Asunto(s)
Densidad Ósea/genética , Fémur/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Mutación Missense , Osteocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Sustitución de Aminoácidos , Animales , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fémur/patología , Técnicas de Sustitución del Gen , Sitios Genéticos , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Tamaño de los Órganos/genética , Osteocitos/patologíaRESUMEN
Spontaneous and induced mutations of neuronal Na+ channels in human patients and mutant mice result in a broad range of neurological-disease. Epilepsy, a disorder of neuronal hyperexcitability, has been associated with delayed inactivation of SCN2A in mice, and with altered kinetics of SCN1A in human patients. Movement disorders including tremor, ataxia, dystonia and paralysis have been observed in mice with mutations of SCN8A. Electrophysiological recordings from neurons isolated from mice with mutations in individual channels reveal the contributions of each channel to in vivo firing patterns. In addition to monogenic disease, Na+ channel mutations are likely to contribute to polygenic disease susceptibility and to normal variation in neuronal function. Advances in molecular methods coupled with genomic sequences from the Human Genome Project will permit identification of many new patient mutations and generation of animal models to dissect their physiological and cellular consequences.