RESUMEN
Bruck syndrome is an autosomal recessive form of osteogenesis imperfecta (OI) caused by biallelic variants in PLOD2 or FKBP10 and is characterized by joint contractures, bone fragility, short stature, and scoliosis. PLOD2 encodes LH2, which hydroxylates type I collagen telopeptide lysines, a critical step for collagen crosslinking. The Plod2 global knockout mouse model is limited by early embryonic lethality, thus the role of PLOD2 in skeletogenesis is not well understood. We generated a novel Plod2 mouse line modeling a variant identified in two unrelated individuals with Bruck syndrome: PLOD2 c.1559dupC, predicting a frameshift and loss of the long isoform LH2b. In the mouse, the duplication led to loss of LH2b mRNA as well as significantly reduced total LH2 protein. This model, Plod2fs/fs, survived up to E18.5 although in non-Mendelian genotype frequencies. The homozygous frameshift model recapitulated the joint contractures seen in Bruck syndrome and had indications of absent type I collagen telopeptide lysine hydroxylation in bone. Genetically labeling tendons with Scleraxis-GFP in Plod2fs/fs mice revealed the loss of extensor tendons in the forelimb by E18.5 and developmental studies showed extensor tendons developed through E14.5 but were absent starting at E16.5. Second harmonic generation showed abnormal tendon type I collagen fiber organization, suggesting structurally abnormal tendons. Characterization of the skeleton by µCT and Raman spectroscopy showed normal bone mineralization levels. This work highlights the importance of properly crosslinked type I collagen in tendon and bone, providing a promising new mouse model to further our understanding of Bruck syndrome.
Bruck syndrome is a rare disease where individuals have brittle bone as well as contracted or stiff joints. Mutations in two genes are associated with Bruck syndrome and, in this work, we focus on PLOD2. Mice without Plod2 die at an early embryonic stage, before they have a chance to fully develop. In this work, we created a mouse with a PLOD2 mutation seen in people with Bruck syndrome. Some of these new Bruck syndrome model mice survived to a later gestational age, but all died at birth. The Bruck syndrome mice were small and had contracted joints. We found they were missing tendons in their arms and had structurally abnormal tendons in their knees. Bone mineralization was normal, but there were indications that the modifications needed for normal type I collagen structure were absent. Overall, this is an advantageous new mouse model of Bruck syndrome that can be used to study this rare disease and highlights the importance of Plod2 in tendon.
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The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.
Asunto(s)
Colágeno , Metaloproteinasa 14 de la Matriz , Animales , Ratones , Ritmo Circadiano , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Homeostasis , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismoRESUMEN
Type V collagen is a regulatory fibrillar collagen essential for type I collagen fibril nucleation and organization and its deficiency leads to structurally abnormal extracellular matrix (ECM). Haploinsufficiency of the Col5a1 gene encoding α(1) chain of type V collagen is the primary cause of classic Ehlers-Danlos syndrome (EDS). The mechanisms by which this initial insult leads to the spectrum of clinical presentation are not fully understood. Using transcriptome analysis of skin and Achilles tendons from Col5a1 haploinsufficient (Col5a1+/-) mice, we recognized molecular alterations associated with the tissue phenotypes. We identified dysregulation of ECM components including thrombospondin-1, lysyl oxidase, and lumican in the skin of Col5a1+/- mice when compared with control. We also identified upregulation of transforming growth factor ß1 (Tgf-ß) in serum and increased expression of pSmad2 in skin from Col5a1+/- mice, suggesting Tgf-ß dysregulation is a contributor to abnormal wound healing and atrophic scarring seen in classic EDS. Together, these findings support altered matrix to cell signaling as a component of the pathogenesis of the tissue phenotype in classic EDS and point out potential downstream signaling pathways that may be targeted for the treatment of this disease.
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Síndrome de Ehlers-Danlos , Animales , Colágeno/genética , Colágeno Tipo V/genética , Modelos Animales de Enfermedad , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patología , Haploinsuficiencia , Ratones , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, although tendons tend to heal faster than ligaments. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we suspect is functionally associated with the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important to improving the design of orthopedic treatment approaches.
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Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.
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Huesos/metabolismo , Proteína Coat de Complejo I/genética , Proteína Coatómero/genética , Discapacidades del Desarrollo/genética , Discapacidad Intelectual/genética , Osteoporosis/genética , Animales , Ácido Ascórbico/farmacología , Huesos/efectos de los fármacos , Huesos/patología , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Niño , Preescolar , Proteína Coat de Complejo I/deficiencia , Proteína Coatómero/química , Proteína Coatómero/deficiencia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Embrión no Mamífero , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación del Desarrollo de la Expresión Génica , Aparato de Golgi , Haploinsuficiencia , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Masculino , Ratones , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Osteoporosis/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Índice de Severidad de la Enfermedad , Pez CebraRESUMEN
Osteogenesis imperfecta (OI) is a genetic disorder that features wide-ranging defects in both skeletal and nonskeletal tissues. Previously, we and others reported that loss-of-function mutations in FK506 Binding Protein 10 (FKBP10) lead to skeletal deformities in conjunction with joint contractures. However, the pathogenic mechanisms underlying joint dysfunction in OI are poorly understood. In this study, we have generated a mouse model in which Fkbp10 is conditionally deleted in tendons and ligaments. Fkbp10 removal substantially reduced telopeptide lysyl hydroxylation of type I procollagen and collagen cross-linking in tendons. These biochemical alterations resulting from Fkbp10 ablation were associated with a site-specific induction of fibrosis, inflammation, and ectopic chondrogenesis followed by joint deformities in postnatal mice. We found that the ectopic chondrogenesis coincided with enhanced Gli1 expression, indicating dysregulated Hedgehog (Hh) signaling. Importantly, genetic inhibition of the Hh pathway attenuated ectopic chondrogenesis and joint deformities in Fkbp10 mutants. Furthermore, Hh inhibition restored alterations in gait parameters caused by Fkbp10 loss. Taken together, we identified a previously unappreciated role of Fkbp10 in tendons and ligaments and pathogenic mechanisms driving OI joint dysfunction.
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Condrocitos/patología , Articulaciones/fisiopatología , Actividad Motora , Osteogénesis Imperfecta/fisiopatología , Osteogénesis , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Animales Recién Nacidos , Condrogénesis/genética , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Marcha , Eliminación de Gen , Regulación de la Expresión Génica , Proteínas Hedgehog/metabolismo , Hidroxilación , Inflamación/genética , Inflamación/patología , Articulaciones/patología , Ligamentos/patología , Lisina/metabolismo , Ratones , Modelos Biológicos , Osificación Heterotópica/complicaciones , Osificación Heterotópica/genética , Osificación Heterotópica/patología , Osificación Heterotópica/fisiopatología , Osteogénesis/genética , Osteogénesis Imperfecta/complicaciones , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/patología , Péptidos/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Proteínas de Unión a Tacrolimus/genética , Tendones/patologíaRESUMEN
Osteogenesis imperfecta (OI) is characterized by short stature, skeletal deformities, low bone mass, and motor deficits. A subset of OI patients also present with joint hypermobility; however, the role of tendon dysfunction in OI pathogenesis is largely unknown. Using the Crtap-/- mouse model of severe, recessive OI, we found that mutant Achilles and patellar tendons were thinner and weaker with increased collagen cross-links and reduced collagen fibril size at 1- and 4-months compared to wildtype. Patellar tendons from Crtap-/- mice also had altered numbers of CD146+CD200+ and CD146-CD200+ progenitor-like cells at skeletal maturity. RNA-seq analysis of Achilles and patellar tendons from 1-month Crtap-/- mice revealed dysregulation in matrix and tendon marker gene expression concomitant with predicted alterations in TGF-ß, inflammatory, and metabolic signaling. At 4-months, Crtap-/- mice showed increased αSMA, MMP2, and phospho-NFκB staining in the patellar tendon consistent with excess matrix remodeling and tissue inflammation. Finally, a series of behavioral tests showed severe motor impairments and reduced grip strength in 4-month Crtap-/- mice - a phenotype that correlates with the tendon pathology.
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Tendón Calcáneo/patología , Proteínas de la Matriz Extracelular/deficiencia , Actividad Motora , Osteogénesis Imperfecta/patología , Osteogénesis Imperfecta/fisiopatología , Ligamento Rotuliano/patología , Tendón Calcáneo/metabolismo , Actinas/metabolismo , Factores de Edad , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Proteínas de la Matriz Extracelular/genética , Colágenos Fibrilares/genética , Colágenos Fibrilares/metabolismo , Genes Recesivos , Predisposición Genética a la Enfermedad , Fuerza de la Mano , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Chaperonas Moleculares/genética , FN-kappa B/metabolismo , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Ligamento Rotuliano/metabolismo , Fenotipo , Fosforilación , Resistencia Física , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Bruck syndrome (BS) is a congenital disorder characterized by joint flexion contractures, skeletal dysplasia, and increased bone fragility, which overlaps clinically with osteogenesis imperfecta (OI). On a genetic level, BS is caused by biallelic mutations in either FKBP10 or PLOD2. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of cross-linking lysine residues in fibrillar collagen telopeptide domains. This modification enables collagen to form chemically stable (permanent) intermolecular cross-links in the extracellular matrix. Normal bone collagen develops a unique mix of such stable and labile lysyl-oxidase-mediated cross-links, which contribute to bone strength, resistance to microdamage, and crack propagation, as well as the ordered deposition of mineral nanocrystals within the fibrillar collagen matrix. Bone from patients with BS caused by biallelic FKBP10 mutations has been shown to have abnormal collagen cross-linking; however, to date, no direct studies of human bone from BS caused by PLOD2 mutations have been reported. Here the results from a study of a 4-year-old boy with BS caused by compound heterozygous mutations in PLOD2 are discussed. Diminished hydroxylation of type I collagen telopeptide lysines but normal hydroxylation at triple-helical sites was found. Consequently, stable trivalent cross-links were essentially absent. Instead, allysine aldol dimeric cross-links dominated as in normal skin collagen. Furthermore, in contrast to the patient's bone collagen, telopeptide lysines in cartilage type II collagen cross-linked peptides from the patient's urine were normally hydroxylated. These findings shed light on the complex mechanisms that control the unique posttranslational chemistry and cross-linking of bone collagen, and how, when defective, they can cause brittle bones and related connective tissue problems. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
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While details remain unclear, initiation of woven bone mineralization is believed to be mediated by collagen and potentially nucleated by bone sialoprotein (BSP). Interestingly, our recent publication showed that BSP and type XI collagen form complexes in mineralizing osteoblastic cultures. To learn more, we examined the protein composition of extracellular sites of de novo hydroxyapatite deposition which were enriched in BSP and Col11a1 containing an alternatively spliced "6b" exonal sequence. An alternate splice variant "6a" sequence was not similarly co-localized. BSP and Col11a1 co-purify upon ion-exchange chromatography or immunoprecipitation. Binding of the Col11a1 "6b" exonal sequence to bone sialoprotein was demonstrated with overlapping peptides. Peptide 3, containing three unique lysine-triplet sequences, displayed the greatest binding to osteoblastic cultures; peptides containing fewer lysine triplet motifs or derived from the "6a" exon yielded dramatically lower binding. Similar results were obtained with 6-carboxyfluorescein (FAM)-conjugated peptides and western blots containing extracts from osteoblastic cultures. Mass spectroscopic mapping demonstrated that FAM-peptide 3 bound to 90 kDa BSP and its 18 to 60 kDa fragments, as well as to 110 kDa nucleolin. In osteoblastic cultures, FAM-peptide 3 localized to biomineralization foci (site of BSP) and to nucleoli (site of nucleolin). In bone sections, biotin-labeled peptide 3 bound to sites of new bone formation which were co-labeled with anti-BSP antibodies. These results establish the fluorescent peptide 3 conjugate as the first nonantibody-based method to identify BSP on western blots and in/on cells. Further examination of the "6b" splice variant interactions will likely reveal new insights into bone mineralization during development.
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Calcificación Fisiológica/fisiología , Colágeno Tipo XI/metabolismo , Osteopontina/metabolismo , Animales , Huesos/metabolismo , Calcificación Fisiológica/genética , Colágeno/metabolismo , Colágeno Tipo XI/genética , Fluoresceínas/química , Sialoproteína de Unión a Integrina/metabolismo , Masculino , Osteoblastos/metabolismo , Osteopontina/genética , Péptidos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ratas , Sialoglicoproteínas/metabolismo , NucleolinaRESUMEN
To date 45 autosomal recessive disease-causing variants are reported in the FKBP10 gene. Those variant were found to be associated with Osteogenesis Imperfecta (OI) for which the hallmark phenotype is bone fractuers or Bruck Syndrome (BS) where bone fractures are accompanied with contractures. In addition, a specific homozygous FKBP10 mutation (p.Tyr293del) has been described in Yup'ik Inuit population to cause Kuskokwim syndrome (KS) in which contractures without fractures are observed. Here we present an extended Palestinian family with 10 affected individuals harboring a novel homozygous splice site mutation, c.391+4A > T in intron 2 of the FKBP10 gene, in which the three above mentioned syndromes segregate as a result of skipping of exon 2 and absence of the FKBP65 protein. At the biochemical level, Hydroxylysyl pyridinoline (HP)/lysyl pyridinoline (LP) values were inversely correlated with OI phenotypes, a trend we could confirm in our patients. Our findings illustrate that single familial FKBP10 mutations can result in a phenotypic spectrum, ranging from fractures without contractures, to fractures and contractures and even to only contractures. This broad intra-familial clinical variability within one single family is a new finding in the field of bone fragility.
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Artrogriposis/genética , Mutación , Osteogénesis Imperfecta/genética , Fenotipo , Proteínas de Unión a Tacrolimus/genética , Adolescente , Adulto , Aminoácidos/metabolismo , Artrogriposis/patología , Células Cultivadas , Niño , Femenino , Homocigoto , Humanos , Masculino , Osteogénesis Imperfecta/patología , Linaje , Sitios de Empalme de ARNRESUMEN
Null mutations in CRTAP or P3H1, encoding cartilage-associated protein and prolyl 3-hydroxylase 1, cause the severe bone dysplasias, types VII and VIII osteogenesis imperfecta. Lack of either protein prevents formation of the ER prolyl 3-hydroxylation complex, which catalyzes 3Hyp modification of types I and II collagen and also acts as a collagen chaperone. To clarify the role of the A1 3Hyp substrate site in recessive bone dysplasia, we generated knock-in mice with an α1(I)P986A substitution that cannot be 3-hydroxylated. Mutant mice have normal survival, growth, femoral breaking strength and mean bone mineralization. However, the bone collagen HP/LP crosslink ratio is nearly doubled in mutant mice, while collagen fibril diameter and bone yield energy are decreased. Thus, 3-hydroxylation of the A1 site α1(I)P986 affects collagen crosslinking and structural organization, but its absence does not directly cause recessive bone dysplasia. Our study suggests that the functions of the modification complex as a collagen chaperone are thus distinct from its role as prolyl 3-hydroxylase.
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Sustitución de Aminoácidos , Colágeno Tipo I/genética , Osteoblastos/citología , Osteogénesis Imperfecta/genética , Animales , Células Cultivadas , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Fibroblastos/citología , Fibroblastos/metabolismo , Técnicas de Sustitución del Gen , Humanos , Hidroxilación , Masculino , Ratones , Osteoblastos/metabolismo , Osteogénesis Imperfecta/metabolismo , FenotipoAsunto(s)
Colágeno Tipo IX/biosíntesis , Colágeno Tipo IX/genética , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Animales , Técnicas de Inactivación de Genes , Estudios de Asociación Genética/métodos , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Ratones , Ratones NoqueadosRESUMEN
Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1⯱â¯PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.
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Colágeno Tipo I/genética , Retículo Endoplásmico/metabolismo , Procolágeno/metabolismo , Rastreo Diferencial de Calorimetría , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Microscopía Fluorescente , Mutación Missense , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Estructura Terciaria de ProteínaRESUMEN
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders1-5, but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported. Here we describe a neomorphic seed region mutation in the chondrocyte-specific, super-enhancer-associated MIR140 gene encoding microRNA-140 (miR-140) in a novel autosomal dominant human skeletal dysplasia. Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice6. This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects. In chondrocytes, the mutation causes widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, indicating that the mutation produces both loss-of-function and gain-of-function effects. Furthermore, the mutant miR-140-5p seed competes with the conserved RNA-binding protein Ybx1 for overlapping binding sites. This finding may explain the potent target repression and robust in vivo effect by this mutant miRNA even in the absence of evolutionary selection of miRNA-target RNA interactions, which contributes to the strong regulatory effects of conserved miRNAs7,8. Our study presents the first case of a pathogenic gain-of-function miRNA mutation and provides molecular insight into neomorphic actions of emerging and/or mutant miRNAs.
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Enfermedades del Desarrollo Óseo/genética , Mutación con Ganancia de Función/genética , MicroARNs/genética , Animales , Secuencia de Bases , Condrocitos/metabolismo , Femenino , Homocigoto , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , MicroARNs/metabolismo , Linaje , Fenotipo , Transcriptoma/genéticaRESUMEN
Lysyl oxidase-generated intermolecular cross-links are essential for the tensile strength of collagen fibrils. Two cross-linking pathways can be defined, one based on telopeptide lysine aldehydes and another on telopeptide hydroxylysine aldehydes. Since the 1970s it has been accepted that the mature cross-linking structures on the lysine aldehyde pathway, which dominates in skin and cornea, incorporate histidine residues. Here, using a range of MS-based methods, we re-examined this conclusion and found that telopeptide aldol dimerization is the primary mechanism for stable cross-link formation. The C-telopeptide aldol dimers formed labile addition products with glucosylgalactosyl hydroxylysine at α1(I)K87 in adjacent collagen molecules that resisted borohydride reduction and after acid hydrolysis produced histidinohydroxylysinonorleucine (HHL), but only from species with a histidine in their α1(I) C-telopeptide sequence. Peptide MS analyses and the lack of HHL formation in rat and mouse skin, species that lack an α1(I) C-telopeptide histidine, revealed that HHL is a laboratory artifact rather than a natural cross-linking structure. Our experimental results also establish that histidinohydroxymerodesmosine is produced by borohydride reduction of N-telopeptide allysine aldol dimers in aldimine intermolecular linkage to nonglycosylated α1(I) K930. Borohydride reduction of the aldimine promotes an accompanying base-catalyzed Michael addition of α1(I) H932 imidazole to the α,ß-unsaturated aldol. These aldehydes are intramolecular at the N terminus but at the C terminus they can be both intramolecular and intermolecular according to present and earlier findings.
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Aldehídos/análisis , Colágeno Tipo I/análisis , Dipéptidos/análisis , Histidina/análogos & derivados , Hidroxilisina/análogos & derivados , Péptidos/análisis , Piel/química , Aldehídos/química , Animales , Artefactos , Bovinos , Colágeno Tipo I/química , Histidina/análisis , Hidroxilisina/análisis , Hidroxilisina/química , Péptidos/química , Proteína-Lisina 6-Oxidasa/químicaRESUMEN
Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.
Asunto(s)
Enfermedades Óseas/metabolismo , Enfermedades Óseas/patología , Condrocitos/metabolismo , Colágeno/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Animales , Cartílago/metabolismo , Matriz Extracelular/metabolismo , Glucosa/metabolismo , Glutamina/metabolismo , Placa de Crecimiento/metabolismo , Hidroxilación , Prolina Dioxigenasas del Factor Inducible por Hipoxia/deficiencia , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Ácidos Cetoglutáricos/metabolismo , Lisina/metabolismo , Masculino , Ratones , Osteogénesis , Oxidación-Reducción , Prolina/metabolismoRESUMEN
The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.
Asunto(s)
Colágeno Tipo I , Modelos Animales de Enfermedad , Síndrome de Ehlers-Danlos , Osteogénesis Imperfecta , Pez Cebra , Animales , Animales Modificados Genéticamente , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/patología , Humanos , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Nonenzymatic glycation of collagen has long been associated with the progressive secondary complications of diabetes. How exactly such random glycations result in impaired tissues is still poorly understood. Because of the slow turnover rate of most fibrillar collagens, they are more susceptible to accumulate time-dependent glycations and subsequent advanced glycation end-products. The latter are believed to include cross-links that stiffen host tissues. However, diabetic animal models have also displayed weakened tendons with reduced stiffness. Strikingly, not a single experimentally identified specific molecular site of glycation in a collagen has been reported. Here, using targeted MS, we have identified partial fructosyl-hydroxylysine glycations at each of the helical domain cross-linking sites of type I collagen that are elevated in tissues from a diabetic mouse model. Glycation was not found at any other collagen lysine residues. Type I collagen in mouse tendons is cross-linked intermolecularly by acid-labile aldimine bonds formed by the addition of telopeptide lysine aldehydes to hydroxylysine residues at positions α1(I)Lys87, α1(I)Lys930, α2(I)Lys87, and α2(I)Lys933 of the triple helix. Our data reveal that site-specific glycations of these specific lysines may significantly impair normal lysyl oxidase-controlled cross-linking in diabetic tendons. We propose that such N-linked glycations can hinder the normal cross-linking process, thus altering the content and/or placement of mature cross-links with the potential to modify tissue material properties.
Asunto(s)
Colágeno Tipo I/química , Diabetes Mellitus Tipo 2/metabolismo , Lisina/química , Obesidad/metabolismo , Tendones/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Glucemia/metabolismo , Colágeno Tipo I/metabolismo , Reactivos de Enlaces Cruzados/química , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Hemoglobina Glucada/metabolismo , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Hidroxilación , Hidroxilisina/química , Hidroxilisina/metabolismo , Lisina/metabolismo , Masculino , Espectrometría de Masas , Ratones , Obesidad/patología , Proteína-Lisina 6-Oxidasa/química , Proteína-Lisina 6-Oxidasa/metabolismo , Cola (estructura animal) , Tendones/química , Tendones/patologíaRESUMEN
Tandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both Sc65-/- and P3h3-/- skin revealed the same abnormal chain pattern on SDS-PAGE with an overabundance of a γ112 cross-linked trimer. The latter proved to be from native molecules that had intramolecular aldol cross-links at each end. The lysine under-hydroxylation was shown to alter the divalent aldimine cross-link chemistry of mutant skin collagen. Furthermore, the ratio of mature HP/LP cross-links in bone of both P3h3-/- and Sc65-/- mice was reversed compared with wild type, consistent with the level of lysine under-hydroxylation seen in individual chains at cross-linking sites. The effect on cross-linking lysines was quantitatively very similar to that previously observed in EDS VIA human and Plod1-/- mouse tissues, suggesting that P3H3 and/or SC65 mutations may cause as yet undefined EDS variants.