RESUMEN
Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.
Asunto(s)
Ciclofilinas/química , Lisina/química , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/química , Piel/enzimología , Animales , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Ciclofilinas/genética , Ciclofilinas/ultraestructura , Retículo Endoplásmico/química , Retículo Endoplásmico/enzimología , Glicosilación , Heterocigoto , Hidroxilación , Lisina/genética , Espectrometría de Masas , Ratones , Ratones Noqueados , Microscopía de Fuerza Atómica , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procesamiento Proteico-Postraduccional/genética , Piel/químicaRESUMEN
Intraflagellar transport (IFT) is essential for assembling primary cilia required for bone formation. Disruption of IFT frequently leads to bone defects in humans. While it has been well studied about the function of IFT in osteogenic cell proliferation and differentiation, little is known about its role in collagen biosynthesis during bone formation. Here we show that IFT20, the smallest IFT protein in the IFT-B complex, is important for collagen biosynthesis in mice. Deletion of Ift20 in craniofacial osteoblasts displayed bone defects in the face. While collagen protein levels are unaffected by loss of Ift20, collagen cross-linking was significantly altered. In both Ift20:Wnt1-Cre and Ift20:Ocn-Cre mice the bones exhibit increased hydroxylysine-aldehyde deived cross-linking, and decreased lysine-aldehyde derived cross-linking. To obtain insight into the molecular mechanisms, we examined the expression levels of telopeptidyl lysyl hydroxylase 2 (LH2), and associated chaperone complexes. The results demonstrated that, while LH2 levels were unaffected by loss of Ift20, its chaperone, FKBP65, was significantly increased in Ift20:Wnt1-Cre and Ift20:Ocn-Cre mouse calvaria as well as femurs. These results suggest that IFT20 plays a pivotal role in collagen biosynthesis by regulating, in part, telopeptidyl lysine hydroxylation and cross-linking in bone. To the best of our knowledge, this is the first to demonstrate that the IFT components control collagen post-translational modifications. This provides a novel insight into the craniofacial bone defects associated with craniofacial skeletal ciliopathies.
Asunto(s)
Proteínas Portadoras/metabolismo , Colágeno/biosíntesis , Huesos Faciales/metabolismo , Osteoblastos/metabolismo , Osteogénesis/genética , Animales , Proteínas Portadoras/genética , Colágeno/metabolismo , Huesos Faciales/crecimiento & desarrollo , Eliminación de Gen , Inmunohistoquímica , Ratones , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión a Tacrolimus/metabolismo , Microtomografía por Rayos XRESUMEN
Glycosylation in type I collagen occurs as O-linked galactosyl- (G-) lesser and glucosylgalactosyl-hydroxylysine (GG-Hyl); however, its biological significance is still not well understood. To investigate the function of this modification in bone, we have generated preosteoblast MC3T3-E1 (MC)-derived clones, short hairpin (Sh) clones, in which Glt25d1 gene expression was stably suppressed. In Sh clones, the GLT25D1 protein levels were markedly diminished in comparison to controls (MC and those transfected with the empty vector). In Sh collagen, levels of both G- and GG-Hyl were significantly diminished with a concomitant increase in the level of free-Hyl. In addition, the level of immature divalent cross-links significantly diminished while the level of the mature trivalent cross-link increased. As determined by mass spectrometric analysis, seven glycosylation sites were identified in type I collagen and the most predominant site was at the helical cross-linking site, α1-87. At all of the glycosylation sites, the relative levels of G- and GG-Hyl were markedly diminished, i.e., by â¼50-75%, in Sh collagen, and at five of these sites, the level of Lys hydroxylation was significantly increased. The collagen fibrils in Sh clones were larger, and mineralization was impaired. These results indicate that GLT25D1 catalyzes galactosylation of Hyl throughout the type I collagen molecule and that this modification may regulate maturation of collagen cross-linking, fibrillogenesis, and mineralization.
Asunto(s)
Colágeno Tipo I/metabolismo , Galactosiltransferasas/metabolismo , Fenotipo , Células 3T3 , Animales , Biocatálisis , Colágeno Tipo I/química , Glicosilación , Lisina/metabolismo , RatonesRESUMEN
Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.
Asunto(s)
Colágeno Tipo I , Ciclofilinas/deficiencia , Dentina/ultraestructura , Animales , Colágeno Tipo I/ultraestructura , Ciclofilinas/fisiología , Dentina/patología , Matriz Extracelular/patología , Matriz Extracelular/ultraestructura , Glicosilación , Hidroxilación , Lisina/metabolismo , Espectrometría de Masas , Ratones , Ratones Noqueados , Osteogénesis Imperfecta , Procolágeno-Prolina Dioxigenasa/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation.
Asunto(s)
Colágeno Tipo I/química , Lisina/química , Animales , Colágeno/química , Ciclofilinas/metabolismo , Hidroxilación , Tendones/metabolismoRESUMEN
Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147-1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.
Asunto(s)
Colágeno/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Línea Celular Tumoral , Colágeno/genética , Matriz Extracelular/genética , Humanos , Ratones , Proteínas de Neoplasias/genética , Neoplasias/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genéticaRESUMEN
Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations.
Asunto(s)
Aorta/metabolismo , Colágeno/biosíntesis , Proteínas de la Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Animales , Colágeno/metabolismo , Elastina/metabolismo , Eliminación de Gen , Homocigoto , Ratones , Músculo Liso/metabolismo , Oxidación-Reducción , Proteína-Lisina 6-Oxidasa/metabolismo , ProteómicaRESUMEN
Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.
Asunto(s)
Colágeno Tipo I/genética , Ciclofilinas/genética , Osteogénesis Imperfecta/genética , Procesamiento Proteico-Postraduccional/genética , Animales , Colágeno/química , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/patología , Genes Recesivos , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Mutación , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Pliegue de ProteínaRESUMEN
Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosyl-hydroxylysine. Furthermore, key glycosylated hydroxylysine residues, α1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-cross-linked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving α1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998-23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen.
Asunto(s)
Huesos/química , Colágeno Tipo I/química , Hidroxilisina/química , Animales , Bovinos , Cromatografía Liquida , Glicosilación , Espectrometría de Masas , Estabilidad ProteicaRESUMEN
Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846-8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization.
Asunto(s)
Calcificación Fisiológica/fisiología , Colágeno Tipo I/metabolismo , Osteoblastos/enzimología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Animales , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/ultraestructura , Reactivos de Enlaces Cruzados/metabolismo , Ácido Glucárico/metabolismo , Glicosilación , Hidroxilisina/metabolismo , Isoenzimas/metabolismo , Espectrometría de Masas , Ratones , Microscopía Electrónica de Transmisión , Osteoblastos/citología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Liver fibrosis is characterized by excessive deposition of extracellular matrix proteins by myofibroblasts derived from hepatic stellate cells and portal fibroblasts. Activation of these precursors to myofibroblasts requires matrix stiffness, which results in part from increased collagen cross-linking mediated by lysyl oxidase (LOX) family proteins. The aims of this study were to characterize the mechanical changes of early fibrosis, to identify the cells responsible for LOX production in early injury, and to determine which cells in normal liver produce collagens and elastins, which serve as substrates for LOXs early after injury. Hepatocytes and liver nonparenchymal cells were isolated from normal and early-injured liver and examined immediately for expression of LOXs and matrix proteins. We found that stellate cells and portal fibroblasts were the major cellular sources of fibrillar collagens and LOXs in normal liver and early after injury (1 day after bile duct ligation and 2 and 7 days after CCl(4) injury). Activity assays using stellate cells and portal fibroblasts in culture demonstrated significant increases in LOX family enzymatic activity as cells became myofibroblastic. LOX family-mediated deoxypyridinoline and pyridinoline cross-links increased after CCl(4)-mediated injury. There was a significant association between liver stiffness (as quantified by the shear storage modulus G') and deoxypyridinoline levels; increased deoxypyridinoline levels were also coincident with significantly increased elastic resistance to large strain deformations, consistent with increased cross-linking of the extracellular matrix. These data suggest a model in which the liver is primed to respond quickly to injury, activating potential mechanical feed-forward mechanisms.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Colágeno , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Células Estrelladas Hepáticas/metabolismo , Hígado , Proteína-Lisina 6-Oxidasa/metabolismo , Aminoácidos/análisis , Animales , Colágeno/biosíntesis , Colágeno/metabolismo , Módulo de Elasticidad/fisiología , Matriz Extracelular/metabolismo , Fibrosis/metabolismo , Hepatocitos/metabolismo , Macrófagos del Hígado/metabolismo , Hígado/patología , Hígado/fisiología , Hígado/fisiopatología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder affecting tissues of mesenchymal origin. Most individuals with HGPS harbor a de novo c.1824C > T (p.G608G) mutation in the gene encoding lamin A (LMNA), which activates a cryptic splice donor site resulting in production of the toxic "progerin" protein. Clinical manifestations include growth deficiency, lipodystrophy, sclerotic dermis, cardiovascular defects, and bone dysplasia. Here we utilized the LmnaG609G knock-in (KI) mouse model of HGPS to further define mechanisms of bone loss associated with normal and premature aging disorders. Newborn skeletal staining of KI mice revealed altered rib cage shape and spinal curvature, and delayed calvarial mineralization with increased craniofacial and mandibular cartilage content. MicroCT analysis and mechanical testing of adult femurs indicated increased fragility associated with reduced bone mass, recapitulating the progressive bone deterioration that occurs in HGPS patients. We investigated mechanisms of bone loss in KI mice at the cellular level in bone cell populations. Formation of wild-type and KI osteoclasts from marrow-derived precursors was inhibited by KI osteoblast-conditioned media in vitro, suggesting a secreted factor(s) responsible for decreased osteoclasts on KI trabecular surfaces in vivo. Cultured KI osteoblasts exhibited abnormal differentiation characterized by reduced deposition and mineralization of extracellular matrix with increased lipid accumulation compared to wild-type, providing a mechanism for altered bone formation. Furthermore, quantitative analyses of KI transcripts confirmed upregulation of adipogenic genes both in vitro and in vivo. Thus, osteoblast phenotypic plasticity, inflammation and altered cellular cross-talk contribute to abnormal bone formation in HGPS mice.
Asunto(s)
Envejecimiento Prematuro , Enfermedades del Desarrollo Óseo , Progeria , Ratones , Animales , Progeria/genética , Progeria/metabolismo , Mutación , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Diferenciación CelularRESUMEN
INTRODUCTION: We evaluated the 3-dimensional craniofacial skeletal and pharyngeal airway morphology in growing patients with and without cleft lip and palate. METHODS: Our juvenile subjects consisted of 34 girls (ages, 9-12 years); 15 had cleft lip and palate, and 19 did not. The adolescent subjects consisted of 32 girls (ages, 13-17 years); 14 had cleft lip and palate, and 18 did not. Each subject was examined with cone-beam computed tomography. The dimensions of the craniofacial skeleton and pharyngeal airway were measured. The Scheffé method of multiple comparisons was used to identify relationships among skeletal and pharyngeal variables. RESULTS: The pharyngeal airway and mandibular size variables did not differ significantly between the juvenile and adolescent cleft lip and palate groups. Significant differences were observed between each cleft lip and palate group and its corresponding control group. FHN-A, FHN-B, FH-NA, FH-NB, and Co-Me were significantly smaller in the cleft lip and palate groups than in the corresponding control groups. Anteroposterior and lateral widths, heights, and volumes of the superior oropharyngeal airway were significantly smaller in the adolescent cleft lip and palate group than in the adolescent controls. CONCLUSIONS: The mandible and the oropharyngeal airway were larger in the adolescent controls than in the juvenile controls without cleft lip and palate, but there were no significant differences between the adolescent and juvenile patients with cleft lip and palate.
Asunto(s)
Fisura del Paladar/patología , Imagenología Tridimensional/métodos , Mandíbula/patología , Desarrollo Maxilofacial , Faringe/patología , Adolescente , Análisis de Varianza , Pueblo Asiatico , Estudios de Casos y Controles , Cefalometría , Vértebras Cervicales/diagnóstico por imagen , Vértebras Cervicales/crecimiento & desarrollo , Niño , Labio Leporino/patología , Labio Leporino/cirugía , Fisura del Paladar/diagnóstico por imagen , Fisura del Paladar/cirugía , Tomografía Computarizada de Haz Cónico , Epiglotis/anatomía & histología , Epiglotis/diagnóstico por imagen , Epiglotis/patología , Femenino , Humanos , Japón , Mandíbula/anatomía & histología , Mandíbula/diagnóstico por imagen , Cavidad Nasal/anatomía & histología , Cavidad Nasal/diagnóstico por imagen , Cavidad Nasal/patología , Orofaringe/anatomía & histología , Orofaringe/diagnóstico por imagen , Orofaringe/patología , Paladar Blando/anatomía & histología , Paladar Blando/diagnóstico por imagen , Paladar Blando/patología , Faringe/anatomía & histología , Faringe/diagnóstico por imagen , Radiografía Dental Digital , Estadísticas no ParamétricasRESUMEN
Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.
Asunto(s)
Colágeno Tipo I , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Hidroxilación , Lisina/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Lysyl hydroxylase 2 (LH2) is an enzyme that catalyzes the hydroxylation of lysine (Lys) residues in fibrillar collagen telopeptides, a critical post-translational modification for the stability of intermolecular cross-links. Though abnormal LH2 activities have been implicated in various diseases including Bruck syndrome, the molecular basis of the pathologies is still not well understood. Since LH2 null mice die at early embryonic stage, we generated LH2 heterozygous (LH2+/-) mice in which LH2 level is significantly diminished, and characterized collagen and bone phenotypes using femurs. Compared to the wild-type (WT), LH2+/- collagen showed a significant decrease in the ratio of hydroxylysine (Hyl)- to the Lys-aldehyde-derived collagen cross-links without affecting the total number of aldehydes involved in cross-links. Mass spectrometric analysis revealed that, in LH2+/- type I collagen, the extent of hydroxylation of all telopeptidyl Lys residues was significantly decreased. In the helical domain, Lys hydroxylation at the cross-linking sites was either unaffected or slightly lower, but other sites were significantly diminished compared to WT. In LH2+/- femurs, mineral densities of cortical and cancellous bones were significantly decreased and the mechanical properties of cortical bones evaluated by nanoindentation analysis were compromised. When cultured, LH2+/- osteoblasts poorly produced mineralized nodules compared to WT osteoblasts. These data provide insight into the functionality of LH2 in collagen molecular phenotype and its critical role in bone matrix mineralization and mechanical properties.
Asunto(s)
Osteogénesis Imperfecta , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Animales , Colágeno/química , Colágeno Tipo I/genética , Ratones , Fenotipo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/química , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/farmacocinéticaRESUMEN
In spite of major advances over the past several decades in diagnosis and treatment, breast cancer remains a global cause of morbidity and premature death for both human and veterinary patients. Due to multiple shared clinicopathological features, dogs provide an excellent model of human breast cancer, thus, a comparative oncology approach may advance our understanding of breast cancer biology and improve patient outcomes. Despite an increasing awareness of the critical role of fibrillar collagens in breast cancer biology, tumor-permissive collagen features are still ill-defined. Here, we characterize the molecular and morphological phenotypes of type I collagen in canine mammary gland tumors. Canine mammary carcinoma samples contained longer collagen fibers as well as a greater population of wider fibers compared to non-neoplastic and adenoma samples. Furthermore, the total number of collagen cross-links enriched in the stable hydroxylysine-aldehyde derived cross-links was significantly increased in neoplastic mammary gland samples compared to non-neoplastic mammary gland tissue. The mass spectrometric analyses of type I collagen revealed that in malignant mammary tumor samples, lysine residues, in particular those in the telopeptides, were markedly over-hydroxylated in comparison to non-neoplastic mammary tissue. The extent of glycosylation of hydroxylysine residues was comparable among the groups. Consistent with these data, expression levels of genes encoding lysyl hydroxylase 2 (LH2) and its molecular chaperone FK506-binding protein 65 were both significantly increased in neoplastic samples. These alterations likely lead to an increase in the LH2-mediated stable collagen cross-links in mammary carcinoma that may promote tumor cell metastasis in these patients.
Asunto(s)
Colágeno/metabolismo , Enfermedades de los Perros/metabolismo , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/metabolismo , Aminoácidos/metabolismo , Animales , Colágeno Tipo I/metabolismo , Enfermedades de los Perros/patología , Perros , Femenino , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/patología , Fenotipo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and incidence rates are continuing to rise globally. HNSCC patient prognosis is closely related to the occurrence of tumor metastases, and collagen within the tumor microenvironment (TME) plays a key role in this process. Lysyl hydroxylase 2 (LH2), encoded by the Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) gene, catalyzes hydroxylation of telopeptidyl lysine (Lys) residues of fibrillar collagens which then undergo subsequent modifications to form stable intermolecular cross-links that change the biomechanical properties (i.e. quality) of the TME. While LH2-catalyzed collagen modification has been implicated in driving tumor progression and metastasis in diverse cancers, little is known about its role in HNSCC progression. Thus, using gain- and loss-of-function studies, we examined the effects of LH2 expression levels on collagen cross-linking and cell behavior in vitro and in vivo using a tractable bioluminescent imaging-based orthotopic xenograft model. We found that LH2 overexpression dramatically increases HNSCC cell migratory and invasive abilities in vitro and that LH2-driven changes in collagen cross-linking robustly induces metastasis in vivo. Specifically, the amount of LH2-mediated collagen cross-links increased significantly with PLOD2 overexpression, without affecting the total quantity of collagen cross-links. Conversely, LH2 knockdown significantly blunted HNSCC cells invasive capacity in vitro and metastatic potential in vivo. Thus, regardless of the total "quantity" of collagen crosslinks, it is the "quality" of these cross-links that is the key driver of HNSCC tumor metastatic dissemination. These data implicate LH2 as a key regulator of HNSCC tumor invasion and metastasis by modulating collagen cross-link quality and suggest that therapeutic strategies targeting LH2-mediated collagen cross-linking in the TME may be effective in controlling tumor progression and improving disease outcomes.
Asunto(s)
Colágeno/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Imagen Molecular , Metástasis de la Neoplasia , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/diagnóstico por imagen , Microambiente Tumoral/genéticaRESUMEN
Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.
Asunto(s)
Adenocarcinoma del Pulmón/fisiopatología , Progresión de la Enfermedad , Glucosiltransferasas/metabolismo , Neoplasias Pulmonares/fisiopatología , Animales , RatonesRESUMEN
Collagenous stromal accumulations predict a worse clinical outcome in a variety of malignancies. Better tools are needed to elucidate the way in which collagen influences cancer cells. Here, we report a method to generate collagenous matrices that are deficient in key post-translational modifications and evaluate cancer cell behaviors on those matrices. We utilized genetic and biochemical approaches to inhibit lysine hydroxylation and glucosylation on collagen produced by MC-3T3-E1 murine osteoblasts (MC cells). Seeded onto MC cell-derived matrix surface, multicellular aggregates containing lung adenocarcinoma cells alone or in combination with cancer-associated fibroblasts dissociated with temporal and spatial patterns that were influenced by collagen modifications. These findings demonstrate the feasibility of generating defined collagen matrices that are suitable for cell culture studies.