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1.
Am J Pathol ; 187(10): 2300-2311, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28734943

RESUMEN

Classic Ehlers-Danlos syndrome (cEDS) is characterized by fragile, hyperextensible skin and hypermobile joints. cEDS can be caused by heterozygosity for missense mutations in genes COL5A2 and COL5A1, which encode the α2(V) and α1(V) chains, respectively, of collagen V, and is most often caused by COL5A1 null alleles. However, COL5A2 null alleles have yet to be associated with cEDS or other human pathologies. We previously showed that mice homozygous null for the α2(V) gene Col5a2 are early embryonic lethal, whereas haploinsufficiency caused aberrancies of adult skin, but not a frank cEDS-like phenotype, as skin hyperextensibility at low strain and dermal cauliflower-contoured collagen fibril aggregates, two cEDS hallmarks, were absent. Herein, we show that ubiquitous postnatal Col5a2 knockdown results in pathognomonic dermal cauliflower-contoured collagen fibril aggregates, but absence of skin hyperextensibility, demonstrating these cEDS hallmarks to arise separately from loss of collagen V roles in control of collagen fibril growth and nucleation events, respectively. Col5a2 knockdown also led to loss of dermal white adipose tissue (WAT) and markedly decreased abdominal WAT that was characterized by miniadipocytes and increased collagen deposition, suggesting α2(V) to be important to WAT development/maintenance. More important, Col5a2 haploinsufficiency markedly increased the incidence and severity of abdominal aortic aneurysms, and caused aortic arch ruptures and dissections, indicating that α2(V) chain deficits may play roles in these pathologies in humans.


Asunto(s)
Tejido Adiposo/anomalías , Aneurisma de la Aorta Torácica/genética , Colágeno Tipo V/deficiencia , Colágeno/deficiencia , Predisposición Genética a la Enfermedad , Anomalías Cutáneas/metabolismo , Piel/patología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/patología , Animales , Aneurisma de la Aorta Torácica/patología , Colágeno/metabolismo , Colágeno Tipo V/metabolismo , Dermis/patología , Modelos Animales de Enfermedad , Síndrome de Ehlers-Danlos/patología , Colágenos Fibrilares/metabolismo , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Integrasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Reproducibilidad de los Resultados , Piel/efectos de los fármacos , Piel/ultraestructura , Anomalías Cutáneas/patología , Tamoxifeno/farmacología , Cicatrización de Heridas/efectos de los fármacos
2.
J Biol Chem ; 291(7): 3359-70, 2016 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-26721885

RESUMEN

We have shown previously that collagen V (col(V)) autoimmunity is a consistent feature of atherosclerosis in human coronary artery disease and in the Apoe(-/-) mouse model. We have also shown sensitization of Apoe(-/-) mice with col(V) to markedly increase the atherosclerotic burden, providing evidence of a causative role for col(V) autoimmunity in atherosclerotic pathogenesis. Here we sought to determine whether induction of immune tolerance to col(V) might ameliorate atherosclerosis, providing further evidence for a causal role for col(V) autoimmunity in atherogenesis and providing insights into the potential for immunomodulatory therapeutic interventions. Mucosal inoculation successfully induced immune tolerance to col(V) with an accompanying reduction in plaque burden in Ldlr(-/-) mice on a high-cholesterol diet. The results therefore demonstrate that inoculation with col(V) can successfully ameliorate the atherosclerotic burden, suggesting novel approaches for therapeutic interventions. Surprisingly, tolerance and reduced atherosclerotic burden were both dependent on the recently described IL-35 and not on IL-10, the immunosuppressive cytokine usually studied in the context of induced tolerance and amelioration of atherosclerotic symptoms. In addition to the above, using recombinant protein fragments, we were able to localize two epitopes of the α1(V) chain involved in col(V) autoimmunity in atherosclerotic Ldlr(-/-) mice, suggesting future courses of experimentation for the characterization of such epitopes.


Asunto(s)
Aterosclerosis/prevención & control , Autoinmunidad , Colágeno Tipo V/uso terapéutico , Hipersensibilidad Tardía/prevención & control , Tolerancia Inmunológica , Interleucinas/metabolismo , Administración Intranasal , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/metabolismo , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Bovinos , Células Cultivadas , Colágeno Tipo V/administración & dosificación , Colágeno Tipo V/química , Colágeno Tipo V/genética , Dieta Occidental/efectos adversos , Mapeo Epitopo , Humanos , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/metabolismo , Hipersensibilidad Tardía/fisiopatología , Inmunidad Mucosa , Interleucinas/antagonistas & inhibidores , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bazo/inmunología , Bazo/metabolismo , Bazo/patología
3.
Cell Tissue Res ; 370(3): 461-476, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28936615

RESUMEN

Procollagen C-proteinase enhancer 1 (PCPE-1) has been characterized as a protein capable of enhancing the activity of bone morphogenetic protein 1/tolloid-like proteinases in the biosynthetic processing of C-propeptides from procollagens I-III. This processing step is thought necessary to the formation of collagen I-III monomers capable of forming fibrils. Thus, PCPE-1 is predicted to play an important role in scarring, as scar tissue is predominantly composed of fibrillar collagen. Corneal scarring is of great clinical importance, as it leads to loss of visual acuity and, in severe cases, blindness. Here, we investigate a possible role for PCPE-1 in corneal scarring. Although differences in corneal opacity associated with scarring following injury of Pcolce -/- and wild-type (WT) mice using full-thickness excision or alkali burn models of corneal injury were not grossly apparent, differences in procollagen I processing levels between Pcolce -/- and WT primary corneal keratocytes were consistent with a role for PCPE-1 in corneal collagen deposition. An unexpected finding was that neoangiogenesis, which follows alkali burn cornea injury, was strikingly increased in Pcolce -/- cornea, compared to WT. A series of aortic ring assays confirmed the anti-angiogenic effects of PCPE-1. Another unexpected finding was of abnormalities of epithelial basement membrane and of re-epithelialization following Pcolce -/- corneal injury. Thus, PCPE-1 appears to be of importance as an anti-angiogenic factor and in re-epithelialization following injury in cornea and perhaps in other tissues as well.


Asunto(s)
Lesiones de la Cornea/patología , Queratocitos de la Córnea/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Neovascularización Fisiológica/fisiología , Animales , Células Cultivadas , Colágeno/metabolismo , Córnea/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cicatrización de Heridas/fisiología
4.
Nat Genet ; 40(9): 1119-23, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18677313

RESUMEN

Geleophysic dysplasia is an autosomal recessive disorder characterized by short stature, brachydactyly, thick skin and cardiac valvular anomalies often responsible for an early death. Studying six geleophysic dysplasia families, we first mapped the underlying gene to chromosome 9q34.2 and identified five distinct nonsense and missense mutations in ADAMTSL2 (a disintegrin and metalloproteinase with thrombospondin repeats-like 2), which encodes a secreted glycoprotein of unknown function. Functional studies in HEK293 cells showed that ADAMTSL2 mutations lead to reduced secretion of the mutated proteins, possibly owing to the misfolding of ADAMTSL2. A yeast two-hybrid screen showed that ADAMTSL2 interacts with latent TGF-beta-binding protein 1. In addition, we observed a significant increase in total and active TGF-beta in the culture medium as well as nuclear localization of phosphorylated SMAD2 in fibroblasts from individuals with geleophysic dysplasia. These data suggest that ADAMTSL2 mutations may lead to a dysregulation of TGF-beta signaling and may be the underlying mechanism of geleophysic dysplasia.


Asunto(s)
Anomalías Múltiples/genética , Proteínas de la Matriz Extracelular/genética , Trastornos del Crecimiento/genética , Válvulas Cardíacas/anomalías , Factor de Crecimiento Transformador beta/metabolismo , Disponibilidad Biológica , Línea Celular , Niño , Preescolar , Deformidades Congénitas de la Mano/genética , Cardiopatías Congénitas/genética , Humanos , Mutación
5.
Hum Mol Genet ; 23(12): 3085-101, 2014 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-24419319

RESUMEN

Osteogenesis imperfecta (OI), or brittle bone disease, is most often caused by dominant mutations in the collagen I genes COL1A1/COL1A2, whereas rarer recessive OI is often caused by mutations in genes encoding collagen I-interacting proteins. Recently, mutations in the gene for the proteinase bone morphogenetic 1 (BMP1) were reported in two recessive OI families. BMP1 and the closely related proteinase mammalian tolloid-like 1 (mTLL1) are co-expressed in various tissues, including bone, and have overlapping activities that include biosynthetic processing of procollagen precursors into mature collagen monomers. However, early lethality of Bmp1- and Tll1-null mice has precluded use of such models for careful study of in vivo roles of their protein products. Here we employ novel mouse strains with floxed Bmp1 and Tll1 alleles to induce postnatal, simultaneous ablation of the two genes, thus avoiding barriers of Bmp1(-/-) and Tll1(-/-) lethality and issues of functional redundancy. Bones of the conditionally null mice are dramatically weakened and brittle, with spontaneous fractures-defining features of OI. Additional skeletal features include osteomalacia, thinned/porous cortical bone, reduced processing of procollagen and dentin matrix protein 1, remarkably high bone turnover and defective osteocyte maturation that is accompanied by decreased expression of the osteocyte marker and Wnt-signaling inhibitor sclerostin, and by marked induction of canonical Wnt signaling. The novel animal model presented here provides new opportunities for in-depth analyses of in vivo roles of BMP1-like proteinases in bone and other tissues, and for their roles, and for possible therapeutic interventions, in OI.


Asunto(s)
Proteína Morfogenética Ósea 1/genética , Fémur/patología , Técnicas de Silenciamiento del Gen/métodos , Osteogénesis Imperfecta/patología , Metaloproteinasas Similares a Tolloid/genética , Animales , Proteína Morfogenética Ósea 1/metabolismo , Modelos Animales de Enfermedad , Fémur/ultraestructura , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Osteogénesis Imperfecta/genética , Metaloproteinasas Similares a Tolloid/metabolismo
6.
Am J Pathol ; 185(7): 2000-11, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25987251

RESUMEN

Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2(-/-) homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2(-/-) embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2(-/-) and Col5a1(-/-) fibril formation and embryonic survival suggest that α1(V)3 homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of α2(V) chains, serve functional roles that partially compensate for loss of the most common collagen V isoform. Col5a2(+/-) adults have skin with marked hyperextensibility and reduced tensile strength at high strain but not at low strain. Col5a2(+/-) adults also have aortas with increased compliance and reduced tensile strength. Results thus suggest that COL5A2(+/-) humans, although unlikely to present with frank classic Ehlers-Danlos syndrome, are likely to have fragile connective tissues with increased susceptibility to trauma and certain chronic pathologic conditions.


Asunto(s)
Colágeno Tipo V/genética , Colágeno/genética , Síndrome de Ehlers-Danlos/genética , Adulto , Alelos , Animales , Colágeno/metabolismo , Colágeno Tipo V/metabolismo , Tejido Conectivo/anomalías , Tejido Conectivo/patología , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/patología , Femenino , Heterocigoto , Homocigoto , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación , Fenotipo , Piel/patología
7.
Am J Hum Genet ; 90(4): 661-74, 2012 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-22482805

RESUMEN

Bone morphogenetic protein 1 (BMP1) is an astacin metalloprotease with important cellular functions and diverse substrates, including extracellular-matrix proteins and antagonists of some TGFß superfamily members. Combining whole-exome sequencing and filtering for homozygous stretches of identified variants, we found a homozygous causative BMP1 mutation, c.34G>C, in a consanguineous family affected by increased bone mineral density and multiple recurrent fractures. The mutation is located within the BMP1 signal peptide and leads to impaired secretion and an alteration in posttranslational modification. We also characterize a zebrafish bone mutant harboring lesions in bmp1a, demonstrating conservation of BMP1 function in osteogenesis across species. Genetic, biochemical, and histological analyses of this mutant and a comparison to a second, similar locus reveal that Bmp1a is critically required for mature-collagen generation, downstream of osteoblast maturation, in bone. We thus define the molecular and cellular bases of BMP1-dependent osteogenesis and show the importance of this protein for bone formation and stability.


Asunto(s)
Proteína Morfogenética Ósea 1/fisiología , Osteogénesis/genética , Osteogénesis/fisiología , Animales , Secuencia de Bases , Conservadores de la Densidad Ósea/uso terapéutico , Proteína Morfogenética Ósea 1/genética , Proteína Morfogenética Ósea 1/metabolismo , Huesos/metabolismo , Diferenciación Celular , Preescolar , Colágeno/biosíntesis , Difosfonatos/uso terapéutico , Exoma , Femenino , Fracturas Óseas/tratamiento farmacológico , Fracturas Óseas/prevención & control , Sitios Genéticos , Proteínas de Choque Térmico , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Osteogénesis/efectos de los fármacos , Fragmentos de Péptidos , Procesamiento Proteico-Postraduccional , Pez Cebra/genética , Pez Cebra/metabolismo
8.
bioRxiv ; 2024 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-38585782

RESUMEN

Mitochondrial dysfunction has been linked to both idiopathic and familial forms of Parkinson's disease (PD). We have previously identified RCC1-like (RCC1L) as a protein of the inner mitochondrial membrane important to mitochondrial fusion. Herein, to test whether deficits in RCC1L mitochondrial function might be involved in PD pathology, we have selectively ablated the Rcc1l gene in the dopaminergic (DA) neurons of mice. A PD-like phenotype resulted that includes progressive movement abnormalities, paralleled by progressive degeneration of the nigrostriatal tract. Experimental and control groups were examined at 2, 3-4, and 5-6 months of age. Animals were tested in the open field task to quantify anxiety, exploratory drive, locomotion, and immobility; and in the cylinder test to quantify rearing behavior. Beginning at 3-4 months, both female and male Rcc1l knockout mice show rigid muscles and resting tremor, kyphosis and a growth deficit compared with heterozygous or wild type littermate controls. Rcc1l knockout mice begin showing locomotor impairments at 3-4 months, which progress until 5-6 months of age, at which age the Rcc1l knockout mice die. The progressive motor impairments were associated with progressive and significantly reduced tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta (SNc), and dramatic loss of nigral DA projections in the striatum. Dystrophic spherical mitochondria are apparent in the soma of SNc neurons in Rcc1l knockout mice as early as 1.5-2.5 months of age and become progressively more pronounced until 5-6 months. Together, the results reveal the RCC1L protein to be essential to in vivo mitochondrial function in DA neurons. Further characterization of this mouse model will determine whether it represents a new model for in vivo study of PD, and the putative role of the human RCC1L gene as a risk factor that might increase PD occurrence and severity in humans.

9.
J Biol Chem ; 287(48): 40598-610, 2012 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-23060441

RESUMEN

BACKGROUND: α1(V) is an extensively modified collagen chain important in disease. RESULTS: Comprehensive mapping of α1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets. CONCLUSION: The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties. SIGNIFICANCE: Positions, numbers, and occupancy of modified sites can provide insights into α1(V) biological properties. Aberrant expression of the type V collagen α1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the α1(V) chain are linked to lung transplant rejection and atherosclerosis. The α1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the α1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental α1(V) and human recombinant pro-α1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these α1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed α1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of α1(V) chain and type V collagen properties.


Asunto(s)
Colágeno Tipo V/química , Hidroxiprolina/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Bovinos , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Humanos , Hidroxiprolina/genética , Hidroxiprolina/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Mapeo Peptídico
10.
Am J Physiol Lung Cell Mol Physiol ; 304(6): L401-14, 2013 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23262228

RESUMEN

Obliterative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death after lung transplantation. Type V collagen [col(V)] overexpression and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here, we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) α1 chains [α1 (V)] in normal airway epithelial cells in vitro and detected α1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-ß mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-ßRI tyrosine kinase, p38 MAPK, or focal adhesion kinase prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-ß mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-ß, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation.


Asunto(s)
Bronquitis/metabolismo , Colágeno Tipo V/metabolismo , Transición Epitelial-Mesenquimal , Regulación de la Expresión Génica , Interleucina-17/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Autoanticuerpos/metabolismo , Bronquitis/inmunología , Bronquitis/patología , Bronquitis/cirugía , Movimiento Celular , Células Cultivadas , Colágeno Tipo V/genética , Colágeno Tipo V/inmunología , Femenino , Expresión Génica , Humanos , Trasplante de Pulmón , Masculino , Ratones , Persona de Mediana Edad , Cultivo Primario de Células , Ratas , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal
11.
J Biol Chem ; 286(49): 41905-41911, 2011 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-22027825

RESUMEN

Many secreted proteins are synthesized as precursors with propeptides that must be cleaved to yield the mature functional form of the molecule. In addition, various growth factors occur in extracellular latent complexes with protein antagonists and are activated upon cleavage of such antagonists. Research in the separate fields of embryonic patterning and extracellular matrix formation has identified members of the BMP1/Tolloid-like family of metalloproteinases as key players in these types of biosynthetic processing events in species ranging from Drosophila to humans.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Metaloproteasas/metabolismo , Animales , Proteína Morfogenética Ósea 1/metabolismo , Colágeno/metabolismo , Drosophila melanogaster , Matriz Extracelular , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Estructura Terciaria de Proteína , Factor de Crecimiento Transformador beta/metabolismo , Xenopus laevis
12.
J Biol Chem ; 286(33): 29014-29025, 2011 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-21697095

RESUMEN

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor ß1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.


Asunto(s)
Proteína Morfogenética Ósea 1/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Animales , Proteína Morfogenética Ósea 1/genética , Línea Celular Tumoral , Proliferación Celular , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Mutantes , Transducción de Señal/fisiología , Proteínas Smad , Metaloproteinasas Similares a Tolloid/genética , Metaloproteinasas Similares a Tolloid/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo
13.
Circ Res ; 107(9): 1106-16, 2010 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-20814021

RESUMEN

RATIONALE: Considerable evidence shows atherosclerosis to be a chronic inflammatory disease in which immunity to self-antigens contributes to disease progression. We recently identified the collagen type V [col(V)] α1(V) chain as a key autoantigen driving the Th17-dependent cellular immunity underlying another chronic inflammatory disease, obliterative bronchiolitis. Because specific induction of α1(V) chains has previously been reported in human atheromas, we postulated involvement of col(V) autoimmunity in atherosclerosis. OBJECTIVE: To determine whether col(V) autoimmunity may be involved in the pathogenesis of atherosclerosis. METHODS AND RESULTS: Here, we demonstrate Th17-dependent anti-col(V) immunity to be characteristic of atherosclerosis in human coronary artery disease (CAD) patients and in apolipoprotein E-null (ApoE(-/-)) atherosclerotic mice. Responses were α1(V)-specific in CAD with variable Th1 pathway involvement. In early atherosclerosis in ApoE(-/-) mice, anti-col(V) immunity was tempered by an interleukin (IL)-10-dependent mechanism. In support of a causal role for col(V) autoimmunity in the pathogenesis of atherosclerosis, col(V) sensitization of ApoE(-/-) mice on a regular chow diet overcame IL-10-mediated inhibition of col(V) autoimmunity, leading to increased atherosclerotic burden in these mice and local accumulation of IL-17-producing cells, particularly in the col(V)-rich adventitia subjacent to the atheromas. CONCLUSIONS: These findings establish col(V) as an autoantigen in human CAD and show col(V) autoimmunity to be a consistent feature in atherosclerosis in humans and mice. Furthermore, data are consistent with a causative role for col(V) in the pathogenesis of atherosclerosis.


Asunto(s)
Aterosclerosis/inmunología , Enfermedades Autoinmunes/inmunología , Colágeno Tipo V/fisiología , Interleucina-17/fisiología , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Bovinos , Colágeno Tipo V/efectos adversos , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/patología
14.
Dev Biol ; 341(2): 444-58, 2010 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-20226780

RESUMEN

Chordin is the prototype of a group of cysteine-rich domain-containing proteins that bind and modulate signaling of various TGFbeta-like ligands. Chordin-like 1 and 2 (CHL1 and 2) are two members of this group that have been described in human, mouse, and chick. However, in vivo roles for CHL1 and 2 in early development are unknown due to lack of loss-of-function analysis. Here we identify and characterize zebrafish, Danio rerio, CHL (Chl). The chl gene is on a region of chromosome 21 syntenic with the area of murine chromosome 7 bearing the CHL2 gene. Inability to identify a separate zebrafish gene corresponding to the mammalian CHL1 gene suggests that Chl may serve roles in zebrafish distributed between CHL1 and CHL2 in other species. Chl is a maternal factor that is also zygotically expressed later in development and has spatiotemporal expression patterns that differ from but overlap those of zebrafish chordin (Chd), suggesting differences but also possible overlap in developmental roles of the two proteins. Chl, like Chd, dorsalizes embryos upon overexpression and is cleaved by BMP1, which antagonizes this activity. Loss-of-function experiments demonstrate that Chl serves as a BMP antagonist with functions that overlap and are redundant with those of Chd in forming the dorsoventral axis.


Asunto(s)
Tipificación del Cuerpo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 1/metabolismo , Embrión no Mamífero/metabolismo , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Pez Cebra/genética , Proteínas de Pez Cebra/genética
15.
J Cell Biol ; 175(1): 111-20, 2006 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-17015622

RESUMEN

Transforming growth factor beta1 (TGFbeta1), an important regulator of cell behavior, is secreted as a large latent complex (LLC) in which it is bound to its cleaved prodomain (latency-associated peptide [LAP]) and, via LAP, to latent TGFbeta-binding proteins (LTBPs). The latter target LLCs to the extracellular matrix (ECM). Bone morphogenetic protein 1 (BMP1)-like metalloproteinases play key roles in ECM formation, by converting precursors into mature functional proteins, and in morphogenetic patterning, by cleaving the antagonist Chordin to activate BMP2/4. We provide in vitro and in vivo evidence that BMP1 cleaves LTBP1 at two specific sites, thus liberating LLC from ECM and resulting in consequent activation of TGFbeta1 via cleavage of LAP by non-BMP1-like proteinases. In mouse embryo fibroblasts, LAP cleavage is shown to be predominantly matrix metalloproteinase 2 dependent. TGFbeta1 is a potent inducer of ECM formation and of BMP1 expression. Thus, a role for BMP1-like proteinases in TGFbeta1 activation completes a novel fast-forward loop in vertebrate tissue remodeling.


Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Proteínas de Unión a TGF-beta Latente/metabolismo , Metaloendopeptidasas/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Proteína Morfogenética Ósea 1 , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Ratones , Modelos Biológicos , Estructura Terciaria de Proteína
16.
J Periodontol ; 92(7): 1018-1029, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33169406

RESUMEN

BACKGROUND: Periodontitis is caused by multiple factors involving a bacterial challenge and a susceptible host, although there is no report on gene mutation directly linked to this common disease. Mutations in the proteinase bone morphogenetic protein 1 (BMP1) were identified in patients with osteogenesis imperfecta, who display some dentin defects and alveolar bone loss. We previously reported essential roles of BMP1 and tolloid-like 1 (TLL1), two closely related extracellular proteinases with overlapping functions, in mouse periodontium growth by simultaneous knockout (KO) of both genes, although the separate roles of BMP1 and TLL1 have remained unclear. Here, we have investigated whether and how BMP1 and TLL1 separately maintain periodontal homeostasis by comparing single Bmp1 KO and Tll1 KO with double KO (dKO) phenotypes. METHODS: Floxed Bmp1 and/or Tll1 alleles were deleted in transgenic mice via ubiquitously expressed CreERT2 induced by tamoxifen treatment starting at 4-weeks of age (harvested at 18-weeks of age). Multiple approaches, including X-ray, micro-CT, calcein and alizarin red double-labeling, scanning electron microscopy, and histological and immunostaining assays, were used to analyze periodontal phenotypes and molecular mechanisms. RESULTS: Both Bmp1 KO and double KO mice exhibited severe periodontal defects, characterized by periodontal ligament (PDL) fiber loss and ectopic ossification in the expanded PDL area, and drastic reductions in alveolar bone and cementum volumes, whereas Tll1 KO mice displayed very mild phenotypes. Mechanistic studies revealed a sharp increase in the uncleaved precursor of type I collagen (procollagen I), leading to defective extracellular matrices. CONCLUSIONS: BMP1, but not TLL1, is essential for maintaining periodontal homeostasis. This occurs at least partly via biosynthetic processing of procollagen I, thereby maintaining appropriate levels of procollagen I and its activated products such as mature collagen I.


Asunto(s)
Péptido Hidrolasas , Metaloproteinasas Similares a Tolloid , Animales , Proteína Morfogenética Ósea 1/genética , Proteína Morfogenética Ósea 1/metabolismo , Homeostasis , Humanos , Ratones , Proteolisis , Metaloproteinasas Similares a Tolloid/genética , Metaloproteinasas Similares a Tolloid/metabolismo
17.
J Biol Chem ; 284(38): 25879-88, 2009 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-19617627

RESUMEN

Bone morphogenetic protein-1-like proteinases play key roles in formation of the extracellular matrix (ECM) in vertebrates via biosynthetic processing of precursors into mature functional proteins involved in ECM assembly. Such processing includes proteolytic activation of the zymogen for lysyl oxidase. Fibronectin (FN) is an abundant protein component of the ECM that is capable of regulating manifold cellular functions through its interactions with various ECM and cell surface proteins. It was previously shown that proteolytic activation of lysyl oxidase is much reduced in cultures of FN-null mouse embryo fibroblasts (MEFs). Here we demonstrate that cellular fibronectin, the form produced by fibroblasts and various other tissue cell types, and plasma fibronectin bind BMP1 with dissociation constants (KD) of approximately 100 nM, consistent with a physiological role. Also consistent with such a role, cellular fibronectin FN is shown to positively regulate BMP1 processing activity against Chordin, probiglycan, and type I procollagen in vitro. Endogenous FN and BMP1 are demonstrated to co-localize in cell layers and to form complexes in culture medium. In addition, processing of endogenous BMP1 substrates Chordin, probiglycan, and procollagen is demonstrated to be strikingly reduced in cultures of FN(-/-) MEFs compared with FN(+/-) MEF cultures despite similar levels of endogenous BMP1. These data support the conclusion that FN binds BMP1-like proteinases in vivo and that FN is an important determinant of the in vivo activity levels of BMP1-like proteinases.


Asunto(s)
Proteína Morfogenética Ósea 1/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Animales , Biglicano , Proteína Morfogenética Ósea 1/genética , Línea Celular Tumoral , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibronectinas/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Noqueados , Procolágeno/genética , Procolágeno/metabolismo , Unión Proteica/fisiología , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , Proteoglicanos/genética , Proteoglicanos/metabolismo , Especificidad por Sustrato/fisiología
18.
J Clin Invest ; 117(11): 3498-506, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17965778

RESUMEN

Bronchiolitis obliterans syndrome (BOS), a process of fibro-obliterative occlusion of the small airways in the transplanted lung, is the most common cause of lung transplant failure. We tested the role of cell-mediated immunity to collagen type V [col(V)] in this process. PBMC responses to col(II) and col(V) were monitored prospectively over a 7-year period. PBMCs from lung transplant recipients, but not from healthy controls or col(IV)-reactive Goodpasture's syndrome patients after renal transplant, were frequently col(V) reactive. Col(V)-specific responses were dependent on both CD4+ T cells and monocytes and required both IL-17 and the monokines TNF-alpha and IL-1beta. Strong col(V)-specific responses were associated with substantially increased incidence and severity of BOS. Incidences of acute rejection, HLA-DR mismatched transplants, and induction of HLA-specific antibodies in the transplant recipient were not as strongly associated with a risk of BOS. These data suggest that while alloimmunity initiates lung transplant rejection, de novo autoimmunity mediated by col(V)-specific Th17 cells and monocyte/macrophage accessory cells ultimately causes progressive airway obliteration.


Asunto(s)
Bronquiolitis Obliterante/inmunología , Colágeno Tipo V/inmunología , Susceptibilidad a Enfermedades , Rechazo de Injerto/inmunología , Inmunidad Celular , Interleucina-17/inmunología , Trasplante de Pulmón , Antígenos CD/inmunología , Colágeno Tipo II/inmunología , Humanos , Interferón gamma/inmunología , Interleucina-1beta/inmunología , Trasplante de Pulmón/inmunología , Trasplante de Pulmón/patología , Estudios Prospectivos , Factores de Riesgo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factor de Necrosis Tumoral alfa/inmunología
19.
J Immunol ; 181(8): 5738-47, 2008 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-18832733

RESUMEN

Primary graft dysfunction (PGD) is a major complication following lung transplantation. We reported that anti-type V collagen (col(V)) T cell immunity was strongly associated with PGD. However, the role of preformed anti-col(V) Abs and their potential target in PGD are unknown. Col(V) immune serum, purified IgG or B cells from col(V) immune rats were transferred to WKY rat lung isograft recipients followed by assessments of lung pathology, cytokines, and PaO(2)/FiO(2), an index of lung dysfunction in PGD. Immune serum, purified IgG, and B cells all induced pathology consistent with PGD within 4 days posttransfer; up-regulated IFN-gamma, TNF-alpha, and IL-1beta locally; and induced significant reductions in PaO(2)/FiO(2). Depleting anti-col(V) Abs before transfer demonstrated that IgG2c was a major subtype mediating injury. Confocal microscopy revealed strong apical col(V) expression on lung epithelial, but not endothelial cells; which was consistent with the ability of col(V) immune serum to induce complement-dependent cytotoxicity only in the epithelial cells. Examination of plasma from patients with or without PGD revealed that higher levels of preformed anti-col(V) Abs were strongly associated with PGD development. This study demonstrates a major role for anti-col(V) humoral immunity in PGD, and identifies the airway epithelium as a target in PGD.


Asunto(s)
Formación de Anticuerpos/inmunología , Autoanticuerpos/inmunología , Colágeno Tipo V/inmunología , Inmunoglobulina G/inmunología , Trasplante de Pulmón , Pulmón/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/farmacología , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos B/trasplante , Bovinos , Citocinas/inmunología , Células Endoteliales , Regulación de la Expresión Génica/inmunología , Pulmón/patología , Trasplante de Pulmón/patología , Ratas , Ratas Endogámicas WKY , Trasplante Isogénico
20.
Transplant Direct ; 6(10): e607, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-33062840

RESUMEN

Individuals harbor preexisting HLA-DR/DQ-restricted responses to collagen type V (ColV) mediated by Th17 cells under Treg control, both specific to peptides that bind to inherited HLA class II antigens. Yet after transplant, the donor-DR type somehow influences graft outcome. We hypothesized that, long after a lung or heart allograft, the particular HLA-DR type of the mismatched transplant donor transforms the specificity of the "anti-self" response. This could explain why, over long term, certain donor DRs could be more immunogenic than others. METHODS: We analyzed 7 HLA-DR15neg patients who had received a lung allograft from a DR15+ donor. To determine the mechanism of acquired specificity in self-reactivity, we analyzed the kinetics of DR1 (host) and DR15 (donor) peptide restriction in a heart transplant model using DR-transgenic mice. RESULTS: Beyond 1.5 years post-lung transplant, all patients tested had acquired DR15-restricted immune responses to ColV peptides. These responses were either unrestrained Th17 type (n = 4) or Th17 controlled by Treg arising early (<5 y) or late (>7 y) after transplant (n = 4). Treg suppression via conventional (transforming growth factor-ß [TGF-ß]) and extracellular vesicle-associated (IL-35) cytokines correlated with superior outcomes. Naïve DR1 and DR15 transgenic mice had preexisting DR-restricted responses, exclusively to ColV fragments containing DR1- or DR15-binding peptides. When HLA-DR1 transgenic recipients of a HLA-DR15 heart developed ColV reactivity post-transplant, mice that acutely rejected (20-25 d) responded only to the DR1-restricted ColV peptide epitope. In animals whose grafts survived long term, we could detect acquisition of DR from the transplant donor onto the surface of recipient dendritic cells, and immune responses against a donor DR15-restricted ColV peptide. CONCLUSIONS: These results might explain how certain donor HLA-DR types redirect host immune responses to novel peptides of critical self-antigens. Unless regulated, such responses may predispose the allograft to chronic rejection.

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