RESUMEN
Although the mechanism for activation of latent TGFß1 and TGFß3 is understood to involve the binding of the TGFß propeptide (LAP) to both an integrin and an insoluble substrate, the activation of latent TGFß2 has been unclear because the TGFß2 LAP does not have the classical integrin binding sequence found in the other two TGFß isoform LAPs. To assess the potential requirement for covalent linkage with a matrix or cell surface protein for the activation of latent TGFß2, we generated mice in which the TGFß2 Cys residue predicted to be involved in binding was mutated to Ser (Tgfb2C24S). We reasoned that, if covalent interaction with a second molecule is required for latent TGFß2 activation, mutant mice should display a Tgfb2 null (Tgfb2-/-)-like phenotype. Tgfb2C24S mice closely phenocopy Tgfb2-/- mice with death in utero between E18 and P1 and with congenital heart and kidney defects similar to those described for Tgfb2-/- mice. The mutant latent TGFß2 is secreted at levels similar to WT, yet TGFß signaling monitored as nuclear pSmad2 is suppressed. We conclude that, like latent TGFß1, latent TGFß2 activation requires binding to an immobilized matrix or plasma membrane molecule.
RESUMEN
Marfan syndrome causes a hereditary form of thoracic aortic aneurysms with worse outcomes in male compared to female patients. In this study, we examine the effects of 17 ß-estradiol on aortic dilation and rupture in a Marfan mouse model. Marfan male mice were administered 17 ß-estradiol, and the growth in the aortic root, along with the risk of aortic rupture, was measured. Transcriptomic profiling was used to identify enriched pathways from 17 ß-estradiol treatments. Aortic smooth muscle cells were then treated with cytokines to validate functional mechanisms. We show that 17 ß-estradiol decreased the size and rate of aortic root dilation and improved survival from rupture. The Marfan transcriptome was enriched in inflammatory genes, and the addition of 17 ß-estradiol modulated a set of genes that function through TNFα mediated NF-κB signaling. In addition, 17 ß-estradiol suppressed the induction of these TNFα induced genes in aortic smooth muscle cells in vitro in an NF-κB dependent manner, and 17 ß-estradiol decreased the formation of adventitial inflammatory foci in aortic roots in vivo. In conclusion, 17 ß-estradiol protects against the dilation and rupture of aortic roots in Marfan male mice through the inhibition of TNFα-NF-κB signaling.
Asunto(s)
Estradiol , Síndrome de Marfan , Femenino , Masculino , Animales , Ratones , Estradiol/farmacología , Factor de Necrosis Tumoral alfa/genética , Aorta Torácica , FN-kappa B , Dilatación , Síndrome de Marfan/tratamiento farmacológico , Síndrome de Marfan/genéticaRESUMEN
Marfan syndrome causes a hereditary form of thoracic aortic aneurysms with dilation of the aortic root. Human and animal models suggest a worse phenotype for males compared to females with respect to aneurysm size and risk of dissection. In this study we examine the effects of 17 ß-estradiol on aortic dilation and rupture in a Marfan mouse model. Marfan male mice were administered 17 ß-estradiol and the growth in aortic root size along with the risk of aortic rupture or dissection with the addition of angiotensin II was measured. Transcriptomic profiling was used to identify enriched pathways from 17 ß-estradiol treatment. Aortic smooth muscle cells were then treated with cytokines in order to validate the mechanism of 17 ß-estradiol protection. We show that 17 ß-estradiol decreased the size and rate of aortic root dilation and improved survival from rupture and dissection after treatment with angiotensin II. The Marfan transcriptome was enriched in inflammatory genes and the addition of 17 ß-estradiol modulated a set of genes that function through TNFα mediated NF-κB signaling. These included many proteins known to play a role in the phenotypic shift of aortic smooth muscle cells from a contractile to a more inflammatory-like state such as Vcam-1, Mcp-1, Lgals3, Il-6, Il-1b, and C3. In addition, 17 ß-estradiol suppressed the induction of these TNFα induced genes in aortic smooth muscle cells in vitro and this effect appears to be NF-κB dependent. In conclusion, 17 ß-estradiol protects against the dilation and rupture of aortic roots in Marfan male mice through the inhibition of TNFα -NF-κB signaling and thus prevents the phenotypic switch of aortic smooth muscle cells from a contractile to an inflammatory state.
RESUMEN
To assess the contribution of individual TGF-ß isoforms to aortopathy in Marfan syndrome (MFS), we quantified the survival and phenotypes of mice with a combined fibrillin1 (the gene defective in MFS) hypomorphic mutation and a TGF-ß1, 2, or 3 heterozygous null mutation. The loss of TGF-ß2, and only TGF-ß2, resulted in 80% of the double mutant animals dying earlier, by postnatal day 20, than MFS only mice. Death was not from thoracic aortic rupture, as observed in MFS mice, but was associated with hyperplastic aortic valve leaflets, aortic regurgitation, enlarged aortic root, increased heart weight, and impaired lung alveolar septation. Thus, there appears to be a relationship between loss of fibrillin1 and TGF-ß2 in the postnatal development of the heart, aorta and lungs.
Asunto(s)
Haploinsuficiencia , Síndrome de Marfan , Animales , Ratones , Aorta , Fibrilina-1/genética , Síndrome de Marfan/genética , Fenotipo , Factor de Crecimiento Transformador beta2/genéticaRESUMEN
The major diseases affecting the thoracic aorta are aneurysms and acute dissections, and pathogenic variants in 11 genes are confirmed to lead to heritable thoracic aortic disease. However, many families in which multiple members have thoracic aortic disease do not have alterations in the known aortopathy genes. Genes highly expressed in the aorta were assessed for rare variants in exome sequencing data from such families, and compound rare heterozygous variants (p.Pro45Argfs∗25 and p.Glu750∗) in LTBP3 were identified in affected members of one family. A homozygous variant (p.Asn678_Gly681delinsThrCys) that introduces an additional cysteine into an epidermal growth factor (EGF)-like domain in the corresponding protein, latent TGF-ß binding protein (LTBP-3), was identified in a second family. Individuals with compound heterozygous or homozygous variants in these families have aneurysms and dissections of the thoracic aorta, as well as aneurysms of the abdominal aorta and other arteries, along with dental abnormalities and short stature. Heterozygous carriers of the p.Asn678_Gly681delinsThrCys variant have later onset of thoracic aortic disease, as well as dental abnormalities. In these families, LTBP3 variants segregated with thoracic aortic disease with a combined LOD score of 3.9. Additionally, heterozygous rare LTBP3 variants were found in individuals with early onset of acute aortic dissections, and some of these variants disrupted LTBP-3 levels or EGF-like domains. When compared to wild-type mice, Ltbp3-/- mice have enlarged aortic roots and ascending aortas. In summary, homozygous LTBP3 pathogenic variants predispose individuals to thoracic aortic aneurysms and dissections, along with the previously described skeletal and dental abnormalities.
Asunto(s)
Aneurisma de la Aorta Torácica/genética , Disección Aórtica/genética , Predisposición Genética a la Enfermedad , Proteínas de Unión a TGF-beta Latente/genética , Mutación/genética , Adulto , Anciano de 80 o más Años , Animales , Presión Sanguínea/genética , Femenino , Homocigoto , Humanos , Masculino , Ratones , Persona de Mediana Edad , LinajeRESUMEN
Latent transforming growth factor ß (TGFß)-binding proteins (LTBPs) are important for the secretion, activation, and function of mature TGFß, especially so in cancer cell physiology. However, specific roles of the LTBPs remain understudied in the context of the primary tumor microenvironment. Herein, we investigated the role of LTBP3 in the distinct processes involved in cancer metastasis. By using three human tumor cell lines of different tissue origin (epidermoid HEp-3 and prostate PC-3 carcinomas and HT-1080 fibrosarcoma) and several metastasis models conducted in both mammalian and avian settings, we show that LTBP3 is involved in the early dissemination of primary cancer cells, namely in the intravasation step of the metastatic cascade. Knockdown of LTBP3 in all tested cell lines led to significant inhibition of tumor cell intravasation, but did not affect primary tumor growth. LTBP3 was dispensable in the late steps of carcinoma cell metastasis that follow tumor cell intravasation, including vascular arrest, extravasation, and tissue colonization. However, LTBP3 depletion diminished the angiogenesis-inducing potential of HEp-3 cells in vivo, which was restorable by exogenous delivery of LTBP3 protein. A similar compensatory approach rescued the dampened intravasation of LTBP3-deficient HEp-3 cells, suggesting that LTBP3 regulates the induction of the intravasation-supporting angiogenic vasculature within developing primary tumors. Using our recently developed microtumor model, we confirmed that LTBP3 loss resulted in the development of intratumoral vessels with an abnormal microarchitecture incompatible with efficient intravasation of HEp-3 carcinoma cells. Collectively, these findings demonstrate that LTBP3 represents a novel oncotarget that has distinctive functions in the regulation of angiogenesis-dependent tumor cell intravasation, a critical process during early cancer dissemination. Our experimental data are also consistent with the survival prognostic value of LTBP3 expression in early-stage head and neck squamous cell carcinomas, further indicating a specific role for LTBP3 in cancer progression toward metastatic disease.
Asunto(s)
Proteínas de Unión a TGF-beta Latente/fisiología , Neoplasias/genética , Neoplasias/patología , Animales , Línea Celular Tumoral , Embrión de Pollo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Unión a TGF-beta Latente/antagonistas & inhibidores , Proteínas de Unión a TGF-beta Latente/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/mortalidad , Neovascularización Patológica/genética , Neovascularización Patológica/patología , ARN Interferente Pequeño/farmacología , Análisis de SupervivenciaRESUMEN
The latent transforming growth factor (TGF) ß binding proteins (LTBP) are crucial mediators of TGFß function, as they control growth factor secretion, matrix deposition, presentation and activation. Deficiencies in specific LTBP isoforms yield discrete phenotypes representing defects in bone, lung and cardiovascular development mediated by loss of TGFß signaling. Additional phenotypes represent loss of unique TGFß-independent features of LTBP effects on elastogenesis and microfibril assembly. Thus, the LTBPs act as sensors for the regulation of both growth factor activity and matrix function.
Asunto(s)
Proteínas de Unión a TGF-beta Latente/deficiencia , Proteínas de Unión a TGF-beta Latente/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Enfermedades Óseas/metabolismo , Enfermedades Cardiovasculares/metabolismo , Matriz Extracelular/metabolismo , Humanos , Enfermedades Pulmonares/metabolismo , Transducción de SeñalRESUMEN
Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, caused by mutations of the microfibrillar protein fibrillin-1, that predisposes affected individuals to aortic aneurysm and rupture and is associated with increased TGFß signaling. TGFß is secreted from cells as a latent complex consisting of TGFß, the TGFß propeptide, and a molecule of latent TGFß binding protein (LTBP). Improper extracellular localization of the latent complex can alter active TGFß levels, and has been hypothesized as an explanation for enhanced TGFß signaling observed in MFS. We previously reported the absence of LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFß signaling in MFS might be due to defective interaction of latent TGFß complexes containing LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we genetically suppressed Ltbp3 expression in a mouse model of progressively severe MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved survival, essentially no aneurysms, reduced disruption and fragmentation of medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their aortas. These data suggest that, in MFS, improper localization of latent TGFß complexes composed of LTBP-3 and TGFß contributes to aortic disease progression.
Asunto(s)
Aneurisma de la Aorta Torácica/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Síndrome de Marfan/complicaciones , Síndrome de Marfan/genética , Complejos Multiproteicos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Análisis de Varianza , Animales , Aneurisma de la Aorta Torácica/etiología , ADN Complementario/biosíntesis , Fibrilina-1 , Fibrilinas , Inmunohistoquímica , Proteínas de Unión a TGF-beta Latente/deficiencia , Ratones , Proteínas de Microfilamentos/genética , Músculo Liso Vascular/citología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The LTBPs (or latent transforming growth factor ß binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFß by covalently binding the TGFß propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFß precursor in the trans-golgi network but LAP and TGFß remain strongly bound through non-covalent interactions. LAP, TGFß, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFß latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFß activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFß regulation, TGFß-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.
Asunto(s)
Proteínas de Unión a TGF-beta Latente/fisiología , Animales , Matriz Extracelular/fisiología , Fibrilinas , Humanos , Proteínas de Microfilamentos/fisiología , Isoformas de Proteínas/fisiología , Transducción de Señal , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Mice deficient in Latent TGFß Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFß2 levels. However, the elastic fiber assembly is not improved in Tgfb2(-/-) ;Ltbp4S(-/-) compared to Ltbp4S(-/-) lungs. We found that decreased levels of TGFß1 or TGFß3 did not improve lung septation indicating that the TGFß isoform elevated in Ltbp4S(-/-) lungs is TGFß2. Expression of a form of Ltbp4 that could not bind latent TGFß did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFß complexes. Therefore, the change in TGFß-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S(-/-) mutation with a fibulin-5 null mutant in Fbln5(-/-) ;Ltbp4S(-/-) mice improves the lung septation compared to Ltbp4S(-/-) lungs. Large globular elastin aggregates characteristic for Ltbp4S(-/-) lungs do not form in Fbln5(-/-) ;Ltbp4S(-/-) lungs and EM studies showed that elastic fibers in Fbln5(-/-) ;Ltbp4S(-/-) lungs resemble those found in Fbln5(-/-) mice. These results are consistent with a role for TGFß2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly.
Asunto(s)
Tipificación del Cuerpo/genética , Tejido Elástico/embriología , Proteínas de la Matriz Extracelular/fisiología , Proteínas de Unión a TGF-beta Latente/fisiología , Pulmón/embriología , Factor de Crecimiento Transformador beta2/metabolismo , Animales , Tejido Elástico/anomalías , Elastina/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Fibrilinas , Proteínas de Unión a TGF-beta Latente/genética , Pulmón/anomalías , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Proteínas Recombinantes/genética , Transducción de Señal/genética , Factor de Crecimiento Transformador beta2/genéticaRESUMEN
Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-ß binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-ß because TGF-ß-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-ß-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Proteínas de Unión a TGF-beta Latente/fisiología , Proteínas Recombinantes/metabolismo , Animales , Células HEK293 , Humanos , Proteínas de Unión a TGF-beta Latente/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Interferencia de ARNRESUMEN
Fibrillin microfibrils are extracellular matrix structures with essential functions in the development and the organization of tissues including blood vessels, bone, limbs and the eye. Fibrillin-1 and fibrillin-2 form the core of fibrillin microfibrils, to which multiple proteins associate to form a highly organized structure. Defining the components of this structure and their interactions is crucial to understand the pathobiology of microfibrillopathies associated with mutations in fibrillins and in microfibril-associated molecules. In this study, we have analyzed both in vitro and in vivo the role of fibrillin microfibrils in the matrix deposition of latent TGF-ß binding protein 1 (LTBP-1), -3 and -4; the three LTBPs that form a complex with TGF-ß. In Fbn1(-/-) ascending aortas and lungs, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. In addition, in cultures of Fbn1(-/-) smooth muscle cells or lung fibroblasts, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. Fibrillin-2 is not involved in the deposition of LTBP-1 in Fbn1(-/-) extracellular matrix as cells deficient for both fibrillin-1 and fibrillin-2 still incorporate LTBP-1 in their matrix. However, blocking the formation of the fibronectin network in Fbn1(-/-) cells abrogates the deposition of LTBP-1. Together, these data indicate that LTBP-3 and LTBP-4 association with the matrix depends on fibrillin-1 microfibrils, whereas LTBP-1 association depends on a fibronectin network.
Asunto(s)
Fibronectinas/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Unión a TGF-beta Latente/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , ADN Complementario/genética , ADN Complementario/metabolismo , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Fibroblastos/metabolismo , Fibronectinas/genética , Proteínas de Unión a TGF-beta Latente/genética , Pulmón/citología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , ARN/genética , ARN/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The latent TGF-ß binding proteins (LTBP-1 -3, and -4) assist in the secretion and localization of latent TGF-ß molecules. Ltbp3(-/-) and Ltbp4S(-/-) mice have distinct phenotypes and only in the lungs does deficiency of either Ltbp-3 or Ltbp-4 cause developmental abnormalities. To determine if these two LTBPs have additional common functions, we generated mice deficient for both Ltbp-3 and Ltbp-4S. The only novel defect in Ltbp3(-/-);Ltbp4S(-/-) mice was an early lethality compared to mice with single mutations. In addition lung abnormalities were exacerbated and the terminal air sac septation defect was more severe in Ltbp3(-/-);Ltbp4S(-/-) mice than in Ltbp4S(-/-) mice. Decreased cellularity of Ltbp3(-/-);Ltbp4S(-/-) lungs was correlated with higher rate of apoptosis in newborn lungs of Ltbp3(-/-);Ltbp4S(-/-) animals compared to WT, Ltbp3(-/-), and Ltbp4S(-/-) mice. No differences in the maturation of the major lung cell types were discerned between the single and double mutant mice. However, the distribution of type 2 cells and myofibroblasts was abnormal, and myofibroblast segregation in some areas might be an indication of early fibrosis. We also observed differences in ECM composition between Ltbp3(-/-);Ltbp4S(-/-) and Ltbp4S(-/-) lungs after birth, reflected in decreased incorporation of fibrillin-1 and -2 in Ltbp3(-/-);Ltbp4S(-/-) matrix. The function of the lungs of Ltbp3(-/-);Ltbp4S(-/-) mice after the first week of life was potentially further compromised by macrophage infiltration, as proteases secreted from macrophages might exacerbate developmental emphysema. Together these data indicate that LTBP-3 and -4 perform partially overlapping functions only in the lungs.
Asunto(s)
Proteínas de Unión a TGF-beta Latente/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Animales , Apoptosis , Diferenciación Celular , Elastina/biosíntesis , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Inflamación/metabolismo , Inflamación/patología , Proteínas de Unión a TGF-beta Latente/deficiencia , Proteínas de Unión a TGF-beta Latente/genética , Pulmón/patología , Ratones , Microfibrillas/metabolismo , FenotipoRESUMEN
Transforming Growth Factor ß (TGF-ß) is crucial for valve development and homeostasis. The long form of Latent TGF-ß binding protein 1 (LTBP1L) covalently binds all TGF-ß isoforms and regulates their bioavailability. Ltbp1L expression analysis during valvulogenesis revealed two patterns of Ltbp1L production: an early one (E9.5-11.5) associated with endothelial-to-mesenchymal transformation (EMT); and a late one (E12.5 to birth) contemporaneous with valve remodeling. Similarly, histological analysis of Ltbp1L(-/-) developing valves identified two different pathologies: generation of hypoplastic endocardial cushions in early valvulogenesis, followed by development of hyperplastic valves in late valvulogenesis. Ltbp1L promotes valve EMT, as Ltbp1L absence yields hypoplastic endocardial cushions in vivo and attenuated EMT in vitro. Ltbp1L(-/-) valve hyperplasia in late valvuogenesis represents a consequence of prolonged EMT. We demonstrate that Ltbp1L is a major regulator of Tgf-ß activity during valvulogenesis since its absence results in a perturbed Tgf-ß pathway that causes all Ltbp1L(-/-) valvular defects.
Asunto(s)
Válvulas Cardíacas/embriología , Proteínas de Unión a TGF-beta Latente/fisiología , Animales , Embrión de Mamíferos , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Cardiopatías Congénitas/genética , Válvulas Cardíacas/anomalías , Válvulas Cardíacas/metabolismo , Hiperplasia , Proteínas de Unión a TGF-beta Latente/química , Proteínas de Unión a TGF-beta Latente/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Válvula Mitral/anomalías , Válvula Mitral/embriología , Válvula Mitral/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Crecimiento Transformadores/genética , Factores de Crecimiento Transformadores/metabolismoRESUMEN
We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.
Asunto(s)
Dermis/anomalías , Intestinos/anomalías , Proteínas de Unión a TGF-beta Latente/genética , Pulmón/anomalías , Mutación , Sistema Urinario/anomalías , Células Cultivadas , Niño , Preescolar , Técnicas de Cocultivo , Medios de Cultivo Condicionados/química , ADN/genética , ADN/aislamiento & purificación , Dermis/metabolismo , Dermis/ultraestructura , Femenino , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Homocigoto , Humanos , Inmunohistoquímica , Lactante , Mucosa Intestinal/metabolismo , Proteínas de Unión a TGF-beta Latente/química , Pulmón/metabolismo , Masculino , Sistema Musculoesquelético , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Piel/citología , Síndrome , Sistema Urinario/metabolismoRESUMEN
Latent transforming growth factor (TGF) beta-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFbeta. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit "exquisite specificities," a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Sitios de Unión/genética , Unión Competitiva , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Células Cultivadas , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Fibrilina-1 , Fibrilinas , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Ratones , Ratones Noqueados , Microfibrillas/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Excessive transforming growth factor-beta (TGF-beta) signaling characterizes the progression of aortic aneurysm in mouse models of Marfan syndrome, a systemic disorder of the connective tissue that is caused by mutations in the gene encoding the extracellular matrix protein fibrillin-1. Fibrillin-1 mutations are believed to promote abnormal Smad2/3 signaling by impairing the sequestration of latent TGF-beta complexes into the extracellular matrix. Here we report that promiscuous Smad2/3 signaling is the cell-autonomous phenotype of primary cultures of vascular smooth muscle cells (VSMC) explanted from the thoracic aortas of Fbn1 mutant mice with either neonatal onset or progressively severe aortic aneurysm. This cellular phenotype was characterized in VSMC isolated from Fbn1-null (mgN/mgN) mice, which recapitulate the most severe form of Marfan syndrome. We found that loss of fibrillin-1 deposition promotes the production of intracellular reactive oxygen species and abnormal accumulation of phosphorylated TGF-beta-activated kinase 1 and p38 MAPK, in addition to increasing the levels of endogenous phospho-Smad2. We showed that improper Smad2/3 signaling in Fbn1-null VSMC is in part stimulated by phospho-p38 MAPK, which is in turn activated in response to signals other than those mediated by the kinase activity of the ALK5 receptor. Consistent with these cell culture data, in vivo analyses documented that phospho-p38 MAPK accumulates earlier than phospho-Smad2 in the aortic wall of mgN/mgN mice and that systemic inhibition of phospho-p38 MAPK activity lowers the levels of phospho-Smad2 in this tissue. Collectively, these findings indicate that improper activation of p38 MAPK is a precursor of constitutive Smad2/3 signaling in the aortic wall of a mouse model of neonatal lethal Marfan syndrome.
Asunto(s)
Aorta Torácica/metabolismo , Proteínas de Microfilamentos/fisiología , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Aorta Torácica/citología , Células Cultivadas , Fibrilina-1 , Fibrilinas , Regulación del Desarrollo de la Expresión Génica , Immunoblotting , Técnicas para Inmunoenzimas , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína Smad2/genética , Proteína smad3/genéticaRESUMEN
Transforming growth factor-beta (TGF-beta) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-beta is often released as part of an inactive tripartite complex consisting of TGF-beta, the TGF-beta propeptide, and a molecule of latent TGF-beta binding protein (LTBP). The interaction of TGF-beta and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-beta from this state is crucial for signaling. To examine the contribution of LTBP to TGF-beta function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1(C33S/C33S) mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-beta1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1(-/-) animals. Tgfb1(C33S/C33S) mice exhibited decreased levels of active TGF-beta1, decreased TGF-beta signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-beta complex is important for proper TGF-beta1 function and that Tgfb1(C33S/C33S) mice are hypomorphs for active TGF-beta1. Moreover, although mechanisms exist to activate latent TGF-beta1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.
Asunto(s)
Inflamación/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Neoplasias/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Células Cultivadas , Fibroblastos/citología , Mucosa Gástrica/citología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Inflamación/patología , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Células de Langerhans/citología , Células de Langerhans/metabolismo , Proteínas de Unión a TGF-beta Latente/genética , Ratones , Ratones Noqueados , Neoplasias/patología , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily and were originally identified as proteins that induce ectopic bone formation. BMPs were shown subsequently to be involved in several biological processes during development and in adult tissues through the regulation of the growth, differentiation and apoptosis of various cell types. An alkaline phosphatase (ALP)-based assay is the most widely used assay to evaluate BMP activity. However, the ALP assay is not rapid and not sensitive enough to measure BMP activity at physiological concentrations. In this paper, we describe a highly sensitive, rapid, and specific cell-based assay for the quantification of BMP activity. RESULTS: Two cells lines, C2C12 and HepG2 were stably transfected with a reporter plasmid consisting of BMP-responsive elements from the Id1 promoter fused to a luciferase reporter gene. Exposure of cells containing this construct to BMPs induces the expression of luciferase, which can be quantified with a luminometer. The bioassay is specific for BMPs and can detect BMP-4 activity at a concentration as low as 3 pM. Related family members, such as TGF-beta1, TGF-beta2 and TGF-beta3, do not induce the reporter gene. CONCLUSION: The assay is rapid (less than 24 hours) and can be used, as described in this paper, to measure BMP activity in complex solutions and in cell culture in a simple and efficient way.
Asunto(s)
Bioensayo/métodos , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/análisis , Proteínas Morfogenéticas Óseas/metabolismo , Animales , Línea Celular , Medios de Cultivo/química , Regulación de la Expresión Génica , Genes Reporteros , Hepatocitos/metabolismo , Humanos , Luciferasas/química , Mediciones Luminiscentes , Ratones , Mioblastos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/química , Sensibilidad y Especificidad , Transducción de Señal/fisiología , Proteínas Smad/química , TransactivadoresRESUMEN
Blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. We describe here the structure and the biological action of a new cyclic peptide derived from vascular endothelial growth factor (VEGF). This 17-amino acid molecule designated cyclopeptidic vascular endothelial growth inhibitor (cyclo-VEGI, CBO-P11) encompasses residues 79-93 of VEGF which are involved in the interaction with VEGF receptor-2. In aqueous solution, cyclo-VEGI presents a propensity to adopt a helix conformation that was largely unexpected because only beta-sheet structures or random coil conformations have been observed for macrocyclic peptides. Cyclo-VEGI inhibits binding of iodinated VEGF165 to endothelial cells, endothelial cells proliferation, migration, and signaling induced by VEGF165. This peptide also exhibits anti-angiogenic activity in vivo on the differentiated chicken chorioallantoic membrane. Furthermore, cyclo-VEGI significantly blocks the growth of established intracranial glioma in nude and syngeneic mice and improves survival without side effects. Taken together, these results suggest that cyclo-VEGI is an attractive candidate for the development of novel angiogenesis inhibitor molecules useful for the treatment of cancer and other angiogenesis-related diseases.