RESUMO
A disproportionate tall stature is the most evident manifestation in Marfan syndrome (MFS), a multisystem condition caused by mutations in the extracellular protein and TGFß modulator, fibrillin-1. Unlike cardiovascular manifestations, there has been little effort devoted to unravel the molecular mechanism responsible for long bone overgrowth in MFS. By combining the Cre-LoxP recombination system with metatarsal bone cultures, here we identify the outer layer of the perichondrium as the tissue responsible for long bone overgrowth in MFS mice. Analyses of differentially expressed genes in the fibrillin-1-deficient perichondrium predicted that loss of TGFß signaling may influence chondrogenesis in the neighboring epiphyseal growth plate (GP). Immunohistochemistry revealed that fibrillin-1 deficiency in the outer perichondrium is associated with decreased accumulation of latent TGFß-binding proteins (LTBPs)-3 and -4, and reduced levels of phosphorylated (activated) Smad2. Consistent with these findings, mutant metatarsal bones grown in vitro were longer and released less TGFß than the wild-type counterparts. Moreover, addition of recombinant TGFß1 normalized linear growth of mutant metatarsal bones. We conclude that longitudinal bone overgrowth in MFS is accounted for by diminished sequestration of LTBP-3 and LTBP-4 into the fibrillin-1-deficient matrix of the outer perichondrium, which results in less TGFß signaling locally and improper GP differentiation distally.
Assuntos
Síndrome de Marfan , Animais , Fibrilina-1/genética , Fibrilina-2 , Fibrilinas , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Síndrome de Marfan/genética , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can sequester BMP complexes in a latent state.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas dos Microfilamentos/genética , Doenças Musculares/genética , Animais , Animais Recém-Nascidos , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Creatina Quinase/sangue , Feminino , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Regulação da Expressão Gênica , Deformidades Congênitas dos Membros/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas dos Microfilamentos/metabolismo , Músculo Esquelético/anormalidades , Músculo Esquelético/patologia , Doenças Musculares/fisiopatologia , Distrofias Musculares/genética , Técnicas de Cultura de Órgãos , Transdução de Sinais/genéticaRESUMO
Cancer is one of the most common causes of death among adults. Chemotherapy is crucial in determining patient survival and quality of life. However, the development of multidrug resistance (MDR) continues to pose a significant challenge in the management of cancer. In this study, we analyzed the role of human ribosomal protein uL3 (formerly rpL3) in multidrug resistance. Our studies revealed that uL3 is a key determinant of multidrug resistance in p53-mutated lung cancer cells by controlling the cell redox status. We established and characterized a multidrug resistant Calu-6 cell line. We found that uL3 down-regulation correlates positively with multidrug resistance. Restoration of the uL3 protein level re-sensitized the resistant cells to the drug by regulating the reactive oxygen species (ROS) levels, glutathione content, glutamate release, and cystine uptake. Chromatin immunoprecipitation experiments and luciferase assays demonstrated that uL3 coordinated the expression of stress-response genes acting as transcriptional repressors of solute carrier family 7 member 11 (xCT) and glutathione S-transferase α1 (GST-α1), independently of Nuclear factor erythroid 2-related factor 2 (Nrf2). Altogether our results describe a new function of uL3 as a regulator of oxidative stress response genes and advance our understanding of the molecular mechanisms underlying multidrug resistance in cancers.
Assuntos
Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/genética , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Proteína Ribossômica L3RESUMO
OBJECTIVE: Studies of mice with mild Marfan syndrome (MFS) have correlated the development of thoracic aortic aneurysm (TAA) with improper stimulation of noncanonical (Erk-mediated) TGFß signaling by the angiotensin type I receptor (AT1r). This correlation was largely based on comparable TAA modifications by either systemic TGFß neutralization or AT1r antagonism. However, subsequent investigations have called into question some key aspects of this mechanism of arterial disease in MFS. To resolve these controversial points, here we made a head-to-head comparison of the therapeutic benefits of TGFß neutralization and AT1r antagonism in mice with progressively severe MFS (Fbn1(mgR/mgR) mice). APPROACH AND RESULTS: Aneurysm growth, media degeneration, aortic levels of phosphorylated Erk and Smad proteins and the average survival of Fbn1(mgR/mgR) mice were compared after a ≈3-month-long treatment with placebo and either the AT1r antagonist losartan or the TGFß-neutralizing antibody 1D11. In contrast to the beneficial effect of losartan, TGFß neutralization either exacerbated or mitigated TAA formation depending on whether treatment was initiated before (postnatal day 16; P16) or after (P45) aneurysm formation, respectively. Biochemical evidence-related aneurysm growth with Erk-mediated AT1r signaling, and medial degeneration with TGFß hyperactivity that was in part AT1r dependent. Importantly, P16-initiated treatment with losartan combined with P45-initiated administration of 1D11 prevented death of Fbn1(mgR/mgR) mice from ruptured TAA. CONCLUSIONS: By demonstrating that promiscuous AT1r and TGFß drive partially overlapping processes of arterial disease in MFS mice, our study argues for a therapeutic strategy against TAA that targets both signaling pathways although sparing the early protective role of TGFß.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Anticorpos Neutralizantes/farmacologia , Aorta Torácica/efeitos dos fármacos , Aneurisma da Aorta Torácica/prevenção & controle , Losartan/farmacologia , Síndrome de Marfan/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Ruptura Aórtica/genética , Ruptura Aórtica/metabolismo , Ruptura Aórtica/patologia , Ruptura Aórtica/prevenção & controle , Modelos Animais de Doenças , Progressão da Doença , Fibrilina-1 , Fibrilinas , Humanos , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas dos Microfilamentos/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Fosforilação , Receptor Tipo 1 de Angiotensina/metabolismo , Proteína Smad2/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
EHD {EH [Eps15 (epidermal growth factor receptor substrate 15) homology]-domain-containing} proteins participate in several endocytic events, such as the internalization and the recycling processes. There are four EHD proteins in mammalian cells, EHD1-EHD4, each with diverse roles in the recycling pathway of endocytosis. EHD2 is a plasma-membrane-associated member of the EHD family that regulates internalization. Since several endocytic proteins have been shown to undergo nucleocytoplasmic shuttling and have been assigned roles in regulation of gene expression, we tested the possibility that EHD proteins also shuttle to the nucleus. Our results showed that, among the three EHD proteins (EHD1-EHD3) that were tested, only EHD2 accumulates in the nucleus under nuclear export inhibition treatment. Moreover, the presence of a NLS (nuclear localization signal) was essential for its entry into the nucleus. Nuclear exit of EHD2 depended partially on its NES (nuclear export signal). Elimination of a potential SUMOylation site in EHD2 resulted in a major accumulation of the protein in the nucleus, indicating the involvement of SUMOylation in the nuclear exit of EHD2. We confirmed the SUMOylation of EHD2 by employing co-immunoprecipitation and the yeast two-hybrid system. Using GAL4-based transactivation assay as well as a KLF7 (Krüppel-like factor 7)-dependent transcription assay of the p21WAF1/Cip1 [CDKN1A (cyclin-dependent kinase inhibitor 1A)] gene, we showed that EHD2 represses transcription. qRT-PCR (quantitative real-time PCR) of RNA from cells overexpressing EHD2 or of RNA from cells knocked down for EHD2 confirmed that EHD2 represses transcription of the p21WAF1/Cip1 gene.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas Repressoras/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células COS , Proteínas de Transporte/genética , Núcleo Celular/genética , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteínas Repressoras/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Loss-of-function experiments in mice have yielded invaluable mechanistic insights into the pathogenesis of Marfan syndrome (MFS) and implicitly, into the multiple roles fibrillin-1 microfibrils play in the developing and adult organism. Unfortunately, neonatal death from aortic complications of mice lacking fibrillin-1 (Fbn1(-/-) mice) has limited the scope of these studies. Here, we report the creation of a conditional mutant allele (Fbn1(fneo) ) that contains loxP sites bordering exon1 of Fbn1 and an frt-flanked neo expression cassette downstream of it. Fbn1(fneo/+) mice were crossed with FLPeR mice and the resulting Fbn1(Lox/+) progeny were crossed with Fbn1(+/-) ;CMV-Cre mice to generate Fbn1(CMV-/-) mice, which were found to phenocopy the vascular abnormalities of Fbn1(-/-) mice. Furthermore, mating Fbn1(Lox/+) mice with Prx1-Cre or Osx-Cre mice revealed an unappreciated role of fibrillin-1 microfibrils in restricting osteoprogenitor cell recruitment. Fbn1(Lox/+) mice are, therefore, an informative genetic resource to further dissect MFS pathogenesis and the role of extracellular fibrillin-1 assemblies in organ development and homeostasis.
Assuntos
Microfibrilas/genética , Proteínas dos Microfilamentos/genética , Osteoblastos/metabolismo , Osteogênese/genética , Animais , Densidade Óssea/genética , Diferenciação Celular , Fibrilina-1 , Fibrilinas , Técnicas de Silenciamento de Genes , Ordem dos Genes , Marcação de Genes/métodos , Genótipo , Camundongos , Camundongos Knockout , Microfibrilas/metabolismo , Mutação , Osteoblastos/citologia , FenótipoRESUMO
Reduced bone mineral density (osteopenia) is a poorly characterized manifestation of pediatric and adult patients afflicted with Marfan syndrome (MFS), a multisystem disorder caused by structural or quantitative defects in fibrillin-1 that perturb tissue integrity and TGFß bioavailability. Here we report that mice with progressively severe MFS (Fbn1(mgR/mgR) mice) develop osteopenia associated with normal osteoblast differentiation and bone formation. In vivo and ex vivo experiments, respectively, revealed that adult Fbn1(mgR/mgR) mice respond more strongly to locally induced osteolysis and that Fbn1(mgR/mgR) osteoblasts stimulate pre-osteoclast differentiation more than wild-type cells. Greater osteoclastogenic potential of mutant osteoblasts was largely attributed to Rankl up-regulation secondary to improper TGFß activation and signaling. Losartan treatment, which lowers TGFß signaling and restores aortic wall integrity in mice with mild MFS, did not mitigate bone loss in Fbn1(mgR/mgR) mice even though it ameliorated vascular disease. Conversely, alendronate treatment, which restricts osteoclast activity, improved bone quality but not aneurysm progression in Fbn1(mgR/mgR) mice. Taken together, our findings shed new light on the pathogenesis of osteopenia in MFS, in addition to arguing for a multifaceted treatment strategy in this congenital disorder of the connective tissue.
Assuntos
Alendronato/uso terapêutico , Aneurisma Aórtico/complicações , Aneurisma Aórtico/tratamento farmacológico , Doenças Ósseas Metabólicas/complicações , Doenças Ósseas Metabólicas/tratamento farmacológico , Losartan/uso terapêutico , Síndrome de Marfan/complicações , Alendronato/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Aorta/fisiopatologia , Aneurisma Aórtico/fisiopatologia , Doenças Ósseas Metabólicas/fisiopatologia , Proteínas Morfogenéticas Ósseas/metabolismo , Reabsorção Óssea/complicações , Reabsorção Óssea/fisiopatologia , Modelos Animais de Doenças , Fibrilina-1 , Fibrilinas , Losartan/farmacologia , Síndrome de Marfan/tratamento farmacológico , Síndrome de Marfan/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Mutação/genética , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/patologia , Coluna Vertebral/fisiopatologia , Tomografia Computadorizada por Raios X , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mutations in fibrillin-1 or fibrillin-2, the major structural components of extracellular microfibrils, cause pleiotropic manifestations in Marfan syndrome and congenital contractural arachnodactyly, respectively. We recently found that fibrillin-1 and fibrillin-2 control bone formation by regulating osteoblast differentiation through the differential modulation of endogenous TGFß and bone morphogenetic protein signals. Here, we describe in vivo and ex vivo experiments that implicate the fibrillins as negative regulators of bone resorption. Adult Fbn2(-/-) mice display a greater than normal osteolytic response to locally implanted lipopolysaccharide-coated titanium particles. Although isolated cultures of Fbn2(-/-) preosteoclasts exhibited normal differentiation and activity, these features were substantially augmented when mutant or wild-type preosteoclasts were co-cultured with Fbn2(-/-) but not wild-type osteoblasts. Greater osteoclastogenic potential of Fbn2(-/-) osteoblasts was largely accounted for by up-regulation of the Rankl gene secondary to heightened TGFß activity. This conclusion was based on the findings that blockade of TGFß signaling blunts Rankl up-regulation in Fbn2(-/-) osteoblasts and bones and that systemic TGFß antagonism improves locally induced osteolysis in Fbn2(-/-) mice. Abnormally high Rankl expression secondary to elevated TGFß activity was also noted in cultured osteoblasts from Fbn1(-/-) mice. Collectively our data demonstrated that extracellular microfibrils balance local catabolic and anabolic signals during bone remodeling in addition to implying distinct mechanisms of bone loss in Marfan syndrome and congenital contractural arachnodactyly.
Assuntos
Regulação da Expressão Gênica , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Ligante RANK/biossíntese , Fator de Crescimento Transformador beta1/biossíntese , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Técnicas de Cocultura , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microfibrilas/metabolismo , Proteínas dos Microfilamentos/química , Osteoclastos/citologia , Transdução de SinaisRESUMO
Fibrillin-1 and fibrillin-2 are structural components of the extracellular matrix which are also involved in modulating local TGFß and BMP bioavailability. Loss of fibrillin-1 or fibrillin-2 is associated with perturbed osteoblast maturation principally as the result of unbalanced TGFß and BMP signaling. Here, we demonstrated that stable expression of small hairpin RNAs against fibrillin-1(Fbn1) or fibrillin-2 (Fbn2) transcripts in the clonal osteoprogenitor cell line Kusa-A1 led to the same phenotypic and molecular manifestations as germline Fbn1- or Fbn2-null mutations in primary calvarial osteoblast cultures. Proof-of-concept experiments are also presented showing that Fbn1- or Fbn2-silenced Kusa-A1 cell lines are suitable models to identify candidate determinants of osteogenesis which are under the control of extracellular microfibrils. Specific findings included: the inference of a potential role for fibrillin-1-mediated cell-matrix interactions in regulating Kusa-A1 proliferation; the possibility of fibrillin-2 involvement in modulating the activity of transcription factor Runx2 by restricting microRNA expression and/or processing; and the suggestion that fibrillin-1 and fibrillin-2 influence Notch signaling indirectly by differentially regulating BMP signaling. Collectively, the data reiterated the notion that fibrillin-1 and fibrillin-2 exert opposite effects on osteoblast differentiation through the discrete modulation of a broad network of interacting signaling molecules.
Assuntos
Matriz Extracelular/metabolismo , Proteínas dos Microfilamentos/deficiência , Osteoblastos/citologia , Osteoblastos/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Previously we reported the identification of a homozygous COL27A1 (c.2089G>C; p.Gly697Arg) missense variant and proposed it as a founder allele in Puerto Rico segregating with Steel syndrome (STLS, MIM #615155); a rare osteochondrodysplasia characterized by short stature, congenital bilateral hip dysplasia, carpal coalitions, and scoliosis. We now report segregation of this variant in five probands from the initial clinical report defining the syndrome and an additional family of Puerto Rican descent with multiple affected adult individuals. We modeled the orthologous variant in murine Col27a1 and found it recapitulates some of the major Steel syndrome associated skeletal features including reduced body length, scoliosis, and a more rounded skull shape. Characterization of the in vivo murine model shows abnormal collagen deposition in the extracellular matrix and disorganization of the proliferative zone of the growth plate. We report additional COL27A1 pathogenic variant alleles identified in unrelated consanguineous Turkish kindreds suggesting Clan Genomics and identity-by-descent homozygosity contributing to disease in this population. The hypothesis that carrier states for this autosomal recessive osteochondrodysplasia may contribute to common complex traits is further explored in a large clinical population cohort. Our findings augment our understanding of COL27A1 biology and its role in skeletal development; and expand the functional allelic architecture in this gene underlying both rare and common disease phenotypes.
Assuntos
Anormalidades Múltiplas/genética , Colágenos Fibrilares/genética , Efeito Fundador , Luxação do Quadril/genética , Escoliose/genética , Anormalidades Múltiplas/patologia , Adolescente , Animais , Desenvolvimento Ósseo , Criança , Pré-Escolar , Consanguinidade , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Colágenos Fibrilares/metabolismo , Frequência do Gene , Luxação do Quadril/patologia , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Linhagem , Escoliose/patologia , SíndromeRESUMO
Fibrillin-rich microfibrils have emerged recently as an informative model system in which to study fundamental questions related to extracellular matrix biology and connective tissue pathophysiology. As a result, these studies have yielded novel clinical concepts and promising therapeutic strategies. These achievements have been based on the realization from studies of genetically engineered mice that mutations in fibrillin-rich microfibrils impair both the structural integrity of connective tissues and signaling events by TGF-beta/BMP superfamily members. In this view, fibrillin-rich microfibrils represent architectural assemblies that specify the concentration and timely release of local effectors of morphogenesis and tissue remodeling, in addition to conferring structural integrity to individual organ systems. This review summarizes the evidence supporting our current understanding of the structural and instructive roles that fibrillin-rich microfibrils play during embryonic development and in human diseases.
Assuntos
Tecido Conjuntivo/fisiopatologia , Desenvolvimento Embrionário/fisiologia , Matriz Extracelular/fisiologia , Mamíferos/fisiologia , Microfibrilas/genética , Proteínas dos Microfilamentos/fisiologia , Animais , Aorta/citologia , Aorta/fisiologia , Osso e Ossos/embriologia , Osso e Ossos/fisiologia , Fibrilinas , Microfibrilas/fisiologiaRESUMO
The Krüppel-like transcription factors (KLFs) are important regulators of cell proliferation and differentiation in several different organ systems. The mouse Klf7 gene is strongly active in postmitotic neuroblasts of the developing nervous system, and the corresponding protein stimulates transcription of the cyclin-dependent kinase inhibitor p21waf/cip gene. Here we report that loss of KLF7 activity in mice leads to neonatal lethality and a complex phenotype which is associated with deficits in neurite outgrowth and axonal misprojection at selected anatomical locations of the nervous system. Affected axon pathways include those of the olfactory and visual systems, the cerebral cortex, and the hippocampus. In situ hybridizations and immunoblots correlated loss of KLF7 activity in the olfactory epithelium with significant downregulation of the p21waf/cip and p27kip1 genes. Cotransfection experiments extended the last finding by documenting KLF7's ability to transactivate a reporter gene construct driven by the proximal promoter of p27kip1. Consistent with emerging evidence for a role of Cip/Kip proteins in cytoskeletal dynamics, we also documented p21waf/cip and p27kip1 accumulation in the cytoplasm of differentiating olfactory sensory neurons. KLF7 activity might therefore control neuronal morphogenesis in part by optimizing the levels of molecules that promote axon outgrowth.
Assuntos
Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Morfogênese , Neurônios/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Axônios/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Sistema Nervoso Central/crescimento & desenvolvimento , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p21 , Perda do Embrião/genética , Regulação da Expressão Gênica no Desenvolvimento , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios/citologia , Mucosa Olfatória/citologia , Regiões Promotoras Genéticas , Retina/citologia , Distribuição Tecidual , Transcrição GênicaRESUMO
MoKA is a novel F-box containing protein that interacts with and stimulates the activity of transcription factor KLF7, a regulator of neuronal differentiation. MoKA accumulates throughout the cell and predominantly in the cytosol, consistent with the presence of several putative nuclear localization and export signals (NLSs and NESs). The present study was designed to refine the identity and location of the sequences responsible for MoKA intracellular shuttling and transcriptional activity. Forced expression of fusion proteins in mammalian cells demonstrated that only one of three putative NLSs potentially recognized by karyopherin receptors is involved in nuclear localization of MoKA. By contrast, three distinct sequences were found to participate in mediating cytoplasmic accumulation. One of them is structurally and functionally related to the leucine-rich export signal that interacts with the exportin 1 (CRM1) receptor. The other two export signals instead display either a novel leucine-rich sequence or an undefined peptide motif, and both appear to act through CRM1-independent pathways. Finally, transcriptional analyses using the chimeric GAL4 system mapped the major activation domain of MoKA to a highly acidic sequence that resides between the NLS and NES clusters.
Assuntos
Proteínas F-Box/química , Proteínas F-Box/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Transativadores/química , Transativadores/metabolismo , Animais , Sítios de Ligação , Células COS , Núcleo Celular/química , Chlorocebus aethiops , Citoplasma/química , Proteínas F-Box/análise , Carioferinas/metabolismo , Camundongos , Células NIH 3T3 , Sinais de Exportação Nuclear , Sinais de Localização Nuclear , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Transativadores/análise , Proteína Exportina 1RESUMO
KLF7, a member of the Krüppel-like transcription factor family, is believed to regulate neurogenesis and cell cycle progression. Here, a yeast two-hybrid screen for KLF7 cofactors in the developing nervous system identified a novel 140-kDa protein named MoKA, for modulator of KLF7 activity. Interaction between MoKA and KLF7 was confirmed by the in vitro glutathione S-transferase pull-down assay and by coimmunoprecipitation of the proteins overexpressed in mammalian cells. Functional assays documented that MoKA is a KLF7 coactivator, and in situ hybridizations identified the developing nervous system and the adult testes as two sites of MoKA and Klf7 coexpression. Chromatin immunoprecipitation experiments demonstrated KLF7 binding to the p21(WAF1/Cip1) gene while transient transfection assays documented KLF7 stimulation of the p21(WAF1/Cip1) proximal promoter. Additional tests revealed that distinct structural motifs of MoKA direct interaction with KLF7 and shuttling between the nucleus and cytoplasm of asynchronously cycling cells. Altogether, our results strongly suggest that MoKA and KLF7 interact functionally to regulate gene expression during cell differentiation and identify the cell cycle regulator p21(WAF1/Cip1) as one of the targeted genes.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , Proteínas de Ligação a DNA/genética , Proteínas F-Box/genética , Fatores de Transcrição Kruppel-Like , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The severe skeletal abnormalities associated with Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA) underscore the notion that fibrillin assemblies (microfibrils and elastic fibers) play a critical role in bone formation and function in spite of representing a low abundance component of skeletal matrices. Studies of MFS and CCA mice have correlated the skeletal phenotypes of these mutant animals with distinct pathophysiological mechanisms that reflect the contextual contribution of fibrillin-1 and -2 scaffolds to TGFß and BMP signaling during bone patterning, growth and metabolism. Illustrative examples include the unique role of fibrillin-2 in regulating BMP-dependent limb patterning and the distinct impact of the two fibrillin proteins on the commitment and differentiation of marrow mesenchymal stem cells. Collectively, these findings have important implication for our understanding of the pathophysiological mechanisms that drive age- and injury-related processes of bone degeneration.
Assuntos
Aracnodactilia/patologia , Contratura/patologia , Fibrilina-1/genética , Fibrilina-2/genética , Síndrome de Marfan/patologia , Animais , Aracnodactilia/genética , Aracnodactilia/metabolismo , Padronização Corporal , Diferenciação Celular , Contratura/genética , Contratura/metabolismo , Modelos Animais de Doenças , Fibrilina-1/metabolismo , Fibrilina-2/metabolismo , Humanos , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Mutação , Osteogênese , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
We have recently demonstrated that fibrillin-1 assemblies regulate the fate of skeletal stem cells (aka, mesenchymal stem cells [MSCs]) by modulating TGFß activity within the microenvironment of adult bone marrow niches. Since MSCs can also influence hematopoietic stem cell (HSC) activities, here we investigated adult hematopoiesis in mice with Cre-mediated inactivation of the fibrillin-1 (Fbn1) gene in the mesenchyme of the forming limbs (Fbn1(Prx1-/-) mice). Analyses of 3-month-old Fbn1(Prx1-/-) mice revealed a statistically significant increase of circulating red blood cells, which a differentiation assay correlated with augmented erythropoiesis. This finding, together with evidence of fibrillin-1 deposition in erythroblastic niches, supported the notion that this extracellular matrix protein normally restricts differentiation of erythroid progenitors. Whereas flow cytometry measurements identified a decreased HSC frequency in mutant relative to wild type mice, no appreciable differences were noted with regard to the relative abundance and differentiation potential of myeloid progenitor cells. Together these findings implied that fibrillin-1 normally promotes HSC expansion but does not influence cell lineage commitment. Since local TGFß hyperactivity has been associated with abnormal osteogenesis in Fbn1(Prx1-/-) mice, 1-month-old mutant and wild type animals were systemically treated for 8weeks with either a pan-TGF-ß-neutralizing antibody or an antibody of the same IgG1 isotype. The distinct outcomes of these pharmacological interventions strongly suggest that fibrillin-1 differentially modulates TGFß activity in HSC vs. erythroid niches.
Assuntos
Células-Tronco Adultas/metabolismo , Células da Medula Óssea/citologia , Fibrilina-1/genética , Hematopoese , Animais , Diferenciação Celular , Fibrilina-1/metabolismo , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Camundongos , Transdução de Sinais , Nicho de Células-Tronco , Fator de Crescimento Transformador beta/metabolismoRESUMO
A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age-related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFß modulator fibrillin-1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin-1 (Fbn1(Prx1-/-) mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1(Prx1-/-) mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1(Prx1-/-) mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1-silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin-1 modulation of TGFß bioavailability, cultures of marrow stromal cells from Fbn1(Prx1-/-) limb bones showed improper overactivation of latent TGFß. In line with this finding, systemic TGFß neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin-1 regulates MSC activity by modulating TGFß bioavailability within the microenvironment of marrow niches.
Assuntos
Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Nicho de Células-Tronco/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Fibrilina-1 , Fibrilinas , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
The Krüppel-like transcription factors (KLFs) represent a family of 15 different zinc finger proteins of the C(2)H(2) type that are involved in vertebrate development and which control cell proliferation, growth and differentiation. Structural-functional considerations have segregated KLF6 and KLF7 into a phylogenetically distinct group. Here we report the identification of Luna, the Drosophila progenitor of the mammalian KLF6/KLF7 group. This conclusion is based on the near sequence identity, as well as the comparable location of the DNA-binding domains and nuclear localization signals of the insect and mammalian proteins. The homology extends to the composition and function of the amino-terminal segment of Luna which, similarly to the mammalian counterparts, stimulates transcription in a reporter gene assay. We also present preliminary in vivo evidence of Luna involvement in embryonic development and cell differentiation. First, luna RNA interference and luna overexpression during early Drosophila embryogenesis leads to developmental arrest at different embryonic stages. Second, targeted perturbation of luna expression in the forming compound eye interferes with terminal cell differentiation, but not cell specification. We therefore propose that Luna is a novel transcriptional determinant of Drosophila development.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Proto-Oncogênicas , Transativadores/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Mutação , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Hu-Surf5 is included within the Surfeit locus, a cluster of six genes originally identified in mouse. In the present study, we have cloned and characterized the Hu-Surf5 gene and its mRNA multiple transcripts. Comparison of the most abundant cDNA and genomic sequence shows that the Hu-Surf5 is spread over a region of approximately 7.5 kb and consists of five exons separated by four introns. The nucleotide sequence of the genomic region flanking the 3'-end of the Hu-Surf5 gene revealed the presence of a processed pseudogene of human ribosomal protein L21 followed by Hu-Surf6 gene. Only 110 bp separate the transcription start site of Hu-Surf5 and Hu-Surf3/L7a gene and the transcription direction is divergent. Earlier studies defined the 110 bp region essential for promoter activity of Hu-Surf3/L7a. Here, we show that this region stimulates transcription with a slightly different efficiency in both directions. The bidirectional promoter lacks an identifiable TATA box and is characterized by a CpG island that extends through the first exon into the first intron of both genes. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for Hu-Surf5 expression. Hu-Surf5 encodes three different transcripts, Surf-5a, Surf-5b, and Surf-5c, which result from alternative splicing. Two protein products, SURF-5A and SURF-5B have been characterized. Production of chimaeras between the full-length SURF-5A or SURF-5B and the green fluorescent protein (GFP) allowed to localize both proteins in the cytoplasm.
Assuntos
Processamento Alternativo , Proteínas/genética , Sequência de Bases , Clonagem Molecular , Éxons , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Íntrons , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Complexo Mediador , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição , Transcrição Gênica , Células Tumorais CultivadasRESUMO
BACKGROUND: Scleroderma (systemic sclerosis; SSc) is a clinically heterogeneous and often lethal acquired disorder of the connective tissue that is characterized by vascular, immune/inflammatory and fibrotic manifestations. Tissue fibrosis is the main cause of morbidity and mortality in SSc and an unmet medical challenge, mostly because of our limited understanding of the molecular factors and signalling events that trigger and sustain disease progression. Recent evidence has correlated skin fibrosis in SSc with stabilization of proto-oncogene Ha-Ras secondary to auto-antibody stimulation of reactive oxygen species production. The goal of the present study was to explore the molecular connection between Ha-Ras stabilization and collagen I production, the main read-out of fibrogenesis, in a primary dermal fibroblast culture system that replicates the early stages of disease progression in SSc. RESULTS: Forced expression of proto-oncogene Ha-Ras in dermal fibroblasts demonstrated the promotion of an immediate collagen I up-regulation, as evidenced by enhanced activity of a collagen I-driven luciferase reporter plasmid and increased accumulation of endogenous collagen I proteins. Moreover, normal levels of Tgfß transcripts and active transforming growth factor-beta (TGFß) implied Ha-Ras stimulation of the canonical Smad2/3 signalling pathway independently of TGFß production or activation. Heightened Smad2/3 signalling was furthermore correlated with greater Smad3 phosphorylation and Smad3 protein accumulation, suggesting that Ha-Ras may target both Smad2/3 activation and turnover. Additional in vitro evidence excluded a contribution of ERK1/2 signalling to improper Smad3 activity and collagen I production in cells that constitutively express Ha-Ras. CONCLUSIONS: Our study shows for the first time that constitutively elevated Ha-Ras protein levels can directly stimulate Smad2/3 signalling and collagen I accumulation independently of TGFß neo-synthesis and activation. This finding therefore implicates the Ha-Ras pathway with the early onset of fibrosis in SSc and implicitly identifies new therapeutic targets in SSc.