RESUMEN
Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-ß3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.
Asunto(s)
Queratocitos de la Córnea/patología , Queratocono/patología , Estrés Oxidativo , Arginina/metabolismo , Ácido Ascórbico/farmacología , Diferenciación Celular , Células Cultivadas , Colágeno Tipo I/biosíntesis , Colágeno Tipo III/biosíntesis , Colágeno Tipo IV/biosíntesis , Córnea/citología , Córnea/patología , Queratocitos de la Córnea/citología , Matriz Extracelular , Fibroblastos/citología , Glutatión/metabolismo , Humanos , Ácido Láctico/metabolismo , Malatos/metabolismo , Metabolómica , Miofibroblastos/citología , Proteoglicanos/biosíntesis , Ácido Pirúvico/metabolismo , Factor de Crecimiento Transformador beta3/farmacologíaRESUMEN
Previously, we have demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in confluent rat lung fibroblasts via the binding of a Fra-1-c-Jun heterodimer to an activator protein-1-cAMP response element in the distal region of the elastin promoter. In the present study, we show that bFGF activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2, resulting in the translocation of phosphorylated extracellular signal-regulated kinase 1/2 into the nucleus followed by increased binding of Elk-1 to the serum response element of the c-Fos promoter, transient induction of c-Fos mRNA, and sustained induction of Fra-1 mRNA. The addition of PD-98059, an inhibitor of mitogen-activated protein kinase kinase, abrogates the bFGF-dependent repression of elastin mRNA expression. Comparative analyses of confluent and subconfluent fibroblast cultures reveal significant differences in elastin mRNA levels and activator protein-1 protein factors involved in the regulation of elastin transcription. These findings suggest that bFGF modulates specific cellular events that are dependent on the state of the cell and provide a rationale for the differential responses that can be expected in development and injury or repair situations.
Asunto(s)
Proteínas de Unión al ADN , Elastina/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/enzimología , Pulmón/citología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factores de Transcripción , Animales , Elastina/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Expresión Génica/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína Elk-1 con Dominio etsRESUMEN
Previous studies demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in pulmonary fibroblasts. In this study we pursue the identification of the element and the trans-acting factors responsible. Gel shift analyses show that bFGF increases protein binding to a sequence located at -564 to -558 base pairs (bp), which possesses homology to both AP1 and cAMP-response consensus elements yet displays a unique affinity for heterodimer binding. Site-directed mutation of the -564- to -558-bp sequence results in an increase in promoter activity and abrogates the effect of bFGF. Western blot analysis shows that bFGF induces a sustained increase in the steady-state levels of Fra 1, and co-transfection of a Fra 1 expression vector with an elastin promoter reporter construct results in an inhibition of elastin promoter activity. Overall the results suggest that bFGF represses elastin gene transcription by increasing the amount of the Fra 1 that subsequently binds to the -564- to -558-bp as a heterodimer with c-Jun to form an inhibitory complex. We propose that the identified bFGF response element can serve to down-regulate elastin transcription in elastogenic cells and, conversely, can serve to up-regulate elastogenesis in cells where endogenous bFGF signaling is attenuated or altered.
Asunto(s)
AMP Cíclico/metabolismo , Elastina/genética , Factor 2 de Crecimiento de Fibroblastos/fisiología , Regiones Promotoras Genéticas , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Dimerización , Regulación hacia Abajo , Mutagénesis , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
In this communication we offer data to suggest that the zebrafish swimbladder may provide a simple model of elastin injury and repair which is amenable to genetic analysis and pertinent to lung physiology. In situ hybridization of zebrafish embryos illustrated that elastin gene expression is evident in the developing gut tract prior to swimbladder morphogenesis. Northern blot analysis demonstrated that the major zebrafish elastin mRNA is 2.0 kb which is significantly smaller than its higher vertebrate counterpart. Amino acid analysis of alkali-resistant protein from the anterior chamber of the adult zebrafish swimbladder showed a composition similar to higher vertebrate elastins including significant amounts of desmosine crosslinks. Electron microscopic investigations of the swimbladder wall indicate a simple structure with an inner layer of elastin fibers. Elastase delivery to the swimbladder in vitro resulted in significant fragmentation of elastin in the anterior chamber providing an environment for studying elastin repair within the tissue.
Asunto(s)
Sacos Aéreos/metabolismo , Elastina/metabolismo , Enfermedades Pulmonares/metabolismo , Modelos Biológicos , Cicatrización de Heridas/fisiología , Pez Cebra/metabolismo , Sacos Aéreos/efectos de los fármacos , Sacos Aéreos/ultraestructura , Aminoácidos/análisis , Animales , Northern Blotting , Elastina/genética , Expresión Génica , Hibridación in Situ , Intestinos/embriología , Morfogénesis , Elastasa Pancreática/farmacología , ARN/aislamiento & purificación , ARN Mensajero/biosíntesis , Pez Cebra/embriologíaRESUMEN
The overall goals of this study were to establish the level at which elastin gene expression is regulated during chick lung embryogenesis and to identify the temporal and spatial relationships among elastogenesis, smooth muscle cell differentiation, and cell proliferation. A comparison of lung elastin mRNA and transcriptional levels during embryogenesis shows that elastin expression is developmentally regulated at the transcriptional level. The increase in elastogenic activity occurs during the late stages of lung embryogenesis and coincides with terminal maturation of the tertiary bronchi. In situ hybridization analysis demonstrates that the increase in elastin mRNA expression is confined to the tertiary bronchial respiratory subunits, connective tissue septa, and supporting vasculature of the lung parenchyma. Immunohistochemical localization of smooth muscle cell alpha-actin and tropoelastin suggests that alpha-actin-immunoreactive cells of the lung parenchyma are a major contributor to the increase in elastin expression during embryogenesis. This observation is also reflected by Northern blot analysis, which demonstrates a temporal coincidence in the increase of both alpha-actin and elastin mRNA levels. Histone mRNA expression, which was used as an index of cellular proliferation, reveals a level and spatial pattern inversely related to that of the elastin transcript. Tissue transfections of chick lungs isolated from 18-day embryos with various elastin gene deletion/reporter constructs illustrate that the elastin promoter is not promiscuous within a tissue environment and that sequences spanning the -500 to +2 region are capable of directing promoter activity spatially comparable to the endogenous elastin gene.
Asunto(s)
Elastina/biosíntesis , Elastina/genética , Pulmón/embriología , Pulmón/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Inmunohistoquímica , Hibridación in Situ , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Transfección , Tropoelastina/metabolismoRESUMEN
BACKGROUND: Evidence from in vitro and in vivo studies indicates that damaged elastic fibers can be repaired. METHODS: Lipid interstitial pulmonary fibroblasts were cultured for 6 weeks. Cultures were then exposed to 25 microg of porcine pancreatic elastase and fixed in pairs (control, elastase-treated) immediately after exposure and at 1, 2, 3, 4, 7, 10, 14, and 22 days for ultrastructural examination. Elastin was also analyzed biochemically for resistance to hot alkali, an indicator of repair. Steady-state levels of tropoelastin and lysyl oxidase mRNA at 2, 4, and 7 days after elastase treatment were determined by Northern blot analysis. RESULTS: Immediately after exposure to elastase, damaged elastic fibers exhibited a frayed, porous appearance and a granular texture. Through day 4, fibers showed no evidence of repair. By day 7, the granular texture of damaged fibers was no longer evident and a gradual filling-in of porous areas appeared to be taking place. By 22 days, elastic fibers were indistinguishable from elastic fibers in control cultures. The ultrastructural changes were paralleled by changes in hot alkali resistance. Through day 4, there was no change in the level of hot alkali resistant elastin. Between day 4 and day 7, resistance to hot alkali increased sharply and continued to increase at a slower rate, reaching 84% of the control level by day 22. Steady-state levels of tropoelastin and lysyl oxidase mRNA showed no increase over control levels at 2, 4, and 7 days after elastase treatment. CONCLUSIONS: Elastic fibers synthesized by lipid interstitial pulmonary fibroblasts in culture were repaired after damage by elastase. This type of repair may have relevance to the prevention of pathological conditions, such as emphysema.
Asunto(s)
Tejido Elástico/lesiones , Tejido Elástico/ultraestructura , Lesión Pulmonar , Pulmón/ultraestructura , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Tejido Elástico/metabolismo , Elastina/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Pulmón/metabolismo , Microscopía Electrónica , Elastasa Pancreática/farmacología , Proteína-Lisina 6-Oxidasa/genética , Enfisema Pulmonar/etiología , Enfisema Pulmonar/patología , Enfisema Pulmonar/prevención & control , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Factores de Tiempo , Tropoelastina/genéticaRESUMEN
Previous studies have demonstrated that insulin-like growth factor-I (IGF-I) increases elastin gene transcription in aortic smooth muscle cells and that this up-regulation is accompanied by a loss of protein binding to the proximal promoter. Sp1 has been identified as one of the factors whose binding is lost, and in the present study we show that Sp3 binding is also abrogated by IGF-I, but in a selected manner. In functional analyses using Drosophila SL-2 cells, Sp1 expression can drive transcription from the elastin proximal promoter, while co-expression of Sp3 results in a repression of Sp1 activity. Footprint and gel shift analyses position the IGF-I responsive sequences to a putative retinoblastoma control element (RCE). Mutation of the putative RCE sequence as assessed by transient transfection of smooth muscle cells results in an increase in reporter activity equal in magnitude to that conferred by IGF-I on the wild type promoter. Together these results support the hypothesis that IGF-I-mediated increase in elastin transcription occurs via a mechanism of derepression involving the abrogation of a repressor that appears to be Sp3 binding to the RCE.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Elastina/genética , Factor I del Crecimiento Similar a la Insulina/fisiología , Retinoblastoma/genética , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Células Cultivadas , Drosophila melanogaster , Humanos , Mutagénesis , Unión Proteica , Ratas , Ratas Sprague-Dawley , Factor de Transcripción Sp3 , TransfecciónRESUMEN
We have reported previously that a factor released by elastase treatment of pulmonary fibroblast cultures is capable of down-regulating elastin gene expression. In the present study we have pursued the identification of the factor released by elastase treatment and the characterization of the level of elastin gene expression at which this factor exerts its effect. We have found by immunologic and biochemical procedures that elastase treatment results in the release of basic fibroblast growth factor (bFGF) that is bound within the matrix. Both purified bFGF and bFGF released by elastase from cell matrices decrease the transcriptional level of the elastin gene by 70-80% within 24 h. Transient transfections of pulmonary fibroblasts with a series of elastin promoter deletion constructs show that the region of the elastin gene responsive to bFGF is located within sequences spanning -900 to -200 base pairs. The biological implications of these findings coupled with our previous report are significant, since they demonstrate that elastase digestion of pulmonary fibroblast matrices not only results in the proteolysis of elastin but also results in the release of a potent regulator of elastin gene transcription whose activity can influence repair mechanisms.
Asunto(s)
Elastina/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica , Pulmón/metabolismo , Elastasa Pancreática/farmacología , Animales , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/biosíntesis , Elastina/genética , Factor 2 de Crecimiento de Fibroblastos/aislamiento & purificación , Fibroblastos/citología , Fibroblastos/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Fragmentos de Péptidos/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción GenéticaRESUMEN
We have recently identified a novel element (EFE 5/6) in the human elastin gene promoter that modulates the ability of insulin-like growth factor I (IGF-I) to up-regulate elastin gene transcription in aortic smooth muscle cells. In the present study, we have pursued the identification of those nuclear proteins binding to the EFE 5/6 element and affected by IGF-I treatment. Chelation inactivation and metal reactivation experiments together with supershift gel analyses demonstrated that Sp1 was one of the proteins affected by IGF-I. Southwestern and Western analyses showed that Sp1 was present in IGF-I nuclear extracts and capable of binding DNA after fractionation. Addition of retinoblastoma gene product (Rb) antibody mimicked the effect of IGF-I in gel shift analysis, suggesting that Sp1 binding may be regulated by an inhibitor normally associated with Rb. The fact that the phosphorylation state of Rb was affected by IGF-I was shown by Western blot analysis. The control smooth muscle cells transcribed the elastin gene at a high level without addition of IGF-I, so it is likely that disruption of Sp1 binding is the first step in allowing the binding of a more potent activating factor.
Asunto(s)
Elastina/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Músculo Liso Vascular/metabolismo , Proteína de Retinoblastoma/fisiología , Factor de Transcripción Sp1/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Aorta , Secuencia de Bases , ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Zinc/farmacologíaRESUMEN
Nuclear proteins were isolated from different aged chick embryonic aorta and examined by gel mobility shift and footprint analyses using the -195 to +2 bp region of the human elastin gene. A major developmental change in DNA/protein complexes involved the loss of binding complexes present initially in the 8- to 11-day nuclear extracts. Both chemical and enzymatic footprinting demonstrated that the deprotected binding complexes corresponded to positions -167 to -137 bp within the elastin promoter fragment. Gel shift competition assays confirmed the identity of the specific DNA sequences affected. These results demonstrate that increased elastogenesis during aortic embryogenesis involves the derepression of a negative element functionality previously shown to be affected by IGF-I in vitro.
Asunto(s)
Aorta/metabolismo , ADN/metabolismo , Elastina/biosíntesis , Elastina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Factor II del Crecimiento Similar a la Insulina/farmacología , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Animales , Aorta/embriología , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Embrión de Pollo , Desoxirribonucleasa I , Desarrollo Embrionario y Fetal , Datos de Secuencia Molecular , Músculo Liso Vascular/embriología , Proteínas Nucleares/aislamiento & purificación , Oligodesoxirribonucleótidos , Unión ProteicaRESUMEN
Neonatal rat aortic smooth muscle cell cultures were used to investigate the mechanisms by which insulin-like growth factor-I (IGF-I) up-regulates aortic elastogenesis. The addition of IGF-I (50 ng/ml) to quiescent smooth muscle cell cultures resulted in a 5-fold increase in the steady-state levels of tropoelastin mRNA beginning between 2 and 4 h and reaching maximal levels at 8 h. Addition of cycloheximide blocked the effect of IGF-I. Nuclear run-on transcription analyses of nuclei isolated from IGF-I-treated cells showed increased synthesis of new tropoelastin transcripts indicating that transcriptional activation is a major component of IGF-I up-regulation. Transient transfections with deletion constructs containing different portions of the elastin 5'-upstream region localized the IGF-I-responsive area to sequences between -195 and -136 base pairs and further showed that this region contains a negative element. Gel retardation assays using nuclear proteins extracted from control and IGF-I-treated cells demonstrated that IGF-I treatment results in the loss of binding complexes. Footprint analyses of specific binding complexes affected by IGF-I show the deprotection of two closely positioned sequences spanning positions -165 to -137 base pairs. These results suggest that IGF-I up-regulation of elastogenesis involves the abrogation of a negative element functionality.
Asunto(s)
Aorta/metabolismo , Elastina/biosíntesis , Elastina/genética , Regulación de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/farmacología , Músculo Liso Vascular/metabolismo , ARN Mensajero/metabolismo , Tropoelastina/biosíntesis , Animales , Animales Recién Nacidos , Secuencia de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/biosíntesis , ADN/biosíntesis , Cinética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesis , Timidina/metabolismo , Transcripción Genética/efectos de los fármacos , Tritio , Tropoelastina/genéticaRESUMEN
The complete primary structure of chick lysyl oxidase was determined by recombinant DNA techniques. The nucleotide sequence of contiguous chick lysyl oxidase cDNA clones contained an open reading frame of 1260 bases which encodes a predicted protein of 420 amino acid residues (48,150 Da). In comparison to the deduced primary structure of rat lysyl oxidase, the chick enzyme is larger in size and exhibits a strong conservation of sequence within the latter two thirds of the molecule (92% identity) and a high degree of divergence in the first 150 amino acid residues (60% identity allowing for several insertions in both sequences). The developmental steady-state levels of lysyl oxidase mRNA together with the mRNAs encoding two of the enzyme's substrates (tropoelastin and type I collagen) increased between 8 and 16 days of embryonic development. Although levels of lysyl oxidase mRNA increased during aortic embryogenesis, the specific activity of the enzyme remained fairly constant suggesting that lysyl oxidase activity increases in direct proportion to total protein synthesis and cell number. In situ hybridization showed that the spatial expressions of lysyl oxidase and tropoelastin transcripts differ suggesting that the enzyme and substrate genes are differentially regulated within the cells of the arterial wall.
Asunto(s)
Aorta/embriología , Aorta/enzimología , Regulación Enzimológica de la Expresión Génica , Músculo Liso Vascular/embriología , Músculo Liso Vascular/enzimología , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Pollos , Colágeno/genética , Colágeno/metabolismo , ADN/genética , Hibridación in Situ , Hígado/enzimología , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transcripción Genética , Tropoelastina/genética , Tropoelastina/metabolismoRESUMEN
Rat neonatal aortic smooth muscle and pulmonary fibroblast cell cultures were exposed to different amounts of insulin-like growth factor-I (IGF-I, 1-100 ng/ml of medium) for 24 h. Aortic smooth muscle cells exhibited an increase in both steady-state levels of tropoelastin mRNA and soluble elastin with increasing amounts of IGF-I, suggesting that the growth factor is acting by increasing transcription or transcript stability. In contrast, pulmonary fibroblast cultures did not exhibit an elastogenic response to IGF-I because neither the steady-state levels of tropoelastin mRNA nor soluble elastin were affected. Transient transfection of the two cell cultures with a chimeric construct containing 500 bp of the elastin gene 5'-flanking region fused to the chloramphenicol acetyltransferase reporter gene showed that reporter activity was increased threefold in smooth muscle cells treated with IGF-I, whereas activity remains essentially the same in control and growth factor-treated pulmonary fibroblast cells. Receptor binding analyses revealed that both cell types possess the type I IGF-I receptor. Therefore, the lack of an elastogenic response in the lung cells cannot be attributed to lack of the appropriate receptor. These data, obtained in vitro with cell types that are principal producers of lung and aortic elastin, agree with results obtained in vivo. This agreement suggests that the regulation of elastin gene expression varies among cells derived from different tissues and furthermore provides model systems to investigate differential regulation of the elastin gene.
Asunto(s)
Aorta/crecimiento & desarrollo , Tejido Elástico/crecimiento & desarrollo , Factor I del Crecimiento Similar a la Insulina/fisiología , Pulmón/crecimiento & desarrollo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Aorta/citología , Aorta/metabolismo , Células Cultivadas , Quimera , Elastina/genética , Elastina/metabolismo , Fibroblastos/metabolismo , Genes , Factor I del Crecimiento Similar a la Insulina/farmacología , Pulmón/citología , Pulmón/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Receptores de Superficie Celular/metabolismo , Receptores de Somatomedina , Tropoelastina/genéticaRESUMEN
The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.
Asunto(s)
Proteínas Contráctiles/genética , ADN Recombinante/metabolismo , Proteínas de la Matriz Extracelular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bovinos , Embrión de Pollo , Codón , Proteínas Contráctiles/inmunología , Proteínas Contráctiles/aislamiento & purificación , Biblioteca de Genes , Sueros Inmunes , Inmunohistoquímica , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Poli A/genética , Poli A/aislamiento & purificación , Conformación Proteica , ARN/genética , ARN/aislamiento & purificación , Factores de Empalme de ARN , ARN Mensajero , Mapeo Restrictivo , Transcripción Genética , Tropoelastina/genéticaRESUMEN
When beta-aminopropionitrile (BAPN) is added to neonatal rat aortic smooth muscle cell cultures there is a decrease in insoluble elastin accumulation with a concomitant increase in tropoelastin and tropoelastin fragments in the culture medium. The experiments described here examine the biological significance of this fragmentation. BAPN, as well as purified tropoelastin fragments isolated from spent medium of cells grown in the presence of BAPN, were added to cultures. A decrease in elastin mRNA was observed in cultures grown in the presence of BAPN and also in those cultures to which the purified tropoelastin moieties were added. These studies indicate that the inhibition of lysyl oxidase by BAPN prevents elastin crosslinking which results in an increase in tropoelastin moieties, thus leading to a down regulation of the steady state levels of elastin mRNA.
Asunto(s)
Aminopropionitrilo/farmacología , Elastina/metabolismo , Músculo Liso Vascular/citología , Animales , Northern Blotting , Células Cultivadas , Elastina/genética , Regulación de la Expresión Génica , Músculo Liso Vascular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Tropoelastina/metabolismo , Tropoelastina/farmacologíaRESUMEN
Disruption and degradation of interstitial elastic fibers are significant characteristics of pulmonary emphysema. In order to examine the responses of elastogenic cells to the conditions mimicking degradation of interstitial pulmonary elastin, rat pulmonary fibroblast cultures were used as an in vitro model. Second passage fibroblasts were divided into two different environmental situations to represent cells adjacent to and remote from the site of elastase-digested matrix. One set of cell cultures was briefly digested with pancreatic elastase. The resultant digest was then added back incrementally to the medium of elastase-digested cell cultures and to the medium of a second set of undigested cultures. Both sets of cell cultures remained viable and metabolically active during these treatments (96 h of incubation) as judged by protein synthesis, cell number, and steady-state levels of beta-actin mRNA. However, the two sets of cultures exhibited opposite responses in elastin gene expression with addition of increasing amounts of the elastase digest. The elastase-digested cultures exhibited a 200% increase in extractable soluble elastin and a 186% increase in tropoelastin mRNA with the addition of increasing amounts of the elastase digest to the medium. Conversely, the amount of soluble elastin recovered from the undigested cultures decreased 75%, and the steady-state level of tropoelastin mRNA decreased 63%. Soluble elastin peptides generated from oxalic acid treatment of purified elastin were shown to decrease tropoelastin mRNA in undigested cell cultures in the same manner as the elastase digest. Based on these data, we propose that pulmonary fibroblast elastin gene expression can be controlled coordinately by the state of the extracellular matrix and solubilized peptides derived from that matrix. Such integrated regulation may serve to localize elastin repair mechanisms.
Asunto(s)
Elastina/genética , Enfisema/genética , Regulación de la Expresión Génica , Pulmón/metabolismo , ARN Mensajero/genética , Actinas/genética , Animales , Animales Recién Nacidos , Células Cultivadas , ADN/genética , Modelos Animales de Enfermedad , Fibroblastos/citología , Fibroblastos/metabolismo , ARN Mensajero/metabolismo , Ratas , Tropoelastina/genéticaRESUMEN
An age-related decrease in elasticity of arteries has been found in clinical and experimental studies done during the past two decades. We have investigated molecular and endocrine aspects of that decrease by examining the effects of age and insulin-like growth factor-I (IGF-I) on rat aorta elastogenesis. For comparison, pulmonary elastogenesis was examined in the same experimental animals. Different aged groups of male Fischer 344 rats (barrier protected) were implanted with minipumps for a two-week infusion of either 0.1 N acetic acid (vehicle solution) or IGF-I (1.2 mg/kg/day). The DNA content (micrograms DNA/g tissue) decreased with age in aorta but remained fairly constant in lung. Administration of IGF-I increased the aortic DNA content in all but the oldest rats. Conversely, the DNA content of pulmonary tissue was significantly increased in only the youngest animals. The steady-state levels of tropoelastin mRNA decreased dramatically in both aorta and lung with increased age. The decrease was greater in lung than aorta. Administration of IGF-I elevated aortic tropoelastin mRNA steady-state levels, whereas lung tropoelastin mRNA levels were unaffected by IGF-I administration. Aortic tissue synthesized decreased amounts of insoluble elastin with increased age. These results establish a direct relationship between aortic tropoelastin mRNA levels and the synthesis of insoluble elastin in aging. Administration of IGF-I increased aortic elastin synthesis throughout the life span of the rat, although the proportionate increase diminished with age.
Asunto(s)
Envejecimiento/efectos de los fármacos , Aorta/efectos de los fármacos , Elastina/biosíntesis , Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Somatomedinas/farmacología , Animales , Aorta/análisis , Aorta/metabolismo , ADN/análisis , Elastina/genética , Bombas de Infusión , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/análisis , Pulmón/análisis , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , ARN/análisis , Radioinmunoensayo , Ratas , Ratas Endogámicas F344RESUMEN
Studies from several laboratories have demonstrated the existence of multiple tropoelasting mRNAs and protein isoforms. The present study was designed to examine the developmental expression of a specific tropoelastin mRNA, its encoded isoform, and the fate of that isoform in the extracellular matrix. A chick genomic DNA library was screened with a chick tropoelastin cDNA. Seven unique, overlapping clones spanning 39 kilobases were isolated. A synthetic oligonucleotide complementary to a variable tropoelastin mRNA sequence was used to identify a 1.5-kilobase PstI-BamHI genomic fragment. Nucleotide sequence data revealed that the putative exon was surrounded by intron sequences possessing canonical splice sites at the exon/intron borders. Using both immunologic and molecular probes specific to the tropoelastin isoform and mRNA, quantitative protein and RNA analyses were performed. Results demonstrate that total tropoelastin mRNAs increased significantly during aortic embryogenesis whereas the amount of mRNA containing the variable exon remained relatively constant. The amount of total tropoelastins within the same developmental period reflect the level of total tropoelastin mRNA. The amount of the tropoelastin isoform containing the variable exon essentially mirrored the corresponding mRNA with the exception that a decrease in the isoform at day 15 was not seen in the mRNA level. Immunoelectron micrographs of 13-day chick aortic tissue using both total and isoform-specific antisera showed ultrastructural localization to definable elastic fibers. Antibodies to the variable tropoelastin isoform occurred preferentially at sites where elastic fiber microfibril structures were evident.
Asunto(s)
Elastina/análogos & derivados , Matriz Extracelular/metabolismo , Genes , Variación Genética , Tropoelastina/genética , Secuencia de Aminoácidos , Animales , Antígenos , Aorta/metabolismo , Secuencia de Bases , Embrión de Pollo , Pollos , Clonación Molecular , Sueros Inmunes , Hígado/metabolismo , Datos de Secuencia Molecular , Músculo Liso Vascular/metabolismo , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
The potential role of insulin-like growth factor-I (IGF-I) as a modulator of chick aortic embryogenesis was examined. Studies were designed to investigate the in vivo relationships between embryonic IGF-I serum concentrations, liver and aortic IGF-I mRNA steady-state levels and the inception and perpetuation of aortic elastogenesis. In addition to aortic tissue, elastogenesis was measured in heart and lung tissues in order to compare the responses of functionally unique elastin-containing tissues to the developmental appearance of IGF-I in embryonic serum. Our results demonstrate that the induction of aortic tropoelastin mRNA steady-state levels coincides with a major increase in serum IGF-I concentration. This is not the case with either lung or heart elastogenic responses. All three tissues examined (aorta, lung, and heart) exhibited different developmental patterns of tropoelastin mRNA steady-state levels during the embryonic ages studied (8- through 10-day). Only aortic tropoelastin mRNA levels paralleled the rise and fall of IGF-I serum levels. Steady-state levels of liver IGF-I mRNA peaked one day (9-day) prior to detectable IGF-I serum levels but otherwise mirrored the gradual, but steady decrease in IGF-I serum levels through 16-day. Aortic tissue also expresses IGF-I mRNA beginning at 8-day and continuing throughout the embryonic ages examined (16-day). Although the relative levels of aortic IGF-I mRNA are very low in comparison to corresponding mRNA levels in liver, the fact that IGF-I mRNA is transcribed in the aorta points to the possibility that autocrine and/or paracrine mechanisms of IGF-I action may be operative in aortic elastogenesis.
Asunto(s)
Aorta/embriología , Regulación de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/fisiología , Somatomedinas/fisiología , Tropoelastina/biosíntesis , Animales , Aorta/metabolismo , Embrión de Pollo , Elasticidad , Elastina , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/genética , Hígado/embriología , Hígado/metabolismo , Hibridación de Ácido Nucleico , ARN Mensajero/biosíntesis , Somatomedinas/análisis , Tropoelastina/genéticaRESUMEN
Several overlapping rat tropoelastin cDNA clones were isolated from a lambda gt11 rat heart cDNA library and their nucleotide sequence was determined. The corresponding deduced amino acid sequence of rat tropoelastin revealed strong homology to bovine and human tropoelastins although possessing some unique features including greater size (18%) and composition of repetitive units. Comparison of the amino acid sequence of rat tropoelastin to four other tropoelastin species reveals that the hydrophobic peptide repeat regions in the middle of each molecule and the crosslinking areas containing three lysine residues are remarkably conserved. A possible function for the clustering of three lysine residues in providing a mechanism for the in vivo reduction of dehydrolysinonorleucine via a redox shuttle with dihydrodesmosine is proposed. In addition, the COOH-terminal sequence of the rat tropoelastin is virtually identical to tropoelastins of other species in possessing a cysteine/arginine/lysine containing segment. There are no obvious amino acid insertions or substitutions in the COOH-terminal half of the rat tropoelastin molecule which would signal unique cleavage or glycosylation sites. Examination of the steady-state levels of rat tropoelastin mRNA in 8- and 12-day neonatal lung, heart, and aortic tissues showed that the amount of tropoelastin mRNA was abundant and of similar size (3.9 kb) in all three tissues.