RESUMEN
Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the -112/-61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.
Asunto(s)
Colágeno Tipo I/biosíntesis , Proteínas de Unión al ADN/metabolismo , Dermis/metabolismo , Fibroblastos/metabolismo , Elementos de Respuesta , Esclerodermia Localizada/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Factor de Transcripción ReIA/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Adulto , Niño , Preescolar , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Proteínas de Unión al ADN/genética , Dermis/patología , Fibroblastos/patología , Regulación de la Expresión Génica/genética , Humanos , Masculino , Esclerodermia Localizada/patología , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/genética , Factor de Transcripción ReIA/genética , Factores de Transcripción/genéticaRESUMEN
The in vivo expression levels of four rRNA promoter pairs (rrnp(1)p(2)) of Bacillus subtilis were determined by employing single-copy lacZ fusions integrated at the amyE locus. The rrnO, rrnJ, rrnD, and rrnB promoters displayed unique growth rate regulation and stringent responses. Both lacZ activity and mRNA levels were highest for rrnO under all growth conditions tested, while rrnJ, rrnB, and rrnD showed decreasing levels of activity. During amino acid starvation induced by serine hydroxamate (SHX), only the strong rrnO and rrnJ promoters demonstrated stringent responses. Under the growth conditions used, the rrn promoters showed responses similar to the responses to carbon source limitation induced by α-methyl glucoside (α-MG). The ratio of P2 to P1 transcripts, determined by primer extension analysis, was high for the strong rrnO and rrnJ promoters, while only P2 transcripts were detected for the weak rrnD and rrnB promoters. Cloned P1 or P2 promoter fragments of rrnO or rrnJ were differentially regulated. In wild-type (relA(+)) and suppressor [relA(S)] strains under the conditions tested, only P2 responded to carbon source limitation by a decrease in RNA synthesis, correlating with an increase in (p)ppGpp levels and a decrease in the GTP concentration. The weak P1 promoter elements remain relaxed in the three genetic backgrounds [relA(+), relA, relA(S)] in the presence of α-MG. During amino acid starvation, P2 was stringently regulated in relA(+) and relA(S) cells, while only rrnJp(1) was also regulated, but to a lesser extent. Both the relA(+) and relA(S) strains showed (p)ppGpp accumulation after α-MG treatment but not after SHX treatment. These data reveal the complex nature of B. subtilis rrn promoter regulation in response to stress, and they suggest that the P2 promoters may play a more prominent role in the stringent response.
Asunto(s)
Bacillus subtilis/fisiología , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , ARN Ribosómico/biosíntesis , Estrés Fisiológico , Fusión Artificial Génica , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Genes Reporteros , Guanosina Tetrafosfato , Transcripción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Despite several investigations, the transcriptional mechanisms which regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. In this study, we determined the effects of both native ichtyan chondroïtin sulphate (CS) and its derived hydrolytic fragments (CSf) on human normal (NF) and scleroderma (SF) fibroblasts. Here, we demonstrate for the first time that CS and CSf exert an inhibitory effect on type I collagen protein synthesis and decrease the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in NF and SF. These glycosaminoglycan molecules repress COL1A1 gene transcription through a -112/-61 bp sequence upstream the start site of transcription and imply hc-Krox and Sp1 transcription factors. In addition, CS and CSf induced a down-regulation of TbetaRI expression. As a conclusion, our findings highlight a possible new role for CS and CSf as anti-fibrotic molecules and could help in elucidating the mechanisms of action by which CS and CSf exert their inhibitory effect on type I collagen synthesis.
Asunto(s)
Sulfatos de Condroitina/farmacología , Colágeno/biosíntesis , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Esclerodermia Localizada/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Emparejamiento Base , Secuencia de Bases , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esclerodermia Localizada/patología , Proteínas Smad/metabolismo , Factor de Transcripción Sp3/metabolismoRESUMEN
Despite several investigations, the transcriptional mechanisms that regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF). Forced hc-Krox expression was found to up-regulate COL1A1 transcription through a -112/-61-bp sequence in FF, ANF, and SF. Knockdown of hc-Krox by short interfering RNA and decoy strategies confirmed the transactivating effect of hc-Krox and decreased substantially COL1A1 transcription levels in all fibro-blast types. The -112/-61-bp sequence bound specifically hc-Krox but also Sp1 and CBF. Attempts to elucidate the potential interactions between hc-Krox, Sp1, and Sp3 revealed that all of them co-immunoprecipitate from FF cellular extracts when a c-Krox antibody was used and bind to the COL1A1 promoter in chromatin immunoprecipitation assays. Moreover, hc-Krox DNA binding activity to its COL1A1-responsive element is increased in SF, cells producing higher amounts of type I collagen compared with ANF and FF. These data suggest that the regulation of COL1A1 gene transcription in human dermal fibroblasts involves a complex machinery that implicates at least three transcription proteins, hc-Krox, Sp1, and Sp3, which could act in concert to up-regulate COL1A1 transcriptional activity and provide evidence for a pro-fibrotic role of hc-Krox.
Asunto(s)
Colágeno Tipo I/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Adulto , Secuencia de Bases , Niño , Proteínas de Unión al ADN/genética , Prepucio/citología , Prepucio/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Interferencia de ARN , ARN Interferente Pequeño , Esclerodermia Sistémica , Piel/citología , Piel/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Considerable evidence indicates that the amyloid-beta (Abeta) peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Abeta, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Abeta of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl peptide hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Abeta-degrading enzyme, and we show that the degradation of Abeta by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Abeta peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.
Asunto(s)
Aciltransferasas/metabolismo , Péptidos beta-Amiloides/metabolismo , Medios de Cultivo Condicionados/química , Serina/metabolismo , Aciltransferasas/genética , Animales , Autorradiografía/métodos , Línea Celular , Chlorocebus aethiops , Inhibidores de la Colinesterasa , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Isoflurofato/farmacocinética , Mutación/fisiología , Neuroblastoma/enzimología , Neuroblastoma/patología , Unión Proteica/efectos de los fármacos , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Serina/genética , Espectrometría de Masas en Tándem/métodos , Transfección , Tritio/farmacocinéticaRESUMEN
Cartilage oligomeric matrix protein (COMP) is an extracellular glycoprotein that belongs to the thrombospondin gene family. It is found predominantly in cartilage, tendon, ligament, and bone. Mutations in the COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphysial dysplasia. COMP influences the organization of collagen fibrils by interacting with collagens I, II and IX. Gene expression profiling of cultured skin fibroblasts suggested that COMP mRNA levels were elevated in scleroderma. We therefore examined COMP expression in SSc and normal skin biopsies. Immunohistochemistry confirmed that COMP protein accumulates in SSc but not normal skin, with SSc skin showing striking deposition in the papillary and deeper dermis. Significant staining was also seen in non-lesional skin from patients. Due to its involvement in the development of fibrosis, TGFbeta was examined for a possible role in regulating COMP expression. Cultured SSc fibroblasts demonstrated greater staining for COMP compared to normal controls prior to stimulation, and TGFbeta-1 induced a large increase in mRNA and protein. Murine fibroblasts engineered to overexpress human COMP demonstrated increased levels of fibronectin and collagen in the extracellular matrix. Taken together, these data demonstrate that COMP is overexpressed in SSc skin and cultured fibroblasts possibly due to autocrine TGFbeta stimulation, and COMP overexpression is sufficient to stimulate excess matrix deposition. By interactions with other matrix proteins and cells, COMP may play a role in pathogenic matrix deposition.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Expresión Génica , Glicoproteínas/metabolismo , Esclerodermia Sistémica/patología , Piel/patología , Animales , Proteína de la Matriz Oligomérica del Cartílago , Células Cultivadas , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/genética , Fibronectinas/metabolismo , Glicoproteínas/genética , Humanos , Proteínas Matrilinas , Ratones , ARN Mensajero , Piel/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Chondrocyte glycosaminoglycan (GAG) synthesis is regulated by the availability of UDP-glucuronate, the substrate of glucuronosyl transferases which form the GAG chains in proteoglycans and hyaluronan. UDP-glucose dehydrogenase (UDPGD) is therefore a key enzyme in the synthesis of UDP-glucuronate from glucose. However, the mechanisms regulating its expression in chondrocytes are not fully understood. We investigated the effect of c-Krox, a zinc-finger transcription factor previously shown to modulate several matrix genes, on the synthesis of GAG and transcriptional activity of several UDPGD gene promoter constructs, using transient transfection and decoy experiments in rabbit articular chondrocytes (RACs). We show that overexpression of c-Krox inhibits radiosulfate incorporation into neosynthesized GAG and that the effect was mediated by a cis-sequence located between +18 and +39bp of the UDPGD gene. Since that sequence can also bind Sp1/Sp3 factors, it is likely that c-Krox acts in concert with these proteins to modulate the UDPGD gene expression in articular chondrocytes.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Proteínas de Unión al ADN/metabolismo , Glicosaminoglicanos/metabolismo , Factores de Transcripción/metabolismo , Uridina Difosfato Glucosa Deshidrogenasa/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Conejos , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Uridina Difosfato Glucosa Deshidrogenasa/genéticaRESUMEN
Collagen type I is composed of three polypeptide chains transcribed from two separate genes (COL1A1 and COL1A2) with different promoters requiring coordinate regulation. Our recent publications, centering on COL1A2 regulation, demonstrate that methylation in the first exon of COL1A2 at a regulatory factor for X box (RFX) site (at -1 to +20) occurs in human cancer cells and correlates with increased RFX1 binding and decreased collagen transcription (Sengupta, P. K., Erhlich, M., and Smith, B. D. (1999) J. Biol. Chem. 274, 36649-36655; Sengupta, S., Smith, E. M., Kim, K., Murnane, M. J., and Smith, B. D. (2003) Cancer Res. 63, 1789-1797). In normal cells, RFX5 complex along with major histocompatibility class II transactivator (CIITA) is induced by interferon-gamma to occupy this site and repress collagen transcription (Xu, Y., Wang, L., Buttice, G., Sengupta, P. K., and Smith, B. D. (2004) J. Biol. Chem. 279, 41319-41332). In this paper, we demonstrate that COL1A1 has an RFX consensus binding site surrounding the transcription start site (-11 to +10) that contains three methylation sites rather than one in the COL1A2 gene RFX binding site. RFX1 interacts weakly with the unmethylated COL1A1 site, and binds with higher affinity to the methylated site. RFX1 represses the unmethylated COL1A1 less efficiently than COL1A2. COL1A1 promoter activity is sensitive to DNA methylation and the COL1A1 gene is methylated in human cancer cells with coordinately decreased collagen expression. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (aza-dC) increases collagen gene expression with time in human cancer cells. On the other hand, RFX5 interacts with both collagen type I genes with a similar binding affinity and represses both promoters equally in transient transfections. Two dominant negative forms of RFX5 activate both collagen genes coordinately. Finally, CIITA RNA interference experiments indicate that CIITA induction is required for interferon gamma-mediated repression of both collagen type I genes.