RESUMEN
Keloids are benign collagenous tumors that occur during dermal wound healing in genetically predisposed individuals. The lesions are characterized by over-proliferation of fibroblasts, some leukocyte infiltration, and prolonged high rates of collagen synthesis. To determine whether leukocyte chemoattractants or chemokines are participating in this disease process, immunohistochemical staining for the CXC chemokine, MGSA/GROalpha, and its receptor, CXCR2, was performed on tissue from keloids, hypertrophic scars and normal skin. Immunoreactive MGSA/GROalpha was not observed in hypertrophic scars or normal dermis, but was present in some myofibroblasts and lymphocytes in nodular areas of the keloid samples. This staining positively correlated with the degree of inflammatory infiltrate in the lesions. Keloids, but not hypertrophic scars or normal dermis, also exhibited intensive immunoreactivity for the CXCR2 receptor in endothelial cells and inflammatory infiltrates with occasional staining of myofibroblasts. In contrast, cultured fibroblasts from either keloids or normal skin did not express detectable amounts of mRNA for MGSA/GRO or CXCR2, although interleukin-1 strongly induced MGSA/GRO mRNA in both cell types. Interleukin-1 induction of MGSA/GRO was inhibited by glucocorticoid in normal and keloid fibroblasts, and the effect was more pronounced in keloid fibroblasts. This event was not correlated with inhibition of nuclear activation of NF-kappaB, AP-1 or Sp1, and might therefore be mediated by another mechanism such as decreased mRNA stability or transcriptional repression through the glucocorticoid response element in the MGSA/GRO promoter. Data from in vitro wounding experiments with cultured normal and keloid fibroblasts indicate that there were no significant differences in MGSA/GRO or CXCR2 receptor levels between normal and keloid fibroblasts. We also show that cultured keloid fibroblasts exhibit a delayed wound healing response. We postulate that the inflammatory component is important in development of keloid lesions and chemotactic cytokines may participate in this process.
Asunto(s)
Quimiocinas CXC , Quimiocinas/análisis , Quimiocinas/genética , Factores Quimiotácticos/análisis , Factores Quimiotácticos/genética , Cicatriz/patología , Fibroblastos/química , Expresión Génica/genética , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Queloide/patología , Receptores de Citocinas/análisis , Receptores de Citocinas/genética , Receptores de Interleucina-8B/análisis , Receptores de Interleucina-8B/genética , Northern Blotting , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CXCL1 , Cicatriz/genética , Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad/genética , Humanos , Inmunohistoquímica , Interleucina-1/farmacología , Queloide/genética , Receptores de Citocinas/efectos de los fármacos , Receptores de Interleucina-8B/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
Heme deficiency precipitated by CoCl2 administration to rats leads to a striking decrease in the inducibility of CYP2B1/B2 mRNA levels and its transcription by phenobarbitone (PB), besides decreasing the basal levels. Exogenous hemin administration counteracts the effects of CoCl2 administration. The binding of nuclear proteins to labeled positive cis-acting element (-69 to -98 nucleotides) in the near 5'-upstream region of the gene is inhibited by CoCl2 administration to saline or PB-treated rats, as assessed in gel shift assays. Administration of exogenous hemin to the animal or addition in vitro to the extracts is able to overcome the effects of CoCl2 treatment. The protein mediating this effect has been purified from CoCl2 administered nuclear extracts by heparin-agarose, positive element oligonucleotide affinity, and heme affinity column chromatography. This 65-kDa protein manifests very little binding to the positive element, but in the presence of certain other nuclear proteins, shows a strong heme-responsive binding. The purified protein binds heme. It is also able to stimulate transcription of a minigene construct of the CYP2B1/B2 gene containing -179 nucleotides of the 5'-upstream region and the I exon in a cell-free system, manifesting heme response. It is concluded that the 65-kDa protein mediates the constitutive requirement of heme for the transcription of CYP2B1/B2 gene.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP2B1/genética , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemo/metabolismo , Hígado/enzimología , Proteínas Nucleares/metabolismo , Esteroide Hidroxilasas/genética , Animales , Cloruros , Cobalto/farmacología , Citocromo P-450 CYP2B1/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática/efectos de los fármacos , Compuestos Férricos/farmacología , Hemina/farmacología , Peso Molecular , Fenobarbital/farmacología , Protoporfirinas/farmacología , ARN Mensajero/metabolismo , Ratas , Esteroide Hidroxilasas/biosíntesis , Transcripción GenéticaRESUMEN
The synthesis and phosphorylation of protein factor(s) that bind to the positive cis-acting element (-69 to -98 nt) of the CYP2B1/B2 gene have been examined in vivo in the rat. Treatment of rats with cycloheximide, a protein synthetic inhibitor, suppresses basal as well as phenobarbitone-induced levels of CYP2B1/B2 mRNA and its run-on transcription. Under these conditions, complex formation of the nuclear extract with the positive element is also inhibited, as judged by gel shift assays. Treatment of rats with 2-aminopurine, a general protein kinase inhibitor, blocks the phenobarbitone-mediated increase in CYP2B1/B2 mRNA, cell-free transcription of a minigene construct containing the positive element, pP450e179DNA, and binding of nuclear proteins to the positive element. Treatment of rats with okadaic acid, a protein phosphatase inhibitor, mimics the effects of phenobarbitone, but only partially. Thus, both phenobarbitone and okadaic acid individually enhance binding of the nuclear protein(s) to the positive element, cell-free transcription of the minigene construct, and phosphorylation of the approximately 26- and 94-kDa proteins binding to the positive element. But unlike phenobarbitone, okadaic acid is not an inducer of CYP2B1/B2 mRNA or its run-on transcription. Thus, phenobarbitone-responsive positive element interactions constitute only a minimal requirement, and okadaic acid is perhaps not able to bring about the total requirement for activation of CYP2B1/B2 gene transcription that should include interaction between the minimal promoter and further upstream elements. An intriguing feature is the antagonistic effect of okadaic acid on phenobarbitone-mediated effects on CYP21B1/B2 mRNA levels, cell-free and run-on transcription, and nuclear protein binding to the positive element. The reason for this antagonism is not clear. It is concluded that phenobarbitone treatment enhances in vivo the synthesis and phosphorylation of protein factors binding to the positive element and these constitute a minimal requirement for the transcriptional activation of the CYP2B1/B2 gene.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , ADN/metabolismo , Proteínas Nucleares/biosíntesis , Fenobarbital/farmacología , Esteroide Hidroxilasas/genética , Transcripción Genética/efectos de los fármacos , 2-Aminopurina/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Cicloheximida/farmacología , Inhibidores Enzimáticos/farmacología , Éteres Cíclicos/farmacología , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Ácido Ocadaico , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Inhibidores de Proteínas Quinasas , ARN Mensajero/metabolismo , RatasRESUMEN
The phenobarbitone-responsive minimal promoter has been shown to lie between nt -179 and nt + 1 in the 5' (upstream) region of the CYP2B1/B2 gene in rat liver, on the basis of the drug responsiveness of the sequence linked to human growth hormone gene as reporter and targeted to liver as an asialoglycoprotein-DNA complex in vivo. Competition analyses of the nuclear protein-DNA complexes formed in gel shift assays with the positive (nt -69 to -98) and negative (nt -126 to -160) cis elements (PE and NE, respectively) identified within this region earlier indicate that the same protein may be binding to both the elements. The protein species purified on PE and NE affinity columns appear to be identical based on SDS/PAGE analysis, where it migrates as a protein of 26-28 kDa. Traces of a high molecular weight protein (94-100 kDa) are also seen in the preparation obtained after one round of affinity chromatography. The purified protein stimulates transcription of a minigene construct containing the 179 nt on the 5' side of the CYP2B1/B2 gene linked to the I exon in a cell-free system from liver nuclei. The purified protein can give rise to all the three complexes (I, II, and III) with the PE, just as the crude nuclear extract, under appropriate conditions. Manipulations in vitro indicate that the NE has a significantly higher affinity for the dephosphorylated form than for the phosphorylated form of the protein. The PE binds both forms. Phenobarbitone treatment of the animal leads to a significant increase in the phosphorylation of the 26- to 28-kDa and 94-kDa proteins in nuclear labeling experiments followed by isolation on a PE affinity column. We propose that the protein binding predominantly to the NE in the dephosphorylated state characterizes the basal level of transcription of the CYP2B1/B2 gene. Phenobarbitone treatment leads to phosphorylation of the protein, shifting the equilibrium toward binding to the PE. This can promote interaction with an upstream enhancer through other proteins such as the 94-kDa protein and leads to a significant activation of transcription.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Hígado/metabolismo , Esteroide Hidroxilasas/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Adenosina Trifosfato/metabolismo , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Sondas de ADN , Marcación de Gen/métodos , Hígado/efectos de los fármacos , Hígado/enzimología , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fenobarbital/farmacología , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Ratas , Factores de Transcripción/aislamiento & purificaciónRESUMEN
The region -160 to -127 nt of the upstream of CYP2B1/B2 gene has been found to function as a negative cis-acting element on the basis of DNase-I footprint and gel mobility shift assays as well as cell-free transcriptional assays using Bal-31 mutants. A reciprocal relationship in the interaction of the negative and the recently characterized positive elements with their respective protein factors has been found under repressed and induced conditions of the gene. The negative element also harbors the core glucocorticoid responsive sequence, TGTCCT. It is concluded that the negative element mediates the repressed state of the gene under the uninduced condition and also mediates the repressive effect of dexamethasone, when given along with the inducer phenobarbitone in rats. Dexamethasone is able to antagonize the effects of phenobarbitone at as low a concentration as 100 micrograms/kg body wt in these animals.