RESUMEN
Pathological conditions such as ischaemic stroke and inflammatory disorders cause c-fos activation in the brain. This activation contributes to the initiation of the brain's inflammatory response, orchestrated by activated glial cells. The inflammatory signalling cascades leading to c-fos activation in glial cells are not well characterized. Thus, we have attempted a detailed analysis of the cis-acting elements, transcription factors and upstream kinase pathways involved in the activation of c-fos by lipopolysaccharide (LPS) in primary rat cortical glial cells. We found that (1) LPS-induced c-fos mRNA levels were sensitive to p38 mitogen-activated protein kinase (MAPK) inhibitors but not to mitogen-activated/extracellular signal-regulated kinase (ERK) or calcium-calmodulin-dependent kinase inhibitors, (2) LPS activated both serum response element (SRE) and cyclic AMP/calcium response element (CRE)-driven luciferase reporters in transient transfection assays, (3) LPS induced the phosphorylation of Elk1 CRE-binding protein (CREB)/activated transcription factor-1 (ATF-1) and the activation of GAL4-Elk1 and GAL4-CREB chimeric proteins, and (4) mutation of both SRE and CRE elements was necessary and sufficient to completely abolish LPS induction of a rat c-fos proximal promoter-luciferase reporter. Thus, c-fos activation by LPS in glial cells occurs via the SRE or CRE in an independent manner, and involves the Elk1 or CREB/ATF-1 transcription factors. Elk1-mediated transactivation was dependent on p38 MAPK, suggesting a crucial role of these factors in mediating inflammatory responses in the CNS.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Regulación de la Expresión Génica/fisiología , Genes fos/fisiología , Lipopolisacáridos/farmacología , Neuroglía/enzimología , Elemento de Respuesta al Suero/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Genes fos/efectos de los fármacos , Neuroglía/efectos de los fármacos , Ratas , Elemento de Respuesta al Suero/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Cytochrome P450 2E1 (CYP2E1) exhibits a pronounced oxidase activity that may mediate apoptotic injury in glial cells as well as hepatocytes. Strict regulation of CYP2E1 and it's activity is therefore thought to be crucial. We have studied CYP2E1 transcriptional regulation in primary cortical glial cells and have identified a novel repressor element at +1452/+1460 in intron 2 of the rat CYP2E1 gene. The element very potently repressed CYP2E1 and SV40 promoters and consisted of the non-palindromic core sequence 5'-TTCCACTCA-3'. Jun proteins were found to interact with the site. The protein complexes were also found to contain an as yet unidentified protein of approximately 60 kDa, probably with DNA binding properties similar to G-box binding factors found in, e.g. Arabidopsis thaliana. Stimulation with lipopolysaccharide, or overexpression of the mitogen-activated protein kinase kinase kinase, MEKK-1, further deepened the repression in primary cortical glial cells. It is suggested that this novel Jun binding repressor helps to control basal expression levels of CYP2E1, and modulates the response to inflammatory factors. Future in vivo experiments will, however, be required for a full appreciation of the role of this repressor in the complex regulation of CYP2E1 during inflammatory conditions.
Asunto(s)
Citocromo P-450 CYP2E1/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Intrones/fisiología , Lipopolisacáridos/farmacología , Neuroglía/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencias de Aminoácidos , Animales , Células Cultivadas , Corteza Cerebral/citología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Intrones/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas Quinasa Quinasa PAM/efectos de los fármacos , Quinasas Quinasa Quinasa PAM/metabolismo , Sustancias Macromoleculares , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Neuroglía/citología , Neuroglía/efectos de los fármacos , Unión Proteica/genética , Ratas , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Análisis de Secuencia de ADN , TransfecciónRESUMEN
Acute systemic lipopolysaccharide (endotoxin, LPS) exposure, which can lead to septic shock, enhances the hepatic expression of inflammatory and acute-phase proteins (APPs). To better understand how LPS aggravates damage, changes in hepatic gene expression after a single LPS dose was screened by using microarrays for 1176 rat genes. We detected more than 20 new potential LPS-induced APPs. Following acute LPS challenge, significant up-regulation of the steady-state mRNA levels of several important early transcription factors, such as c-jun and STAT3, and cytokine-associated genes, was observed. In contrast, RT-PCR analysis revealed marked down-regulation of the nuclear receptors RXRalpha, PXR, FXR, LXR, PPARalpha and CAR. Also genes encoding lipolytic, antioxidant as well as drug- and alcohol-metabolizing enzymes were down-regulated. These data suggest that acute LPS treatment induces important early transcription factors and co-ordinately down-regulates nuclear receptors, and that this results in altered expression of a large number of downstream genes.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Endotoxinas/farmacología , Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Fase Aguda/genética , Animales , Regulación hacia Abajo/efectos de los fármacos , Hígado/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide (LPS) or interleukin-1beta. We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3 h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h. The induction was sensitive to several tyrosine kinase inhibitors, and was further modulated by inhibitors of p38 MAP kinase. MAP kinase kinase-3 (MKK3) was phosphorylated in response to LPS, and expression of constitutively active MKK3, but not the MAP kinase kinases MEKK1 or MKK1, activated CYP2E1. Transcriptional activation was mediated through a C/EBPbeta and -delta binding element situated at -486/-474, and appeared to involve activation of prebound factors as well as recruitment of newly synthesized C/EBPbeta and -delta. It is thus suggested that LPS induces MKK3 activation in astrocytes, which in turn stimulates a C/EBPbeta and -delta binding element to mediate transcriptional activation of CYP2E1.
Asunto(s)
Astrocitos/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Citocromo P-450 CYP2E1/biosíntesis , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Células Cultivadas , Inducción Enzimática/efectos de los fármacos , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 3 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , RatasRESUMEN
Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide or interleukin-1beta. In the present study, phorbol-12,13-dibutyrate (PDBu) was found to induce catalytically active CYP2E1 more than fourfold in cortical glial cultures. Little induction was seen up to 12 h, and full effects only at 21-24 h of PDBu treatment. CYP2E1 expression in PDBu-treated cells was enriched in a subset of astrocytes. The protein kinase C inhibitors, staurosporine and calphostin C, and the tyrosine kinase inhibitor genistein, but not its inactive analogue daidzein, prevented the induction of CYP2E1 by PDBu. It is suggested that CYP2E1, together with interleukin-6 and ciliary neurotrophic factor, is part of a response of astrocytes to cellular stress elicited by, e.g. cerebral injury, cytokines or phorbol ester, and mediated in part through protein kinase C.
Asunto(s)
Astrocitos/enzimología , Citocromo P-450 CYP2E1/metabolismo , Ésteres del Forbol/farmacología , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Clorzoxazona/metabolismo , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Forbol 12,13-Dibutirato/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , RatasRESUMEN
Chlomethiazole and pyridinyl imidazole compounds, exemplified by SB203580, are structurally distinct p38 mitogen-activated protein kinase inhibitors with neuroprotective properties in models of cerebral ischaemia. We have examined their effects in interleukin-1beta (IL-1beta) synthesis, release and signalling in rat cortical glial cells, given the important role of IL-1beta in cerebral ischaemia. We analysed (i) IL-1beta mRNA expression by northern blot, (ii) IL-1beta protein precursor levels within the cells by western blot, and (iii) the levels of the mature IL-1beta protein secreted into the medium by enzyme-linked immunosorbent assay (ELISA) after treatment of rat cortical glial cells with lipopolysaccharide. While the induction of IL-1beta expression by lipopolysaccharide or by IL-1beta itself was very sensitive to nuclear factor kappa B (NF-kappaB) inhibitors, chlomethiazole or SB203580 were nearly without effect, indicating a differential regulation as compared to peripheral cells, e.g. monocytes. In contrast, chlomethiazole and SB203580 potently inhibited the IL-1beta-induced expression of c-fos and inducible nitric oxide synthase, as monitored by northern blot and quantitative RT-PCR, respectively. Because IL-1beta-induced expression of c-fos and inducible nitric oxide synthase is believed to directly contribute to the pathology of cerebral ischaemic injury, the results suggest a direct mechanism for the neuroprotective effects of chlomethiazole and SB203580, and further establish the anti-inflammatory properties of chlomethiazole.