佛波酯诱导内皮素和FOS／JUN基因在血管内皮细胞中的表达及AP－1结合活性

佛波酯诱导内皮素和ＦＯＳ／ＪＵＮ基因在血管内皮细胞中的表达及ＡＰ－１结合活性温进坤,魏素珍（河北医学院生化教研室,石家庄０５００１７）张晨晖,姚阿卿,周爱儒,汤健（北京医科大学心血管基础研究所,北京,１０００８３）关键词内皮素基因表达;ＡＰ－１转录因...     

A new fluorescent histological marker for ischemic neurons,EA 50: Correlated with Fos and Jun/ AP-1 immunoreactivity

Cerebral ischemia/hypoxia induces ischemic neuronal changes characterized by nuclear pyknosis, cytoplasmic shrinkage, and basophilia. The ischemic neurons were shown to exhibit strong and persistent c-fos proto-oncogene. The ischemic neuronal changes and c-fos expression are thought to be the consequence of release of excessive glutamate by the ischemic neurons. In the present study, we investigated with immunohistochemistry the subcellular distribution of Fos and Jun/AP-1, the protein products of c-fos and c-jun proto-oncogenes, and compared them with histological changes show by hematoxylin-eosin and by EA 50 stains. The latter is a stain mixture used traditionally in the Papanicolaou procedure and has a specific affinity for ischemic neurons. The active ingredient is eosin Y, a tetrabrominated derivative of fluorescein. With EA 50, the ischemic neurons stain red and emit a yellow fluorescence, while the non-ischemic neurons are green and non-fluorescent. The subcellular site of cosin Y binding corresponds with Fos and Jun/AP-1; all are concentrated in the nuclei and spread into the perikaryon, dendrites, and axons. The eosin Y-binding appears in neurons that have shown advanced ischemic changes. The dye is thus a good histological marker for damaged neurons, but requires freshly fixed tissues and paraffin sections of less than 4 m thick. Preincubation of tissue sections in antibodies against Fos and Jun abolishes the eosin Y binding, suggesting that the dye may interact with Fos/Jun/AP-1 protein or other protein products in the ischemic neurons.     

Hormone-independent repression of AP-1-inducible collagenase promoter activity by glucocorticoid receptors.

The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200- to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.     

Uncoupling of apoptosis and Jun/AP-1 activity in human promonocytic cells treated with DNA-damaging and stress-inducing agents

Earlier studies have indicated that Jun/AP-1 activity is associated with, and probably required for apoptosis induction by DNA-damaging and stress-inducing agents in human myeloid cells. To investigate this possibility, we examined the capacity of continuous treatments with etoposide (10 microM) and camptothecin (0.4 microM), and pulse treatments with X-rays (20 Gy), heat (2 h at 42.5 C) and cadmium chloride (2 h at 200 microM) followed by recovery, to provoke apoptosis and to simulate c-jun and c-fos expression and AP-1 binding in U-937 human promonocytic cells. All these treatments generated apoptosis with similar efficacy (50-60% apoptotic cells at 6 h of treatment or recovery). However, the capacity to increase c-jun and c-fos mRNA levels and to stimulate AP-1 binding was very different, ranging from more than a twelve-fold increase in the case of cadmium, to almost no increase in the case of heat-shock and etoposide. When the cells were pre-conditioned with a soft heat shock (1 h at 42 degrees C) the cadmium-provoked apoptosis was greatly inhibited, but the stimulation of AP-1 binding was not affected. The administration of cAMP-increasing agents also reduced the etoposide- and cadmium-provoked apoptosis. However, cAMP greatly stimulated c-jun and c-fos expression and AP-1 binding when applied together with etoposide (which itself was ineffective), and potentiated the cadmium-induced AP-1 binding. Conversely, retinoic acid abrogated the cadmium-provoked stimulation of AP-1 binding and transactivation capacity, and greatly inhibited the stimulation of binding caused by camptothecin and X-rays. However, retinoic acid did not inhibit the induction of apoptosis by these agents. These results indicate that Jun/AP-1 activity is not necessarily coupled with apoptosis, nor required for apoptosis induction by DNA-damaging and stress-inducing agents in human promonocytic cells.     

Three prime exonuclease I (TREX1) is Fos/AP-1 regulated by genotoxic stress and protects against ultraviolet light and benzo(a)pyrene-induced DNA damage

Cells respond to genotoxic stress with the induction of DNA damage defence functions. Aimed at identifying novel players in this response, we analysed the genotoxic stress-induced expression of DNA repair genes in mouse fibroblasts proficient and deficient for c-Fos or c-Jun. The experiments revealed a clear up-regulation of the three prime exonuclease I (trex1) mRNA following ultraviolet (UV) light treatment. This occurred in the wild-type but not c-fos and c-jun null cells, indicating the involvement of AP-1 in trex1 induction. Trex1 up-regulation was also observed in human cells and was found on promoter, RNA and protein level. Apart from UV light, TREX1 is induced by other DNA damaging agents such as benzo(a)pyrene and hydrogen peroxide. The mouse and human trex1 promoter harbours an AP-1 binding site that is recognized by c-Fos and c-Jun, and its mutational inactivation abrogated trex1 induction. Upon genotoxic stress, TREX1 is not only up-regulated but also translocated into the nucleus. Cells deficient in TREX1 show reduced recovery from the UV and benzo(a)pyrene-induced replication inhibition and increased sensitivity towards the genotoxins compared to the isogenic control. The data revealed trex1 as a novel DNA damage-inducible repair gene that plays a protective role in the genotoxic stress response.