Regulation of CYP1A1 expression.

CYP1A1 is considered to be involved mainly in oxidative metabolism of exogenous chemicals and drugs. Synthesis of this hemoprotein is induced in livers, lungs, and other tissues of experimental animals by the administration of these chemicals. Regulatory mechanisms of the induction process of the protein have been investigated by the DNA transfer method using the isolated genomic DNA. At least two kinds of cis-acting regulatory DNA sequences are localized 5' upstream of the gene. One is distributed five times in a relatively wide range from -0.5 to -3.5 kb and functions as an inducible enhancer-designated xenobiotic responsive element or XRE. The other is localized just upstream of the TATA sequence and acts as a regulatory element for the constitutive expression. The two DNA elements are required for a high level of the inducible expression. Their cognate DNA binding factors are recognized in the nuclear extracts of Hepa-1 cells and rat liver cells which show the inducible expression of CYP1A1 in response to the inducer. This paper discusses the regulatory mechanisms of CYP1A1 gene expression by summarizing the present state of knowledge about properties of the DNA regulatory elements and their cognate DNA-binding factors.     

Caveolin-1真核表达载体的构建及其Hepa1-6细胞中的表达

Caveolin-1在不同肿瘤中发挥作用不同,既发挥抑癌基因样作用又发挥癌基因样作用.旨在分析caveolin-1 在小鼠肝癌细胞系中的表达情况及建立稳定表达外源caveolin-1的Hepa1-6细胞.利用RT-PCR和Western-blot方法检测caveolin-1在小鼠肝癌H22、Hea-F和Hepa1-6细胞中的表达;通过分子克隆构建小鼠caveolin-1 cDNA真核表达栽体,利用脂质体转染等方法建立稳定表达外源caveolin-1的Hepa1-6细胞株;通过RT-PCR、Western-blot、免疫细胞化学等方法鉴定其稳定表达细胞株.结果显示,caveolin-1在Hepa1-6细胞中表达呈阴性,在H22和Hca-F 中高表达;成功获得小鼠caveolin-1 cDNA真核表达载体pEGFP-N2/Cav-1,筛选并鉴定出高表达外源caveolin-1的Hepa1-6稳定细胞株C1和C4,为进一步分析caveolin-1在肝癌中所发挥的作用奠定了一定的研究基础.     

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.     

Effect of uncoupling protein-1 expression on 3T3-L1 adipocyte gene expression

The mitochondrial respiratory uncoupling protein 1 (UCP1) partially uncouples substrate oxidation and oxidative phosphorylation to promote the dissipation of cellular biochemical energy as heat in brown adipose tissue. We have recently shown that expression of UCP1 in 3T3-L1 white adipocytes reduces the accumulation of triglycerides. Here, we investigated the molecular basis underlying UCP1 expression in 3T3-L1 adipocytes. Gene expression data showed that forced UCP1 expression down-regulated several energy metabolism pathways; but ATP levels were constant. A metabolic flux analysis model was used to reflect the gene expression changes onto metabolic processes and concordance was observed in the down-regulation of energy consuming pathways. Our data suggest that adipocytes respond to long-term mitochondrial uncoupling by minimizing ATP utilization.     

DEC1 expression in 1p-aberrant oligodendroglial neoplasms

BACKGROUND: Expression of hypoxia-related tissue factors in 1p-aberrant oligodendroglial neoplasms diminishes patient outcome. Differentiated embryo-chondrocyte expressed gene 1 (DEC1) has been described as novel hypoxia-related tissue factor. In our study, we assessed the expression of DEC1 in 1p aberrant oligodendroglial neoplasms and its association with necrosis and expression of hypoxia-inducible factor 1alpha (HIF-1alpha), carbonic anhydrase-9 (CA9), and vascular endothelial growth factor-mRNA (VEGF). MATERIALS AND METHODS: 44 primary and 16 recurrent oligodendroglial neoplasms with 1p-aberrations were investigated immunohistochemically for the expression of DEC1, HIF-1alpha, and CA9. Expression of VEGF was investigated using in situ hybridization. DEC1 expression was correlated with necrosis and with expression of HIF-1alpha, CA9, and VEGF. RESULTS: DEC1 was expressed in tumor cell nuclei, and occasionally in nuclei of endothelial cells, and glial and neuronal cells of surrounding brain tissue. High expression (>10% of tumor cells immunolabeled) of DEC1 was found in 56 cases, low expression (<10% of tumor cells immunolabeled) was found in 3 cases. In 1 case no expression of DEC1 was evident. DEC1 expression showed no topographical association with necrosis or expression of HIF-1alpha, CA9, or VEGF. CONCLUSION: DEC1 expression is found in the majority of 1p-aberrant oligodendroglial neoplasms and does not correlate with necrosis or expression of HIF-1alpha, CA9, VEGF. Thus, immunohistochemical analysis of DEC1 expression is in our hands not suitable for detection of tissue hypoxia in this type of primary brain tumor.     

Dissociation of insulin receptor expression and signaling from caveolin-1 expression

The presence of cell surface caveolin/caveolae has been postulated to influence the localization, expression levels, and kinase activity of numerous receptors, including the insulin receptor. However, there are conflicting data concerning the effects of caveolin on insulin receptor expression and function. To help clarify this issue, we created a gain of function situation by expressing caveolin-1 at various levels in HEK-293 cells where the endogenous level of caveolin-1 is very low. We generated four permanent lines of this cell expressing amounts of caveolin-1 ranging from 10 to 40 times that of parental cells. The amount of caveolin-1 in the human embryonic kidney cells expressing the highest caveolin levels is comparable with that of adipocytes, cells that naturally express one of the highest levels of caveolin-1. We measured insulin receptor amount and insulin-dependent receptor autophosphorylation as well as insulin receptor substrate 1 (IRS1) tyrosine phosphorylation as an index of insulin signaling. We found that the insulin receptor level was essentially the same in the parental and all four derived cell lines. Likewise, we determined that insulin-dependent insulin receptor and IRS1 tyrosine phosphorylation was not significantly different in the four cell lines representing parental, low, medium, and high levels of caveolin-1 expression. We conclude that insulin receptor expression and ligand-dependent signaling is independent of caveolin-1 expression.     

Induction of OGG1 gene expression by HIV-1 Tat

To identify the cellular gene target for Tat, we performed gene expression profile analysis and found that Tat up-regulates the expression of the OGG1 (8-oxoguanine-DNA glycosylase-1) gene, which encodes an enzyme responsible for repairing the oxidatively damaged guanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). We observed that Tat induced OGG1 gene expression by enhancing its promoter activity without changing its mRNA stability. We found that the upstream AP-4 site within the OGG1 promoter is responsible and that Tat interacted with AP-4 and removed AP-4 from the OGG1 promoter by in vivo chromatin immunoprecipitation assay. Thus, Tat appears to activate OGG1 expression by sequestrating AP-4. Interestingly, although Tat induces oxidative stress known to generate 8-oxo-dG, which causes the G:C to T:A transversion, we observed that the amount of 8-oxo-dG was reduced by Tat. When OGG1 was knocked down by small interfering RNA, Tat increased the amount of 8-oxo-dG, thus confirming the role of OGG1 in preventing the formation of 8-oxo-dG. These findings collectively indicate the possibility that Tat may play a role in maintenance of the genetic integrity of the proviral and host cellular genomes by up-regulating OGG1 as a feed-forward mechanism.     

SDF-1-induced activation of ERK enhances HIV-1 expression

Chemokine receptors are not only able to bind chemokines but, together with CD4, they serve as an entry door for the human immunodeficiency virus type 1 (HIV-1). The signalling capacity of chemokine receptors, which is of fundamental importance for chemokine-induced chemotaxis, is not used by HIV-1 to enter a target cell, nor by chemokines or chemokine-derived ligands to inhibit viral entry. In addition, an ill-defined signal triggered by chemokines can, under some circumstances, lead to an increase in HIV-1 expression. We show here that, in infected cells, exposure to SDF-1 leads to an increased expression of a X4 strain of HIV-1. A similar increase can be induced by an N-terminal peptide of SDF-1 which had previously been shown to elicit an intracellular calcium response and to inhibit the entry of X4 strains of HIV-1. We demonstrate the involvement of extracellular signal-regulated kinases (ERK) in this phenomenon. SDF-1 activates ERK-1 and ERK-2 in Jurkat cells. In HeLa cells, ERK-2 only is activated by SDF-1 or by a SDF-derived peptide. This ERK activation can be blocked by pertussis toxin and by the MEK inhibitor U0126. Most importantly, SDF-1-dependent HIV-1 expression is abolished by pretreating the cells with pertussis toxin or with U0126. The consequences of this SDF-1-induced, ERK-dependent modulation of HIV-1 expression in infected cells may have a clinical relevance for eradicating latent viruses.     

Effects of tripterine on mRNA expression of TGF-beta1 and collagen IV expression in BW F1 mice

Tripterine is a chemical isolated from a traditional Chinese herb which had been testified for its anti-inflammatory and immunosuppressive activities in a previous study. However, little is known about the effects and mechanism of action of Tripterine on treating lupus nephritis. In the present study we investigated the effect of Tripterine on the F1 hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice which functioned as a model of human systemic lupus erythematosus (BW F1 mice) and evaluated the possible mechanism implicated in the mRNA expression of TGF-beta1 and collagen IV expression of the BW F1 mice kidney tissue. Different doses of Tripterine were injected peritoneally to BW F1 mice at different stages to study the preventive effects of Tripterine on lupus nephritis glomerulosclerosis and its mechanisms. Twenty-four hour urine protein excretion, serum anti-dsDNA antibodies and the expression of collagen type IV were examined by immunohistochemistry while the expression of TGF-beta1 mRNA was detected by RT nested PCR. Tripterine decreased urine protein excretion and the level of serum anti-dsDNA antibodies and also suppressed the expression of collagen type IV and TGF-beta1 mRNA in the murine kidney tissue. Administration of Tripterine before the occurrence of proteinuria had much greater protective effects than if it was administered after the occurrence of proteinuria. No significant difference was found between the 3 mg/kg/week Tripterine-treated-group and the 6 mg/kg/week Tripterine-treated-group. Tripterine had a definite protective effect on glomerulosclerosis of the lupus murine model. Tripterine could significantly reduce the amount of urine protein excretion, suppress the formation of serum anti-dsDNA antibodies, it could also efficiently decrease the expression of renal collagen type IV probably due to its suppressive effect on the expressions of local TGF-(1 mRNA) in this model.