Gene profiles of a human alveolar epithelial cell line after in vitro exposure to respiratory (non-)sensitizing chemicals: Identification of discriminating genetic markers and pathway analysis

There are currently no accepted biological prediction models for assessing the potential of a substance to cause respiratory sensitization. New tests should be based on mechanistic understanding and should be preferentially restricted to in vitro assays. The major goal of this study was to investigate the alterations in gene expression of human alveolar epithelial (A549) cells after exposure to respiratory sensitizing and non-respiratory sensitizing chemicals, and to identify genes that are able to discriminate between both groups of chemicals. A549 cells were exposed during 6, 10, and 24 h to the respiratory sensitizers ammonium hexachloroplatinate IV, hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene. Overall changes in gene expression were evaluated using Agilent Whole Human Genome 4x44K oligonucleotide arrays. A Fisher linear discriminant analysis was used to obtain a ranking of genes that reflects their potential to discriminate between respiratory sensitizing and respiratory non-sensitizing chemicals. Among the 20 most discriminating genes, which were categorized into molecular and biological gene ontology (GO) terms, CTLA4 could be associated with asthma and/or respiratory sensitization. When categorizing the top-1000 genes into biological GO terms, 22 genes were associated with immune function. Using a pathway analysis tool to identify possible underlying mechanisms of respiratory sensitization, no known canonical signaling pathway was observed to be activated in the A549 cell line.     

视黄酸受体和类视黄醇X受体激动剂对脐血单个核细胞IL-4和IFN-γ表达的影响

目的观察视黄酸受体(RARs)激动剂四氢四甲基萘丙烯苯甲酸乙酯(TTNPB)和类视黄醇X受体(RXRs)激动剂甲氧普烯酸(MA)对脐血单个核细胞表达IL-4和IFN-γ的影响。方法采用ELISA和RT-PCR,分别从蛋白质和基因水平上观察TTNPB和MA对脐血单个核细胞表达IL-4和IFN-γ的影响。结果 ELISA显示10-8、10-6、10-5、10-4mol/L的TTNPB和10-8、10-5、10-4mol/L的MA显著降低IFN-γ/IL-4蛋白含量比值(P0.05);RT-PCR显示10-4mol/L的MA显著降低IFN-γ/IL-4mRNA比值(P0.05),而10-5mol/L的TTNPB和10-7、10-6mol/L的MA显著升高IFN-γ/IL-4mRNA比值(P0.05)。结论适当浓度的TTNPB和MA可以在蛋白质水平上下调IFN-γ/IL-4的比值;不同浓度的TTNPB和MA在基因水平上对IFN-γ/IL-4的比值有不同影响。     

Prevention of acute and chronic allograft rejection by a novel retinoic acid receptor-alpha-selective agonist

To investigate the involvement of retinoic acid receptor (RAR)-alpha in allograft rejection, we investigated the effect of a novel selective agonist to the receptor, ER-38925, in a mouse cardiac allograft model. Prophylactic treatment with ER-38925 inhibited the acute rejection of the mouse cardiac allograft (BALB/c --> C3H/HeN) at 0.3 and 3 mg/kg, and its effect was enhanced in combination with tacrolimus. In this model, ER-38925 remarkably inhibited cytotoxic T lymphocyte induction and alloantigen-stimulated production of cytokines, i.e. IL-2, IL-12 and IFN-gamma. In the chronic rejection model, combined treatment with tacrolimus and ER-38925 reduced the grade and incidence of arteriosclerosis in the cardiac allografts significantly more potently than tacrolimus monotherapy. ER-38925 inhibited the proliferation of rat aortic smooth muscle cells stimulated in vitro, presumably through the induction of a cyclin-dependent kinase inhibitor, p27(kip-1). Those results provide a rationale for using RAR-alpha agonists as immunosuppressants in human organ transplantation.     

Cyclic AMP enhances progesterone action in human myometrial cells

Cyclic AMP (cAMP) has been shown to promote progesterone and glucocorticoid action in a variety of cellular settings. In this study, we have used human myometrial cells to investigate whether cAMP potentiates the ability of progesterone to repress IL-1β-driven COX-2 expression. We found that forskolin enhanced progesterone-repression of IL-1β-driven COX-2 expression in association with delayed IL-1β-induced nuclear phospho-p65 entry and reduced NF-κB binding to the COX-2 promoter. Further, forskolin enhanced the progesterone-induced expression of FKBP5 and 11βHSD1, progesterone-driven activity of a progesterone response element (PRE) and progesterone receptor (PR)-B binding to a transfected PRE. In addition, forskolin treatment increased PR-B levels and reduced the PR-A:PR-B ratio while acutely decreasing the association between PR and nuclear receptor co-repressor (NCoR) and reducing NCoR levels after 6 h. These findings are of importance in situations where enhancing progesterone activity is desirable, for example in the management of endometrial cancer, the promotion of endometrial receptivity or the maintenance of myometrial quiescence during pregnancy.     

The CYP2B2 5' flank contains a complex glucocorticoid response unit

Rat CYP2B1 and CYP2B2 and mouse CYP2B10 are dramatically induced by phenobarbital (PB) in liver. PB responsiveness requires the constitutive androstane receptor (CAR). However, dexamethasone treatment can also induce CYP2B genes in both rat and mouse liver. Three regions have been shown to be involved in conferring dexamethasone responsiveness on CYP2B2 reporter constructs. They are the PB response unit, a functional glucocorticoid response element at −1.3 kb in the 5′ flank and a weak element in the basal promoter. We report here the identification, by deletion analysis of the CYP2B2 5′ flank, of new glucocorticoid response elements or accessory factor sites. Moreover, we show that CAR acts as an accessory factor in the dexamethasone response in vivo of CYP2B10 protein in mice, by increasing both the basal and induced levels. We propose a model to explain the dexamethasone responsiveness of the CYP2B2 gene in which induction is mediated by a complex glucocorticoid response unit.     

Myeloid differentiation is impaired in transgenic mice with targeted expression of a dominant negative form of retinoid X receptor β

To investigate the in vivo function of retinoid X receptor (RXR) on myelopoiesis, we generated transgenic (Tg) mice with targeted expression of a dominant negative form of RXR β in myeloid cells. In these Tg mice the transgene is expected to suppress the function of heterodimeric receptors composed of RXR and its counterparts, such as retinoic acid receptor. Out of 12 mice analysed, one Tg mouse exhibited a severe maturation arrest at the promyelocytic stage. Three other Tg mice showed a mild inhibition of myeloid differentiation, which was further augmented when mice were treated with 5-fluorouracil (5-FU). Furthermore, four Tg mice showed impaired myeloid differentiation in response to the treatment by 5-FU or granulocyte-colony stimulating factor (G-CSF), although they exhibited apparently normal myelopoiesis in the untreated state. The phenotype of Tg mice observed after G-CSF treatment correlated with the expression level of the transgene, although the correlation was not found in untreated mice. These results indicated that myeloid differentiation is perturbed in the Tg mice by the dominant negative effect of the transgenic RXR, indicating that RXR plays a role in myelopoiesis.     

De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity

Previous studies could demonstrate, that the naturally occuring polyphenol resveratrol inhibits cell growth of colon carcinoma cells at least in part by inhibition of protooncogene ornithine decarboxylase (ODC). The objective of this study was to provide several lines of evidence suggesting that the induction of ceramide synthesis is involved in this regulatory mechanisms. Cell growth was determined by BrdU incorporation and crystal violet staining. Ceramide concentrations were detected by HPLC-coupled mass-spectrometry. Protein levels were examined by Western blot analysis. ODC activity was assayed radiometrically measuring [(14)CO(2)]-liberation. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Antiproliferative effects of resveratrol closely correlate with a dose-dependent increase of endogenous ceramides (p<0.001). Compared to controls the cell-permeable ceramide analogues C2- and C6-ceramide significantly inhibit ODC-activity (p<0.001) in colorectal cancer cells. C6-ceramide further diminished protein levels of protooncogenes c-myc (p<0.05) and ODC (p<0.01), which is strictly related to the ability of ceramides to inhibit cell growth in a time- and dose-dependent manner. These results were further confirmed using inhibitors of sphingolipid metabolism, where only co-incubation with a serine palmitoyltransferase (SPT) inhibitor could significantly counteract resveratrol-mediated actions. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrol-mediated inhibition of ODC. In contrast to the regulation of catabolic spermidine/spermine acetyltransferase by resveratrol, inhibitory effects on ODC occur PPARgamma-independently, indicating independent pathways of resveratrol-action. Due to our findings resveratrol could show great chemopreventive and therapeutic potential in the treatment of colorectal cancers.     

视黄酸联合视网膜细胞共培养对胚胎干细胞体外分化的诱导作用

目的 探讨胚胎干细胞(ESC)在视黄酸联合视网膜细胞共培养诱导条件下的分化特征。方法 将ESC自液氮中复苏、培养，传1代后进行拟胚体培养。将部分3．5d拟胚体离心重悬后加入含有视黄酸的24孔板中进行诱导，另一部分加入已经培养有视网膜混合细胞的培养瓶中，培养液中同时也加入视黄酸。视网膜混合细胞中仅加入视黄酸未加拟胚体作为对照。倒置相差显微镜下观察细胞在整个诱导过程中形态学的改变并使用免疫细胞化学检测诱导细胞中巢蛋白、胶质纤维酸性蛋白、广谱细胞角蛋白、微管相关蛋白-2、视紫质蛋白表达情况。结果 (1)形态学改变仅由视黄酸诱导，诱导细胞呈多种形态；在共培养条件下，绝大多数拟胚体分化出来的细胞形态非常单一，呈透明圆形。部分原贴壁的视网膜细胞出现了明显的网状结构。(2)免疫细胞化学显示两种诱导条件均可见大部分诱导细胞MAP-2阳性，并可见Nestin阳性细胞。共培养诱导尚可见GFAP阳性、Cytokeratin阳性和Rhodopsin阳性的细胞。结论 视黄酸可以诱导大部分细胞成为神经细胞，在视黄酸和与视网膜细胞共培养诱导条件下，可以获得更为纯化的形态一致的神经样细胞，部分诱导细胞表达视网膜细胞的特性。