Induction of tolerance against grafted organs is achieved by the immunosuppressive agent cyclosporine, a prominent member of the calcineurin inhibitors. Unfortunately, its lifetime use is associated with hypertension and nephrotoxicity. Several mechanism for cyclosporine induced hypertension have been proposed, i.e. activation of the sympathetic nervous system, endothelin-mediated systemic vasoconstriction, impaired vasodilatation secondary to reduction in prostaglandin and nitric oxide, altered cytosolic calcium translocation, and activation of the renin-angiotensin system (RAS). In this regard the molecular basis for undue RAS activation and an increased signaling of the vasoactive oligopeptide angiotensin II (AngII) remain elusive. Notably, angiotensinogen (AGT) is the precursor of AngII and transcriptional regulation of AGT is controlled by the hepatic nuclear factor HNF4alpha. To better understand the molecular events associated with cyclosporine induced hypertension, we investigated the effect of cyclosporine on HNF4alpha expression and activity and searched for novel HNF4alpha target genes among members of the RAS cascade. Using bioinformatic algorithm and EMSA bandshift assays we identified angiotensin II receptor type 1 (AGTR1), angiotensin I converting enzyme (ACE), and angiotensin I converting enzyme 2 (ACE2) as genes targeted by HNF4alpha. Notably, cyclosporine represses HNF4alpha gene and protein expression and its DNA-binding activity at consensus sequences to AGT, AGTR1, ACE, and ACE2. Consequently, the gene expression of AGT, AGTR1, and ACE2 was significantly reduced as evidenced by quantitative real-time RT-PCR. While RAS is composed of a sophisticated interplay between multiple factors we propose a decrease of ACE2 to enforce AngII signaling via AGTR1 to ultimately result in vasoconstriction and hypertension. Taken collectively we demonstrate cyclosporine to repress HNF4alpha activity through calcineurin inhibitor mediated inhibition of nuclear factor of activation of T-cells (NFAT) which in turn represses HNF4alpha that leads to a disturbed balance of RAS.

Background

Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time.

Methods

Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis.

Results

We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed.

Conclusions

Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals.

Background

Lung cancer is a leading cause of cancer morbidity. To improve an understanding of molecular causes of disease a transgenic mouse model was investigated where targeted expression of the serine threonine kinase c-Raf to respiratory epithelium induced initialy dysplasia and subsequently adenocarcinomas. This enables dissection of genetic events associated with precancerous and cancerous lesions.

Methodology/Principal Findings

By laser microdissection cancer cell populations were harvested and subjected to whole genome expression analyses. Overall 473 and 541 genes were significantly regulated, when cancer versus transgenic and non-transgenic cells were compared, giving rise to three distinct and one common regulatory gene network. At advanced stages of tumor growth predominately repression of gene expression was observed, but genes previously shown to be up-regulated in dysplasia were also up-regulated in solid tumors. Regulation of developmental programs as well as epithelial mesenchymal and mesenchymal endothelial transition was a hall mark of adenocarcinomas. Additionaly, genes coding for cell adhesion, i.e. the integrins and the tight and gap junction proteins were repressed, whereas ligands for receptor tyrosine kinase such as epi- and amphiregulin were up-regulated. Notably, Vegfr- 2 and its ligand Vegfd, as well as Notch and Wnt signalling cascades were regulated as were glycosylases that influence cellular recognition. Other regulated signalling molecules included guanine exchange factors that play a role in an activation of the MAP kinases while several tumor suppressors i.e. Mcc, Hey1, Fat3, Armcx1 and Reck were significantly repressed. Finally, probable molecular switches forcing dysplastic cells into malignantly transformed cells could be identified.

Conclusions/Significance

This study provides insight into molecular pertubations allowing dysplasia to progress further to adenocarcinoma induced by exaggerted c-Raf kinase activity.

Background

Decoding of the liver proteome is subject of intense research, but hampered by methodological constraints. We recently developed an improved protocol for studying rat liver proteins based on 2-DE-MALDI-TOF-MS peptide mass finger printing.

This methodology was now applied to develop a mouse liver protein database.

Results

Liver proteins were extracted by two different lysis buffers in sequence followed by a liquid-phase IEF pre-fractionation and separation of proteins by 2 DE at two different pH ranges, notably 5-8 and 7-10. Based on 9600 in gel digests a total of 643 mouse liver proteins with high sequence coverage (> 20 peptides per protein) could be identified by MALDI-TOF-MS peptide mass finger printing. Notably, 255 proteins are novel and have not been reported so far by conventional two-dimensional electrophoresis proteome mapping. Additionally, the results of the present findings for mouse liver were compared to published data of the rat proteome to compile as many proteins as possible in a rodent liver database.

Conclusion

Based on 2-DE MALDI-TOF-MS a significantly improved proteome map of mouse liver was obtained. We discuss some prominent members of newly identified proteins for a better understanding of liver biology.

Background

The choroid plexus consists of highly differentiated epithelium and functions as a barrier at the interface of the blood-cerebrospinal-fluid (CSF). This tissue may therefore determine the bioavailability and transport of drugs to the brain. Little is known about the expression of drug and xenobiotic metabolizing enzymes (DME) and of drug transporters in the human choroid plexus. Notably, the transcription factor and zinc finger protein HNF4alpha is a master regulator of DMEs and of drug transporters. As of today its activity in the blood-CSF barrier is unknown. Here we report our efforts in determining HNF4alpha activity in the regulation of ABC transporters in the human and rat choroid plexus.

Results

We report expression of HNF4alpha by qRT-PCR and by immunohistochemistry and evidence transcript expression of the ATP-binding cassette transporters ABCB1, ABCB4, ABCC1-6 in choroid plexus. Additionally, HNF4alpha DNA binding activity at regulatory sequences of ABCB4 and ABCC1 was determined by EMSA bandshift assays with a specific antibody. We then performed siRNA mediated functional knock down of HNF4alpha in Caco-2 cells and found ABCC1 gene expression to be repressed in cell culture experiments.

Conclusion

Our study evidences activity of HNF4alpha in human and rat choroid plexus. This transcription factor targets DMEs and drug transporters and may well determine availability of drugs at the blood-CSF barrier.

Background

Lung cancer is a multistage process with poor prognosis and high morbidity. Importantly, the genetics of dysplasia, a facultative cancer, at the edge of malignant transformation is unknown.

Methodology/Principal Findings

We employed laser microdissection to harvest c-Raf1- induced dysplastic as opposed to transgenic but otherwise morphologically unaltered epithelium and compared findings to non-transgenic lung. We then employed microarrays to search genome wide for gene regulatory networks. A total of 120 and 287 genes were significantly regulated, respectively. Dysplasia was exclusive associated with up-regulation of genes coding for cell growth and proliferation, cell-to-cell signalling and interaction, lipid metabolism, development, and cancer. Likewise, when dysplasia was compared with non-transgenic cells up-regulation of cancer associated genes, tight junction proteins, xenobiotic defence and developmental regulators was observed. Further, in a comparison of the data sets of dysplasia vs transgenic and dysplasia vs non-transgenic 114 genes were regulated in common. We additionally confirmed regulation of some genes by immunohistochemistry and therefore demonstrate good concordance between gene regulation and coded protein.

Conclusion

Our study identified transcriptional networks at successive stages of tumor-development, i.e. from histological unaltered but transgenic lungs to nuclear atypia. Our SP-C/c-raf transgenic mouse model revealed interesting and novel candidate genes and pathways that provide clues on the mechanism forcing respiratory epithelium into dysplasia and subsequently cancer, some of which might also be useful in the molecular imaging and flagging of early stages of disease.

Posttransplantation diabetes mellitus (PTDM) is a frequent complication in immunosuppressive therapy. To better understand the molecular events associated with PTDM we investigated the effect of cyclosporine on expression and activity of hepatic nuclear factor (HNF)1alpha and 4alpha and on genes coding for glucose metabolism in cultures of the rat insulinoma cell line INS-1E, the human epithelial cell line Caco-2 and with Zucker diabetic fatty (ZDF) rats. In the pancreas of untreated but diabetic animals expression of HNF4alpha, insulin1, insulin2 and of phosphoenolpyruvate carboxykinase was significantly repressed. Furthermore, cyclosporine treatment of the insulinoma-1E cell line resulted in remarkable reduction in HNF4alpha protein and INS1 as well as INS2 gene expression, while transcript expression of HNF4alpha, apolipoprotein C2, glycerolkinase, pyruvatekinase and aldolase B was repressed in treated Caco-2 cells. Furthermore, with nuclear extracts of cyclosporine treated cell lines protein expression and DNA binding activity of hepatic nuclear factors was significantly repressed. As cyclosporine inhibits the calcineurin dependent dephosphorylation of nuclear factor of activated T-cells (NFAT) we also searched for binding sites for NFAT in the pancreas specific P2 promoter of HNF4alpha. Notably, we observed repressed NFAT binding to a novel DNA binding site in the P2 promoter of HNF4alpha. Thus, cyclosporine caused inhibition of DNA binding of two important regulators for insulin signaling, i.e. NFAT and HNF4alpha. We further investigated HNF4alpha transcript expression and observed >200-fold differences in abundance in n = 14 patients. Such variability in expression might help to identify individuals at risk for developing PTDM. We propose cyclosporine to repress HNF4alpha gene and protein expression, DNA-binding to targeted promoters and subsequent regulation of genes coding for glucose metabolism and of pancreatic beta-cell function.

Background

Unravelling the serum proteome is the subject of intensified research. In this regard, two-dimensional electrophoresis coupled with MALDI MS analysis is still one of the most commonly used method. Despite some improvements, there is the need for better protocols to enable comprehensive identification of serum proteins.

Here we report a combination of two proteomic strategies, zoom in acidic and neutral part of 2-D gels and an application of two optimised matrix preparations for MALDI-MS analyses to simplify serum proteome mapping.

Results

Mouse serum proteins were separated by 2-D electrophoresis at the pH ranges 3–10 and 4–7, respectively. Then in gel tryptic digests were analysed by MALDI-MS. Notably, sample-matrix preparations consisted of either a thin-layer α-ciano-4-hydroxycinnamic acid (CHCA) matrix deposition or a matrix-layer 2,5-dihydroxybenzoic acid (DHB). This enabled an identification of 90 proteins. The herein reported method enhanced identification of proteins by 32% when compared with previously published studies of mouse serum proteins, using the same approaches. Furthermore, experimental improvements of matrix preparations enabled automatic identification of mouse proteins, even when one of the two matrices failed.

Conclusion

We report a simple and reliable protocol for serum proteome analysis that combines an optimized resolution of 2-D gels spots and improved sample-matrix preparations for MALDI-MS analysis. The protocol allowed automated data acquisition for both CHCA and DHB and simplified the MS data acquisition therefore avoiding time-consuming procedures. The simplicity and reliability of the developed protocol may be applied universally.

Background

The phenomenon of co-localization of transcriptionally upregulated genes showing similar expression levels is known across all eukaryotic genomes. We recently mapped the Aroclor 1254-regulated transcriptome back onto the genome and provided evidence for the statistically significant co-localization of regulated genes. They did, however, not always show similar expression levels, and many of the regulated genes were, in fact, repressed.

Results

In this study, we were able to reproduce this observation with microarray data stemming from 1) human hepatocytes treated with the gyrase and potential topoisomerase II inhibitor trovafloxacin, 2) human hepatocytes treated with the topoisomerase II inhibitor doxorubicin and 3) mouse lymphoma cells treated with the topoisomerase II inhibitor etoposide. We found statistically significant co-localization of regulated gene pairs – induced and repressed – within the window size of 0–100 kbp. Notably, by using microarray data stemming from lung tissue of a mouse transgenic line overexpressing the transcription factor c-myc, which served as a negative control, we found regulated genes to be located with regard to each other nearly in the same way as genes distributed randomly all over the genome (0–100 kbp).

Conclusion

We suggest topoisomerase II inhibition by Aroclor 1254, trovafloxacin, doxorubicin, and etoposide to be responsible for significant co-localization of regulated genes through the inability of the stabilized enzyme complexes to religate DNA. Within the permanently opened chromatin domains, neighbored genes might be allowed to be regulated. Overexpression of c-myc, however, does not inhibit topoisomerase II activity. Consequently, the enzyme is able to perform its normal function of transiently breaking and rejoining the DNA double strand. As a result, exclusively target genes are regulated.

Background

The transcriptional regulator c-Myc is the most frequently deregulated oncogene in human tumors. Targeted overexpression of this gene in mice results in distinct types of lung adenocarcinomas. By using microarray technology, alterations in the expression of genes were captured based on a female transgenic mouse model in which, indeed, c-Myc overexpression in alveolar epithelium results in the development of bronchiolo-alveolar carcinoma (BAC) and papillary adenocarcinoma (PLAC). In this study, we analyzed exclusively the promoters of induced genes by different in silico methods in order to elucidate the c-Myc transcriptional regulatory network.

Results

We analyzed the promoters of 361 transcriptionally induced genes with respect to c-Myc binding sites and found 110 putative binding sites in 94 promoters. Furthermore, we analyzed the flanking sequences (+/- 100 bp) around the 110 c-Myc binding sites and found Ap2, Zf5, Zic3, and E2f binding sites to be overrepresented in these regions. Then, we analyzed the promoters of 361 induced genes with respect to binding sites of other transcription factors (TFs) which were upregulated by c-Myc overexpression. We identified at least one binding site of at least one of these TFs in 220 promoters, thus elucidating a potential transcription factor network. The analysis correlated well with the significant overexpression of the TFs Atf2, Foxf1a, Smad4, Sox4, Sp3 and Stat5a. Finally, we analyzed promoters of regulated genes which where apparently not regulated by c-Myc or other c-Myc targeted TFs and identified overrepresented Oct1, Mzf1, Ppargamma, Plzf, Ets, and HmgIY binding sites when compared against control promoter background.

Conclusion

Our in silico data suggest a model of a transcriptional regulatory network in which different TFs act in concert upon c-Myc overexpression. We determined molecular rules for transcriptional regulation to explain, in part, the carcinogenic effect seen in mice overexpressing the c-Myc oncogene.

An application of machine learning algorithms enables prediction of the functional context of transcription factor binding sites in the human genome.

We report an application of machine learning algorithms that enables prediction of the functional context of transcription factor binding sites in the human genome. We demonstrate that our method allowed de novo identification of hepatic nuclear factor (HNF)4α binding sites and significantly improved an overall recognition of faithful HNF4α targets. When applied to published findings, an unprecedented high number of false positives were identified. The technique can be applied to any transcription factor.

Background

Cytochrome P450 monooxygenases (CYP) play an important role in the defense against inhaled toxicants, and expression of CYP enzymes may differ among various lung cells and tissue compartments.

Methods

We studied the effects of tobacco smoke in volunteers and investigated gene expression of 19 CYPs and 3 flavin-containing monooxygenases, as well as isoforms of gluthathione S-transferases (GST) and uridine diphosphate glucuronosyltransferases (UGT) and the microsomal epoxide hydrolase (EPHX1) in bronchoalveolar lavage cells and bronchial biopsies derived from smokers (n = 8) and nonsmokers (n = 10). We also investigated gene expression of nuclear transcription factors known to be involved in the regulation of xenobiotic metabolism enzymes.

Results

Gene expression of CYP1A1, CYP1B1, CYP2S1, GSTP1, and EPHX1 was induced in bronchoalveolar lavage cells of smokers, whereas expression of CYP2B6/7, CYP3A5, and UGT2A1 was repressed. In bronchial biopsies of smokers, CYP1A1, CYP1B1, CYP2C9, GSTP1, and GSTA2 were induced, but CYP2J2 and EPHX1 were repressed. Induction of CYP1A1 and CYP1B1 transcript abundance resulted in increased activity of the coded enzyme. Finally, expression of the liver X receptor and the glucocorticoid receptor was significantly up-regulated in bronchoalveolar lavage cells of smokers.

Conclusions

We found gene expression of pulmonary xenobiotic metabolizing enzymes and certain key transcription factors to be regulated in bronchoalveolar lavage cells and bronchial biopsies of smokers. The observed changes demonstrate tissue specificity in xenobiotic metabolism, with likely implications for the metabolic activation of procarcinogens to ultimate carcinogens of tobacco smoke.

Background

Aroclor 1254 is a well-known hepatotoxin and consists of a complex mixture of polychlorinated biphenyls (PCBs), some of which have the ability to activate the aryl hydrocarbon receptor (AhR) and other transcription factors (TFs). Altered transcription factor expression enables activation of promoters of many genes, thereby inducing a regulatory gene network. In the past, computational approaches were not applied to understand the combinatorial interplay of TFs acting in concert after treatment of human hepatocyte cultures with Aroclor 1254. We were particularly interested in interrogating promoters for transcription factor binding sites of regulated genes.

Results

Here, we present a framework for studying a gene regulatory network and the large-scale regulation of transcription on the level of chromatin structure. For that purpose, we employed cDNA and oligomicroarrays to investigate transcript signatures in human hepatocyte cultures treated with Aroclor 1254 and found 910 genes to be regulated, 52 of which code for TFs and 47 of which are involved in cell cycle and apoptosis. We identified regulatory elements proximal to AhR binding sites, and this included recognition sites for the transcription factors ETS, SP1, CREB, EGR, NF-kB, NKXH, and ZBP. Notably, ECAT and TBP binding sites were identified for Aroclor 1254-induced and E2F, MAZ, HOX, and WHZ for Aroclor 1254-repressed genes. We further examined the chromosomal distribution of regulated genes and observed a statistically significant high number of gene pairs within a distance of 200 kb. Genes regulated by Aroclor 1254, are much closer located to each other than genes distributed randomly all over the genome. 37 regulated gene pairs are even found to be directly neighbored. Within these directly neighbored gene pairs, not all genes were bona fide targets for AhR (primary effect). Upon further analyses many were targets for other transcription factors whose expression was regulated by Aroclor 1254 (secondary effect).

Conclusion

We observed coordinate events in transcript regulation upon treatment of human hepatocytes with Aroclor 1254 and identified a regulatory gene network of different TFs acting in concert. We determined molecular rules for transcriptional regulation to explain, in part, the pleiotropic effect seen in animals and humans upon exposure to Aroclor 1254.