Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor.

Glucocorticoids are widely used in the treatment of inflammatory and other diseases. However, high-dose or chronic administration often triggers troublesome side effects such as metabolic syndrome and osteoporosis. We recently described that one glucocorticoid receptor gene produces eight translational glucocorticoid receptor isoforms that have distinct gene-regulatory abilities. We show here that specific, but not all, glucocorticoid receptor isoforms induced apoptosis in human osteosarcoma U-2 OS bone cells. Whole human genome microarray analysis revealed that the majority of the glucocorticoid target genes were selectively regulated by specific glucocorticoid receptor isoforms. Real-time PCR experiments confirmed that proapoptotic enzymes necessary for cell death, granzyme A and caspase-6, were induced by specific glucocorticoid receptor isoforms. Chromatin immunoprecipitation assays further suggested that glucocorticoid receptor isoform-dependent induction of proapoptotic genes was likely due to selective coregulator recruitment and chromatin modification. Interestingly, the capabilities to transrepress proinflammatory genes were similar among glucocorticoid receptor isoforms. Together, these findings provide new evidence that translational glucocorticoid receptor isoforms can elicit distinct glucocorticoid responses and may be useful for the development of safe glucocorticoids with reduced side effects.

Transcriptomic analysis of pathways regulated by toll-like receptor 4 in a murine model of chronic pulmonary inflammation and carcinogenesis.

BACKGROUND: Therapeutic strategies exist for human pulmonary neoplasia, however due to the heterogeneity of the disease, most are not very effective. The innate immunity gene, toll-like receptor 4 (TLR4), protects against chronic pulmonary inflammation and tumorigenesis in mice, but the mechanism is unclear. This study was designed to identify TLR4-mediated gene expression pathways that may be used as prognostic indicators of susceptibility to lung tumorigenesis in mice and provide insight into the mechanism. METHODS: Whole lung mRNA was isolated from C.C3H-Tlr4(Lps-d) (BALB(Lps-d); Tlr4 mutant) and BALB/c (Tlr4 normal) mice following butylated hydroxytoluene (BHT)-treatment (four weekly ip. injections; 150-200 mg/kg/each; "promotion"). mRNA from micro-dissected tumors (adenomas) and adjacent uninvolved tissue from both strains were also compared 27 wks after a single carcinogen injection (3-methylcholanthrene (MCA), 10 microg/g; "control") or followed by BHT (6 weekly ip. injections; 125-200 mg/kg/each; "progression"). Bronchoalveolar lavage fluid was analyzed for inflammatory cell content and total protein determination, a marker of lung hyperpermeability; inflammation was also assessed using immunohistochemical staining for macrophages (F4/80) and lymphocytes (CD3) in mice bearing tumors (progression). RESULTS: During promotion, the majority of genes identified in the BALB(Lps-d) compared to BALB/c mice (P < 0.05) were involved in epithelial growth factor receptor (EGFR) signaling (e.g. epiregulin (Ereg)), secreted phosphoprotein 1(Spp1)), which can lead to cell growth and eventual tumor development. Inflammation was significantly higher in BALB(Lps-d) compared to BALB/c mice during progression, similar to the observed response during tumor promotion in these strains. Increases in genes involved in signaling through the EGFR pathway (e.g. Ereg, Spp1) were also observed during progression in addition to continued inflammation, chemotactic, and immune response gene expression in the BALB(Lps-d) versus BALB/c mice (P < 0.05), which appears to provide more favorable conditions for cell growth and tumor development. In support of these findings, the BALB/c mice also had significantly reduced expression of many immune response and inflammatory genes in both the tumors and uninvolved tissue. CONCLUSION: This transcriptomic study determined the protective effect of TLR4 in lung carcinogenesis inhibition of multiple pathways including EGFR (e.g. Ereg), inflammatory response genes (e.g. Cxcl5), chemotaxis (e.g. Ccr1) and other cell proliferation genes (e.g. Arg1, Pthlh). Future studies will determine the utility of these pathways as indicators of immune system deficiencies and tumorigenesis.

Profile of estrogen-responsive genes in an estrogen-specific mammary gland outgrowth model.

Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify estrogen-responsive genes associated with pubertal ductal growth in the mouse mammary gland in the absence of other ovarian hormones and at different stages of development. We hypothesized that the estrogen-induced genes and their associated functions at early stages of ductal elongation would be distinct from those induced after significant ductal elongation had occurred. Therefore, ovariectomized prepubertal mice were exposed to 17beta-estradiol from two to 28 days, and mammary gland global gene expression analyzed by microarray analysis at various times during this period. We found that: (a) gene expression changes in our estrogen-only model mimic those changes that occur in normal pubertal development in intact mice, (b) both distinct and overlapping gene profiles were observed at varying extents of ductal elongation, and (c) cell proliferation, the immune response, and metabolism/catabolism were the most common functional categories associated with mammary ductal growth. Particularly striking was the novel observation that genes active during carbohydrate metabolism were rapidly and robustly decreased in response to estradiol. Lastly, we identified mammary estradiol-responsive genes that are also co-expressed with estrogen receptor alpha in human breast cancer. In conclusion, our genomic data support the physiological observation that estradiol is one of the primary hormonal signals driving ductal elongation during pubertal mammary development.

Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts.

Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice and cells derived from them showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target mRNAs for TTP include tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-2beta. Here we used microarray analysis of RNA from wild-type and TTP-deficient fibroblast cell lines to identify transcripts with different decay rates, after serum stimulation and actinomycin D treatment. Of 250 mRNAs apparently stabilized in the absence of TTP, 23 contained two or more conserved TTP binding sites; nine of these appeared to be stabilized on Northern blots. The most dramatically affected transcript encoded the protein Ier3, recently implicated in the physiological control of blood pressure. The Ier3 transcript contained several conserved TTP binding sites that could bind TTP directly and conferred TTP sensitivity to the mRNA in cell transfection studies. These studies have identified several new, physiologically relevant TTP target transcripts in fibroblasts; these target mRNAs encode proteins from a variety of functional classes.

Farnesol-induced apoptosis in human lung carcinoma cells is coupled to the endoplasmic reticulum stress response.

Farnesol (FOH) and other isoprenoid alcohols induce apoptosis in various carcinoma cells and inhibit tumorigenesis in several in vivo models. However, the mechanisms by which they mediate their effects are not yet fully understood. In this study, we show that FOH is an effective inducer of apoptosis in several lung carcinoma cells, including H460. This induction is associated with activation of several caspases and cleavage of poly(ADP-ribose) polymerase (PARP). To obtain insight into the mechanism involved in FOH-induced apoptosis, we compared the gene expression profiles of FOH-treated and control H460 cells by microarray analysis. This analysis revealed that many genes implicated in endoplasmic reticulum (ER) stress signaling, including ATF3, DDIT3, HERPUD1, HSPA5, XBP1, PDIA4, and PHLDA1, were highly up-regulated within 4 h of FOH treatment, suggesting that FOH-induced apoptosis involves an ER stress response. This was supported by observations showing that treatment with FOH induces splicing of XBP1 mRNA and phosphorylation of eIF2alpha. FOH induces activation of several mitogen-activated protein kinase (MAPK) pathways, including p38, MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK, and c-jun NH2-terminal kinase (JNK). Inhibition of MEK1/2 by U0126 inhibited the induction of ER stress response genes. In addition, knockdown of the MEK1/2 and JNK1/2 expression by short interfering RNA (siRNA) effectively inhibited the cleavage of caspase-3 and PARP and apoptosis induced by FOH. However, only MEK1/2 siRNAs inhibited the induction of ER stress-related genes, XBP1 mRNA splicing, and eIF2alpha phosphorylation. Our results show that FOH-induced apoptosis is coupled to ER stress and that activation of MEK1/2 is an early upstream event in the FOH-induced ER stress signaling cascade.

Genome wide transcriptional profiling in breast cancer cells reveals distinct changes in hormone receptor target genes and chromatin modifying enzymes after proteasome inhibition.

Steroid hormone receptors, like glucocorticoid (GR) and estrogen receptors (ER), are master regulators of genes that control many biological processes implicated in health and disease. Gene expression is dependent on receptor levels which are tightly regulated by the ubiquitin-proteasome system. Previous studies have shown that proteasome inhibition increases GR, but decreases ER-mediated gene expression. At the gene expression level this divergent role of the proteasome in receptor-dependent transcriptional regulation is not well understood. We have used a genomic approach to examine the impact of proteasome activity on GR- and ER-mediated gene expression in MCF-7 breast cancer cells treated with dexamethasone (DEX) or 17beta-estradiol (E2), the proteasome inhibitor MG132 (MG) or MG132 and either hormone (MD or ME2) for 24 h. Transcript profiling reveals that inhibiting proteasome activity modulates gene expression by GR and ER in a similar manner in that several GR and ER target genes are upregulated and downregulated after proteasome inhibition. In addition, proteasome inhibition modulates receptor-dependent genes involved in the etiology of a number of human pathological states, including multiple myeloma, leukemia, breast/prostate cancer, HIV/AIDS, and neurodegenerative disorders. Importantly, our analysis reveals that a number of transcripts encoding histone and DNA modifying enzymes, prominently histone/DNA methyltransferases and demethylases, are altered after proteasome inhibition. As proteasome inhibitors are currently in clinical trials as therapy for multiple myeloma, HIV/AIDS and leukemia, the possibility that some of the target molecules are hormone regulated and chromatin modifying enzymes is intriguing in this era of epigenetic therapy.

Gene expression profiling reveals a regulatory role for ROR alpha and ROR gamma in phase I and phase II metabolism.

Retinoid-related orphan receptors alpha (ROR alpha) and gamma (ROR gamma) are both expressed in liver; however, their physiological functions in this tissue have not yet been clearly defined. The ROR alpha1 and ROR gamma 1 isoforms, but not ROR alpha 4, show an oscillatory pattern of expression during circadian rhythm. To obtain insight into the physiological functions of ROR receptors in liver, we analyzed the gene expression profiles of livers from WT, ROR alpha-deficient staggerer (sg) mice (ROR alpha(sg/sg)), ROR gamma(-/-), and ROR alpha(sg/sg)ROR gamma(-/-) double knockout (DKO) mice by microarray analysis. DKO mice were generated to study functional redundancy between ROR alpha and ROR gamma. These analyses demonstrated that ROR alpha and ROR gamma affect the expression of a number of genes. ROR alpha and ROR gamma are particularly important in the regulation of genes encoding several phase I and phase II metabolic enzymes, including several 3beta-hydroxysteroid dehydrogenases, cytochrome P450 enzymes, and sulfotransferases. In addition, our results indicate that ROR alpha and ROR gamma each affect the expression of a specific set of genes but also exhibit functional redundancy. Our study shows that ROR alpha and ROR gamma receptors influence the regulation of several metabolic pathways, including those involved in the metabolism of steroids, bile acids, and xenobiotics, suggesting that RORs are important in the control of metabolic homeostasis.

NABP1, a novel RORgamma-regulated gene encoding a single-stranded nucleic-acid-binding protein.

RORgamma2 (retinoid-related orphan receptor gamma2) plays a critical role in the regulation of thymopoiesis. Microarray analysis was performed in order to uncover differences in gene expression between thymocytes of wild-type and RORgamma-/- mice. This analysis identified a novel gene encoding a 22 kDa protein, referred to as NABP1 (nucleic-acid-binding protein 1). This subsequently led to the identification of an additional protein, closely related to NABP1, designated NABP2. Both proteins contain an OB (oligonucleotide/oligosaccharide binding) motif at their N-terminus. This motif is highly conserved between the two proteins. NABP1 is highly expressed in the thymus of wild-type mice and is greatly suppressed in RORgamma-/- mice. During thymopoiesis, NABP1 mRNA expression is restricted to CD4+CD8+ thymocytes, an expression pattern similar to that observed for RORgamma2. These observations appear to suggest that NABP1 expression is regulated either directly or indirectly by RORgamma2. Confocal microscopic analysis showed that the NABP1 protein localizes to the nucleus. Analysis of nuclear proteins by size-exclusion chromatography indicated that NABP1 is part of a high molecular-mass protein complex. Since the OB-fold is frequently involved in the recognition of nucleic acids, the interaction of NABP1 with various nucleic acids was examined. Our results demonstrate that NABP1 binds single-stranded nucleic acids, but not double-stranded DNA, suggesting that it functions as a single-stranded nucleic acid binding protein.

Estren behaves as a weak estrogen rather than a nongenomic selective activator in the mouse uterus.

A proposed membrane-mediated mechanism of rapid nongenomic response to estrogen has been the intense focus of recent research. Estren, a synthetic steroid, is reported to act selectively through a rapid membrane-mediated pathway, rather than through the classical nuclear estrogen receptor (ER)-mediated pathway, to maintain bone density in ovariectomized mice without uterotropic effects. To evaluate the mechanism and physiological effects of estren, we studied responses in adult ovariectomized mice. In a 3-d uterine bioassay, we found that 300 microg estren significantly increased uterine weight; in comparison, a more maximal response was seen with 1 mug estradiol (E2). The estren response was partly ERalpha independent, because ERalpha knockout (alphaERKO) uteri also exhibited a more moderate weight increase. Estren induced epithelial cell proliferation in wild-type, but not alphaERKO, mice, indicating ERalpha dependence of the epithelial growth response. Examination of estren-regulated uterine genes by microarray indicated that early (2 h) changes in gene expression are similar to the early responses to E2. These gene responses are ERalpha dependent, because they are not seen in alphaERKO mice. Later estren-induced changes in gene expression (24 h) are blunted compared with those seen 24 h after E2. In contrast to early genes, these later estren responses are independent of ERalpha, because the alphaERKO shows a similar response to estren at 24 h. We found that E2 or estren treatments lead to depletion of ERalpha in the uterine cytosol fraction and accumulation in the nuclear fraction within 30-60 min, consistent with the ability of estren to regulate genes through a nuclear ERalpha rather than a nongenomic mechanism. Interestingly, estren, but not E2, induces accumulation of androgen receptor (AR) in the nuclear fraction of both wild-type and alphaERKO samples, suggesting that AR might be involved in the later ERalpha-independent genomic responses to estren. In conclusion, our studies suggest that estren is weakly estrogenic in the mouse uterus and might induce nuclear ERalpha- and AR-mediated responses. Given its activity in our uterine model, the use of estren as a bone-selective clinical compound needs to be reconsidered.

Global gene expression associated with hepatocarcinogenesis in adult male mice induced by in utero arsenic exposure.

Our previous work has shown that exposure to inorganic arsenic in utero produces hepatocellular carcinoma (HCC) in adult male mice. To explore further the molecular mechanisms of transplacental arsenic hepatocarcinogenesis, we conducted a second arsenic transplacental carcinogenesis study and used a genomewide microarray to profile arsenic-induced aberrant gene expression more extensively. Briefly, pregnant C3H mice were given drinking water containing 85 ppm arsenic as sodium arsenite or unaltered water from days 8 to 18 of gestation. The incidence of HCC in adult male offspring was increased 4-fold and tumor multiplicity 3-fold after transplacental arsenic exposure. Samples of normal liver and liver tumors were taken at autopsy for genomic analysis. Arsenic exposure in utero resulted in significant alterations (p < 0.001) in the expression of 2,010 genes in arsenic-exposed liver samples and in the expression of 2,540 genes in arsenic-induced HCC. Ingenuity Pathway Analysis revealed that significant alterations in gene expression occurred in a number of biological networks, and Myc plays a critical role in one of the primary networks. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis of selected genes/proteins showed > 90% concordance. Arsenic-altered gene expression included activation of oncogenes and HCC biomarkers, and increased expression of cell proliferation-related genes, stress proteins, and insulin-like growth factors and genes involved in cell-cell communications. Liver feminization was evidenced by increased expression of estrogen-linked genes and altered expression of genes that encode gender-related metabolic enzymes. These novel findings are in agreement with the biology and histology of arsenic-induced HCC, thereby indicating that multiple genetic events are associated with transplacental arsenic hepatocarcinogenesis.

Global uterine genomics in vivo: microarray evaluation of the estrogen receptor alpha-growth factor cross-talk mechanism.

Cross-talk between growth factor receptors and the estrogen receptor (ER) has been proposed as a signaling mechanism in estrogen target tissues, with ER(alpha) as a direct target of growth factor receptor-activated signals, leading to regulation of estrogen target genes and estrogen-like biological responses to growth factors. We evaluated whether global genomic changes in the mouse uterus in response to epidermal growth factor or IGF-I mimic those of estradiol (E2), reflecting the cross-talk mechanism. Overlapping responses to growth factors and E2 were expected in the wild type (WT) whereas no response was expected in mice lacking ER(alpha) (ER(alpha) knockout). Surprisingly, although most of the E2 response in the WT also occurred after growth factor treatment, some genes were induced only by E2. Second, although E2 did not induce gene changes in the ER(alpha) knockout, the growth factor response was almost indistinguishable from that of the WT. Differences in response of some genes to IGF-I or epidermal growth factor indicated selective regulation mechanisms, such as phosphatidylinositol 3-kinase or MAPK-dependent responses. The robust ER(alpha)-independent genomic response to growth factor observed here is surprising considering that the biological growth response is ER(alpha) dependent. We propose two mechanisms as alternatives to the cross-talk mechanism for uterine gene regulation. First, E2 increases uterine growth factors, which activate downstream signaling cascades, resulting in gene regulation. Second, growth factors and estrogen regulate similar genes. Our results suggest that the estrogen response in the uterus involves E2-specific ER(alpha)-mediated responses as well as responses resulting from convergence of growth factor and ER-initiated activities.

Functional genomic characterization of delipidation elicited by trans-10, cis-12-conjugated linoleic acid (t10c12-CLA) in a polygenic obese line of mice.

Gene expression was measured during t10c12-CLA-induced body fat reduction in a polygenic obese line of mice. Adult mice (n = 185) were allotted to a 2 x 2 factorial experiment consisting of either nonobese (ICR-control) or obese (M16-selected) mice fed a 7% fat, purified diet containing either 1% linoleic acid (LA) or 1% t10c12-CLA. Body weight (BW) by day 14 was 12% lower in CLA- compared with LA-fed mice (P < 0.0001). By day 14, t10c12-CLA reduced weights of epididymal, mesenteric, and brown adipose tissues, as a percentage of BW, in both lines by 30, 27, and 58%, respectively, and increased liver weight/BW by 34% (P < 0.0001). Total RNA was isolated and pooled (4 pools per tissue per day) from epididymal adipose (days 5 and 14) of the obese mice to analyze gene expression profiles using Agilent mouse oligo microarray slides representing > 20,000 genes. Numbers of genes differentially expressed by greater than or equal to twofold in epididymal adipose (days 5 and 14) were 29 and 125, respectively. It was concluded that, in adipose tissue, CLA increased expression of uncoupling proteins (1 and 2), carnitine palmitoyltransferase system, tumor necrosis factor-alpha (P < 0.05), and caspase-3 but decreased expression of peroxisome proliferator-activated receptor-gamma, glucose transporter-4, perilipin, caveolin-1, adiponectin, resistin, and Bcl-2 (P < 0.01). In conclusion, this experiment has revealed candidate genes that will be useful in elucidating mechanisms of adipose delipidation.

A qualitative assessment of direct-labeled cDNA products prior to microarray analysis.

BACKGROUND: The success of the microarray process in determining differential gene expression of thousands of genes is dependent upon the quality and integrity of the starting RNA, this being particularly true of direct labeling via a reverse transcription procedure. Furthermore, an RNA of reasonable quality still may not yield reliable hybridization data if the labeling efficiency was poor. RESULTS: Here we present a novel assay for assessing the quality of directly labeled fluorescent cDNA prior to microarray hybridization utilizing the Agilent 2100 Bioanalyzer, which employs microfluidic technology for the analysis of nucleic acids and proteins. Using varying amounts of RNase to simulate RNA degradation, we show the strength of this un-advertised assay in determining the relative amounts of cDNA obtained from a direct labeling reaction. CONCLUSION: Utilization of this method in the lab will help to prevent the costly mistake of hybridizing poor quality direct labeled products to expensive arrays.

Dexamethasone blocks the rapid biological effects of 17beta-estradiol in the rat uterus without antagonizing its global genomic actions.

Estrogens and glucocorticoids have opposing effects on the female reproductive tract, but the molecular basis for this antagonism is poorly understood. We therefore examined the biological and transcriptional programs induced by estrogens and glucocorticoids in the uterus of immature female rats. Estradiol 17beta (E2) rapidly induced morphological changes reminiscent of an acute inflammatory response, including infiltration of eosinophils, edema in the stroma and myometrium, and a decrease in the height of luminal epithelial cells, whereas dexamethasone (Dex) only altered stromal cell morphology. When coadministered with E2, Dex completely blocked the proinflammatory effects of E2. Surprisingly, examination of E2 and Dex effects on gene expression using cDNA microarrays and real-time PCR revealed that these hormones had similar effects on the expression of many genes and that very few genes displayed antagonistic regulation. Together, these results indicate strong discord between the early biologic and genomic actions of estrogens and glucocorticoids and highlight a complex regulatory role for glucocorticoids and GR in the mammalian uterus.

Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen.

The physiological responses of the rodent uterus to acute estrogen (E) dosing can be divided into early and late events. Examples of early responses include increased RNA transcription, hyperemia, and water imbibition 2 and 6 h following E administration respectively, whereas later responses include cycles of DNA synthesis and mitosis of epithelial cells beginning 10 and 16 h after E. The development of estrogen receptor (ER) knockout (ERKO) mice, combined with microarray technology, has allowed us to design a genomic approach to study the acute response of the rodent reproductive tract to E. To determine whether early and late biological responses are correlated with altered regulation of a single set of genes or distinct sets of genes characteristic of early and late responses, uterine RNA was obtained from ovariectomized mice that were treated with vehicle or with estradiol for 2 h (early) or 24 h (late). Samples were also prepared from identically treated mice that lacked either ERalpha (alphaERKO) or ERbeta (betaERKO) to address the relative contributions of the ERs in the uterine responses. Microarray analysis of the relative expression of 8700 mouse cDNAs indicated distinct clusters of genes that were regulated both positively and negatively by E in the early or late phases as well as clusters of genes regulated at both times. Both early and late responses by the betaERKO samples were indistinguishable from those of WT samples, whereas the alphaERKO showed little change in gene expression in response to E, indicating the predominant role for ERalpha in the genomic response. Further studies indicated that the genomic responses in samples from intermediate time points (6 h, 12 h) fall within the early or late clusters, rather than showing unique clusters regulated in the intermediary period. The use of this genomic approach has illustrated how physiological responses are reflected in genomic patterns. Furthermore, the identification of functional gene families that are regulated by E in the uterus combined with the utilization of genetically altered experimental animal models can help to uncover and define novel mechanisms of E action.

Identification of putative gene based markers of renal toxicity.

This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants--cisplatin, gentamicin, and puromycin--to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg). Groups of rats were sacrificed at various times after administration of these compounds for standard clinical chemistry, urine analysis, and histological evaluation of the kidney. RNA was extracted from the kidney for microarray analysis. Principal component analysis and gene expression-based clustering of compound effects confirmed sample separation based on dose, time, and degree of renal toxicity. In addition, analysis of the profile components revealed some novel changes in the expression of genes that appeared to be associated with injury in specific portions of the nephron and reflected the mechanism of action of these various nephrotoxicants. For example, although puromycin is thought to specifically promote injury of the podocytes in the glomerulus, the changes in gene expression after chronic exposure of this compound suggested a pattern similar to the known proximal tubular nephrotoxicants cisplatin and gentamicin; this prediction was confirmed histologically. We conclude that renal gene expression profiling coupled with analysis of classical end points affords promising opportunities to reveal potential new mechanistic markers of renal toxicity.

Platforms for biomarker analysis using high-throughput approaches in genomics, transcriptomics, proteomics, metabolomics, and bioinformatics.

Global biological responses that reflect disease or exposure biology are kinetic and highly dynamic phenomena. While high-throughput DNA sequencing continues to drive genomics, the possibility of more broadly measuring changes in gene expression has been a recent development manifested by a diversity of technical platforms. Such technologies measure transcripts, proteins and small biological molecules, or metabolites, and respectively define the fields of transcriptomics, proteomics and metabolomics that can be performed at a cell-, tissue-, or organism-wide basis. Bioinformatics is the discipline that derives knowledge from the large quantity and diversity of biological, genetic, genomic and gene expression data by integrating computer science, mathematics, statistics and graphic arts. Gene, protein and metabolite expression profiles can be thought of as snapshots of the current, poorly-mapped molecular landscape. The ultimate aim of genomic platforms is to fully map this landscape to more completely describe all of the biological interactions within a living system, during disease and toxicity, and define the behaviour and relationships of all the components of a biological system. The development of databases and knowledge bases will support the integration of data from multiple domains, as well as computational modelling. This chapter will describe the technical platform methods involving DNA sequencing, mass spectrometry, nuclear magnetic resonance combined with separation systems, and bioinformatics to derive genomic and gene expression data and include the relevant bioinformatic tools for analysis. These genomic, or omics platforms should have wide application to epidemiological studies.

Trans-10, cis-12-conjugated linoleic acid alters hepatic gene expression in a polygenic obese line of mice displaying hepatic lipidosis.

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) causes a rapid reduction of body and adipose mass in mice. In addition to changes in adipose tissue, numerous studies have reported alterations in hepatic lipid metabolism. Livers of CLA-fed mice gain mass, partly due to lipid accumulation; however, the precise molecular mechanisms are unknown. To elucidate these mechanisms, we examined fatty acid composition and gene expression profiles of livers from a polygenic obese line of mice fed 1% trans-10, cis-12-CLA for 14 days. Analysis of gene expression data led to the identification of 1393 genes differentially expressed in the liver of CLA-fed male mice at a nominal P value of .01, and 775 were considered significant using a false discovery rate (FDR) threshold of .05. While surprisingly few genes in lipid metabolism were impacted, pathway analysis found that protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP) pathways signaling pathways were affected by CLA treatment and 98 of the 775 genes were found to be regulated by hepatocyte nuclear factor 4alpha, a transcription factor important in controlling liver metabolic status.

Estrogen receptor beta is required for optimal cAMP production in mouse granulosa cells.

Granulosa cells of preovulatory follicles differentiate in response to FSH, and this differentiation is augmented by estradiol. We have previously shown that FSH-mediated granulosa cell differentiation requires functional estrogen receptor-beta (ERbeta) by demonstrating that the granulosa cells of ERbeta(-/-) FSH-treated mice are unable to maximally induce expression of the LH receptor (an indicator of granulosa cell differentiation) compared with ERbeta(+/+) controls. As a result, FSH-primed ERbeta(-/-) granulosa cells exhibit a reduced response to a subsequent ovulatory dose of LH. In this study, we further characterized the attenuated response of ERbeta(-/-) granulosa cells to stimulation by LH and FSH using isolated mouse granulosa cells and primary granulosa cell cultures. We observed a 50% reduction in cAMP levels in cultured ERbeta(-/-) granulosa cells exposed to LH compared with ERbeta(+/+) controls. We also observed an attenuated genomic response in granulosa cells isolated from FSH-primed ERbeta(-/-) mice compared with ERbeta(+/+) controls. Our data indicate that this attenuated response may result from inadequate levels of cAMP, because cAMP levels in cultured ERbeta(-/-) granulosa cells exposed to forskolin were approximately 50% lower than in ERbeta(+/+) granulosa cells. Phosphorylation of cAMP regulatory element binding protein, an indicator of protein kinase A activity, was also reduced in FSH-treated ERbeta(-/-) granulosa cells compared with ERbeta(+/+) controls. These are the first data to indicate that ERbeta plays a role in the induction of the cAMP pathway in mouse granulosa cells and that disruption of proper ERbeta signaling associated with this pathway may cause negative effects on ovulation and fertility.

Kruppel-like zinc finger protein Glis2 is essential for the maintenance of normal renal functions.

To obtain insight into the physiological functions of the Krüppel-like zinc finger protein Gli-similar 2 (Glis2), mice deficient in Glis2 expression were generated. Glis2 mutant (Glis2(mut)) mice exhibit significantly shorter life spans than do littermate wild-type (WT) mice due to the development of progressive chronic kidney disease with features resembling nephronophthisis. Glis2(mut) mice develop severe renal atrophy involving increased cell death and basement membrane thickening in the proximal convoluted tubules. This development is accompanied by infiltration of lymphocytic inflammatory cells and interstitial/glomerular fibrosis. The severity of the fibrosis, inflammatory infiltrates, and glomerular and tubular changes progresses with age. Blood urea nitrogen and creatinine increase, and Glis2(mut) mice develop proteinuria and ultimately die prematurely of renal failure. A comparison of the gene expression profiles of kidneys from 25-day-old/60-day-old WT and Glis2(mut) mice by microarray analysis showed increased expressions of many genes involved in immune responses/inflammation and fibrosis/tissue remodeling in kidneys of Glis2(mut) mice, including several cytokines and adhesion and extracellular matrix proteins. Our data demonstrate that a deficiency in Glis2 expression leads to tubular atrophy and progressive fibrosis, similar to nephronophthisis, that ultimately results in renal failure. Our study indicates that Glis2 plays a critical role in the maintenance of normal kidney architecture and functions.