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Complex modulation of androgen responsive gene expression by methoxyacetic acid

ABSTRACT: Optimal androgen signaling is critical for testicular development and spermatogenesis. Methoxyacetic acid (MAA), the primary active metabolite of the industrial chemical ethylene glycol monomethyl ether, disrupts spermatogenesis and causes testicular atrophy. Transcriptional trans-activation studies have indicated that MAA can enhance androgen receptor activity, however, whether MAA actually impacts the expression of androgen-responsive genes in vivo, and which genes might be affected is not known. A mouse TM3 Leydig cell line that stably expresses androgen receptor (TM3-AR) was prepared and analyzed by transcriptional profiling to identify target gene interactions between MAA and testosterone on a global scale. MAA is shown to have widespread effects on androgen-responsive genes, affecting processes ranging from apoptosis to ion transport, cell adhesion, phosphorylation and transcription, with MAA able to enhance, as well as antagonize, androgenic responses. Moreover, testosterone is shown to exert both positive and negative effects on MAA gene responses. Motif analysis indicated that binding sites for FOX, HOX, LEF/TCF, STAT5 and MEF2 family transcription factors are among the most highly enriched in genes regulated by testosterone and MAA. Notably, 65 FOXO targets were repressed by testosterone or showed repression enhanced by MAA when combined with testosterone; these include 16 genes associated with developmental processes, six of which are Hox genes. These findings highlight the complex interactions between testosterone and MAA, and provide insight into the effects of MAA exposure on androgen-dependent processes in a Leydig cell model. TM3-AR cells (see below) were grown in DMEM-F12 medium containing 5% horse serum and 2.5% FBS. LNCaP cells were maintained in RPMI 1640 containing 10% FBS. RNA was isolated using TRIzol reagent using the manufacturer’s protocol.

ORGANISM(S): Mus musculus

SUBMITTER: David Waxman

PROVIDER: E-GEOD-27410 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Complex modulation of androgen responsive gene expression by methoxyacetic acid.

Bagchi Gargi G Zhang Yijing Y Stanley Kerri A KA Waxman David J DJ

Reproductive biology and endocrinology : RB&E 20110331

<h4>Background</h4>Optimal androgen signaling is critical for testicular development and spermatogenesis. Methoxyacetic acid (MAA), the primary active metabolite of the industrial chemical ethylene glycol monomethyl ether, disrupts spermatogenesis and causes testicular atrophy. Transcriptional trans-activation studies have indicated that MAA can enhance androgen receptor activity, however, whether MAA actually impacts the expression of androgen-responsive genes in vivo, and which genes might be ...[more]

PMID: 21453523

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Project description:Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, an established testicular toxicant. MAA induces the degradation of testicular germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and global gene expression was monitored by microarray analysis. A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes and 60 DNA-binding proteins that responded to MAA rapidly but transiently, and which may contribute to the downstream effects of MAA seen for large numbers of mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. These findings on the progressive changes in gene expression induced by MAA in Leydig cells may help elucidate the signaling pathways perturbed by this testicular toxicant and explain its mechanism of toxicity at the gene level. Mouse TM3 Leydig cells (American Type Culture Collection, Manassas, VA) were grown in DMEM-F12 medium containing 5% horse serum and 2.5% FBS. Cells were grown to ~60% confluence and treated with culture medium alone, or with culture medium containing 1 mM or 5 mM MAA for either 3, 8 or 24 h. Total RNA was then isolated using TRIzol reagent, followed by incubation with RQ1 RNAse-free DNAse for 1 h at 37Â°C and then heating at 75Â°C for 5 min using the manufacturerâs protocol. A total of 6 cultures of TM3 cells were independently treated with MAA under each of the 6 treatment conditions specified above (i.e., 1 mM or 5 mM MAA for either 3, 8 or 24 h), and the corresponding 6 sets of RNA samples were validated by RNA integrity analysis (Agilent Bioanalyzer). Each RNA sample was also validated by qPCR analysis using SYBR Green I-based chemistry and primers specific for 3 genes known to respond to MAA (Cyp17a1, Shbg, and Igfbp3) to verify consistency of the MAA responses. The 6 RNA samples were then used to prepare two independent pools (n=3 RNA samples each) for microarray analysis with dye swaps. Sample labeling, hybridization to microarrays, scanning and calculation of normalized expression ratios were carried out at the Wayne State University Institute of Environmental Health Sciences microarray facility using Alexa 555 and Alexa 647 aminoallyl-aRNA samples

Project description:The role of androgen in breast cancer development is not fully understood although androgen receptors (AR) have been identified in breast cancer clinical samples and cell lines. However the whole spectra of androgen actions cannot be accounted to the classic AR mode of action and the possible existence of a cell surface AR has been suggested. Indeed androgens like all steroids have been reported to trigger membrane initiated signaling activity and exert specific actions. Androgens acting on the membrane can rapidly activate kinase signaling pathways and ultimately could affect gene expression. However, the molecular nature of membrane androgen binding sites represents another major persisting question. In the present study, we investigated early transcriptional effects of testosterone and the impermeable testosterone-BSA conjugate, in two breast cancer cell lines, in an attempt to decipher specific genes modified in each case, providing evidences about specific membrane initiating actions. Our data indicate that the two agents tested affect the expression of several genes. A group of genes were commonly affected while others were uniquely modified by each agent. In MDA-MB-231 cells, that are AR negative, the majority of genes affected by testosterone were also affected by testosterone-BSA indicating a membrane action. Subsequent analysis revealed that the two agents trigger different molecular pathways and cellular/molecular functions, suggestive of a molecular heterogeneity of membrane and intracellular AR. In addition, the phenotypic interactions of membrane-acting androgen with growth factor were verified at the transcriptomic level. Finally an interesting interplay between membrane-acting androgen with inflammation-related molecules, with potential clinical implications was revealed. Experiment Overall Design: Cell lines and culture conditions Experiment Overall Design: The hormone-sensitive breast cancer cell line T47D (expressing ERÎ±, ERÎ² and AR) and the hormone insensitive breast cancer cell line MDA-MB-231 (expressing only ERÎ²) were purchased from the European Collection of Cell Cultures (Salisbury, UK). T47D cells and MDA-MB-231 were cultured in RPMI-1640 medium, supplemented with 10% charcoal treated fetal bovine serum (FBS), in a humidified atmosphere of 5% CO2 in air, at 37Â°C. Culture media and serum were from Gibco BRL (Life Technologies, Paisley, UK). Experiment Overall Design: Treatment- Gene expression assay Experiment Overall Design: Cells were serum starved for 6 hours and then treated with 10-7M Testosterone or charcoal-treated Testosterone-BSA, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, DÃ¼ren, Germany), labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.

Project description:A series of two color gene expression profiles obtained using Agilent 44K expression microarrays was used to examine sex-dependent and growth hormone-dependent differences in gene expression in rat liver. This series is comprised of pools of RNA prepared from untreated male and female rat liver, hypophysectomized (‘Hypox’) male and female rat liver, and from livers of Hypox male rats treated with either a single injection of growth hormone and then killed 30, 60, or 90 min later, or from livers of Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart and killed 30 min after the second injection. The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. A comparison of untreated male liver and untreated female liver liver gene expression profiles showed that of the genes that showed significant expression differences in at least one of the 6 data sets, 25% were sex-specific. Moreover, sex specificity was lost for 88% of the male-specific genes and 94% of the female-specific genes following hypophysectomy. 25-31% of the sex-specific genes whose expression is altered by hypophysectomy responded to short-term growth hormone treatment in hypox male liver. 18-19% of the sex-specific genes whose expression decreased following hypophysectomy were up-regulated after either one or two growth hormone injections. Finally, growth hormone suppressed 24-36% of the sex-specific genes whose expression was up-regulated following hypophysectomy, indicating that growth hormone acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression. For full details, see V. Wauthier and D.J. Waxman, Molecular Endocrinology (2008) This series is comprised of pools of liver RNA prepared from untreated male, hypophysectomized (‘Hypox’) male, untreated female and Hypox female rats (3-4 livers/pool), as well as liver RNA prepared from Hypox male rats treated with a single growth hormone injection and killed either 30, 60, or 90 minutes later (pool of n = 4 livers) or from Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart (pool of n = 5 livers). The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. Dye swapping experiments were carried out for each of the six hybridization experiments, as follows. The Alexa 555-labeled cDNA from one of the two untreated male pools was mixed with the Alexa 647-labeled cDNA from one of the two untreated female pools. Similarly, Alexa 647-labeled cDNA from the second untreated male pool was mixed with the Alexa 555-labeled cDNA from the second untreated female pool. Together, these two mixed cDNA samples comprise a fluorescent reverse pair (dye swap). Dye swaps were similarly carried out for each of the five other competitive hybridization experiments, except that for experiments 5 and 6, a single pool of M-Hypox + GH liver cDNA, or a single pool of M-Hypox + 2GH liver cDNA, was used in each half of the fluorescent reverse pair. Two microarrays, one for each mixed cDNA sample, were hybridized for each of the six fluorescent reverse pairs, giving a total of 12 microarrays.

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Complex modulation of androgen responsive gene expression by methoxyacetic acid

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Complex modulation of androgen responsive gene expression by methoxyacetic acid.

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