A tumour suppressive relationship between mineralocorticoid and retinoic acid receptors activates a transcriptional program consistent with a reverse Warburg effect in breast cancer.

BACKGROUND: The role of nuclear receptors in both the aetiology and treatment of breast cancer is exemplified by the use of the oestrogen receptor (ER) as a prognostic marker and treatment target. Treatments targeting the oestrogen signalling pathway are initially highly effective for most patients. However, for the breast cancers that fail to respond, or become resistant, to current endocrine treatments, the long-term outlook is poor. ER is a member of the nuclear receptor superfamily, comprising 48 members in the human, many of which are expressed in the breast and could be used as alternative targets in cases where current treatments are ineffective. METHODS: We used sparse canonical correlation analysis to interrogate potential novel nuclear receptor expression relationships in normal breast and breast cancer. These were further explored using whole transcriptome profiling in breast cancer cells after combinations of ligand treatments. RESULTS: Using this approach, we discovered a tumour suppressive relationship between the mineralocorticoid receptor (MR) and retinoic acid receptors (RAR), in particular RARß. Expression profiling of MR expressing breast cancer cells revealed that mineralocorticoid and retinoid co-treatment activated an expression program consistent with a reverse Warburg effect and growth inhibition, which was not observed with either ligand alone. Moreover, high expression of both MR and RARB was associated with improved breast cancer-specific survival. CONCLUSION: Our study reveals a previously unknown relationship between MR and RAR in the breast, which is dependent on menopausal state and altered in malignancy. This finding identifies potential new targets for the treatment of breast cancers that are refractory to existing therapeutic options.

Distinct nuclear receptor expression in stroma adjacent to breast tumors.

The interaction between breast tumor epithelial and stromal cells is vital for initial and recurrent tumor growth. While breast cancer-associated stromal cells provide a favorable environment for proliferation and metastasis, the molecular mechanisms contributing to this process are not fully understood. Nuclear receptors (NRs) are intracellular transcription factors that directly regulate gene expression. Little is known about the status of NRs in cancer-associated stroma. Nuclear Receptor Low-Density Taqman Arrays were used to compare the gene expression profiles of all 48 NR family members in a collection of primary cultured cancer-associated fibroblasts (CAFs) obtained from estrogen receptor (ER)α positive breast cancers (n = 9) and normal breast adipose fibroblasts (NAFs) (n = 7). Thirty-three of 48 NRs were expressed in both the groups, while 11 NRs were not detected in either. Three NRs (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1); estrogen-related receptor beta (ERR-ß); and RAR-related orphan receptor beta (ROR-ß)) were only detected in NAFs, while one NR (liver receptor homolog-1 (LRH-1)) was unique to CAFs. Of the NRs co-expressed, four were significantly down-regulated in CAFs compared with NAFs (RAR-related orphan receptor-α (ROR-α); Thyroid hormone receptor-ß (TR-ß); vitamin D receptor (VDR); and peroxisome proliferator-activated receptor-γ (PPAR-γ)). Quantitative immunohistochemistry for LRH-1, TR-ß, and PPAR-γ proteins in stromal fibroblasts from an independent panel of breast cancers (ER-positive (n = 15), ER-negative (n = 15), normal (n = 14)) positively correlated with mRNA expression profiles. The differentially expressed NRs identified in tumor stroma are key mediators in aromatase regulation and subsequent estrogen production. Our findings reveal a distinct pattern of NR expression that therefore fits with a sustained and increased local estrogen microenvironment in ER-positive tumors. NRs in CAFs may provide a new avenue for the development of intratumoral-targeted therapies in breast cancer.

CARM1/PRMT4 is necessary for the glycogen gene expression programme in skeletal muscle cells.

CARM1 (co-activator-associated arginine methyltransferase 1)/PRMT4 (protein arginine methyltransferase 4), functions as a co-activator for transcription factors that are regulators of muscle fibre type and oxidative metabolism, including PGC (peroxisome-proliferator-activated receptor γ co-activator)-1α and MEF2 (myocyte enhancer factor 2). We observed significantly higher Prmt4 mRNA expression in comparison with Prmt1-Prmt6 mRNA expression in mouse muscle (in vitro and in vivo). Transfection of Prmt4 siRNA (small interfering RNA) into mouse skeletal muscle C2C12 cells attenuated PRMT4 mRNA and protein expression. We subsequently performed additional qPCR (quantitative PCR) analysis (in the context of metabolism) to examine the effect of Prmt4 siRNA expression on >200 critical genes that control (and are involved in) lipid, glucose and energy homoeostasis, and circadian rhythm. This analysis revealed a strikingly specific metabolic expression footprint, and revealed that PRMT4 is necessary for the expression of genes involved in glycogen metabolism in skeletal muscle cells. Prmt4 siRNA expression selectively suppressed the mRNAs encoding Gys1 (glycogen synthase 1), Pgam2 (muscle phosphoglycerate mutase 2) and Pygm (muscle glycogen phosphorylase). Significantly, PGAM, PYGM and GYS1 deficiency in humans causes glycogen storage diseases type X, type V/McArdle's disease and type 0 respectively. Attenuation of PRMT4 was also associated with decreased expression of the mRNAs encoding AMPK (AMP-activated protein kinase) α2/γ3 (Prkaa2 and Prkag3) and p38 MAPK (mitogen-activated protein kinase), previously implicated in Wolff-Parkinson-White syndrome and Pompe Disease (glycogen storage disease type II). Furthermore, stable transfection of two PRMT4-site-specific (methyltransferase deficient) mutants (CARM1/PRMT4 VLD and CARM1E267Q) significantly repressed the expression of Gys1, Pgam2 and AMPKγ3. Finally, in concordance, we observed increased and decreased glycogen levels in PRMT4 (native)- and VLD (methylation deficient mutant)-transfected skeletal muscle cells respectively. This demonstrated that PRMT4 expression and the associated methyltransferase activity is necessary for the gene expression programme involved in glycogen metabolism and human glycogen storage diseases.

Chicken ovalbumin upstream promoter-transcription factor II regulates nuclear receptor, myogenic, and metabolic gene expression in skeletal muscle cells.

We demonstrate that chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) mRNA is more abundantly expressed (than COUP-TFI mRNA) in skeletal muscle C2C12 cells and in (type I and II) skeletal muscle tissue from C57BL/10 mice. Consequently, we have utilized the ABI TaqMan Low Density Array (TLDA) platform to analyze gene expression changes specifically attributable to ectopic COUP-TFII (relative to vector only) expression in muscle cells. Utilizing a TLDA-based platform and 5 internal controls, we analyze the entire NR superfamily, 96 critical metabolic genes, and 48 important myogenic regulatory genes on the TLDA platform utilizing 5 internal controls. The low density arrays were analyzed by rigorous statistical analysis (with Genorm normalization, Bioconductor R, and the Empirical Bayes statistic) using the (integromics) statminer software. In addition, we validated the differentially expressed patho-physiologically relevant gene (identified on the TLDA platform) glucose transporter type 4 (Glut4). We demonstrated that COUP-TFII expression increased the steady state levels of Glut4 mRNA and protein, while ectopic expression of truncated COUP-TFII lacking helix 12 (COUP-TFΔH12) reduced Glut4 mRNA expression in C2C12 cells. Moreover, COUP-TFII expression trans-activated the Glut4 promoter (-997/+3), and ChIP analysis identified selective recruitment of COUP-TFII to a region encompassing a highly conserved SP1 binding site (in mouse, rat, and human) at nt positions -131/-118. Mutation of the SpI site ablated COUP-TFII mediated trans-activation of the Glut4 promoter. In conclusion, this study demonstrates that in skeletal muscle cells, COUP-TFII regulates several nuclear hormone receptors, and critical metabolic and muscle specific genes.

Nuclear receptor expression in human differentiated thyroid tumors.

BACKGROUND: Nuclear receptors (NRs) play a key role in endocrine signaling and metabolism and are important therapeutic targets in a number of hormone-dependent malignancies. Studies on the role of NRs in thyroid cancer are limited. OBJECTIVE: The objective of the study was to examine systematically the expression of the 48 human NRs in a series of benign and malignant thyroid tissues. Within the papillary carcinoma cohort, we sought to determine if NR expression differed significantly by BRAF mutation status. PATIENTS AND METHODS: RNA was isolated from multinodular goiter (MNG; n=6), papillary carcinoma (PTC, n=14), follicular carcinoma (FC; n=5), and Hürthle cell carcinoma (HCC; n=7). The 48 human NRs were profiled in this panel by quantitative real time polymerase chain reaction. Protein expression for selected NRs (Rev-erbα and LXR-ß) was examined by immunohistochemistry (IHC) on tissue microarrays comprising benign and malignant thyroid tissues. RESULTS: Across all groups of benign and malignant thyroid tissue, there was prominent expression of LXR-ß and ROR-γ. Key findings in PTC were marked overexpression of RXR-γ and Rev-erbα compared to MNG. Within the PTC cohort, when BRAF(V600E) tumors were compared with wild type BRAF, there was relative upregulation of RXR-γ and Rev-erbα and downregulation of AR, ERR-γ, and ROR-γ. In FC, EAR-2 was overexpressed, while PPAR-α and PPAR-δ were underexpressed compared to MNG. The NR expression profile of HCC was distinct, characterized by significant downregulation of a wide range of NRs. IHC for Rev-erbα and LXR-ß localized protein expression to the tumor cells. Moderate to strong Rev-erbα immunostaining was seen in 22 out of 23 PTC, and, overall, staining was stronger than in the benign group. CONCLUSIONS: These results represent the first systematic examination of NR expression in thyroid cancer. Our finding of tumor-specific patterns of NR expression, as well as significant differences in NR expression between BRAF(V600E) and wild type BRAF PTC, provides a basis for further mechanistic studies and highlights potential novel therapeutic targets for this malignancy.

Breast cancer prognosis predicted by nuclear receptor-coregulator networks.

Although molecular signatures based on transcript expression in breast cancer samples have provided new insights into breast cancer classification and prognosis, there are acknowledged limitations in current signatures. To provide rational, pathway-based signatures of disrupted physiology in cancer tissues that may be relevant to prognosis, this study has directly quantitated changed gene expression, between normal breast and cancer tissue, as a basis for signature development. The nuclear receptor (NR) family of transcription factors, and their coregulators, are fundamental regulators of every aspect of metazoan life, and were rigorously quantified in normal breast tissues and ERα positive and ERα negative breast cancers. Coregulator expression was highly correlated with that of selected NR in normal breast, particularly from postmenopausal women. These associations were markedly decreased in breast cancer, and the expression of the majority of coregulators was down-regulated in cancer tissues compared with normal. While in cancer the loss of NR-coregulator associations observed in normal breast was common, a small number of NR (Rev-ERBß, GR, NOR1, LRH-1 and PGR) acquired new associations with coregulators in cancer tissues. Elevated expression of these NR in cancers was associated with poorer outcome in large clinical cohorts, as well as suggesting the activation of ERα -related, but ERα-independent, pathways in ERα negative cancers. In addition, the combined expression of small numbers of NR and coregulators in breast cancer was identified as a signature predicting outcome in ERα negative breast cancer patients, not linked to proliferation and with predictive power superior to existing signatures containing many more genes. These findings highlight the power of predictive signatures derived from the quantitative determination of altered gene expression between normal breast and breast cancers. Taken together, the findings of this study identify networks of NR-coregulator associations active in normal breast but disrupted in breast cancer, and moreover provide evidence that signatures based on NR networks disrupted in cancer can provide important prognostic information in breast cancer patients.

PRMT2 and RORγ expression are associated with breast cancer survival outcomes.

Protein arginine methyltransferases (PRMTs) methylate arginine residues on histones and target transcription factors that play critical roles in many cellular processes, including gene transcription, mRNA splicing, proliferation, and differentiation. Recent studies have linked PRMT-dependent epigenetic marks and modifications to carcinogenesis and metastasis in cancer. However, the role of PRMT2-dependent signaling in breast cancer remains obscure. We demonstrate PRMT2 mRNA expression was significantly decreased in breast cancer relative to normal breast. Gene expression profiling, Ingenuity and protein-protein interaction network analysis after PRMT2-short interfering RNA transfection into MCF-7 cells, revealed that PRMT2-dependent gene expression is involved in cell-cycle regulation and checkpoint control, chromosomal instability, DNA repair, and carcinogenesis. For example, PRMT2 depletion achieved the following: 1) increased p21 and decreased cyclinD1 expression in (several) breast cancer cell lines, 2) decreased cell migration, 3) induced an increase in nucleotide excision repair and homologous recombination DNA repair, and 4) increased the probability of distance metastasis free survival (DMFS). The expression of PRMT2 and retinoid-related orphan receptor-γ (RORγ) is inversely correlated in estrogen receptor-positive breast cancer and increased RORγ expression increases DMFS. Furthermore, we found decreased expression of the PRMT2-dependent signature is significantly associated with increased probability of DMFS. Finally, weighted gene coexpression network analysis demonstrated a significant correlation between PRMT2-dependent genes and cell-cycle checkpoint, kinetochore, and DNA repair circuits. Strikingly, these PRMT2-dependent circuits are correlated with pan-cancer metagene signatures associated with epithelial-mesenchymal transition and chromosomal instability. This study demonstrates the role and significant correlation between a histone methyltransferase (PRMT2)-dependent signature, RORγ, the cell-cycle regulation, DNA repair circuits, and breast cancer survival outcomes.

Transgenic muscle-specific Nor-1 expression regulates multiple pathways that effect adiposity, metabolism, and endurance.

The mRNA encoding Nor-1/NR4A3 is rapidly and strikingly induced by ß2-adrenergic signaling in glycolytic and oxidative skeletal muscle. In skeletal muscle cells, Nor-1 expression is important for the regulation of oxidative metabolism. Transgenic skeletal muscle-specific expression of activated Nor-1 resulted in the acquisition of an endurance phenotype, an increase in type IIA/X oxidative muscle fibers, and increased numbers of mitochondria. In the current study, we used dual-energy x-ray absorptiometry and magnetic resonance imaging analysis to demonstrate decreased adiposity in transgenic (Tg) Nor-1 mice relative to that in wild-type littermates. Furthermore, the Tg-Nor-1 mice were resistant to diet-induced weight gain and maintained fasting glucose at normoglycemic levels. Expression profiling and RT-quantitative PCR analysis revealed significant increases in genes involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, and glycogen synthesis, in concordance with the lean phenotype. Moreover, expression profiling identified several Z-disc and sarcomeric binding proteins that modulate fiber type phenotype and endurance, eg, α-actinin-3. In addition, we demonstrated that the Tg-Nor-1 mouse line has significantly higher glycogen content in skeletal muscle relative to that in wild-type littermates. Finally, we identified a decreased NAD(+)/NADH ratio with a concordant increase in peroxisome proliferator-activated receptor γ coactivator-1α1 protein/mRNA expression. Increased NADH was associated with an induction of the genes involved in the malate-aspartate shuttle and a decrease in the glycerol 3-phosphate shuttle, which maximizes aerobic ATP production. In conclusion, skeletal muscle-specific Nor-1 expression regulates genes and pathways that regulate adiposity, muscle fiber type metabolic capacity, and endurance.

Research resource: nuclear receptors as transcriptome: discriminant and prognostic value in breast cancer.

To identify biologically relevant groupings or clusters of nuclear receptors (NR) that are associated with breast neoplasia, with potentially diagnostic, discriminant or prognostic value, we quantitated mRNA expression levels of all 48 members of the human NR superfamily by TaqMan low-density array analysis in 116 curated breast tissue samples, including pre- and postmenopausal normal breast and both ERα(+) and ERα(-) tumor tissue. In addition, we have determined NR levels in independent cohorts of tamoxifen-treated ERα(+) and ERα(-) tissue samples. There were differences in relative NR mRNA expression between neoplastic and normal breast, and between ER(+) and ER(-) tumors. First, there is overexpression of the NUR77 subgroup and EAR2 in neoplastic breast. Second, we identify a signature of five NR (ERα, EAR2, NUR77, TRα, and RARγ) that classifies breast samples with more than 97% cross-validated accuracy into normal or cancer classes. Third, we find a novel negative association between five NR (TRß, NUR77, RORγ, COUP-TFII, and LRH1) and histological grade. Finally, four NR (COUP-TFII, TRß, PPARγ, and MR) are significant predictors of metastasis-free survival in tamoxifen-treated breast cancers, independent of ER expression. The present study highlights the discriminant and prognostic value of NR in breast cancer; identifies novel, clinically relevant, NR signatures; and highlights NR signaling pathways with potential roles in breast cancer pathophysiology and as new therapeutic targets.

The nuclear receptor, Nor-1, markedly increases type II oxidative muscle fibers and resistance to fatigue.

Nuclear hormone receptors (NR) have been implicated as regulators of lipid and carbohydrate metabolism. The orphan NR4A subgroup has emerged as regulators of metabolic function. Targeted silencing of neuron-derived orphan receptor 1 (Nor-1)/NR4A3 in skeletal muscle cells suggested that this NR was necessary for oxidative metabolism in vitro. To investigate the in vivo role of Nor-1, we have developed a mouse model with preferential expression of activated Nor-1 in skeletal muscle. In skeletal muscle, this resulted in a marked increase in: 1) myoglobin expression, 2) mitochondrial DNA and density, 3) oxidative enzyme staining, and 4) genes/proteins encoding subunits of electron transport chain complexes. This was associated with significantly increased type IIA and IIX myosin heavy chain mRNA and proteins and decreased type IIB myosin heavy chain mRNA and protein. The contractile protein/fiber type remodeling driving the acquisition of the oxidative type II phenotype was associated with 1) the significantly increased expression of myocyte-specific enhancer factor 2C, and phospho-histone deacetylase 5, and 2) predominantly cytoplasmic HDAC5 staining in the Tg-Nor-1 mice. Moreover, the Nor-1 transgenic line displayed significant improvements in glucose tolerance, oxygen consumption, and running endurance (in the absence of increased insulin sensitivity), consistent with increased oxidative capacity of skeletal muscle. We conclude that skeletal muscle fiber type is not only regulated by exercise-sensitive calcineurin-induced signaling cascade but also by NR signaling pathways that operate at the nexus that coordinates muscle performance and metabolic capacity in this major mass tissue.

Ski overexpression in skeletal muscle modulates genetic programs that control susceptibility to diet-induced obesity and insulin signaling.

Transgenic mice overexpressing chicken Ski (c-Ski) have marked decrease in adipose mass with skeletal muscle hypertrophy. Recent evidence indicates a role for c-Ski in lipogenesis and energy expenditure. In the present study, wild type (WT) and c-Ski mice were challenged on a high-fat (HF) diet to determine whether c-Ski mice were resistant to diet-induced obesity. During the HF feeding WT mice gained significantly more weight than chow-fed animals, while c-Ski mice were partially resistant to the effects of the HF diet on weight. Body composition analysis confirmed the decreased adipose mass in c-Ski mice compared to WT mice. c-Ski mice possess a similar metabolic rate and level of food consumption to WT littermates, despite lower activity levels and on chow diet show mild glucose intolerance relative to WT littermates. On HF diet, glucose tolerance surprisingly remained unchanged in c-Ski mice, while it became worse in WT mice. Skeletal muscle of c-Ski mice exhibit impaired insulin-stimulated Akt phosphorylation and glucose uptake. In concordance, gene expression profiling of skeletal muscle of chow and HF-fed mice indicated that Ski suppresses gene expression associated with insulin signaling and glucose uptake and alters gene pathways involved in myogenesis and adipogenesis. In conclusion, c-Ski mice are partially resistant to diet-induced obesity and display aberrant insulin signaling and glucose homeostasis which is associated with alterations in gene expression that inhibit lipogenesis and insulin signaling. These results suggest Ski plays a major role in skeletal muscle metabolism and adipogenesis and hence influences risk of obesity and diabetes.

Protein arginine methyltransferase 6-dependent gene expression and splicing: association with breast cancer outcomes.

Protein arginine methyltransferase-6 (PRMT6) regulates steroid-dependent transcription and alternative splicing and is implicated in endocrine system development and function, cell death, cell cycle, gene expression and cancer. Despite its role in these processes, little is known about its function and cellular targets in breast cancer. To identify novel gene targets regulated by PRMT6 in breast cancer cells, we used a combination of small interfering RNA and exon-specific microarray profiling in vitro coupled to in vivo validation in normal breast and primary human breast tumours. This approach, which allows the examination of genome-wide changes in individual exon usage and total transcript levels, demonstrated that PRMT6 knockdown significantly affected i) the transcription of 159 genes and ii) alternate splicing of 449 genes. The PRMT6-dependent transcriptional and alternative splicing targets identified in vitro were validated in human breast tumours. Using the list of genes differentially expressed between normal and PRMT6 knockdown cells, we generated a PRMT6-dependent gene expression signature that provides an indication of PRMT6 dysfunction in breast cancer cells. Interrogation of several well-studied breast cancer microarray expression datasets with the PRMT6 gene expression signature demonstrated that PRMT6 dysfunction is associated with better overall relapse-free and distant metastasis-free survival in the oestrogen receptor (ER (ESR1)) breast cancer subgroup. These results suggest that dysregulation of PRMT6-dependent transcription and alternative splicing may be involved in breast cancer pathophysiology and the molecular consequences identifying a unique and informative biomarker profile.

Nr4a1 siRNA expression attenuates α-MSH regulated gene expression in 3T3-L1 adipocytes.

Several recent investigations have underscored the growing role of melanocortin signaling in the peripheral regulation of lipid, glucose, and energy homeostasis. In addition, the melanocortins play a critical role in the central control of satiety. These observations, and the latest reports highlighting the emerging role of the nuclear hormone receptor (NR) 4A subgroup in metabolism, have prompted us to investigate the cross talk between [Nle(4), d-Phe(7)] (NDP)-α-MSH and Nr4a signaling in adipose. We have shown that NDP-MSH strikingly and preferentially induces the expression of the NR4A subgroup (but not any other members of the NR superfamily) in differentiated 3T3-L1 adipocytes. Utilization of quantitative PCR on custom-designed metabolic TaqMan low-density arrays identified the concomitant and marked induction of the mRNAs encoding Il-6, Cox2, Pdk4, and Pck-1 after NDP-MSH treatment. Similar experiments demonstrated that the mRNA expression profile induced by cAMP and NDP-MSH treatment displayed unique but also overlapping properties and suggested that melanocortin-mediated induction of gene expression involves cAMP-dependent and -independent signaling. Nr4a1/Nur77 small interfering RNA (siRNA) expression suppressed NDP-MSH-mediated induction of Nr4a1/Nur77 and Nr4a3/Nor-1 (but not Nr4a2/Nurr1). Moreover, expression of the siRNA-attenuated NDP-MSH mediated induction of the mRNAs encoding Il-6, Cox2/Ptgs2, and Pck-1 expression. In addition, Nur77 siRNA expression attenuated NDP-MSH-mediated glucose uptake. In vivo, ip administration of NDP-MSH to C57 BL/6J (male) mice significantly induced the expression of the mRNA encoding Nur77 and increased IL-6, Cox2, Pck1, and Pdk4 mRNA expression in (inguinal) adipose tissue. We conclude that Nur77 expression is necessary for MSH-mediated induction of gene expression in differentiated adipocytes. Furthermore, this study demonstrates cross talk between MSH and Nr4a signaling in adipocytes.

Nuclear receptor profiling of ovarian granulosa cell tumors.

Granulosa cell tumors of the ovary (GCT) represent ~5% of malignant ovarian tumors. The adult form is defined by a mutation in the FOXL2 gene. GCT exhibit many of the features of normal proliferating granulosa cells. We have profiled the expression of the 48 human nuclear receptors (NR) by quantitative RT-PCR in a panel of GCT and in two GCT-derived cell lines, COV434 and KGN. The highest level of expression is seen for COUP-TF2 with abundant expression of PPARγ, SF-1, and TR-α. Estrogen receptor (ER)-ß is the most abundant of the steroid receptors with relatively high expression also of AR, ER-α, and PR. The concordance of expression for each NR across the tumors is remarkably high with same discordance between the cell lines and the tumors, particularly the COV434 line. No significant differences were observed with respect to tumor stage for NR expression. These findings provide a full profile of NR expression in GCT which will enable full characterization of their roles and potential as therapeutic targets.

Beta-adrenergic signaling regulates NR4A nuclear receptor and metabolic gene expression in multiple tissues.

The nuclear hormone receptor (NR) 4A subgroup of orphan nuclear receptors includes three members, Nur77 (NR4A1), Nurr1 (NR4A2) and Nor-1 (NR4A3). Previously we have identified the rapid and robust (in vitro and in vivo) induction of the NR4A subgroup following beta-adrenergic stimulation in mouse skeletal muscle. This was concomitant with changes in the expression of genes involved in the regulation of nutrient metabolism. We have isolated mouse tissue of cardiovascular, endocrine and gastrointestinal origin at 1, 4, 8 and 24h after a single intraperitoneal injection of the beta-adrenergic agonist, isoprenaline. We similarly identified the significant induction (between 1 and 4h) of the NR4A genes in many of these tissues. Moreover, we have utilized TaqMan((R)) Low Density Arrays to determine the beta-adrenergic-sensitive metabolic gene expression in liver, white adipose and heart. In summary, cross-talk between beta-adrenergic and NR4A signaling occurs in several tissues, and is accompanied by modulation of metabolic gene expression.