Coactivator SRC-1 Research Articles

Z-guggulsterone regulates MDR1 expression mainly through the pregnane X receptor-dependent manner in human brain microvessel endothelial cells

Recently studies showed that pregnane X receptor (PXR) was expressed in human brain microvessel endothelial cells and coordinately induced multidrug resistance protein 1 (MDR1) expression. The present study aimed to investigate the regulatory effect of Z-guggulsterone on MDR1 in human brain microvessel endothelial cells, and explored whether it involved modulation of PXR. The results showed that Z-guggulsterone (30 μM) simultaneously inhibited the expression of PXR and MDR1 at 24 h in human brain-derived microvessel endothelial cells (hBDMECs). Meanwhile, the levels of PXR and MDR1 expression were simultaneously reduced in PXR siRNA-transfected hBDMECs; MDR-1 siRNA-transfected hBDMECs showed significant decrease in MDR1 expression, but no change in PXR expression. Furthermore, Z-guggulsterone inhibited the activation of PXR in hBDMECs through decreasing the release of cAMP/PKA. Z-guggulsterone reduced the co-activator SRC-1 expression in hBDMECs, as to prevent the activation of MDR1 gene transcription. In addition, Z-guggulsterone (30 μM) at 24 h significantly inhibited the expression of human constitutive androstane receptor (CAR) protein in hBDMECs. However, after treatment with Z-guggulsterone (≤30 μM), the level of MDR1 reporter gene activity was lower in human PXR-transfected cells than that in human CAR-transfected cells. The inhibition effect of Z-guggulsterones on MDR1 reporter gene activation was gradually enhanced with the increase of human PXR to CAR ratio, which was greater extent than that with the increase of human CAR to hPXR ratio. The present study suggested that Z-guggulsterone down-regulating the efflux function and expression of MDR1 in hBDMECs might be mainly through the PXR-dependent manner.

Maternal diets enriched in olive oil regulate lipid metabolism and levels of PPARs and their coactivators in the fetal liver in a rat model of gestational diabetes mellitus

In a rat model of gestational diabetes mellitus (GDM) programmed in the offspring of neonatal streptozotocin-induced (nSTZ) diabetic rats, lipids are accumulated in the fetal liver in a sex-dependent way. Here, we evaluated whether maternal diets enriched in olive oil in rats that will develop GDM ameliorate lipid metabolic impairments in the fetal livers. Pregnant offspring of control and nSTZ diabetic rats (F0) were fed a 6% olive oil-supplemented diet throughout the F1 gestation. We evaluated maternal metabolic parameters as well as lipid content, expression of lipid metabolizing enzymes and protein expression of PLIN2, PPARs and PPAR coactivators in the fetal livers. The offspring of nSTZ diabetic rats developed GDM regardless of the maternal treatment. Hypertriglyceridemia in GDM rats was prevented by the olive oil-enriched maternal treatment. In the livers of male fetuses of GDM rats, the maternal olive oil-supplemented diet prevented lipid overaccumulation and prevented the increase in PPARγ and PPARδ levels. In the livers of female fetuses of GDM rats, the maternal olive oil supplementation prevented the increase in PPARδ levels and the reduction in PGC1α levels, but did not prevent the reduced lipid content. Control and GDM rats showed a reduction of lipid metabolic enzymes in the fetal livers, which was associated with reduced levels of the PPAR coactivators PGC-1α and SRC-1 in males and of SRC-1 in females. These results suggest powerful effects of a maternal olive oil-supplemented diet in the fetal liver, possibly providing benefits in the fetuses and offspring from GDM rats.

Elucidation of Molecular Mechanism of a Selective PPARα Modulator, Pemafibrate, through Combinational Approaches of X-ray Crystallography, Thermodynamic Analysis, and First-Principle Calculations.

The selective PPARα modulator (SPPARMα) is expected to medicate dyslipidemia with minimizing adverse effects. Recently, pemafibrate was screened from the ligand library as an SPPARMα bearing strong potency. Several clinical pieces of evidence have proved the usefulness of pemafibrate as a medication; however, how pemafibrate works as a SPPARMα at the molecular level is not fully known. In this study, we investigate the molecular mechanism behind its novel SPPARMα character through a combination of approaches of X-ray crystallography, isothermal titration calorimetry (ITC), and fragment molecular orbital (FMO) analysis. ITC measurements have indicated that pemafibrate binds more strongly to PPARα than to PPARγ. The crystal structure of PPARα-ligand binding domain (LBD)/pemafibrate/steroid receptor coactivator-1 peptide (SRC1) determined at 3.2 Å resolution indicates that pemafibrate binds to the ligand binding pocket (LBP) of PPARα in a Y-shaped form. The structure also reveals that the conformation of the phenoxyalkyl group in pemafibrate is flexible in the absence of SRC1 coactivator peptide bound to PPARα; this gives a freedom for the phenoxyalkyl group to adopt structural changes induced by the binding of coactivators. FMO calculations have indicated that the accumulation of hydrophobic interactions provided by the residues at the LBP improve the interaction between pemafibrate and PPARα compared with the interaction between fenofibrate and PPARα.

The In Vitro Functional Impairment of Thyroid Hormone Receptor Alpha 1 Isoform Mutants Is Mainly Dictated by Reduced Ligand Sensitivity.

Background: Thyroid hormone (TH) acts on TH receptors (TRs) and regulates gene transcription by binding of TRs to TH response elements (TREs) in target gene promoters. The transcriptional activity of TRs is modulated by interactions with TR-coregulatory proteins. Mutations in TRα cause resistance to thyroid hormone alpha (RTHα). In this study, we analyzed if, beyond reduced triiodothyronine (T3) affinity, altered interactions with cofactors or different TREs could account for the differential impaired transcriptional activity of different mutants. Methods: We evaluated four mutants derived from patients (D211G, M256T, A263S, and R384H) and three artificial mutants at equivalent positions in patients with RTHβ (T223A, L287V, and P398H). The in vitro transcriptional activity was evaluated on TRE-luciferase reporters (DR4, IR0, and ER6). The affinity for T3 and interaction with coregulatory proteins (nuclear receptor corepressor 1 [NCoR1] and steroid receptor coactivator 1 [SRC1]) were also determined. Results: We found that the affinity for T3 was significantly reduced for all mutants, except for TRα1-T223A. The reduction in the T3 sensitivity of the transcriptional activity on three TREs, the dissociation of the corepressor NCoR1, and the association of the coactivator SRC1 recruitment for each mutant correlated with the reduced affinity for T3. We did not observe mutation-specific alterations in interactions with cofactors or TREs. Conclusions: In summary, the degree of impaired transcriptional activity of mutants is mainly determined by their reduced affinity for T3.

Abstract P5-05-01: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive aggressive metastatic breast cancer

Abstract Background: Metabolic rewiring is one of the central hallmarks of cancer progression and survival to support anabolic and energetic demands. Tumor cells constantly alter their metabolic state in response to oncogenic stimuli, nutrient availability, and interaction with immune cells however the precise regulation that precedes the metabolic alteration is poorly understood. Here we report a direct interaction of glycolytic enzyme PFKFB4 with transcriptional coregulator SRC-3. PFKFB4 functions as a critical regulator of Warburg effect and our study reveals that upon glucose stimulation PFKFB4 activates SRC-3 driving an invasive-metastatic breast cancer. Methods: Molecular experiments were performed to understand the transcriptional activation of SRC-3 by PFKFB4 enzyme. Chromatin immunoprecipitation and gene expression studies were performed to investigate the functions of PFKFB4/SRC-3 crosstalk on transcriptional regulation. Metabolomics and isotope tracing studies were performed to identify the metabolic adaptations regulated by PFKFB4/SRC-3 in breast tumors. PFKFB4-knockout was established using CRISPR-Cas9 system and functional studies were carried out to define its role in tumor cell proliferation, invasion-migration, and breast to lung metastasis. Human breast tumor samples were evaluated to identify the clinical importance of PFKFB4/SRC-3 crosstalk in patients. Results:Molecular studies revealed that PFKFB4 enzyme phosphorylates SRC-3 at serine 857 (S857) enhancing its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient SRC-3 mutant S857A (SRC-3S857A) significantly abolished SRC-3-mediated transcriptional output (p<0.000001). Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway enabling purine synthesis by transcriptionally upregulating the expression of enzyme transketolase (TKT). Deletion of PFKFB4 by CRISPR-Cas9 system resulted in significantly reduced proliferation (p<0.05) and migration-invasion (p<0.001) compared to wildtype breast tumor cells. Ablation of SRC-3 or PFKFB4 suppressed in vivo breast tumor growth and prevents metastasis to the lung from an orthotopic setting (p<0.0001). PFKFB4 and phosphorylated SRC-3 levels are significantly increased in breast tumors (p=0.02), whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of TNBC patients (p=0.03). Conclusion:Our data suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumors. It also provides first evidence how Warburg pathway drives aggressive breast tumorigenesis by directly activating powerful oncogene SRC-3. Our work suggests that targeting the PFKFB4–SRC-3 axis may be therapeutically valuable in breast tumors that are notably dependent on glucose metabolism. (This work is funded by grants from Susan G. Komen and NCI to S.D.) Citation Format: Dasgupta S, Anand V, John H, Sawant Dessai A, Katsuta E, Takabe K, O'Malley B. Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive aggressive metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-01.

Abstract P5-05-13: Quantitative combinatory indexed ChIP-seq reveals distinct transcriptional complexes containing estrogen receptor and GREB1 at chromatin in breast cancer cells

Abstract GREB1 is an estrogen-activated gene in estrogen receptor a (ER)-positive breast cancer cells. GREB1 is also required for estrogen-stimulated breast cancer cell growth and its level is highly correlated to ER level in breast cancer cells and tumor samples. In endocrine resistant diseases, GREB1 is often dysregulated. GREB1 has been shown to interact with ER and bind to the same ER binding sites throughout the genome. There is no identified functional domain in GREB1 and it is not completely known how GREB1 exerts its function to regulate the transcription of ER target genes. In order to demonstrate whether GREB1 is present in the ER-containing transcriptional complex at chromatin, we have adapted and developed a quantitative combinatory indexed ChIP-seq assay suitable for dissecting components in a transcriptional cofactor complex in a genome-wide scale. In ER-positive MCF-7 breast cancer cells, we found that almost all GREB1 binding sites are the same sites bound by ER or its bona fide coactivator SRC-3. We further found that GREB1 and SRC-3 are both present in the same ER-containing complex at chromatin. Thus, both GREB1 and SRC-3 are integral members of the ER transcriptional complex at chromatin. Moreover, we discovered that only a portion of GREB1 at chromatin is present in the ER complex while the other portion of GREB1 is present in a different complex lacking ER or SRC-3 at the same genomic loci. Thus, two distinct GREB1-containing complexes are identified in equilibrium at chromatin: one contains ER/SRC-3 and the other one lacks ER/SRC-3. Our results suggest a non-traditional role of GREB1 in transcriptional regulation of ER target genes. The method used in our study can be widely applied for probing components of transcriptional complexes at chromatin. Citation Format: Sun J, Lippman ME. Quantitative combinatory indexed ChIP-seq reveals distinct transcriptional complexes containing estrogen receptor and GREB1 at chromatin in breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-13.

Communication between the Ligand-Binding Pocket and the Activation Function-2 Domain of Androgen Receptor Revealed by Molecular Dynamics Simulations.

Androgen receptor (AR), as a member of the nuclear receptor (NR) superfamily, regulates the gene transcription in response to the sequential binding of diverse agonists and coactivators. Great progress has been made in studies on the pharmacology and structure of AR, but the atomic level mechanism of the bidirectional communications between the ligand-binding pocket (LBP) and the activation function-2 (AF2) region of AR remains poorly understood. Therefore, in this study, molecular dynamics (MD) simulations and free energy calculations were carried out to explore the interactions among water, agonist (DHT) or antagonist (HFT), AR, and coactivator (SRC3). Upon the binding of an agonist (DHT) or antagonist (HFT), the LBP structure would transform to the agonistic or antagonistic state, and the conformational changes of the LBP would regulate the structure of the AF2 surface. As a result, the binding of the androgen DHT could promote the recruitment of the coactivator SRC3 to the AF2, and on the contrary, the binding of the antagonist HFT would induce a perturbation to the shape of the AF2 and then weaken its accommodating capability of the coactivators with the LXXLL motif. The simulation results illustrated that the DHT-AR binding affinity was enhanced by the association of the coactivator SRC3, which would reduce the conformational fluctuation of the AR-LBD and expand the size of the AR LBP. On the other hand, the coactivator-to-HFT allosteric pathway, which involves the SRC3, helix 3 (H3), helix 4 (H4), the loop (L1-3) between helix 1 (H1) and H3, and HFT, was characterized. The HFT's skewness and different interactions between HFT and the LBP were observed in the SRC3-present AR. The mutual communications between the AF2 surface and LBP, together with the processes involving the interplay of the ligand binding and coactivator recruitment events, would help in understanding the association of coactivators and rationally develop potent drugs to inhibit the activity of AR.

Abstract SY01-01: Endocrine resistance in metastatic breast cancer: Mechanisms and new therapeutic strategies

Abstract SY01-01: Endocrine resistance in metastatic breast cancer: Mechanisms and new therapeutic strategies

PO-507 ADAM22 as a therapeutic target for endocrine resistant metastatic breast cancer

IntroductionThe development of endocrine resistance is a major hurdle for the treatment of oestrogen receptor positive (ER+) breast cancer. One of the key contributors to this resistant phenotype is overexpression of the nuclear receptor coactivator SRC-1, which in turn has been shown to promote metastatic disease. Using CHIP-seq and microarray studies, the neuronal protein ADAM22 (A Disintegrin And Metalloproteinase) was identified as a potential pro-metastatic SRC-1 regulated gene in endocrine resistant cell lines. ADAM22 expression promotes both migration and de-differentiation, key hallmarks of metastasis. While clinically, elevated ADAM22 expression predicts poor disease free survival. Here, both the role of ADAM22 in metastatic development and the clinical potential of ADAM22 as a biomarker and therapeutic target were explored.Material and methodsThe functional effect of ADAM22 modulation on anchorage independent growth and mammosphere formation was investigated using CRISPR/Cas9 knockout and lentiviral overexpression in the endocrine resistant LY2 cell line. To examine the clinical relevance of ADAM22 as a biomarker, ADAM22 expression was examined in matched primary breast and metastatic cancer tissues. A small peptide mimetic of the ADAM22 endogenous ligand LGI1 (LGIMIM) was designed and explored as a potential anti-ADAM22 therapeutic both in vitro and in vivo.Results and discussionsADAM22 expression promotes anchorage independent growth and mammosphere formation, suggesting ADAM22 contributes to both the survival of metastatic cells and their ability to colonise distant sites. ADAM22 expression was significantly higher in primary breast tumours which went on to metastasise versus primaries which did not. A significant increase in ADAM22 expression was found in brain metastatic tissue compared to matched primary tissue, suggesting ADAM22 may prime endocrine resistant cancer cells to colonise the brain. LGIMIM treatment was sufficient to inhibit ADAM22 mediated phenotypes in vitro and to reduce metastatic burden in a xenograft model of endocrine resistance in vivo.ConclusionADAM22 is a promising biomarker and therapeutic target for endocrine resistant breast cancer.

SRC-3 Coactivator Governs Dynamic Estrogen-Induced Chromatin Looping Interactions during Transcription

Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter invitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain "poised" for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription.

Gene Expression Pattern of Histone Acetylation Enzymes Changed in the Hypothalamus of Middle-Aged Female Rats: A Putative Mechanism for Female Reproductive Aging

Objective: Female reproductive aging is characterized by reduced responsiveness of the hypothalamus to E2-positive feedback, which can result in alterations of gene expression and luteinizing hormone (LH) surge dysfunction. We hypothesize that age-related changes in E2-responsive gene expression are due to altered histone acetylation by histone deacetylases (HDACs) or estrogen receptor-alpha (ERα) coactivators with histone acetyltransferase (HAT) activity. Methods: In the present study, young and middle-aged female rats were ovariectomized (OVX) and treated with E2 or oil once per day for 2 days. At the time of the expected LH surge, the anterior and posterior hypothalami were dissected, and gene expression of 11 HDACs and 4 ERα coactivators with HAT activity was measured using real-time polymerase chain reaction. Results: In the anterior hypothalamus, age affected the gene expression of 3 HDACs (Hdac3, Hdac5, and Hdac11) and 2 ERα coactivators (Src2 and Crebbp). E2 treatment significantly decreased mRNA levels of 4 HDACs (Hdac4, Hdac5, Hdac10, and Hdac11) and 2 ERα coactivators (Src2 and Crebbp) in young females (3–4 months). However, none of the genes responded to E2 in the middle-aged females (9–11 months), except Hdac10. In the posterior hypothalamus, age influenced Hdac5 and Src1 mRNA expression. E2 treatment increased Hdac4 and Crebbp mRNA levels in the young but not middle-aged females. Conclusions: These data suggest that E2 regulates HDACs and ERα coactivators with HAT activity in an age- and E2-dependent manner, which may contribute to the age-related gene expression changes on the day of LH surge in female reproductive aging. Key words: Aging; Estradiol-Positive Feedback; Estrogen Receptor-Alpha Coactivators; Histone Deacetylases; Hypothalamus

Estrogen receptor alpha and beta regulate actin polymerization and spatial memory through an SRC-1/mTORC2-dependent pathway in the hippocampus of female mice

Aging-related decline of estrogens, especially 17β-estradiol (E2), has been shown to play an important role in the impairment of learning and memory in dementias, such as Alzheimer’s disease (AD), but the underlying molecular mechanisms are poorly understood. In this study, we first demonstrated decreases in E2 signaling (aromatase, classic estrogen receptor ERα and ERβ and their coactivator SRC-1), mTORC2 signaling (Rictor and phospho-AKTser473) and actin polymerization (phospho-Cofilin, Profilin-1 and the F-actin/G-actin ratio) in the hippocampus of old female mice compared with those levels detected in the adult hippocampus. We then showed that ERα and ERβ antagonists induced a significant decrease in SRC-1, mTORC2 signaling, actin polymerization, and CA1 spine density, as well as impairments of learning and memory; however, ovariectomy-induced changes of these parameters could be significantly reversed by treatment with ER agonists. We further showed that expression of SRC-1, mTORC2 signaling and actin polymerization could be upregulated by E2 treatment, and the effects of E2 were blocked by the ER antagonists but mimicked by the agonists. We also showed that the lentivirus-mediated SRC-1 knockdown significantly inhibited the agonist-activated mTORC2 signaling and actin polymerization, and the lentivirus-mediated Rictor knockdown also significantly inhibited the agonist-activated actin polymerization. Finally, we demonstrated that the ERα and ERβ antagonists induced a disruption in actin polymerization and an impairment of spatial memory, which were rescued by activation of mTORC2. Taken together, the above results clearly demonstrated an mTORC2-dependent regulation of actin polymerization that contributed to the effects of ERα and ERβ on spatial learning, which may provide a novel target for the prevention and treatment of E2-related dementia in the aged population.

Targeting Bile Acid Receptors: Discovery of a Potent and Selective Farnesoid X Receptor Agonist as a New Lead in the Pharmacological Approach to Liver Diseases.

Bile acid (BA) receptors represent well-defined targets for the development of novel therapeutic approaches to metabolic and inflammatory diseases. In the present study, we report the generation of novel C-3 modified 6-ethylcholane derivatives. The pharmacological characterization and molecular docking studies for the structure-activity rationalization, allowed the identification of 3β-azido-6α-ethyl-7α-hydroxy-5β-cholan-24-oic acid (compound 2), a potent and selective FXR agonist with a nanomolar potency in transactivation assay and high efficacy in the recruitment of SRC-1 co-activator peptide in Alfa Screen assay. In vitro, compound 2 was completely inactive towards common off-targets such as the nuclear receptors PPARα, PPARγ, LXRα, and LXRβ and the membrane G-coupled BA receptor, GPBAR1. This compound when administered in vivo exerts a robust FXR agonistic activity increasing the liver expression of FXR-target genes including SHP, BSEP, OSTα, and FGF21, while represses the expression of CYP7A1 gene that is negatively regulated by FXR. Collectively these effects result in a significant reshaping of BA pool in mouse. In summary, compound 2 represents a promising candidate for drug development in liver and metabolic disorders.

Differential Activation of a Mouse Estrogen Receptor β Isoform (mERβ2) with Endocrine-Disrupting Chemicals (EDCs).

Background:Endocrine-disrupting chemicals (EDCs) are suspected of altering estrogenic signaling through estrogen receptor (ER) α or β (mERβ1 in mice). Several EDC effects have been reported in animal studies and extrapolated to human studies. Unlike humans, rodents express a novel isoform of ERβ (mERβ2) with a modified ligand-binding domain sequence. EDC activity through this isoform remains uncharacterized.Objectives:We identified the expression pattern of mERβ2 in mouse tissues and assessed the estrogenic activity of EDCs through mERβ2.Methods:mERβ2 mRNA expression was measured in mouse tissues. HepG2 cells were used to assess the transactivation activity of mERβ isoforms with EDCs and ER co-activators. 293A cells transiently transfected with mER isoforms were used to detect EDC-mediated changes in endogenous ER target gene expression.Results:Expression of mERβ2 mRNA was detected in mouse reproductive tissues (ovary, testis, and prostate) and lung and colon tissues from both female and male mice. Five (E2, DES, DPN, BPAF, Coum, 1-BP) of 16 compounds tested by reporter assay had estrogenic activity through mERβ2. mERβ2 had a compound-specific negative effect on ERβ/ligand-mediated activity and ER target genes when co-expressed with mERβ1. mERβ2 recruited coactivators SRC2 or SRC3 in the presence of EDCs, but showed less recruitment than mERβ1.Conclusion:mERβ2 showed weaker estrogenic activity than mERβ1 in our in vitro system, and can dampen mERβ1 activity. In vivo models of EDC activity and ER-mediated toxicity should consider the role of mERβ2, as rodent tissue responses involving mERβ2 may not be reproduced in human biology.Citation:Donoghue LJ, Neufeld TI, Li Y, Arao Y, Coons LA, Korach KS. 2017. Differential activation of a mouse estrogen receptor β isoform (mERβ2) with endocrine-disrupting chemicals (EDCs). Environ Health Perspect 125:634–642; http://dx.doi.org/10.1289/EHP396

Development of yeast reporter assays for the enhanced detection of environmental ligands of thyroid hormone receptors α and β from Xenopus tropicalis

Thyroid hormones (THs) are involved in the regulation of metabolic homeostasis during the development and differentiation of vertebrates, particularly amphibian metamorphosis, which is entirely controlled by internal TH levels. Some artificial chemicals have been shown to exhibit TH-disrupting activities. In order to detect TH disruptors for amphibians, we herein developed a reporter assay using yeast strains expressing the thyroid hormone receptors (TRs) α and β together with the transcriptional coactivator SRC-1, all of which were derived from the frog Xenopus tropicalis (XT). These yeast strains responded to endogenous THs (T2, T3, and T4) in a dose-dependent manner. They detected the TR ligand activities of some artificial chemicals suspected to exhibit TH-disrupting activities, as well as TR ligand activity in river water collected downstream of sewage plant discharges, which may have originated from human excrement. Moreover, the responses of XT TR strains to these endogenous and artificial ligands were stronger than those of yeast strains for human TRα and β assays, which had previously been established in our laboratory. These results indicate that the yeast reporter assay system for XT TRα and β is valuable for assessing TR ligand activities in environmental samples that may be particularly potent in amphibians.

KLF15 Enables Rapid Switching between Lipogenesis and Gluconeogenesis during Fasting

Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulated during the postprandial state) as an adaptation to the nutritional environment. While alterations in the expression level of the transcription factor SREBP-1c are known to be critical for nutritionally regulatedlipogenesis, upstream mechanisms governing Srebf1 expression remain unclear. Here, we show that the fasting-induced transcription factor KLF15, a key regulator of gluconeogenesis, forms a complex with LXR/RXR, specifically on the Srebf1 promoter. This complex recruits the corepressor RIP140 instead of the coactivator SRC1, resulting in reduced Srebf1 and thus downstream lipogenic enzyme expression during the early and euglycemic period of fasting prior to hypoglycemia and PKA activation. Through this mechanism, KLF15 overexpression specifically ameliorates hypertriglyceridemia without affecting LXR-mediated cholesterol metabolism. These findings reveal a key molecular link between glucose and lipid metabolism and have therapeutic implications for the treatment of hyperlipidemia.

Ligand activated progesterone receptor B drives autophagy-senescence transition through a Beclin-1/Bcl-2 dependent mechanism in human breast cancer cells

Loss of progesterone-receptors (PR) expression is associated with breast cancer progression. Herein we provide evidence that OHPg/PR-B through Beclin-1 evoke autophagy-senescence transition, in breast cancer cells. Specifically, OHPg increases Beclin-1 expression through a transcriptional mechanism due to the occupancy of Beclin-1 promoter by PR-B, together with the transcriptional coactivator SRC-2. This complex binds at a canonical half progesterone responsive element, which is fundamental for OHPg effects, as shown by site-directed mutagenesis. Beside, OHPg via non-genomic action rapidly activates JNK, which phosphorylates Bcl-2, producing the functional release from Beclin-1 interaction. This is not linked to an efficient autophagic flux, since p62 levels, marker of degradation via lysosomes, were not reduced after sustained OHPg stimulus. Instead, the cell cycle inhibitor p27 was induced, together with an irreversible G1 arrest, hallmark of cellular senescence. Specifically the increase of senescence-associated β-galactosidase activity was blocked by Bcl-2 siRNA but also by Beclin-1 siRNA. Collectively these findings support the importance of PR-B expression in breast cancer cells, thus targeting PR-B may be a useful strategy to provide additional approaches to existing therapies for breast cancer patients.

Abstract 77: Metabolic Reprograming Driven by Interaction AMP-activated Protein Kinase and Coactivator SRC-2 Regulate Cardiac Stress Response

Flexibility in fuel source for the heart is necessary to meet ATP demands during a high and variable workload such as transaortic constriction (TAC). During such chronic metabolic and hemodynamic stresses, shifts occur in substrate utilization in the heart based on magnitude of the demand, oxygen and nutrient availability. These shifts directly affect the amount of ATP available for other stress responsive pathways and therefore their efficacy. Communication from metabolic changes to these other pathways is crucial. As a result, it is important understand the molecular communication between metabolic and hypertrophic pathways under stress. We have found that chronic loss of cardiomyocyte SRC-2 and transient activation of AMPK both effect metabolic inputs prior to TAC and result in a blunted hypertrophic response. We hypothesized that SRC-2 and AMPK are critical upstream regulators of the cardiac stress response controlling metabolic changes translating into altered hypertrophy. Furthermore, we proposed that they could be regulating each other to control both the temporal changes to the metabolic machinery through AMPK and long-term genetic changes through SRC-2. We identified AMPK as a coregulator of SRC-2 activity and confirmed a direct interaction between them in cardiac cells. We have identified putative AMPK phosphorylation sites on SRC-2 that control SRC-2 stability and activity. Conversely, we found by ChIP that SRC-2 localized to AMPK subunit genes. Additionally, we performed co-immunoprecipitation and luciferase reporters and found that SRC-2 can interact with AMPK subunits and regulate its activity. We believe that disruption of these interactions is a main determinant in efficient hypertrophy in response to metabolic changes. Our results suggest the basis of a mechanism whereby energetic changes are sensed via AMPK and translated to transcriptional machinery of diverse targets via SRC-2 which allows AMPK to effect multiple cardiac transcription pathways. This mechanism provides insights into the role of AMPK-SRC-2 interaction in regulating myocardial function, metabolism, hypertrophy, and the progression to heart failure.

Suppression of Adipocyte Differentiation by Foenumoside B from Lysimachia foenum-graecum Is Mediated by PPARγ Antagonism.

Lysimachia foenum-graecum extract (LFE) and its active component foenumoside B (FSB) have been shown to inhibit adipocyte differentiation, but their mechanisms were poorly defined. Here, we investigated the molecular mechanisms responsible for their anti-adipogenic effects. Both LFE and FSB inhibited the differentiation of 3T3-L1 preadipocytes induced by peroxisome proliferator-activated receptor-γ (PPARγ) agonists, accompanied by reductions in the expressions of the lipogenic genes aP2, CD36, and FAS. Moreover, LFE and FSB inhibited PPARγ transactivation activity with IC50s of 22.5 μg/ml and 7.63 μg/ml, respectively, and showed selectivity against PPARα and PPARδ. Rosiglitazone-induced interaction between PPARγ ligand binding domain (LBD) and coactivator SRC-1 was blocked by LFE or FSB, whereas reduced NCoR-1 binding to PPARγ by rosiglitazone was reversed in the presence of LFE or FSB. In vivo administration of LFE into either ob/ob mice or KKAy mice reduced body weights, and levels of PPARγ and C/EBPα in fat tissues. Furthermore, insulin resistance was ameliorated by LFE treatment, with reduced adipose tissue inflammation and hepatic steatosis. Thus, LFE and FSB were found to act as PPARγ antagonists that improve insulin sensitivity and metabolic profiles. We propose that LFE and its active component FSB offer a new therapeutic strategy for metabolic disorders including obesity and insulin resistance.

The pregnane X receptor down-regulates organic cation transporter 1 (SLC22A1) in human hepatocytes by competing for ("squelching") SRC-1 coactivator.

The organic cation transporter 1 (OCT1) transports cationic drugs into hepatocytes. The high hepatic expression of OCT1 is controlled by the HNF4α and USF transcription factors. Pregnane X receptor (PXR) mediates induction of the principal xenobiotic metabolizing enzymes and transporters in the liver. Here, we have assessed the down-regulation of OCT1 expression by PXR activation. We used primary human hepatocytes and related cell lines to measure OCT1 expression and activity, by assaying MPP(+) accumulation. Western blotting, qRT-PCR, the OCT1 promoter gene reporter constructs and chromatin immunoprecipitation assays were also used. OCT1 mRNA in human hepatocytes was down-regulated along with reduced [(3) H]MPP(+) accumulation in differentiated HepaRG cells after treatment with rifampicin. Rifampicin and hyperforin as well as the constitutively active PXR mutant T248D suppressed activity of the 1.8kb OCT1 promoter construct in gene reporter assays. Silencing of both PXR and HNF4α in HepaRG cells blocked the PXR ligand-mediated down-regulation of OCT1 expression. The mutation of HNF4α and USF1 (E-box) responsive elements reversed the PXR-mediated inhibition in gene reporter assays. Chromatin immunoprecipitation assays indicated that PXR activation sequestrates the SRC-1 coactivator from the HNF4α response element and E-box of the OCT1 promoter. Consistent with these findings, exogenous overexpression of the SRC-1, but not the PGC1α coactivator, relieved the PXR-mediated repression of OCT1 transactivation. PXR ligands reduced the HNF4α-mediated and USF-mediated transactivation of OCT1 gene expression by competing for SRC-1 and decreased delivery of a model OCT1 substrate into hepatocytes.