Expression, Purification and Characterization of CAR/NCOA-1 Tethered Protein in E. coli Using Maltose-Binding Protein Fusion Tag and Gelatinized Corn Starch.

The constitutive active/androstane receptor (CAR) is a nuclear receptor that functions as a xenobiotic sensor, which regulates the expression of enzymes involved in drug metabolism and of efflux transporters. Evaluation of the binding properties between CAR and a drug was assumed to facilitate the prediction of drug-drug interaction, thereby contributing to drug discovery. The purpose of this study is to construct a system for the rapid evaluation of interactions between CAR and drugs. We prepared recombinant CAR protein using the Escherichia coli expression system. Since isolated CAR protein is known to be unstable, we designed a fusion protein with the CAR binding sequence of the nuclear receptor coactivator 1 (NCOA1), which was expressed as a fusion protein with maltose binding protein (MBP), and purified it by several chromatography steps. The thus-obtained CAR/NCOA1 tethered protein (CAR-NCOA1) was used to evaluate the interactions of CAR with agonists and inverse agonists by a thermal denaturation experiment using differential scanning fluorometry (DSF) in the presence and absence of drugs. An increase in the melting temperature was observed with the addition of the drugs, confirming the direct interaction between them and CAR. DSF is easy to set up and compatible with multiwell plate devices (such as 96-well plates). The use of DSF and the CAR-NCOA1 fusion protein together allows for the rapid evaluation of the interaction between a drug and CAR, and is thereby considered to be useful in drug discovery.

The Expression and Contribution of SRCs with Preeclampsia Placenta.

The steroid hormones act by binding to their receptors and subsequently interacting with coactivators. Several classes of coactivators have been identified and shown to be essential in estradiol (E2) responsiveness. The major coregulators are the p160 steroid receptor coactivator (SRC) family. Although the function of SRCs in other organs has been well studied, it has not been thoroughly studied in the placenta. In addition, the correlation between preeclampsia (PE) and SRCs has not been examined previously. Therefore, we compared the expression patterns of SRCs in normal and PE placentas. In human PE placental tissues, SRC-1 mRNA, and protein levels were downregulated in the PE group. In addition, to assess the expression of SRCs in a PE environment, we used Reduced Uterine Perfusion Pressure (RUPP) model and placental cells were cultured in hypoxia condition. SRC-1 proteins were reduced in the placenta of PE-like rat RUPP model. Furthermore, SRCs proteins were significantly downregulated in hypoxia-grown placental cells. To examine the interaction between estrogen receptors (ERs) and SRC-1 protein, we performed co-immunoprecipitation. The interaction of SRC-1 with ERα was significantly stronger than that with ERß. In PE placenta, the interaction of both ERα and ERß with SRC-1 was stronger than that in normal placenta. In summary, our results demonstrate that expression levels of SRC-1, not SRC-2 and SRC-3, were decreased in hypoxia-induced PE placenta, which may further reduce the signaling of sex steroid hormones such as E2. The dysregulated signaling of E2 by SRC-1 expression could be associated with the PE placental symptoms of patients.

Interplay among H3K9-editing enzymes SUV39H1, JMJD2C and SRC-1 drives p66Shc transcription and vascular oxidative stress in obesity.

Aims: Accumulation of reactive oxygen species (ROS) promotes vascular disease in obesity, but the underlying molecular mechanisms remain poorly understood. The adaptor p66Shc is emerging as a key molecule responsible for ROS generation and vascular damage. This study investigates whether epigenetic regulation of p66Shc contributes to obesity-related vascular disease. Methods and results: ROS-driven endothelial dysfunction was observed in visceral fat arteries (VFAs) isolated from obese subjects when compared with normal weight controls. Gene profiling of chromatin-modifying enzymes in VFA revealed a significant dysregulation of methyltransferase SUV39H1 (fold change, -6.9, P < 0.01), demethylase JMJD2C (fold change, 3.2, P < 0.01), and acetyltransferase SRC-1 (fold change, 5.8, P < 0.01) in obese vs. control VFA. These changes were associated with reduced di-(H3K9me2) and trimethylation (H3K9me3) as well as acetylation (H3K9ac) of histone 3 lysine 9 (H3K9) on p66Shc promoter. Reprogramming SUV39H1, JMJD2C, and SRC-1 in isolated endothelial cells as well as in aortas from obese mice (LepOb/Ob) suppressed p66Shc-derived ROS, restored nitric oxide levels, and rescued endothelial dysfunction. Consistently, in vivo editing of chromatin remodellers blunted obesity-related vascular p66Shc expression. We show that SUV39H1 is the upstream effector orchestrating JMJD2C/SRC-1 recruitment to p66Shc promoter. Indeed, SUV39H1 overexpression in obese mice erased H3K9-related changes on p66Shc promoter, while SUV39H1 genetic deletion in lean mice recapitulated obesity-induced H3K9 remodelling and p66Shc transcription. Conclusion: These results uncover a novel epigenetic mechanism underlying endothelial dysfunction in obesity. Targeting SUV39H1 may attenuate oxidative transcriptional programmes and thus prevent vascular disease in obese individuals.

MicroRNA-223-3p inhibits human bladder cancer cell migration and invasion.

The regulation of initiation and progression during carcinogenesis of bladder carcinoma is not completely elucidated. Dysregulation of microRNAs has been detected to play critical roles in the development of various cancers, including bladder carcinoma, whereas the involvement of miR-223-3p in the tumorigenesis of bladder carcinoma has not been studied. Here, we show that significantly higher levels of nuclear receptor coactivator 1 and significantly lower levels of miR-223-3p were detected in bladder carcinoma tissue, compared to the adjacent non-tumor tissue. In addition, the levels of nuclear receptor coactivator 1 and miR-223-3p were inversely correlated. Moreover, low miR-223-3p levels in bladder carcinoma specimens were associated with poor prognosis. In vitro, depletion of miR-223-3p increased bladder carcinoma cell invasion, which was abolished by overexpression of nuclear receptor coactivator 1. Bioinformatics studies demonstrate that miR-223-3p may bind to the 3'-UTR of nuclear receptor coactivator 1 messenger RNA to inhibit its protein translation in bladder carcinoma cells. Together, our study highlights miR-223-3p as a previously unrecognized microRNA that inhibits bladder carcinoma invasiveness via nuclear receptor coactivator 1, and this finding may be important for developing innovative therapeutic targets in treating bladder carcinoma.

Steroid Receptor Coactivator 1 Promotes Human Hepatocellular Carcinoma Progression by Enhancing Wnt/ß-Catenin Signaling.

Steroid receptor coactivator 1 (SRC-1) is a transcriptional coactivator not only for steroid receptors, such as androgen receptor and estrogen receptor, but also for other transcription factors. SRC-1 has been shown to play an important role in the progression of breast cancer and prostate cancer. However, its role in liver cancer progression remains unknown. In this study, we report that SRC-1 was overexpressed in 25 (62.5%) of 40 human hepatocellular carcinoma (HCC) specimens. Down-regulation of SRC-1 decreased HCC cell proliferation and impaired tumor maintenance in HCC xenografts. Knockdown of SRC-1 reduced protein levels of the proliferation marker proliferating cell nuclear antigen (PCNA) and the oncogene c-Myc. Knockout of SRC-1 in mice reduced diethylnitrosamine/CCl4-induced tumor formation in the liver and the expression of c-Myc and PCNA in liver tumors. SRC-1 promoted c-Myc expression, at least in part, by directly interacting with ß-catenin to enhance Wnt/ß-catenin signaling. Consistent with these results, the expression of SRC-1 was positively correlated with PCNA expression in human HCC specimens, and the expression levels of c-Myc in SRC-1-positive HCC specimens were higher than in SRC-1-negative HCC specimens. In addition, SRC-1 and SRC-3 were co-overexpressed in 47.5% of HCC specimens, and they cooperated to promote HCC cell proliferation. Simultaneous down-regulation of SRC-1 and SRC-3 dramatically inhibited HCC cell proliferation. Our results demonstrate that SRC-1 promotes HCC progression by enhancing Wnt/ß-catenin signaling and suggest that SRC-1 is a potential therapeutic molecular target for HCC.

Hormonal regulation of steroid receptor coactivator-1 mRNA in the male and female green anole brain.

Green anole lizards are seasonal breeders, with male sexual behaviour primarily regulated by an annual increase in testosterone. Morphological, biochemical and behavioural changes associated with reproduction are activated by testosterone, generally with a greater effect in the breeding season (BS) than in the nonbreeding season (NBS). The present study investigates the possibility that differences in a steroid receptor coactivator may regulate this seasonal difference in responsiveness to testosterone. In situ hybridisation was used to examine the expression of steroid receptor coactivator-1 (SRC-1) in the brains of gonadally intact male and female green anoles across breeding states. A second experiment examined gonadectomised animals with and without testosterone treatment. Gonadally intact males had more SRC-1 expressing cells in the preoptic area and larger volumes of this region as defined by these cells than females. Main effects of both sex and season (males > females and BS > NBS) were present in cell number and volume of the ventromedial hypothalamus. An interaction between sex and season suggested that high expression in BS males was driving these effects. In hormone-manipulated animals, testosterone treatment increased both the number of SRC-1 expressing cells in and volumes of the preoptic area and amygdala. These results suggest that testosterone selectively regulates SRC-1, and that this coactivator may play a role in facilitating reproductive behaviours across both sexes. However, changes in SRC-1 expression are not likely responsible for the seasonal change in responsiveness to testosterone.

Association of CXCR1 and 2 expressions with gastric cancer metastasis in ex vivo and tumor cell invasion in vitro.

BACKGROUND: CXCR1 and CXCR2, cell surface receptors of interleukin-8, regulate cell migration and alteration of their expression has been associated with poor prognosis of various cancers. The aim of this study was to detect their expression in gastric cancer to identify associations with another cell adhesion molecule, matrix metalloproteinase-9 (MMP9), and with clinicopathological data ex vivo, and then explore their potential role in gastric cancer cells in vitro. MATERIALS AND METHODS: A total of 172 cases of gastric cancer tissue specimens were collected for immunohistochemical analysis of CXCR1, CXCR2, and MMP9 expression. Expression of CXCR1 and CXCR2 proteins was knocked in or down using their cDNA and shRNA, respectively, in gastric cancer cell lines to assess the changed cell phenotypes and gene expression. RESULTS: CXCR1, CXCR2, and MMP9 were expressed in 61.0%, 77.9%, and 75.6% of gastric cancer tissues, respectively. Moreover, CXCR1 and CXCR2 expression was associated with tumor differentiations, advanced clinical stages, lymph node, and distant metastasis of gastric cancer. Similarly, MMP9 expression was associated with CXCR1 and CXCR2. Expression of these three proteins was interrelated. In vitro study showed that levels of CXCR1 and CXCR2 proteins were associated with the capacity of gastric cancer cell migration, while knockdown of their expression inhibited gastric cancer cell migration and invasion abilities in vitro. In contrast, overexpression of CXCR1 and CXCR2 proteins promoted tumor cell migration and invasion. At the gene levels, knockdown of CXCR1 or CXCR2 expression suppressed expression of Ets-1, SRC-1, and JNK proteins and phosphorylated c-Jun and Erk1/2. Conversely, upregulation of CXCR1 or CXCR2 promoted expression of Ets-1, SRC-1, JNK, and c-Jun proteins and phosphorylated JNK, c-Jun and Erk1/2. CONCLUSIONS: These findings suggest that CXCR1 and CXCR2 play an important role in gastric cancer progression. Further study will be performed to investigate whether target of their expression can be used as a novel strategy in clinical control of gastric cancer metastasis.

NCOA1 Directly Targets M-CSF1 Expression to Promote Breast Cancer Metastasis.

In breast cancer, overexpression of the nuclear coactivator NCOA1 (SRC-1) is associated with disease recurrence and resistance to endocrine therapy. To examine the impact of NCOA1 overexpression on morphogenesis and carcinogenesis in the mammary gland (MG), we generated MMTV-hNCOA1 transgenic [Tg(NCOA1)] mice. In the context of two distinct transgenic models of breast cancer, NCOA1 overexpression did not affect the morphology or tumor-forming capability of MG epithelial cells. However, NCOA1 overexpression increased the number of circulating breast cancer cells and the efficiency of lung metastasis. Mechanistic investigations showed that NCOA1 and c-Fos were recruited to a functional AP-1 site in the macrophage attractant CSF1 promoter, directly upregulating colony-simulating factor 1 (CSF1) expression to enhance macrophage recruitment and metastasis. Conversely, silencing NCOA1 reduced CSF1 expression and decreased macrophage recruitment and breast cancer cell metastasis. In a cohort of 453 human breast tumors, NCOA1 and CSF1 levels correlated positively with disease recurrence, higher tumor grade, and poor prognosis. Together, our results define an NCOA1/AP-1/CSF1 regulatory axis that promotes breast cancer metastasis, offering a novel therapeutic target for impeding this process.

Expression of estrogen receptors, androgen receptor and steroid receptor coactivator-3 is negatively correlated to the differentiation of astrocytic tumors.

Astrocytic tumors are the most common primary brain tumors. It has been reported that androgen receptor (AR), estrogen receptors alpha (ERα) and beta (ERß) and their coactivator SRC-1 and SRC-3 are involved in the regulation of the growth and development of many tumors, but their expression profiles and significances in the astrocytic tumors remain largely unknown. In this study, the expression of AR, ERs, and SRCs, and the possible roles of them in astrocytic neoplasm were evaluated and compared to normal brain tissues by nickel-intensified immunohistochemistry with tissue microarrays. The results showed that there were no age- or gender-differences regarding to the levels of these receptors or coactivators in astrocytic or normal brain tissues. In the high-grade astrocytic tissue, the levels of AR, ERs and SRC-3 were significantly decreased when compared to the low-grade astrocytic tissues, but the levels of SRC-1 remain unchanged. Correlation analysis revealed that the levels of AR, ERs and SRC-3 were negatively correlated to tumor differentiation, and the levels of SRC-3 were positively correlated to that of ERα. Furthermore, the decreased levels of SRC-3 were associated with an increase of ERß in astrocytic tumors when compared to that of normal brain tissues. These above results indicate a combination of decreased expression of ERs, AR and SRC-3 but not SRC-1 may be involved in the tumorigenesis of gliomas, ERα/SRC-3 axis may play central role in the regulation these tumors.

Expression of functional full-length hSRC-1 in eukaryotic cells using modified vaccinia virus Ankara and baculovirus.

Purified protein expression level and quality are contingent upon specific host expression systems. This differential production is particularly observed for proteins of high molecular weight, hampering further structural studies. We developed an expression method aimed at producing proteins in Escherichia coli, insect, and mammalian systems. Our novel protocol was used to produce in large scale the full-length 160-kDa steroid receptor coactivator 1 (SRC-1), a coregulator of nuclear receptors. The results indicate that we can produce biologically active human SRC-1 in mammalian and insect cells in large scale.

Steroid receptor coactivator-1 upregulates integrin α5 expression to promote breast cancer cell adhesion and migration.

Metastatic breast cancer remains a lethal disease with poorly understood molecular mechanisms. Steroid receptor coactivator-1 (SRC-1 or NCOA1) is overexpressed in a subset of breast cancers with poor prognosis. It potentiates gene expression by serving as a coactivator for nuclear receptors and other transcription factors. We previously reported that SRC-1 promotes breast cancer metastasis without affecting primary mammary tumor formation. Herein, we found that SRC-1 deficiency in mouse and human breast cancer cells substantially reduced cell adhesion and migration capabilities on fibronectin and significantly extended the time of focal adhesion disassembly and reassembly. In agreement with this phenotype, SRC-1 expression positively correlated with integrin α(5) (ITGA5) expression in estrogen receptor-negative breast tumors whereas SRC-1 deficiency decreased ITGA5 expression. Furthermore, ITGA5 reduction in SRC-1-deficient/insufficient breast cancer cells or knockdown of ITGA5 in SRC-1-expressing breast cancer cells was associated with a disturbed integrin-mediated signaling. Critical downstream changes included reduced phosphorylation and/or dampened activation of focal adhesion kinase, paxillin, Rac1, and Erk1/2 during cell adhesion. Finally, we found that SRC-1 enhanced ITGA5 promoter activity through an AP-1 (activator protein)-binding site proximal to the transcriptional initiation site; both SRC-1 and c-Jun were recruited to this promoter region in breast cancer cells. These results show that SRC-1 can promote breast cancer metastasis by directly enhancing ITGA5 expression and thus promoting ITGA5-mediated cell adhesion and migration. Therefore, targeting ITGA5 in SRC-1-positive breast cancers may result in inhibition of SRC-1-promoted breast cancer metastasis.

Alterations of steroid receptor coactivator-1 (SRC-1) immunoreactivities in specific brain regions of young and middle-aged female Sprague-Dawley rats.

Previous studies have shown that steroid receptor coactivator-1 (SRC-1) is involved in the regulation of Purkinje cell development and motor learning, neural stem cell differentiation and reproductive-related plasticity. It is widely distributed in the adult brain, but the aging-related changes in the brain remain unclear. In this study age-related alterations of SRC-1 expression in female brain were examined. The results showed that striking age-related decreases of SRC-1 were noticed in those regions related to central regulation of motor (substantia nigra, pontine nuclei, lateral reticular nucleus and Purkinje cells, etc.), learning and memory (olfactory bulb, hippocampus, Purkinje cells, etc.), and neural stem cell (olfactory, dentate gyrus, cerebral cortex, etc.). Surprisingly, although SRC-1 immunopositive materials were predominantly detected in the cell nuclei, they were also detected in the extra-nuclear components predominantly in these motor-regulation sub-regions. The above results showing age-related decrease of SRC-1 in specific motor, learning and memory nuclei suggested its potential roles in neurodegenerative disorders, which may be one of the underlying mechanisms of the vulnerability of the aged brain.

Oestradiol and SERM treatments influence oestrogen receptor coregulator gene expression in human skeletal muscle cells.

AIM: Oestrogen receptors (ER) are present in human skeletal muscle (hSkM) cells; however, the function of the receptor is currently unknown. We investigated the influence of oestradiol and selective ER modulators [tamoxifen (TAM), raloxifene (RAL)] on ER coregulator mRNA expression in hSkM. METHODS: Human skeletal muscle cells were treated with 10 nm oestradiol, 5 microm TAM and 10 microm RAL over a 24-h period. Following the treatment period, mRNA expression was quantified using real-time PCR to detect changes in ER-alpha, ER-beta, steroid receptor coactivator (SRC), silencing mediator for retinoid and thyroid hormone receptors (SMRT), MyoD, GLUT4 and c-fos. RESULTS: ER-alpha mRNA expression increased with all three drug treatments (P < 0.05) while there was no change in mRNA expression of ER-beta in hSkM cells. mRNA expression of SRC increased and SMRT decreased with oestradiol, TAM and RAL in hSkM cells (P < 0.05). Importantly, mRNA expression of MyoD increased with oestradiol and decreased with TAM and RAL in hSkM cells (P < 0.05). mRNA expression of GLUT4 increased with oestradiol and RAL and decreased with TAM in hSkM cells (P < 0.05). CONCLUSIONS: These findings are novel in that they provide the first evidence that oestradiol and selective ER modulators influence ER-alpha function in hSkM cells. This demonstrates the importance of the ER and alterations in its coregulators, to potentially prevent sarcopenia and promote muscle growth in postmenopausal women using these forms of hormone replacement therapy.