P160 Steroid Receptor Coactivator Family Research Articles

Wound Healing-related Functions of the p160 Steroid Receptor Coactivator Family.

Multicellular organisms have evolved sophisticated mechanisms to recover and maintain original tissue functions following injury. Injury responses require a robust transcriptomic response associated with cellular reprogramming involving complex gene expression programs critical for effective tissue repair following injury. Steroid receptor coactivators (SRCs) are master transcriptional regulators of cell–cell signaling that is integral for embryogenesis, reproduction, normal physiological function, and tissue repair following injury. Effective therapeutic approaches for facilitating improved tissue regeneration and repair will likely involve temporal and combinatorial manipulation of cell-intrinsic and cell-extrinsic factors. Pleiotropic actions of SRCs that are critical for wound healing range from immune regulation and angiogenesis to maintenance of metabolic regulation in diverse organ systems. Recent evidence derived from studies of model organisms during different developmental stages indicates the importance of the interplay of immune cells and stromal cells to wound healing. With SRCs being the master regulators of cell–cell signaling integral to physiologic changes necessary for wound repair, it is becoming clear that therapeutic targeting of SRCs provides a unique opportunity for drug development in wound healing. This review will provide an overview of wound healing–related functions of SRCs with a special focus on cellular and molecular interactions important for limiting tissue damage after injury. Finally, we review recent findings showing stimulation of SRCs following cardiac injury with the SRC small molecule stimulator MCB-613 can promote cardiac protection and inhibit pathologic remodeling after myocardial infarction.

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 ERβ. In PE placenta, the interaction of both ERα and ERβ 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.

Abstract 1565: A novel therapeutic target in lethal prostate cancer in African American males

Abstract Background: The largest US cancer health disparity exists in prostate cancer (PC), with African American (AA) men having a 1.6-fold increased risk of developing PC; younger age and more advanced stage at diagnosis; increased risk for recurrence; and a 2.5-fold increased mortality rate relative to Caucasian American (CA) men. In addition to important socioeconomic and environmental factors, this disparity is linked to incompletely understood genetic and other intrinsic biological factors. Consequently, there is an unmet need to identify actionable targets in AA PC in order to develop more effective therapeutics and, ultimately, improve clinical outcomes for AA PC patients. Rationale/Hypothesis: The p160 Steroid Receptor Coactivator (SRC) family (SRC-1, SRC-2, and SRC-3), are master regulators of transcriptional activity. They cooperate with several oncogenic transcription factors to mediate their gene expression programs, thus representing a critical connecting signaling node in PC. We have hypothesized that the p160 SRCs underlie the higher aggressiveness of AA PC and proposed that the inhibition of the p160 SRCs can target the oncogenic transcriptional programs of AA PC, suppress AA PC cell proliferation, survival, and metastasis, and improve outcomes for AA PC patients. Data from the three p160 SRC knockout mouse models (Src1, Src2, or Src3 knockout mice) show no negative effect on mouse lifespan, suggesting the possibility of a therapeutic window. Experimental approach: We utilized state-of-the-art in vitro assays and multi-Omics datasets to examine these hypotheses and shed light onto the role of the p160 SRCs in the intrinsic biological aggressiveness of AA PC. Recently, our BCM group synthesized an innovative panel of small molecule inhibitors (SMIs) of the p160 SRCs. We examined the efficacy of our novel p160 SRC SMIs against AA PC cell lines and patient-derived xenografts (PDXs). Results: The p160 SRCs exhibit increased activity in AA PC compared to CA PC. They are important regulators of the transcriptional programs that drive AA PC cell proliferation, metabolism, invasion, and metastasis. Importantly, our novel p160 SRC SMIs potently suppress multiple oncogenic signaling programs in AA PC cells and inhibit cell proliferation. They also potently suppress key AA PC driver transcription factors, including AR, SREBP1, and MNX1. The latter two are - so far - undruggable oncogenic transcription factors that our extended BCM group recently discovered to be specifically overexpressed in AA PC. Furthermore, our lead SMI is well tolerated in mice and can inhibit PC cell proliferation and tumor growth in vivo. Conclusions: We propose pharmacologic inhibition of the p160 SRC as an innovative treatment opportunity that will target several signaling pathways simultaneously and improve clinical outcomes in AA PC. Citation Format: Salma Kaochar, Michael Ittmann, Matthew Robertson, Jin Wang, Darlene Skapura, Christel Davis, Erik Ehli, Matthew Freedman, Cristian Coarfa, Bert O'Malley, Nicholas Mitsiades. A novel therapeutic target in lethal prostate cancer in African American males [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1565.

Nuclear Receptor Coactivator 2 Promotes Human Breast Cancer Cell Growth by Positively Regulating the MAPK/ERK Pathway.

As a member of the p160 steroid receptor coactivator (SRC) family, nuclear receptor coactivator 2 (NCOA2) is known to play essential roles in many physiological and pathological processes, including development, endocrine regulation, and tumorigenesis. However, the biological function of NCOA2 in breast cancer is not fully understood. We found that the copy number of the NCOA2 gene was frequently amplified in four breast cancers datasets, varying from 6 to 10%, and the mRNA levels of NCOA2 were also upregulated in 11% of the sequenced cases/patients (TCGA provisional dataset). Next, we confirmed that NCOA2 silencing significantly suppressed cell proliferation in different breast cancer cell lines, by inducing cell cycle arrest and apoptosis. Mechanistically, whole-transcriptome sequencing (RNA-Seq) analysis showed that NCOA2 depletion leads to downregulation of the MAPK/ERK signaling cascade, possibly via downregulating NCOA2's downstream target RASEF. In conclusion, our results suggest NCOA2 as a potential target of therapeutics against breast cancer.

Frequent amplification of AIB1, a critical oncogene modulating major signaling pathways, is associated with poor survival in gastric cancer.

Amplified in breast cancer 1 (AIB1) is a member of p160 steroid receptor coactivator (SRC) family that mediates the transcriptional activities of nuclear receptors and other transcription factors. It acts as a major oncogene in diverse cancers, whereas biological function of AIB1 in gastric cancer remains largely unclear. This study was designed to explore the role of AIB1 in gastric tumorigenesis and its potential as a useful prognostic marker and therapeutic target in this cancer. Our data demonstrated that AIB1 was significantly up-regulated in gastric cancer tissues as compared with control subjects. Moreover, AIB1 amplification was found in 47 of 133 (35.3%) gastric cancer cases, but not in control subjects. AIB1 amplification was positively associated with its protein expression, and was significantly correlated with poor patient survival. AIB1 knockdown in gastric cancer cells dramatically inhibited cell proliferation, invasiveness and tumorigenic potential in nude mice, and induced cell cycle arrest and apoptosis. Mechanically, AIB1 promotes gastric cancer cell proliferation, survival and invasiveness through modulating major signaling pathways such as ErbB and Wnt/β-catenin pathways. Collectively, these findings suggest that AIB1 plays an important role in the pathogenesis of gastric cancer and represents a potential prognostic marker and therapeutic target for this cancer.

Bufalin Is a Potent Small-Molecule Inhibitor of the Steroid Receptor Coactivators SRC-3 and SRC-1

Virtually all transcription factors partner with coactivators that recruit chromatin remodeling factors and interact with the basal transcription machinery. Coactivators have been implicated in cancer cell proliferation, invasion, and metastasis, including the p160 steroid receptor coactivator (SRC) family composed of SRC-1 (NCOA1), SRC-2 (TIF2/GRIP1/NCOA2), and SRC-3 (AIB1/ACTR/NCOA3). Given their broad involvement in many cancers, they represent candidate molecular targets for new chemotherapeutics. Here, we report on the results of a high-throughput screening effort that identified the cardiac glycoside bufalin as a potent small-molecule inhibitor for SRC-3 and SRC-1. Bufalin strongly promoted SRC-3 protein degradation and was able to block cancer cell growth at nanomolar concentrations. When incorporated into a nanoparticle delivery system, bufalin was able to reduce tumor growth in a mouse xenograft model of breast cancer. Our work identifies bufalin as a potentially broad-spectrum small-molecule inhibitor for cancer.

CAMP Response Element-Binding Protein Interacts With and Stimulates the Proteasomal Degradation of the Nuclear Receptor Coactivator GRIP1

The glucocorticoid receptor interacting protein (GRIP1) belongs to the p160 steroid receptor coactivator family that plays essential roles in nuclear receptor-dependent transcriptional regulation. Previously, we reported that the cAMP-dependent protein kinase (PKA) induces ubiquitination leading to degradation of GRIP1. Here we show that the cAMP response element-binding protein (CREB) downregulates GRIP1 and is necessary for the PKA-stimulated degradation of GRIP1, which leads to changes in the expression of a subset of genes regulated by estrogen receptor-α in MCF-7 breast cancer cells. Our data of domain-mapping and ubiquitination analyses suggest that CREB promotes the proteasomal breakdown of ubiquitinated GRIP1 through 2 functionally independent protein domains containing amino acids 347 to 758 and 1121 to 1462. We provide evidence that CREB interacts directly with GRIP1 and that CREB Ser-133 phosphorylation or transcriptional activity is not required for GRIP1 interaction and degradation. The basic leucine zipper domain (bZIP) of CREB is important for the interaction with GRIP1, and deletion of this domain led to an inability to downregulate GRIP1. We propose that CREB mediates the PKA-stimulated degradation of GRIP1 through protein-protein interaction and stimulation of proteasomal degradation of ubiquitinated GRIP1.

Research Resource: Tissue- and Pathway-Specific Metabolomic Profiles of the Steroid Receptor Coactivator (SRC) Family

The rapidly growing family of transcriptional coregulators includes coactivators that promote transcription and corepressors that harbor the opposing function. In recent years, coregulators have emerged as important regulators of metabolic homeostasis, including the p160 steroid receptor coactivator (SRC) family. Members of the SRC family have been ascribed important roles in control of gluconeogenesis, fat absorption and storage in the liver, and fatty acid oxidation in skeletal muscle. To provide a deeper and more granular understanding of the metabolic impact of the SRC family members, we performed targeted metabolomic analyses of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice with global knockouts (KOs) of SRC-1, SRC-2, or SRC-3. We measured amino acids, acyl carnitines, and organic acids in five tissues with key metabolic functions (liver, heart, skeletal muscle, brain, plasma) isolated from SRC-1, -2, or -3 KO mice and their wild-type littermates under fed and fasted conditions, thereby unveiling unique metabolic functions of each SRC. Specifically, SRC-1 ablation revealed the most significant impact on hepatic metabolism, whereas SRC-2 appeared to impact cardiac metabolism. Conversely, ablation of SRC-3 primarily affected brain and skeletal muscle metabolism. Surprisingly, we identified very few metabolites that changed universally across the three SRC KO models. The findings of this Research Resource demonstrate that coactivator function has very limited metabolic redundancy even within the homologous SRC family. Furthermore, this work also demonstrates the use of metabolomics as a means for identifying novel metabolic regulatory functions of transcriptional coregulators.

The role of AIB1 in breast cancer

Amplified in breast cancer 1 (AIB1) is a member of the p160 steroid receptor coactivator family that mediates the transcriptional activities of nuclear receptors including estrogen receptor (ER) and progesterone receptor (PR), as well as certain other transcription factors, including E2F1 and p53. AIB1 is widely implicated in nuclear receptor-mediated diseases, particularly malignant diseases, including breast, prostate, gastric and pancreatic cancers. AIB1 was initially implicated in hormone-dependent breast cancer, where increasing levels of AIB1 mRNA and protein were detected in some of these specimens and the overexpression of AIB1 in mice led to an increased incidence of tumors. More recent studies revealed that AIB1 also affects the growth of hormone-independent breast cancer via signaling pathways such as those of E2F1, IGF-I, EGF and PI3K/Akt/mTOR. The pleiotropic effect of AIB1 and the roles it plays in both normal development and cancer have presented a great challenge to formulating an effective therapeutic strategy for breast cancer. In this review, we highlight the significant progress made with the recent findings and present an overview of the current understanding of the influence of AIB1 on breast cancer via hormone-dependent and -independent signaling pathways.

Expression of AIB1 protein as a prognostic factor in breast cancer

BackgroundAIB1 (amplified in breast cancer I) is a member of the p160 steroid receptor coactivator family. AIB1 is frequently overexpressed in breast cancer and has functions that promote oncogenesis that are independent of estrogen receptor (ER) coactivation. We investigated prognostic significance of AIB1 and relationship between AIB1 and ER, progesterone receptor (PR), androgen receptor (AR), DAX-1, and HER2.MethodsRNA in situ hybridization (ISH) and immunohistochemical (IHC) staining for AIB1, IHC staining for ER and the progesterone receptor (PR) and IHC staining and silver in situ hybridization (SISH) for HER2 were performed for 185 breast cancer cases.ResultsA high level of expression of AIB1 mRNA was observed in 60.0% of tumors. IHC analysis detected AIB1 positivity in 47.3% of tumors, which did not correlate with AIB1 mRNA expression (p = 0.24, r = 0.10). AIB1 protein expression correlated with AR and DAX-1 expression (p = 0.01, r = 0.22 and p = 0.02, r = 0.21, respectively) but not with ER or PR expression (p = 0.14, r = -0.13 and p = 0.16, r = -0.12, respectively). AIB1 protein expression correlated with the amplification of the HER2 gene (p = 0.03, r = 0.19). In contrast to AIB1 protein expression, AIB1 mRNA expression did not correlate with AR, DAX-1, ER, and PR expression, and the amplification of the HER2 gene (p > 0.05 for all).There were trends that strong AIB1 protein expression correlated with poorer disease free survival (p = 0.07). Strong AIB1 protein expression correlated with poorer overall survival (p = 0.04). Among the ER-negative subgroup, strong AIB1 protein expression correlated with poorer disease free survival and overall survival (p = 0.01 and p < 0.01, respectively).ConclusionsStrong AIB1 protein expression was poor prognostic factor in breast cancer, especially in ER-negative breast cancers. Further investigation is essential to determine whether AIB1 might be effective therapeutic targets for ER-negative breast cancers.

SRC-3 Has a Role in Cancer Other Than as a Nuclear Receptor Coactivator

Steroid receptor coactivator-3 (SRC-3), also known as AIB1, is a member of the p160 steroid receptor coactivator family. Since SRC-3 was found to be amplified in breast cancer in 1997, the role of SRC-3 in cancer has been broadly investigated. SRC-3 initially was identified as a transcriptional coactivator for nuclear receptors such as the estrogen receptor (ER), involved in the proliferation of hormone-dependent cancers. However, increasing clinical evidence shows that dysregulation of SRC-3 expression in several human hormone-independent cancers is correlated with pathological factors and clinical prognosis. Recently, both in vivo and in vitro studies demonstrate that SRC-3 may influence a number of cancer cellular processes in several ways independent of nuclear receptor signaling. In addition, an SRC-3 transgenic mice model shows that SRC-3 induces tumors in several mouse tissues. These results indicate that the role of SRC-3 in cancer is not just as a nuclear receptor coactivator. The focus of this review is to examine possible SRC-3 roles in cancer, other than as a nuclear receptor coactivator.

Characterization of steroid receptor coactivator in sea urchin, Strongylocentrotus nudus, and its involvement in embryonic development

Ligand-bound nuclear receptors (NRs) recruit coactivators such as members of the p160 steroid receptor coactivator (SRC) family and cyclic AMP responsive element binding protein (CREB)-binding protein (CBP) to specific enhancer elements and activate target gene transcription. In the present study, we isolated a novel SRC from the sea urchin Strongylocentrotus nudus (SnSRC) by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. The SnSRC and vertebrate SRCs are different in size but share the overall characteristic domains, such as NR interacting domain (NID), CBP-binding and glutamine-rich regions. SnSRC mRNA showed highest expression levels at the 32-cell, 64-cell and pluteus larval stages. Full-length SnSRC (1992 amino acids) interacted with several NRs, including sea urchin estrogen receptor-related receptor (ERR), human and masu salmon estrogen receptors (ERα), mouse ERRγ, rat glucocorticoid receptor α, and rat thyroid receptor β. The SnSRC possesses two functional NIDs, both of which are dependent on their core LxxLL motifs. Furthermore, preferential interacting domains for ERα in the SnSRC are located in the central LxxLL motifs, revealed by the truncation and mutagenesis studies. Strikingly, the SnSRC has a single transcription activation domain, which interacts with CBP, a transcriptional integrator. In addition, transient knockdown of the SnSRC gene in the sea urchin embryo using morpholino antisense RNA induced abnormal phenotypes at gastrulation stage such as the lack of primary invagitation and exogastrulation. These results suggest that the SnSRC is a new member of the SRC family and plays an important role during early embryonic development.

Normal and cancer-related functions of the p160 steroid receptor co-activator (SRC) family

The three homologous members of the p160 SRC family (SRC1, SRC2 and SRC3) mediate the transcriptional functions of nuclear receptors and other transcription factors, and are the most studied of all the transcriptional co-activators. Recent work has indicated that the SRCgenes are subject to amplification and overexpression in various human cancers. Some of the molecular mechanisms responsible for SRC overexpression, along with the mechanisms by which SRCs promote breast and prostate cancer cell proliferation and survival, have been identified, as have the specific contributions of individual SRC family members to spontaneous breast and prostate carcinogenesis in genetically manipulated mouse models. These studies have identified new challenges for cancer research and therapy.

Unique Roles of p160 Coactivators for Regulation of Breast Cancer Cell Proliferation and Estrogen Receptor-α Transcriptional Activity

Each of the three members of the p160 steroid receptor coactivator (SRC) family of coactivators (SRC-1, SRC-2 and SRC-3) stimulates estrogen receptor (ER)-alpha function in trans-activation assays. Consequently, we sought to elucidate their contributions to the ER-regulated processes of cell proliferation, apoptosis, and the expression of ERalpha target genes in MCF-7 breast cancer cells. The small interfering RNA depletion of SRC-2 or SRC-3 but not SRC-1 inhibited growth of MCF-7 cells, and this was reflected in decreased cell cycle progression and increased apoptosis in SRC-2- or SRC-3-depleted cells as well as a reduction in ERalpha transcriptional activity measured on a synthetic reporter gene. However, only SRC-3 depletion blocked estradiol stimulated cell proliferation. Depletion of SRC-1 did not affect these events, and together this reveals functional differences between each of the three SRC family coactivators. Regulation of the endogenous ERalpha target gene, c-myc was not affected by depletion of any of the p160 coactivators although depletion of each of them decreased pS2 mRNA expression in estradiol-treated MCF-7 cells. Moreover, progesterone receptor and cyclin D1 gene expression were decreased in SRC-3 small interfering RNA-treated cells. Expression of mRNA and protein levels for the antiapoptotic gene, Bcl-2 was dependent on SRC-3 expression, whereas Bcl-2 protein but not mRNA expression also was sensitive to SRC-1 depletion. Together these data indicate that the closely related p160 coactivators are not functionally redundant in breast cancer cells because they play gene-specific roles in regulating mRNA and protein expression, and they therefore are likely to make unique contributions to breast tumorigenesis.

Coactivators PGC-1β and SRC-1 Interact Functionally to Promote the Agonist Activity of the Selective Estrogen Receptor Modulator Tamoxifen

PGC-1beta is a transcriptional coactivator that enhances strongly and in a hormone-dependent manner the activity of the estrogen receptor alpha (ERalpha) while having only weak effects on similar steroid hormone receptors, such as ERbeta or the glucocorticoid receptor. Notably, PGC-1beta enhances ERalpha transcriptional activity not only in response to agonist ligands, such as estradiol, but also to selective ER modulators, such as tamoxifen. Here, we dissect the molecular mechanisms underlying the ability of PGC-1beta to act selectively on ERalpha and to promote the agonist activity of tamoxifen. We show that receptor selectivity is achieved by PGC-1beta interactions with not just the ligand binding domain (LBD), which is highly conserved among nuclear receptors, but also the N-terminal domain and the hinge/AF-2a region of ERalpha, which are less well conserved. PGC-1beta interacts directly with the hinge/AF-2a and LBD regions but indirectly and via the coactivator SRC-1 with the N-terminal domain. The three ERalpha surfaces and SRC-1 collectively enable efficient coactivation by PGC-1beta. Similar ERalpha surfaces and interactions enable PGC-1beta to coactivate transcription by tamoxifen-bound ERalpha. Surprisingly, PGC-1beta coactivation of tamoxifen-bound ERalpha depends partially on one of the LXXLL motifs of PGC-1beta and on Lys(362) of the ERalpha LBD (i.e. surfaces implicated in agonist-dependent interactions). Our findings suggest that tamoxifen-induced changes in the ERalpha LBD promote interactions with the coactivator PGC-1beta, which then cooperates with SRC-1 to enable tamoxifen agonism.

Proteomic Analysis of Steady-State Nuclear Hormone Receptor Coactivator Complexes

We report our initial efforts in the analysis of endogenous nuclear receptor coactivator complexes as a research bridging strand of the Nuclear Receptor Signaling Atlas (NURSA) (www.NURSA.org). A proteomic approach is used to systematically isolate a variety of coactivator complexes using HeLa cells as a model cell line and to identify the coactivator-associated proteins with mass spectrometry. We have isolated and identified seven coactivator complexes including the p160 steroid receptor coactivator family, cAMP response element binding protein-binding protein, p300, coactivator of activating protein-1 and estrogen receptors, and E6 papillomavirus-associated protein. The newly identified coactivator-associated proteins provide unbiased clues and links for understanding of the endogenous hormone receptor coregulator network and its regulation. We hope that the electronic availability of these data to the general scientific community will facilitate generation and testing of new hypotheses to further our understanding of nuclear receptor signaling and coactivator functions.

The DNA mismatch repair gene hMSH2 is a potent coactivator of oestrogen receptor α

The DNA mismatch repair gene is a key regulator in the elimination of base–base mismatches and insertion/deletion loops (IDLs). Human MutS homologue 2 (hMSH2), originally identified as a human homologue of the bacterial MutS, is a tumour suppressor gene frequently mutated in hereditary nonpolyposis colorectal cancer. Hereditary nonpolyposis colorectal cancer is characterised by the early onset of colorectal cancer and the development of extracolonic cancers such as endometrial, ovarian, and urological cancers. Oestrogen receptor (ER) α and β are members of a nuclear receptor (NR) superfamily. Ligand-dependent transcription of ER is regulated by the p160 steroid receptor coactivator family, the thyroid hormone receptor-associated proteins/the vitamin D receptor-interacting proteins (TRAP/DRIP) mediator complex, and the TATA box-binding protein (TBP)-free TBP associated factor complex (TFTC) type histone acetyltransferase complex. Here, we report the interaction between ER α/β and hMSH2. Immunoprecipitation and glutathione-S-transferase pulldown assay revealed that ER α and hMSH2 interacted in a ligand-dependent manner, whereas ER β and hMSH2 interacted in a ligand-independent manner. Oestrogen receptor α/β bound to hMSH2 through the hMSH3/hMSH6 interaction domain of hMSH2. In a transient expression assay, hMSH2 potentiated the transactivation function of liganded ER α, but not that of ER β. These results suggest that hMSH2 may play an important role as a putative coactivator in ER α dependent gene expression.

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in VDR transactivation during keratinocyte differentiation

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D (1,25(OH) 2D 3), controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the Vitamin D receptor (VDR) interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the Vitamin D receptor. However, the relationship between these two classes of coactivators is not clear. Using GST–VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP205 and SRC-3 potentiated Vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are differentially involved in Vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in VDR transactivation during keratinocyte differentiation*1

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D3), controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the Vitamin D receptor (VDR) interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the Vitamin D receptor. However, the relationship between these two classes of coactivators is not clear. Using GST–VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP205 and SRC-3 potentiated Vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are differentially involved in Vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Concerted Activation of ETS Protein ER81 by p160 Coactivators, the Acetyltransferase p300 and the Receptor Tyrosine Kinase HER2/Neu

Activator of thyroid and retinoic acid receptor (ACTR) is overexpressed in approximately 60% of primary human breast tumors and belongs to the p160 steroid receptor coactivator family. In this study, we identified a novel interaction partner of ACTR, the ETS transcription factor ER81 that is also heavily implicated in mammary tumor formation. ACTR and related p160 family members (steroid receptor coactivator-1 and glucocorticoid receptor-interacting protein-1 (GRIP-1)) augment ER81-mediated transcription. Although ACTR and GRIP-1 can acetylate ER81, this posttranslational modification of ER81 is not required for its stimulation by ACTR or GRIP-1. In addition, ACTR collaborates with the p300 coactivator, a joint interaction partner of ACTR and ER81, to stimulate ER81 function and the ability of p300 to acetylate ER81 is indispensable for this collaboration. Furthermore, the receptor tyrosine kinase HER2/Neu, an oncoprotein particularly found overexpressed in breast tumors, cooperates with both ACTR and p300 to stimulate ER81-mediated transcription. Thus, oncogenic HER2/Neu and ACTR may synergize to orchestrate mammary tumorigenesis through the dysregulation of the transcription factor ER81 and its target genes.