MMTV Promoter Research Articles

Abstract 3263: Acceleration of mammary tumorigenesis by PDK1 and peroxisome proliferator-activated receptorδ

Abstract The 3-phosphoinositide-dependant kinase-1 (PDK1) is a master regulator of least 23 members of the AGC protein kinase family, including protein kinase B (PKB/Akt). Genetic alterations which activate PI3K/AKT signaling are common in cancer, and elevated expression of PDK1 has been detected in various cancer types, including breast cancer. To assess the effect of PDK1 over-expression in mammary gland function and tumorigenesis, transgenic mice were generated that express PDK1 in the mammary epithelium under the control of the MMTV promoter. PDK1 over-expression resulted in increased pT308Akt and pS9GSK3β and PPARδ expression, but did not affect the phosphorylation of other targets, such as RSK2, S6K and BAD. Transgenic mice exhibited a moderate acceleration of tumor induction by progestin/DMBA in comparison to wild-type mice, and GW501516 treatment accelerated tumorigenesis in both transgenic and wild-type animals. GW501516 promoted the formation of highly keratinized squamous cell carcinomas in both PDK1 transgenic and wild-type mice, and exhibited a profile of elevated pT308Akt and PPARδ expression. Treatment of wild-type mice with PPARδ agonist GW501516 alone increased PDK1 and pT308Akt expression in mammary epithelium. Thus, MMTV-PDK1 mice provide a model in which PDK1 enhances mammary carcinogenesis through activation of Akt and PPARδ to elicit a cooperative effect in promoting tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3263.

Overexpression of VEGF165b in mouse ovary results in reduced litter size

Angiogenesis in the ovary facilitates oxygen, nutrient and hormone delivery. VEGF, which plays a key role in regulation of ovarian follicle development, is differentially spliced in the terminal exon to form two families of proteins, VEGFxxx (angiogenic) and VEGFxxxb (anti‐angiogenic). To identify the function of VEGF165b in the ovary, nine female transgenic mice (TG) overexpressing VEGF165b under the control of the MMTV promoter and 10 wild type (WT) female littermates were mated with WT mice. RT‐PCR and immunohistochemistry indicated strong expression of VEGF165b in ovaries of TG females. TG mothers produced litters of half the size of WT dams (3.3±0.4 vs. 5.8±0.7 pups/litter), or TG males (6.1±0.7 pups/litter). There was no developmental defect identified in either the WT or TG pups from TG mothers. The number of embryos released into oviducts by TG females were reduced to 82±6% of control mice. 38.5% of TG females had embryos lacked the Cumulus Cell‐Oocyte Complex, while none from WT females. Overexpression of VEGF165b appears to reduce fertility, possibly by its anti‐angiogenic actions on follicular development.Supported by the Wellcome Trust and British Heart Foundation.

Glucocorticoid Receptor Phosphorylation Modulates Transcription Efficacy through GRIP-1 Recruitment

The role of GR phosphorylation in modulating GR-mediated transcription is not fully understood. Here we show that the hGR is rapidly phosphorylated at S211 and S226 in response to the synthetic agonist dexamethasone (dex) in COS-1 cells. Using a triple phosphorylation mutant hGR construct, we demonstrate that phosphorylation at one or more S residues (from S203, S211, and S226) is required for maximal hGR-mediated transcriptional activation on the MMTV promoter in response to dex in COS-1 cells, but that this effect is promoter selective. Phosphorylation at these residues does not affect unliganded or agonist-induced hGR degradation, suggesting that the mechanism whereby hGR phosphorylation at these residues regulates GR-mediated transactivation via a GRE does not involve changes in GR half-life. We have previously shown a direct correlation between efficacy for transactivation and interaction of the hGR with glucocorticoid receptor interacting protein-1 (GRIP-1). Here we show by pull-down assays in the absence and presence of glucocorticoid response elements (GREs) that phosphorylation of the hGR is required for GR-GRIP-1 interaction. Chromatin immunoprecipitation (ChIP) assays revealed that hGR phosphorylation at one or more S residues (from S226, S211, and S203) is required for the recruitment of GRIP-1 to the synthetic MMTV promoter as well as to the endogenous GRE-containing glucocorticoid-induced leucine zipper (GILZ) promoter in intact COS-1 cells, but not for nuclear localization. Our results support the conclusion that phosphorylation at S203, S211, and/or S226 of the hGR is required for a maximal transcriptional response via the synthetic MMTV and endogenous GILZ GREs in COS-1 cells, to enable recruitment of GRIP-1 to the hGR.

Identification of Anabolic Selective Androgen Receptor Modulators with Reduced Activities in Reproductive Tissues and Sebaceous Glands

Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC(50), 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5alpha-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands.

Bacillus anthracis Lethal Toxin Represses MMTV Promoter Activity through Transcription Factors

We have recently shown that the anthrax lethal toxin (LeTx) selectively represses nuclear hormone receptors. In this study, we found that LeTx repressed the activation of the mouse mammary tumor virus promoter related to overexpression of the transcription factors hepatocyte nuclear factor 3, octamer-binding protein 1, and c-Jun. LeTx transcriptional repression was associated with a decrease in the protein levels of these transcription factors in a lethal factor protease activity-dependent manner. Early administration of LeTx antagonists partially or completely abolished the repressive effects of LeTx. In contrast to the rapid cleavage of mitogen-activated protein kinase kinases by LeTx, the degradation of these transcription factors occurred at a relatively late stage after LeTx treatment. In addition, LeTx repressed phorbol-12-myristate-13-acetate-induced mouse mammary tumor virus promoter activity and phorbol 12-myristate 13-acetate induction of endogenous c-Jun protein. Collectively, these findings suggest that transcription factors are intracellular targets of LeTx and expand our understanding of the molecular action of LeTx at a later stage of low-dose exposure.

Overexpression of VEGF165b in mouse ovary results in reduced litter size

Physiological angiogenesis occurs in the ovary, and facilitates oxygen, nutrient, and hormone substrate delivery and transfer of synthesized hormones to targeted cells. VEGF plays a key role in the regulation of ovarian follicle development and corpus luteum formation and maintenance.VEGF is differentially spliced in the terminal exon to form two families of proteins, termed VEGFxxx and VEGFxxxb, where xxx denotes the amino acid number. VEGF165b inhibits VEGF165 induced endothelial cell proliferation, migration, vasodilatation and in vivo angiogenesis,To identify the biological function of VEGF165b in the ovary, nine female transgenic mice (TG) overexpressing VEGF165b under the control of the MMTV promoter and 10 wild type (WT) female littermates were mated with WT C57Bl6 mice. RT‐PCR indicated strong expression of VEGF165b in ovaries of TG females. TG mothers produced litters of half the size of WT dams (3.3±0.4 vs. 5.8±0.7 pups/litter), or TG males (6.1±0.7 pups/litter). There was no developmental defect identified in either the WT or TG pups from TG mothers suggesting subfertility due to defects in the female reproductive system. We have therefore shown that over‐expression of VEGF165b reduces fertility, possibly by its anti‐angiogenic actions on follicular development. This project is supported by the Wellcome Trust and British Heart Foundation.

NA-Seq: A Discovery Tool for the Analysis of Chromatin Structure and Dynamics during Differentiation

It is well established that epigenetic modulation of genome accessibility in chromatin occurs during biological processes. Here we describe a method based on restriction enzymes and next-generation sequencing for identifying accessible DNA elements using a small amount of starting material, and use it to examine myeloid differentiation of primary human CD34+ cells. The accessibility of several classes of cis-regulatory elements was a predictive marker of in vivo DNA binding by transcription factors, and was associated with distinct patterns of histone posttranslational modifications. We also mapped large chromosomal domains with differential accessibility in progenitors and maturing cells. Accessibility became restricted during differentiation, correlating with a decreased number of expressed genes and loss of regulatory potential. Our data suggest that a permissive chromatin structure in multipotent cells is progressively and selectively closed during differentiation, and illustrate the use of our method for the identification of functional cis-regulatory elements.

Receptor activator of NF-kB (RANK) and RANK ligand protein expression in invasive ductal carcinoma.

Abstract Abstract #2057 Purpose: Bone metastases are commonly observed in patients with advanced breast cancer. Tumor cells interact with the bone microenvironment to induce osteoclastogenesis via local bone stromal expression of receptor activator of NF-kB ligand (RANKL), leading to bone destruction and release of growth factors. In addition to its critical role in tumor-induced osteolysis, RANKL has been demonstrated to enhance the invasive behavior of epithelial tumor cells that express RANK, and RANK over-expression in transgenic mice using the breast-specific MMTV promoter results in increased mammary carcinoma. This study assessed the expression of human RANK and its ligand (RANKL) in invasive ductal carcinoma (IDC).
 Methods: We studied a total of 57 IDC specimens. Antibodies against human RANK (AF683) and human RANKL (M366; Amgen) were used for immunohistochemistry (IHC) cell staining along with an isotype control. The specificity of the 2 antibodies was substantiated by IHC, flow cytometry and Western blot analysis of positive and negative control cells. In addition, for RANK, mass spectrometry/protein sequencing of immunoprecipitated proteins was performed. The intensity of IHC staining was scored on a semiquantitative scale (1=weak, 2=moderate, 3=intense) or a complex scale (sum of percentages of stained tumor cells x staining intensity (0-3)).
 Results: Using a complex score with a threshold of 10, 20/57 IDC (35%) expressed minimal level of RANK (range 0 to <10, median 1) while 37/57 IDC (65%) expressed RANK protein with a score ranging from 10 to 100 (median 50). The expression in the tumor was heterogeneous, with cells having no expression and cells having intense expression. In addition, RANK was intensely expressed in the basal layer of all normal ducts and lobules. RANKL was observed in rare tumors (7/57; 11%), in rare lymphocytes of most tumor stroma (42/62; 68%), and in rare fibroblast-like cells in the stroma of rare tumors (3/62; 4.8%).
 Conclusion: In this study, RANK expression was observed by IHC in the majority (65%) of IDC. RANKL expression was observed in the epithelial carcinoma element of some tumors (11%) and was detected in 68% of tumors in rare lymphocytes infiltrating the tumor stroma. Correlation studies between prognosis markers ER, PR, Her2, and CK5/6 expression and RANK or RANKL expression in those IDC have been initiated. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2057.

RANKL inhibition decreases the incidence of mammary adenocarcinomas in wild type (WT) and MMTV-RANK transgenic mice.

Abstract Abstract #4167 Purpose: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of mouse mammary gland during pregnancy. Upon treatment with hormone medroxyprogesterone (MPA) and a carcinogen (DMBA), transgenic mice overexpressing RANK in the mammary gland via the MMTV promoter exhibit a higher incidence of ductular hyperplasias, mammary intraepithelial neoplasias (MIN), and mammary adenocarcinomas than WT mice. This study assessed the expression of mouse RANK and RANKL in WT and MMTV-RANK during mammary tumor progression in this model and determined whether RANKL inhibition could inhibit mammary carcinogenesis.
 Methods: At 6 weeks of age WT and MMTV-RANK transgenic mice were implanted with 90-day release 50mg MPA pellets that were replaced at expiration (weeks 19 and 32). DMBA was administered orally at weeks 9, 10, 12, and 13. The expression of RANK and RANKL was determined by immunohistochemistry (IHC). The specificity of the anti-mouse RANKL and anti-mouse RANK antibodies were verified by their lack of reactivity in RANKL -/- or RANK -/- mouse tissues, respectively. Simultaneous with the first DMBA treatment, mice were treated with RANK-Fc (10 mg/kg, 3x/week) or vehicle and the onset of mammary tumor formation was measured. Mammary tumor formation was determined by palpation and confirmed by histologic examination. Preneoplastic lesions were counted by whole mount analysis of the mammary gland at 4 weeks after the last DMBA treatment. Epithelial proliferation was measured by BrdU labeling of the mammary epithelium at multiple timepoints following the last DMBA treatment (2 days after the first DMBA treatment; 4 and 7 weeks following the last).
 Results: In the MPA/DMBA-induced mammary tumor model, MMTV-RANK mice developed palpable mammary tumors earlier than WT mice with a median onset of 86 days for the MMTV-RANK cohort compared with 132 days for the WT mice. At early timepoints, the number of preneoplastic lesions and mammary epithelial proliferation were greater in MMTV-RANK mice relative to WT. IHC demonstrated that MPA increased the number of RANKL-positive epithelial cells and that RANKL was present in preneoplastic MIN lesions and adenocarcinomas in both WT and MMTV-RANK mice. RANK expression was observed in the epithelial component of hyperplasias, MIN lesions, and adenocarcinomas in both WT and MMTV-RANK mice. Inhibition of RANKL with RANK-Fc substantially decreased the presence of preneoplastic lesions and the proliferation of mammary epithelial cells in MMTV-RANK mice. In addition, treatment of either MMTV-RANK or WT mice with RANK-Fc delayed the onset of palpable mammary tumors. Treatment of the MMTV-RANK mice with RANK-Fc decreased the incidence of mammary adenocarcinoma.
 Conclusion: These data suggest an operative role for RANKL in progesterone-dependent mouse mammary tumor development in both transgenic MMTV-RANK and WT mice and support additional studies to determine whether RANKL inhibition will delay tumor progression and metastases. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4167.

Novel mouse model for studying role of ER-nongenomic actions in breast cancer.

Abstract Abstract #601 Back ground: Estradiol (E2) and estrogen receptor (ER) signaling play a key role in development and progression of breast cancer. ER signaling is complex, involves coregulatory proteins and the status of ER coregulators in tumor cells plays an important role in hormonal responsiveness and tumor progression. In addition, ER also participates in non-genomic signaling events in the cytoplasm, however the significance of non-genomic signaling in mammary tumorigenesis remain unknown. PELP1/MNAR is novel ER coregulator that participates in ER genomic and non-genomic actions. PELP1 expression is deregulated in breast tumors and in a subset of tumors PELP1 is predominantly localized in the cytoplasm. Since PELP1 cytoplamsic localization promotes excessive activation of Src and AKT pathways, we hypothesized that PELP1 mediated excessive activation of ER-nongenomic functions may play a role tumorigenesis. To test this, we have generated MMTV-PELP1cyto TG model that uniquely express PELP1 in the cytoplasm of mammary glands that mimics the pathological situation of PELP1 localization seen breast cancer.
 Methods: As a means of targeting the expression of the PELP1 transgene to the mammary gland, we placed the PELP1cyto cDNA under the control of the MMTV promoter. PELP1 transgene integration was verified by PCR and expression levels by Western and IHC in each founder line. Whole-mount preparations and IHC analysis was performed using Tg and age controlled wild type littermates from different developmental stages. Total protein extracts of mammary gland were used for western blot analysis of nongenomic signaling components.
 Results: Preliminary analysis of mammary gland from PELP-cyto mice showed hyperplasia, increased proliferation as analyzed by PCNA staining. Mammary tumors were observed as early as 32 weeks. No spontaneous mammary tumors were found in the wild type cohort. Pathological analysis revealed that these tumor masses represent full blown mammary adenocarcinomas. Mammary tumors showed excessive activation of nongenomic signaling including activation of Src and AKT pathways. A clear induction of aromatase expression was found in PELP1 tumors compared with the wild-type that showed no aromatase expression in the mammary gland.
 Discussion: We have established and characterized a transgenic mouse model that mimics deregulated ER-nongenomic signaling. Our results suggest that PELP1 is a proto-oncogene and demonstrates its in vivo tumorigenic potential. PELP1 driven tumors are ER+ve, express aromatase, thus provide an interesting in vivo model for studying ER-mediated tumorigenesis and to study effect of local E2 on ER-mediated tumorigenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 601.

Essential Roles of mTOR/Akt Pathway in Aurora-A Cell Transformation

We have recently demonstrated that Aurora-A kinase is a potential oncogene to develop mammary gland tumors in mice, when expressed under MMTV promoter. These tumors contain phosphorylated forms of Akt and mTOR, suggesting that Akt-mTOR pathway is involved in transformed phenotype induced by Aurora-A. In the present studies, we discovered that stable cell lines expressing Aurora-A contain phosphorylation of Akt Ser473 after prolonged passages of cell culture, not in cells of the early period of cell culture. Levels of PTEN tumor suppressor are significantly reduced in these late passage cells at least in part due to increased poly ubiquitination of the protein. Akt-activated Aurora-A cells formed larger colonies in soft agar and are resistant to UV-induced apoptosis. Aurora-A inhibitor, VX-680, can cause cell death of Aurora-A cells in which Akt is not activated. siRNA-mediated depletion of mTOR in those cells resulted in decreased phosphorylation of Akt Ser473, suggesting that TORC2 complex phosphorylates Akt in Aurora-A cells. Treatment of late-passage Aurora-A cells with mTOR inhibitor reduced colony formation in soft agar. These results strongly suggest that commitment of cell transformation by Aurora-A is determined by at least co-activation of Akt/mTOR pathway.

Long-term low level glucocorticoid exposure induces persistent repression in chromatin

Environmental exposure to low concentration hormones can have permanent epigenetic effects in animals and humans. The consequence of long-term low concentration glucocorticoid exposure was investigated in cell culture using glucocorticoid responsive genes organized in alternative chromatin structures. The MMTV promoter is induced by short-term glucocorticoid exposure on either an integrated (normal chromatin) or transient (unstructured chromatin) promoter. Longer hormone treatment causes a transient refractory repression of only the integrated promoter. Exposure to low concentrations of hormone for several passages persistently represses the integrated MMTV and endogenous glucocorticoid responsive promoters. The glucocorticoid receptor cannot bind to persistently repressed promoters. Induction by androgens is also inhibited on the repressed MMTV promoter. Similarly, osmotic stress induction of the endogenous Sgk gene is repressed. Persistent repression by glucocorticoids targets glucocorticoid responsive genes using a chromatin-dependent mechanism that disrupts binding of both GR-dependent and GR-independent transcription complexes.

Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter

Brahma (BRM) and Brahma-related gene 1 (BRG1) are the ATP-dependent catalytic subunits of the SWI/SNF family of chromatin-remodeling complexes. These complexes are involved in essential processes such as cell cycle, growth, differentiation, and cancer. Using imaging approaches in a cell line that harbors tandem repeats of stably integrated copies of the steroid responsive MMTV-LTR (mouse mammary tumor virus-long terminal repeat), we show that BRG1 and BRM are recruited to the MMTV promoter in a hormone-dependent manner. The recruitment of BRG1 and BRM resulted in chromatin remodeling and decondensation of the MMTV repeat as demonstrated by an increase in the restriction enzyme accessibility and in the size of DNA fluorescence in situ hybridization (FISH) signals. This chromatin remodeling event was concomitant with an increased occupancy of RNA polymerase II and transcriptional activation at the MMTV promoter. The expression of ATPase-deficient forms of BRG1 (BRG1-K-R) or BRM (BRM-K-R) inhibited the remodeling of local and higher order MMTV chromatin structure and resulted in the attenuation of transcription. In vivo photobleaching experiments provided direct evidence that BRG1, BRG1-K-R, and BRM chromatin-remodeling complexes have distinct kinetic properties on the MMTV array, and they dynamically associate with and dissociate from MMTV chromatin in a manner dependent on hormone and a functional ATPase domain. Our data provide a kinetic and mechanistic basis for the BRG1 and BRM chromatin-remodeling complexes in regulating gene expression at a steroid hormone inducible promoter.

Androgen receptor acetylation sites differentially regulate gene control

Androgen receptor (AR) function is modulated by post-translational modifications such as acetylation, ubiquitylation, sumoylation, and phosphorylation. Concerning acetylation, three lysines residues located in a consensus KxKK motif of the AR hinge domain have been identified. For a better evaluation of the role of this modification, the activity of AR modified at different acetylation sites was determined by comparing the effects on natural and synthetic promoters. We found that mutation of AR acetylation sites affected both potency and efficacy of androgen-dependent response. Remarkably, elimination of all three acetylation sites was still compatible with strong AR activity on the PSA and MMTV promoters, but not on the Pem promoter. This differential effect was seen at various wild-type (wt) to mutant AR receptor ratios and at changing hormone concentrations. Subcellular localization studies showed that both mutated and wt AR efficiently translocated into the cell nucleus. Plasmid immunoprecipitation revealed comparable binding of both receptor forms to the Pem promoter. The differential effects observed for the Pem promoter were partially due to an androgen response element (ARE) named ARE-1 which was only poorly stimulated by the AR acetylation site mutant. Finally, AR mutants impaired in their N/C interaction elicited intact stimulation of the Pem promoter, suggesting that AR acetylation was not influenced by this inter-domain communication. The promoter-selective effects seen for the AR acetylation site mutants strongly suggest this post-translational modification to be important in the fine-tuning of the effects of androgens on different target genes.

Development of a high sensitivity, nested Q-PCR assay for mouse and human aromatase

Measurement of breast tissue estradiol levels could provide a powerful method to predict the risk of developing breast cancer but obtaining sufficient amounts of tissue from women is difficult from a practical standpoint. Assessment of aromatase in ductal lavage fluid or fine needle aspirates from breast might provide a surrogate marker for tissue estrogen levels but highly sensitive methods would be required. These considerations prompted us to develop an ultra-sensitive, "nested" PCR assay for aromatase which is up to one million fold more sensitive than standard PCR methods. We initially validated this assay using multiple tissues from the aromatase transgenic mouse and found that coefficients of variation for measurement of replicate samples averaged less than 5%. We demonstrated a 60-fold enhancement in aromatase message in the transgenic versus the wild type mouse breast but surprisingly, levels in the transgenic animals were highly variable, ranging from 0.4 to 27 relative units. The variability of aromatase expression in the transgenic breast did not correlate with the degree of breast development and did not appear to relate to hormonal manipulation of the MMTV promoter but probably related to lack of exhaustive inbreeding and mixed zygocity of transgenic animals. Extensive validation in mouse tissues provided confidence regarding the assay in human tissues, since nearly identical methods were used. The human assay was sufficiently sensitive to detect aromatase in a single human JAR (choriocarcinoma) cell, in all breast biopsies measured, and in 7/23 ductal lavage fluids.

Mammary Epithelial-specific Deletion of the Focal Adhesion Kinase Gene Leads to Severe Lobulo-Alveolar Hypoplasia and Secretory Immaturity of the Murine Mammary Gland

Integrin-mediated cell adhesion and signaling is required for mammary gland development and functions. As a major mediator of integrin signaling, focal adhesion kinase (FAK) has been implicated to play a role in the survival, proliferation, and differentiation of mammary epithelial cells in previously studies in vitro. To assess the role of FAK in vivo, we created mice in which FAK is selectively deleted in mammary epithelial cells. The mammary gland FAK conditional knock-out (MFCKO) mice are viable, fertile, and macroscopically indistinguishable from the control littermates. In virgin MFCKO mice, mammary ductal elongation is retarded at 5 weeks of age but reaches the full extent by 8 weeks of age compared with the control mice. However, the MFCKO females are unable to nurse their pups due to severe lobulo-alveolar hypoplasia and secretory immaturity during pregnancy and lactation. Analysis of the mammary epithelial cells in MFCKO mice showed reduced Erk phosphorylation, expression of cyclin D1, and a corresponding decrease in proliferative capability compared with the littermate controls. In addition, phosphorylation of STAT5 and expression of whey acidic protein are significantly reduced in the mammary glands of MFCKO mice, suggesting defective secretory maturation in these mice. Therefore, the combination of the severe lobulo-alveolar hypoplasia and defective secretory differentiation is responsible for the inability of the MFCKO females to nurse their pups. Together, these results provide strong support for a role of FAK in the mammary gland development and function in vivo.

The Mediator subunit MED1/TRAP220 is required for optimal glucocorticoid receptor-mediated transcription activation

The MED1/TRAP220 subunit of the Mediator plays a key role in facilitating ligand-dependent interactions of this multisubunit coactivator complex with nuclear receptors through their ligand binding domains. The isolated MED1/TRAP220 protein previously was shown to interact with glucocorticoid receptor (GR) in a ligand-dependent manner. However, the functional role of MED1/TRAP220, within the context of the entire Mediator, is not well studied in GR-mediated transcription. In this study, we show that GR binds directly to the Mediator complex and that both LXXLL motifs of MED1/TRAP220 contribute to its binding to GR. Furthermore, using a Med1/Trap220−/− mouse embryonic fibroblast (MEF) line that lacks entirely MED1/TRAP220, we show that MED1/TRAP220 enhances GR-mediated transcription from an MMTV promoter based-reporter gene and that mutations in the MED1/TRAP220 LXXLL motifs reduce, but do not eliminate, GR-dependent transcription. An analysis of endogenous genes in Med1/Trap220−/− cells has confirmed a variable MED1/TRAP220 requirement for different GR target genes. Taken together, these findings support the idea that Mediator, at least in part through MED1/TRAP220, plays a coregulatory role in ligand-dependent GR-mediated gene expression.

In vitro assessment of transcriptional activation of the estrogen and androgen receptors of mosquitofish, Gambusia affinis affinis

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates and promote the growth and differentiation of the female reproductive system. Androgens play essential roles in the development and functioning of the vertebrate male reproductive system as well as actively supporting spermatogenesis. Importantly, recent studies suggest that androgens and estrogens have important reproductive roles in both males and females. To understand the molecular mechanisms of estrogen and androgen actions and to evaluate estrogen and androgen receptor–ligand interactions in the mosquitofish, Gambusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ERα; ESR1), ERbeta1 (ERβ1) and ERβ2 from the ovary. Full-length mosquitofish ER (mfER) cDNAs were obtained using cDNA library screening and RACE techniques. Amino acid sequences of mfERs showed over-all homology of 46% (α versus β1), 43% (α versus β2), and 52% (β1 versus β2). We applied the ERE-luciferase reporter assay system to characterize these receptors. In this transient transfection assay system using mammalian cells, the mfER proteins displayed estrogen-dependent activation of transcription. In addition to ERs, the transactivation of mosquitofish ARs (mfARs) previously isolated by our group, were examined using an androgen-responsive MMTV-luciferase assay system. Mosquitofish ARs showed androgen-dependent activation of transcription from the MMTV promoter. These data provide a basic tool allowing future studies examining the receptor–ligand interactions and endocrine disrupting mechanisms in mosquitofish and also expands our knowledge of estrogen and androgen receptor evolution.

Coactivation of the N-terminal Transactivation of Mineralocorticoid Receptor by Ubc9

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.

Converging on the Promoter

Steroid hormones modulate gene transcription through interactions of their receptors with hormone-responsive elements on target genes. Noting that steroid receptors also activate the extracellular signal-regulated kinase (ERK) signaling pathway, Vicent et al . used the T47D-MTVL breast cancer cell line--which carries a chromosomally integrated copy of the luciferase gene under the control of the MMTV-LTR promoter--to investigate the role of ERK signaling in progestin action. Treatment with the progesterone receptor (PR) agonist R5020 led to rapid phosphorylation of ERK1/2, the progesterone receptor-B isoform (PR B ), and the ERK target MSK1 (mitogen- and stress-activated protein kinase 1) as well as to transcriptional activation of the MMTV reporter. Hormone-dependent phosphorylation of PR and MSK1 required ERK activation, as did the hormone-dependent induction of MMTV (which also required MSK1 activation) and other progesterone target genes. R5020 stimulated recruitment to the MMTV promoter of PR and a complex containing phosphorylated PR B , phosphorylated ERK, and phosphorylated MSK1. Furthermore, R5020 elicited a MSK1-dependent increase in phosphorylation on serine 10 of histone H3 of the MMTV promoter, associated with the MSK1-dependent loss of HP1γ (heterochromatin protein 1γ) and recruitment of chromatin-remodeling factors and RNA polymerase II. Thus, activation of the ERK signaling pathway appears to play a critical role in progestin-dependent gene transcription that involves MSK1-dependent phosphorylation of histone H3, enabling the loss of repressive complexes from the promoters of target genes and the recruitment of chromatin-remodeling factors. G. P. Vicent, C. Ballaré, A. S. Nacht, J. Clausell, A. Subtil-Rodríguez, I. Quiles, A. Jordan, M. Beato, Induction of progesterone target genes requires activation of Erk and Msk kinases and phosphorylation of histone H3. Mol. Cell. 24 , 367-381 (2006). [PubMed]