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1.
J Mol Cell Cardiol ; 194: 70-84, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38969334

RESUMEN

We recently discovered that steroid receptor coactivators (SRCs) SRCs-1, 2 and 3, are abundantly expressed in cardiac fibroblasts (CFs) and their activation with the SRC small molecule stimulator MCB-613 improves cardiac function and dramatically lowers pro-fibrotic signaling in CFs post-myocardial infarction. These findings suggest that CF-derived SRC activation could be beneficial in the mitigation of chronic heart failure after ischemic insult. However, the cardioprotective mechanisms by which CFs contribute to cardiac pathological remodeling are unclear. Here we present studies designed to identify the molecular and cellular circuitry that governs the anti-fibrotic effects of an MCB-613 derivative, MCB-613-10-1, in CFs. We performed cytokine profiling and whole transcriptome and proteome analyses of CF-derived signals in response to MCB-613-10-1. We identified the NRF2 pathway as a direct MCB-613-10-1 therapeutic target for promoting resistance to oxidative stress in CFs. We show that MCB-613-10-1 promotes cell survival of anti-fibrotic CFs exposed to oxidative stress by suppressing apoptosis. We demonstrate that an increase in HMOX1 expression contributes to CF resistance to oxidative stress-mediated apoptosis via a mechanism involving SRC co-activation of NRF2, hence reducing inflammation and fibrosis. We provide evidence that MCB-613-10-1 acts as a protectant against oxidative stress-induced mitochondrial damage. Our data reveal that SRC stimulation of the NRF2 transcriptional network promotes resistance to oxidative stress and highlights a mechanistic approach toward addressing pathologic cardiac remodeling.


Asunto(s)
Fibroblastos , Miocardio , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Transducción de Señal , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Miocardio/metabolismo , Miocardio/patología , Apoptosis/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Fibrosis , Hemo-Oxigenasa 1/metabolismo , Hemo-Oxigenasa 1/genética , Ratas , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Ratones
2.
Nat Commun ; 15(1): 1947, 2024 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-38431630

RESUMEN

Cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor's transcriptional output. However, the feed-forward mechanisms that shape cell-type-specific transcriptional fulcrums for steroid receptors are unidentified. Herein, we found that a common feed-forward mechanism between GREB1 and steroid receptors regulates the differential effect of GREB1 on steroid hormones in a physiological or pathological context. In physiological (receptive) endometrium, GREB1 controls P4-responses in uterine stroma, affecting endometrial receptivity and decidualization, while not affecting E2-mediated epithelial proliferation. Of mechanism, progesterone-induced GREB1 physically interacts with the progesterone receptor, acting as a cofactor in a positive feedback mechanism to regulate P4-responsive genes. Conversely, in endometrial pathology (endometriosis), E2-induced GREB1 modulates E2-dependent gene expression to promote the growth of endometriotic lesions in mice. This differential action of GREB1 exerted by a common feed-forward mechanism with steroid receptors advances our understanding of mechanisms that underlie cell- and tissue-specific steroid hormone actions.


Asunto(s)
Endometriosis , Proteínas de Neoplasias , Receptores de Esteroides , Animales , Femenino , Humanos , Ratones , Endometriosis/genética , Endometriosis/metabolismo , Endometrio/metabolismo , Estrógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Progesterona/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Esteroides/metabolismo
3.
Proc Natl Acad Sci U S A ; 120(23): e2221707120, 2023 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-37253006

RESUMEN

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were "permanently eradicated" in a genetically engineered tamoxifen-inducible Treg-cell-specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the chemokine (C-C motif) ligand (Ccl) 19/Ccl21/chemokine (C-C motif) receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C motif chemokine ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and natural killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish preestablished breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3-deleted Tregs represents an approach to completely block tumor growth and recurrence without the autoimmune side effects that typically accompany immune checkpoint modulators.


Asunto(s)
Neoplasias de la Mama , Neoplasias Mamarias Animales , Coactivador 3 de Receptor Nuclear , Animales , Femenino , Masculino , Ratones , Ligandos , Ratones Noqueados , Coactivador 3 de Receptor Nuclear/genética , Linfocitos T Reguladores , Tamoxifeno/farmacología
4.
bioRxiv ; 2023 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-37034717

RESUMEN

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were 'permanently eradicated' in a genetically engineered tamoxifen-inducible Treg-cell specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the Chemokine (C-C motif) ligand (Ccl) 19/Ccl21/ Chemokine (C-C motif) Receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C Motif Chemokine Ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and Natural Killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish pre-established breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3 deleted Tregs represents a novel approach to completely block tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators. Significance statement: Tregs are essential in restraining immune responses for immune homeostasis. SRC-3 is a pleiotropic coactivator, the second-most highly expressed transcriptional coactivator in Tregs, and a suspect in Treg function. The disruption of SRC-3 expression in Tregs leads to a 'complete lifetime eradication' of tumors in aggressive syngeneic breast cancer mouse models because deletion of SRC-3 alters the expression of a wide range of key genes involved in efferent and afferent Treg signaling. SRC-3KO Tregs confer this long-lasting protection against cancer recurrence in mice without an apparent systemic autoimmune pathological phenotype. Therefore, treatment with SRC-3 deleted Tregs could represent a novel and efficient future target for eliminating tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators.

5.
Hum Mol Genet ; 32(13): 2152-2161, 2023 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-37000005

RESUMEN

SOX7 is a transcription factor-encoding gene located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with ventricular septal defects (VSDs). We have previously shown that Sox7-/- embryos die of heart failure around E11.5. Here, we demonstrate that these embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. Ablation of Sox7 in the endocardium also resulted in hypocellular endocardial cushions, and we observed VSDs in rare E15.5 Sox7flox/-;Tie2-Cre and Sox7flox/flox;Tie2-Cre embryos that survived to E15.5. In atrioventricular explant studies, we showed that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). RNA-seq studies performed on E9.5 Sox7-/- heart tubes revealed severely reduced Wnt4 transcript levels. Wnt4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of Bmp2 in the myocardium. Both WNT4 and BMP2 have been previously implicated in the development of VSDs in individuals with 46,XX sex reversal with dysgenesis of kidney, adrenals and lungs (SERKAL) syndrome and in individuals with short stature, facial dysmorphism and skeletal anomalies with or without cardiac anomalies 1 (SSFSC1) syndrome, respectively. We now show that Sox7 and Wnt4 interact genetically in the development of VSDs through their additive effects on endocardial cushion development with Sox7+/-;Wnt4+/- double heterozygous embryos having hypocellular endocardial cushions and perimembranous and muscular VSDs not seen in their Sox7+/- and Wnt4+/- littermates. These results provide additional evidence that SOX7, WNT4 and BMP2 function in the same pathway during mammalian septal development and that their deficiency can contribute to the development of VSDs in humans.


Asunto(s)
Cardiopatías Congénitas , Defectos del Tabique Interventricular , Animales , Ratones , Endocardio/metabolismo , Corazón , Cardiopatías Congénitas/genética , Defectos del Tabique Interventricular/genética , Defectos del Tabique Interventricular/metabolismo , Miocardio/metabolismo , Factores de Transcripción SOXF/metabolismo
6.
Biol Reprod ; 105(2): 305-316, 2021 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-34037700

RESUMEN

Current first-line treatment of patients with high-grade serous ovarian cancer (HGSOC) involves the use of cytotoxic drugs that frequently lead to recurrent tumors exhibiting increased resistance to the drugs and poor patient survival. Strong evidence is accumulating to show that HGSOC tumors and cell lines contain a subset of cells called polyploidy giant cancer cells (PGCCs) that act as stem-like, self-renewing cells. These PGCCs appear to play a key role in tumor progression by generating drug-resistant progeny produced, in part, as a consequence of utilizing a modified form of mitosis known as endoreplication. Thus, developing drugs to target PGCCs and endoreplication may be an important approach for reducing the appearance of drug-resistant progeny. In the review, we discuss newly identified regulatory factors that impact mitosis and which may be altered or repurposed during endoreplication in PGCCs. We also review recent papers showing that a single PGCC can give rise to tumors in vivo and spheroids in culture. To illustrate some of the specific features of PGCCs and factors that may impact their function and endoreplication compared to mitosis, we have included immunofluorescent images co-localizing p53 and specific mitotic regulatory, phosphoproteins in xenografts derived from commonly used HGSOC cell lines.


Asunto(s)
Células Gigantes/fisiología , Neoplasias Ováricas/genética , Poliploidía , Animales , Femenino , Humanos , Ratones , Mitosis
7.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-34006643

RESUMEN

HER2-positive (HER2+) breast cancers (BrCs) contain approximately equal numbers of ERα+HER2+ and ERα-HER2+ cases. An enduring obstacle is the unclear cell lineage-related characteristics of these BrCs. Although ERα+HER2+ BrCs could lose ERα to become ERα-HER2+ BrCs, direct evidence is missing. To investigate ERα dependencies and their implications during BrC growth and metastasis, we generated ERαCreRFP-T mice that produce an RFP-marked ERα+ mammary gland epithelial cell (MGEC) lineage. RCAS virus-mediated expression of Erbb2, a rodent Her2 homolog, first produced comparable numbers of ERα+RFP+Erbb2+ and ERα-RFP-Erbb2+ MGECs. Early hyperplasia developed mostly from ERα+RFP+Erbb2+ cells and ERα-RFP-Erbb2+ cells in these lesions were rare. The subsequently developed ductal carcinomas in situ had 64% slow-proliferating ERα+RFP+Erbb2+ cells, 15% fast-proliferating ERα-RFP+Erbb2+ cells derived from ERα+RFP+Erbb2+ cells, and 20% fast-proliferating ERα-RFP-Erbb2+ cells. The advanced tumors had mostly ERα-RFP+Erbb2+ and ERα-RFP-Erbb2+ cells and only a very small population of ERα+RFP+Erbb2+ cells. In ERα-RFP+Erbb2+ cells, GATA3 and FoxA1 decreased expression and ERα promoter regions became methylated, consistent with the loss of ERα expression. Lung metastases consisted of mostly ERα-RFP+Erbb2+ cells, a few ERα-RFP-Erbb2+ cells, and no ERα+RFP+Erbb2+ cells. The high metastatic capacity of ERα-RFP+Erbb2+ cells was associated with ERK1/2 activation. These results show that the slow-proliferating, nonmetastatic ERα+RFP+Erbb2+ cells progressively lose ERα during tumorigenesis to become fast-proliferating, highly metastatic ERα-RFP+Erbb2+ cells. The ERα-Erbb2+ BrCs with an ERα+ origin are more aggressive than those ERα-Erbb2+ BrCs with an ERα- origin, and thus, they should be distinguished and treated differently in the future.


Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Intraductal no Infiltrante/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Receptor ErbB-2/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/secundario , Línea Celular Tumoral , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Proliferación Celular , Transformación Celular Neoplásica , Receptor alfa de Estrógeno/metabolismo , Femenino , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Invasividad Neoplásica , Regiones Promotoras Genéticas , Receptor ErbB-2/metabolismo , Transducción de Señal , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Sci Signal ; 13(652)2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-33023986

RESUMEN

Differences in the relative abundances of the progesterone receptor (PGR) isoforms PGRA and PGRB are often observed in women with reproductive tract cancers. To assess the importance of the PGR isoform ratio in the maintenance of the reproductive tract, we generated mice that overexpress PGRA or PGRB in all PGR-positive tissues. Whereas few PGRA-overexpressing mice developed reproductive tract tumors, all PGRB-overexpressing mice developed ovarian neoplasms that were derived from ovarian luteal cells. Transcriptomic analyses of the ovarian tumors from PGRB-overexpressing mice revealed enhanced AKT signaling and a gene expression signature similar to those of human ovarian and endometrial cancers. Treating PGRB-overexpressing mice with the PGR antagonist RU486 stalled tumor growth and decreased the expression of cell cycle-associated genes, indicating that tumor growth and cell proliferation were hormone dependent in addition to being isoform dependent. Analysis of the PGRB cistrome identified binding events at genes encoding proteins that are critical regulators of mitotic phase entry. This work suggests a mechanism whereby an increase in the abundance of PGRB relative to that of PGRA drives neoplasia in vivo by stimulating cell cycling.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Hormonas/metabolismo , Neoplasias Ováricas/genética , Receptores de Progesterona/genética , Transcriptoma/genética , Animales , Proliferación Celular/genética , Modelos Animales de Enfermedad , Estradiol/sangre , Estradiol/metabolismo , Femenino , Hormonas/sangre , Humanos , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Neoplasias Ováricas/metabolismo , Progesterona/sangre , Progesterona/metabolismo , Receptores de Progesterona/metabolismo
9.
J Steroid Biochem Mol Biol ; 189: 283-290, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30711473

RESUMEN

Mouse studies support a role for endometrial early growth response 1 (EGR1) in uterine receptivity and decidualization, which are processes controlled by estrogen and progesterone. However, the importance of this transcription factor in similar cellular processes in human is unclear. Analysis of clinical samples indicate that endometrial EGR1 levels are decreased in the endometrium of women with recurrent implantation failure, suggesting that tight control of EGR1 levels are necessary for normal endometrial function. Therefore, we used siRNA-mediated knockdown of EGR1 expression in cultured human endometrial stromal cells (hESCs) to assess the functional role of EGR1 in hESC decidualization. Protein expression studies revealed that EGR1 is highly expressed in pre-decidual hESCs. However, EGR1 protein levels rapidly decrease following administration of an established deciduogenic hormone stimulus containing estradiol, medroxyprogesterone acetate, and cyclic adenosine monophosphate. Intriguingly, EGR1 knockdown in pre-decidual hESCs blocks the ability of these cells to decidualize later, indicating that EGR1 is required to transcriptionally program pre-decidual hESCs for decidualization. Support for this proposal comes from the analysis of transcriptome and cistrome datasets, which shows that EGR1 target genes are primarily involved in transcriptional regulation, cell signaling, and proliferation. Collectively, our studies provide translational support for an evolutionary conserved role for human endometrial stromal EGR1 in the early events of pregnancy establishment.


Asunto(s)
Decidua/citología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Endometrio/citología , Células del Estroma/citología , Animales , Células Cultivadas , Decidua/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Implantación del Embrión , Endometrio/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Embarazo , Células del Estroma/metabolismo , Activación Transcripcional
10.
PLoS Genet ; 14(11): e1007787, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30452456

RESUMEN

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, ß-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.


Asunto(s)
Implantación del Embrión/genética , Implantación del Embrión/fisiología , Endometrio/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Animales , Núcleo Celular/metabolismo , Polaridad Celular/genética , Polaridad Celular/fisiología , Decidua/fisiología , Endometrio/citología , Femenino , Proteína Forkhead Box O1/deficiencia , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Embarazo , Receptores de Progesterona/deficiencia , Transducción de Señal
11.
Nat Commun ; 9(1): 4421, 2018 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-30356064

RESUMEN

Mammalian pregnancy depends on the ability of the uterus to support embryo implantation. Previous studies reveal the Sox17 gene as a downstream target of the Pgr-Gata2-dependent transcription network that directs genomic actions in the uterine endometrium receptive for embryo implantation. Here, we report that ablating Sox17 in the uterine epithelium impairs leukemia inhibitory factor (LIF) and Indian hedgehog homolog (IHH) signaling, leading to failure of embryo implantation. In vivo deletion of the SOX17-binding region 19 kb upstream of the Ihh locus by CRISPR-Cas technology reduces Ihh expression specifically in the uterus and alters proper endometrial epithelial-stromal interactions, thereby impairing pregnancy. This SOX17-binding interval is also bound by GATA2, FOXA2, and PGR. This cluster of transcription factor binding is common in 737 uterine genes and may represent a key regulatory element essential for uterine epithelial gene expression.


Asunto(s)
Proteínas HMGB/metabolismo , Factores de Transcripción SOXF/metabolismo , Útero/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Endometrio/metabolismo , Femenino , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Proteínas HMGB/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Inhibidor de Leucemia/genética , Factor Inhibidor de Leucemia/metabolismo , Ratones , Factores de Transcripción SOXF/genética , Transcriptoma/genética
12.
Reproduction ; 156(5): 387-395, 2018 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-30325183

RESUMEN

Establishment of a successful pregnancy requires not only implantation of a healthy embryo into a receptive uterus but also progesterone receptor (PGR)-dependent transformation of endometrial stromal cells (ESCs) into specialized decidual cells. Decidual cells support the developing embryo and are critical for placentation. We have previously shown that a known transcriptional coregulator of the PGR, steroid receptor coactivator-2 (SRC-2), is a critical driver of endometrial decidualization in both human and mouse endometrium. However, the full spectrum of genes transcriptionally controlled by SRC-2 in decidualizing ESCs has not been identified. Therefore, using an RNA- and chromatin immunoprecipitation-sequencing approach, we have identified the transcriptome of decidualizing human ESCs (hESCs) that requires SRC-2. We revealed that the majority of hESC genes regulated by SRC-2 are associated with decidualization. Over 50% of SRC-2-regulated genes are also controlled by the PGR. While ontology analysis showed that SRC-2-dependent genes are functionally linked to signaling processes known to underpin hESC decidualization, cell membrane processes were significantly enriched in this analysis. Follow-up studies showed that retinoid signaling is dependent on SRC-2 during hESC decidualization. Specifically, SRC-2 is required for full induction of the retinol transporter, stimulated by retinoic acid 6 (STRA6), which is essential for hESC decidualization. Together our findings show that a critical subset of genes transcriptionally reprogramed by PGR during hESC decidualization requires SRC-2. Among the multiple genes, pathways and networks that are dependent on SRC-2 during hESC decidualization, first-line analysis supports a critical role for this coregulator in maintaining retinoid signaling during progesterone-driven decidualization.


Asunto(s)
Endometrio/fisiología , Regulación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Coactivador 2 del Receptor Nuclear/fisiología , Transcriptoma , Células Cultivadas , Femenino , Humanos , Receptores de Progesterona/metabolismo , Análisis de Secuencia de ARN
13.
Sci Rep ; 8(1): 13134, 2018 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-30177747

RESUMEN

Steroid receptor coactivator-2 (SRC-2) is a transcriptional coregulator that modulates the activity of many transcription factors. Levels of SRC-2 are elevated in endometrial biopsies from polycystic ovary syndrome patients, a population predisposed to endometrial cancer (EC). Increased expression of SRC-2 is also detected in neoplastic endometrium suggesting a causal link between elevated SRC-2 expression and the emergence of endometrial disorders that can lead to cancer. Here, we reveal that SRC-2 knockdown reduces EC cell proliferation and anchorage-independence. Additionally, SRC-2 is required to maintain cellular glycolytic capacity and oxidative phosphorylation, processes essential for EC cell proliferation. Importantly, SRC-2 is critical for the normal performance of the pentose phosphate pathway (PPP). Perturbation of the PPP due to loss of SRC-2 expression may result from the depletion of ribose-5-P isomerase (RPIA), a key enzyme of the PPP. As with SRC-2, RPIA knockdown reduces EC cell proliferation, which is accompanied by a decrease in glycolytic capacity and oxidative phosphorylation. Glucose metabolite tracking experiments confirmed that knockdown of SRC-2 and RPIA downregulates the metabolic rate of both glycolysis and the PPP, highlighting a novel regulatory cross-talk between glycolysis and the PPP modulated by SRC-2.


Asunto(s)
Isomerasas Aldosa-Cetosa/genética , Endometrio/metabolismo , Regulación Neoplásica de la Expresión Génica , Coactivador 2 del Receptor Nuclear/genética , Vía de Pentosa Fosfato/genética , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/metabolismo , Isótopos de Carbono , Línea Celular Tumoral , Proliferación Celular , Endometrio/patología , Femenino , Glucólisis/genética , Humanos , Metaboloma/genética , Coactivador 2 del Receptor Nuclear/antagonistas & inhibidores , Coactivador 2 del Receptor Nuclear/metabolismo , Fosforilación Oxidativa , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo
14.
Cell Rep ; 24(7): 1852-1864, 2018 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-30110641

RESUMEN

RNA polymerase (pol) III transcribes a variety of small untranslated RNAs involved in transcription, RNA processing, and translation. RNA pol III and its components are altered in various human developmental disorders, yet their roles in cell fate determination and development are poorly understood. Here we demonstrate that Maf1, a transcriptional repressor, promotes induction of mouse embryonic stem cells (mESCs) into mesoderm. Reduced Maf1 expression in mESCs and preadipocytes impairs adipogenesis, while ectopic Maf1 expression in Maf1-deficient cells enhances differentiation. RNA pol III repression by chemical inhibition or knockdown of Brf1 promotes adipogenesis. Altered RNA pol III-dependent transcription produces select changes in mRNAs with a significant enrichment of adipogenic gene signatures. Furthermore, RNA pol III-mediated transcription positively regulates long non-coding RNA H19 and Wnt6 expression, established adipogenesis inhibitors. Together, these studies reveal an important and unexpected function for RNA pol III-mediated transcription and Maf1 in mesoderm induction and adipocyte differentiation.


Asunto(s)
Adipocitos/metabolismo , Adipogénesis/genética , ARN Polimerasa III/genética , Proteínas Represoras/genética , Transcripción Genética , Adipocitos/citología , Animales , Factor 1 de Respuesta al Butirato , Diferenciación Celular , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Masculino , Mesodermo/citología , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Ratones , Ratones Desnudos , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Polimerasa III/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
15.
Mol Cell ; 70(4): 679-694.e7, 2018 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-29775582

RESUMEN

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 in vitro. 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.


Asunto(s)
Neoplasias de la Mama/genética , Cromatina/metabolismo , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica , Coactivador 3 de Receptor Nuclear/metabolismo , Transcripción Genética , Sitios de Unión , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cromatina/genética , Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Coactivador 3 de Receptor Nuclear/genética , Regiones Promotoras Genéticas , Unión Proteica , Células Tumorales Cultivadas
16.
Biol Reprod ; 98(1): 15-27, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-29186366

RESUMEN

Infertility and early embryo miscarriage is linked to inadequate endometrial decidualization. Although transcriptional reprogramming is known to drive decidualization in response to progesterone, the key signaling effectors that directly mediate this hormone response are not fully known. This knowledge gap is clinically significant because identifying the early signals that directly mediate progesterone-driven decidualization will address some of the current limitations in diagnosing and therapeutically treating patients at most risk for early pregnancy loss. We recently revealed that the promyelocytic leukemia zinc finger (PLZF) is a direct target of the progesterone receptor and is essential for decidualization of human endometrial stromal cells (hESCs). The purpose of this current work was to identify the genome-wide transcriptional program that is controlled by PLZF during hESC decidualization using an established in vitro hESC culture model, siRNA-mediated knockdown methods, and RNA-sequencing technology followed by bioinformatic analysis and validation. We discovered that PLZF is critical in the regulation of genes that are involved in cellular processes that are essential for the archetypal morphological and functional changes that occur when hESCs transform into epithelioid decidual cells such as proliferation and cell motility. We predict that the transcriptome datasets identified in this study will not only contribute to a broader understanding of PLZF-dependent endometrial decidualization at the molecular level but may advance the development of more effective molecular diagnostics and therapeutics for the clinical management of female infertility and subfertility that is based on a dysfunctional endometrium.


Asunto(s)
Decidua/fisiología , Endometrio/citología , Proteína de la Leucemia Promielocítica con Dedos de Zinc/metabolismo , Movimiento Celular , Proliferación Celular , Células Cultivadas , Biología Computacional , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Proteína de la Leucemia Promielocítica con Dedos de Zinc/genética , Interferencia de ARN , Células del Estroma/citología , Células del Estroma/metabolismo , Transcriptoma
17.
Biol Reprod ; 96(2): 313-326, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-28203817

RESUMEN

The precise timing of progesterone signaling through its cognate receptor, the progesterone receptor (PGR), is critical for the establishment and maintenance of pregnancy. Loss of PGR expression in the murine uterine epithelium during the preimplantation period is a marker for uterine receptivity and embryo attachment. We hypothesized that the decrease in progesterone receptor A (PGRA) expression is necessary for successful embryo implantation. To test this hypothesis, a mouse model constitutively expressing PGRA (mPgrALsL/+) was generated. Expression of PGRA in all uterine compartments (Pgrcre) or uterine epithelium (Wnt7acre) resulted in infertility with defects in embryo attachment and stromal decidualization. Expression of critical PGRA target genes, indian hedgehog, and amphiregulin (Areg), was maintained through the window of receptivity while the estrogen receptor target gene, the leukemia inhibitory factor (Lif), a key regulator of embryo receptivity, was decreased. Transcriptomic and cistromic analyses of the mouse uterus at day 4.5 of pregnancy identified an altered group of genes regulating molecular transport in the control of fluid and ion levels within the uterine interstitial space. Additionally, LIF and its cognate receptor, the leukemia inhibitory factor receptor (LIFR), exhibited PGR-binding events in regions upstream of the transcriptional start sites, suggesting PGRA is inhibiting transcription at these loci. Therefore, downregulation of the PGRA isoform at the window of receptivity is necessary for the attenuation of hedgehog signaling, transcriptional activation of LIF signaling, and modulation of solutes and fluid, producing a receptive environment for the attaching embryo.


Asunto(s)
Implantación del Embrión , Endometrio , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Alelos , Animales , Clonación Molecular , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica/fisiología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Masculino , Ratones Transgénicos , Receptores OSM-LIF/genética , Receptores OSM-LIF/metabolismo , Receptores de Progesterona/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
18.
Stem Cells ; 35(6): 1554-1565, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28207192

RESUMEN

During neonatal testis development, centrally located gonocytes migrate to basement membrane of the seminiferous cords, where physical contact with a niche established by Sertoli cells is essential for transition of gonocytes into spermatogonial stem cells (SSCs). To provide structural support and signaling stimuli for the gonocyte-to-SSC transition that occurs at a specific location during a finite phase, temporal-spatial establishment of the niche is critical. To date, the factors that guide Sertoli cells to establish the initial stem cell niche remain largely unknown. Using the Sertoli cell-specific Arid4b knockout (Arid4bSCKO) mice, we demonstrated that ablation of AT-rich interaction domain 4B (ARID4B) resulted in abnormal detachment of Sertoli cells from the basement membrane of seminiferous cords during the gonocyte-to-SSC transition phase, suggesting failure to establish a niche for the SSC formation. Without support by a niche environment, gonocytes showed disarranged cell distribution in the Arid4bSCKO testes and underwent apoptosis. The commitment of gonocytes to differentiate into the spermatogonial lineage was broken and the capability of SSCs to self-renew and differentiate was also impaired. Gene expression profiling revealed the molecular mechanisms responsible for the phenotypic changes in the Arid4bSCKO testes, by identifying genes important for stem cell niche function as downstream effectors of ARID4B, including genes that encode gap junction protein alpha-1, KIT ligand, anti-Müllerian hormone, Glial cell-line derived neurotrophic factor, inhibin alpha, inhibin beta, and cytochrome P450 family 26 subfamily b polypeptide 1. Our results identified ARID4B as a master regulator of a signaling network that governs the establishment of a niche during the critical gonocyte-to-SSC transition phase to control the fate of gonocytes and SSCs. Stem Cells 2017;35:1554-1565.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Espermatogonias/citología , Nicho de Células Madre , Células Madre/citología , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Diferenciación Celular , Linaje de la Célula , Autorrenovación de las Células , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Regulación del Desarrollo de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Testículo/embriología , Testículo/metabolismo , Factores de Tiempo
19.
Cell Rep ; 17(5): 1414-1425, 2016 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-27783953

RESUMEN

Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR) expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage.


Asunto(s)
Endometrio/metabolismo , Redes Reguladoras de Genes/genética , Progesterona/metabolismo , Animales , Secuencia de Bases , Secuencia Conservada/genética , Implantación del Embrión , Femenino , Factor de Transcripción GATA2/metabolismo , Humanos , Ratones , Fosfoproteínas/metabolismo , Embarazo , Progesterona/sangre , Unión Proteica/genética , Receptores de Progesterona/metabolismo , Transducción de Señal/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo
20.
Prostate ; 76(14): 1271-84, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27272765

RESUMEN

BACKGROUND: TMPRSS2-ERG fusion occurs in about half of prostate cancers and results in over-expression of the oncogenic ERG protein in the prostate. The mechanism by which ERG contributes to prostate cancer initiation and progression remains largely unknown. Because ERG is a transcriptional activator, we reasoned that the target genes regulated by ERG could contribute to prostate cancer development. METHODS: In a search for ERG target genes, we took advantage of published datasets from the MSKCC Prostate Oncogene Project, in which a comprehensive analysis was applied to define transcriptomes in 150 prostate tumors. We retrieved the mRNA expression dataset, split them based on ERG expression, and identified genes whose expression levels are associated with ERG mRNA levels. RESULTS: mRNA expression levels of 21 genes were found to be significantly increased, while for one gene it was decreased in ERG-positive prostate tumors. Among them, the expression of TDRD1 was the most significantly increased in ERG-positive tumors. Among 131 primary prostate tumors which were primarily from European American patients, TDRD1 is over-expressed in 68% of samples, while ERG is overexpressed in 48% of samples, suggesting an additional ERG-independent mechanism of TDRD1 overexpression. In African American prostate tumors, TDRD1 mRNA is expressed in 44%, while ERG is expressed in 24% of samples. In normal tissues, TDRD1 mRNA is exclusively expressed in germ cells and its protein is also known as cancer/testis antigen 41.1 (CT41.1). We generated a mouse monoclonal antibody that recognizes human TDRD1 protein with high specificity and sensitivity. By Western blot analysis and immunohistochemistry (IHC) staining, we demonstrate that TDRD1 protein is expressed in the majority of human prostate tumors, but not in normal prostate tissue. Finally, TDRD1 is not induced in the prostate of ERG overexpression transgenic mice, suggesting that such model does not fully recapitulate the TMPRSS2/ERG fusion-dependent human prostate cancer development. CONCLUSIONS: Our results suggest TDRD1 as a novel prostate cancer biomarker. As an ERG target gene, TDRD1 might play an important role in human prostate cancer development, and as a cancer/testis antigen, TDRD1 might have long-term potential to be a therapeutic target for prostate cancer immunotherapy. Prostate 76:1271-1284, 2016. © 2016 Wiley Periodicals, Inc.


Asunto(s)
Biomarcadores de Tumor/genética , Proteínas Portadoras/genética , Marcación de Gen/métodos , Células Germinativas , Neoplasias de la Próstata/genética , Animales , Biomarcadores de Tumor/metabolismo , Proteínas Portadoras/biosíntesis , Proteínas de Ciclo Celular , Línea Celular Tumoral , Células Germinativas/metabolismo , Células Germinativas/patología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Regulador Transcripcional ERG/biosíntesis , Regulador Transcripcional ERG/genética
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