RESUMO
Although we have shown that steroid receptor coactivator-2 (SRC-2), a member of the p160/SRC family of transcriptional coregulators, is essential for decidualization of both human and murine endometrial stromal cells, SRC-2's role in the earlier stages of the implantation process have not been adequately addressed. Using a conditional SRC-2 knockout mouse (SRC-2d/d ) in timed natural pregnancy studies, we show that endometrial SRC-2 is required for embryo attachment and adherence to the luminal epithelium. Implantation failure is associated with the persistent expression of Mucin 1 and E-cadherin on the apical surface and basolateral adherens junctions of the SRC-2d/d luminal epithelium, respectively. These findings indicate that the SRC-2d/d luminal epithelium fails to exhibit a plasma membrane transformation (PMT) state known to be required for the development of uterine receptivity. Transcriptomics demonstrated that the expression of genes involved in steroid hormone control of uterine receptivity were significantly disrupted in the SRC-2d/d endometrium as well as genes that control epithelial tight junctional biology and the emergence of the epithelial mesenchymal transition state, with the latter sharing similar biological properties with PMT. Collectively, these findings uncover a new role for endometrial SRC-2 in the induction of the luminal epithelial PMT state, which is a prerequisite for the development of uterine receptivity and early pregnancy establishment.
Assuntos
Implantação do Embrião , Útero , Animais , Feminino , Humanos , Camundongos , Gravidez , Implantação do Embrião/genética , Endométrio/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Camundongos Knockout , Coativador 2 de Receptor Nuclear/genética , Útero/metabolismoRESUMO
Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.
Assuntos
Doxiciclina , Ligante RANK/metabolismo , Transcriptoma , Aglutininas/metabolismo , Animais , Citocinas/metabolismo , Doxiciclina/farmacologia , Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Queratina-5/genética , Queratina-5/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Fenótipo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Timo/metabolismoRESUMO
Although a non-malignant gynecological disorder, endometriosis displays some pathogenic features of malignancy, such as cell proliferation, migration, invasion and adaptation to hypoxia. Current treatments of endometriosis include pharmacotherapy and/or surgery, which are of limited efficacy and often associated with adverse side effects. Therefore, to develop more effective therapies to treat this disease, a broader understanding of the underlying molecular mechanisms that underpin endometriosis needs to be attained. Using immortalized human endometriotic epithelial and stromal cell lines, we demonstrate that the early growth response 1 (EGR1) transcription factor is essential for cell proliferation, migration and invasion, which represent some of the pathogenic properties of endometriotic cells. Genome-wide transcriptomics identified an EGR1-dependent transcriptome in human endometriotic epithelial cells that potentially encodes a diverse spectrum of proteins that are known to be involved in tissue pathologies. To underscore the utility of this transcriptomic data set, we demonstrate that carbonic anhydrase 9 (CA9), a homeostatic regulator of intracellular pH, is not only a molecular target of EGR1 but is also important for maintaining many of the cellular properties of human endometriotic epithelial cells that are also ascribed to EGR1. Considering therapeutic intervention strategies are actively being developed for EGR1 and CAIX in the treatment of other pathologies, we believe EGR1 and its transcriptome (which includes CA9) will offer not only a new conceptual framework to advance our understanding of endometriosis but will also furnish new molecular vulnerabilities to be leveraged as potential therapeutic options in the future treatment of endometriosis.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce , Endometriose , Movimento Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Endometriose/metabolismo , Endométrio/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Células Estromais/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The promyelocytic leukemia zinc finger (PLZF) transcription factor mediates a wide-range of biological processes. Accordingly, perturbation of PLZF function results in a myriad of physiologic defects, the most conspicuous of which is abnormal skeletal patterning. Although whole body knockout of Plzf in the mouse (Plzf KO ) has significantly expanded our understanding of Plzf function in vivo, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has not been developed. Therefore, we used CRISPR/Cas 9 gene editing to generate a mouse model in which exon 2 of the murine Plzf gene is specifically flanked (or floxed) by LoxP sites (Plzf f/f ). Crossing our Plzf f/f mouse with a global cre-driver mouse to generate the Plzf d/d bigenic mouse, we demonstrate that exon 2 of the Plzf gene is ablated in the Plzf d/d bigenic. Similar to the previously reported Plzf KO mouse, the Plzf d/d mouse exhibits a severe defect in skeletal patterning of the hindlimb, indicating that the Plzf f/f mouse functions as designed. Therefore, studies in this short technical report demonstrate that the Plzf f/f mouse will be useful to investigators who wish to explore the role of the Plzf transcription factor in a specific tissue or cell-type.
Assuntos
Sistemas CRISPR-Cas , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Animais , Padronização Corporal , Osso e Ossos/anormalidades , Osso e Ossos/embriologia , Técnicas de Inativação de Genes/métodos , Membro Posterior/anormalidades , Membro Posterior/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína com Dedos de Zinco da Leucemia Promielocítica/deficiênciaRESUMO
Although salivary gland cancers comprise only â¼3-6% of head and neck cancers, treatment options for patients with advanced-stage disease are limited. Because of their rarity, salivary gland malignancies are understudied compared to other exocrine tissue cancers. The comparative lack of progress in this cancer field is particularly evident when it comes to our incomplete understanding of the key molecular signals that are causal for the development and/or progression of salivary gland cancers. Using a novel conditional transgenic mouse (K5:RANKL), we demonstrate that Receptor Activator of NFkB Ligand (RANKL) targeted to cytokeratin 5-positive basal epithelial cells of the salivary gland causes aggressive tumorigenesis within a short period of RANKL exposure. Genome-wide transcriptomic analysis reveals that RANKL markedly increases the expression levels of numerous gene families involved in cellular proliferation, migration, and intra- and extra-tumoral communication. Importantly, cross-species comparison of the K5:RANKL transcriptomic dataset with The Cancer Genome Atlas cancer signatures reveals the strongest molecular similarity with cancer subtypes of the human head and neck squamous cell carcinoma. These studies not only provide a much needed transcriptomic resource to mine for novel molecular targets for therapy and/or diagnosis but validates the K5:RANKL transgenic as a preclinical model to further investigate the in vivo oncogenic role of RANKL signaling in salivary gland tumorigenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Ligante RANK/metabolismo , Neoplasias das Glândulas Salivares/metabolismo , Glândulas Salivares/metabolismo , Transcrição Gênica , Transcriptoma , Animais , Epitélio/metabolismo , Epitélio/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Ligante RANK/genética , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Glândulas Salivares/patologiaRESUMO
Progesterone, via the progesterone receptor (PGR), is essential for endometrial stromal cell decidualization, a cellular transformation event in which stromal fibroblasts differentiate into decidual cells. Uterine decidualization supports embryo implantation and placentation as well as subsequent events, which together ensure a successful pregnancy. Accordingly, impaired decidualization results not only in implantation failure or early fetal miscarriage, but also may lead to potential adverse outcomes in all three pregnancy trimesters. Transcriptional reprogramming on a genome-wide scale underlies progesterone dependent decidualization of the human endometrial stromal cell (hESC). However, identification of the functionally essential signals encoded by these global transcriptional changes remains incomplete. Importantly, this knowledge-gap undercuts future efforts to improve diagnosis and treatment of implantation failure based on a dysfunctional endometrium. By integrating genome-wide datasets derived from decidualization of hESCs in culture, we reveal that the promyelocytic leukemia zinc finger (PLZF) transcription factor is rapidly induced by progesterone and that this induction is indispensable for progesterone-dependent decidualization. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) identified at least ten progesterone response elements within the PLZF gene, indicating that PLZF may act as a direct target of PGR signaling. The spatiotemporal expression profile for PLZF in both the human and mouse endometrium offers further support for stromal PLZF as a mediator of the progesterone decidual signal. To identify functional targets of PLZF, integration of PLZF ChIP-Seq and RNA Pol II RNA-Seq datasets revealed that the early growth response 1 (EGR1) transcription factor is a PLZF target for which its level of expression must be reduced to enable progesterone dependent hESC decidualization. Apart from furnishing essential insights into the molecular mechanisms by which progesterone drives hESC decidualization, our findings provide a new conceptual framework that could lead to new avenues for diagnosis and/or treatment of adverse reproductive outcomes associated with a dysfunctional uterus.
Assuntos
Decídua/fisiologia , Endométrio/citologia , Fatores de Transcrição Kruppel-Like/fisiologia , Células Estromais/citologia , Decídua/citologia , Decídua/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Endométrio/metabolismo , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Progestinas/farmacologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores de Progesterona/fisiologia , Células Estromais/metabolismo , Transcrição Gênica/fisiologiaRESUMO
Using a Rosa26 gene targeting strategy in mouse embryonic stem cells, we have generated a new transgenic mouse (Pgr-B LSL ), which is designed to conditionally express the epitope-tagged mouse progesterone receptor-B (PGR-B) isoform when crossed with a specific cre driver mouse. To functionally validate this transgenic mouse, we crossed the Pgr-B LSL mouse with the MMTV-CREA transgenic mouse to create the MMTV-CREA/Pgr-B LSL bigenic (termed PR-B:OE to denote PGR-B overexpressor). As expected, transgene-derived PGR-B protein was specifically targeted to the virgin mammary gland epithelium. At a functional level, the PR-B:OE bigenic exhibited abnormal mammary morphogenesis-dilated epithelial ducts, precocious alveologenesis and lateral side-branching, along with a prominent proliferative signature-that resulted in pregnant PR-B:OE mice unable to exhibit mammary gland terminal differentiation at parturition. Because of this developmental failure, the PR-B:OE mammary gland was incapable of producing milk resulting in early neonatal death of otherwise healthy litters. This first line of analysis demonstrates the utility of the Pgr-B LSL mouse to examine the role of the PGR-B isoform in different physiologic and pathophysiologic systems that are responsive to progesterone.
Assuntos
Engenharia Genética/métodos , Receptores de Progesterona/genética , Animais , Proliferação de Células , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Masculino , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Morfogênese/genética , Isoformas de Proteínas , Receptores de Progesterona/fisiologiaRESUMO
Speckle-type poz protein (SPOP) is an E3-ubiquitin ligase adaptor for turnover of a diverse number of proteins involved in key cellular processes such as chromatin remodeling, transcriptional regulation, and cell signaling. Genomic analysis revealed that SPOP somatic mutations are found in a subset of endometrial cancers, suggesting that these mutations act as oncogenic drivers of this gynecologic malignancy. These studies also raise the question as to the role of wild-type SPOP in normal uterine function. To address this question, we generated a mouse model (Spopd/d) in which SPOP is ablated in uterine cells that express the PGR. Fertility studies demonstrated that SPOP is required for embryo implantation and for endometrial decidualization. Molecular analysis revealed that expression levels of the PGR at the protein and transcript level are significantly reduced in the Spopd/d uterus. While this result was unexpected, this finding explains in part the dysfunctional phenotype of the Spopd/d uterus. Moderate increased levels of the ESR1, GATA2, and SRC2 were detected in the Spopd/d uterus, suggesting that SPOP is required to maintain the proteome for normal uterine function. With age, the Spopd/d endometrium exhibits large glandular cysts with foci of epithelial proliferation, further supporting a role for SPOP in maintaining a healthy uterus. Collectively, studies on the Spopd/d mouse support an important role for SPOP in normal uterine function and suggest that this mouse model may prove useful to study the role of SPOP-loss-of-function mutations in the etiopathogenesis of endometrial cancer.
Assuntos
Implantação do Embrião/fisiologia , Infertilidade/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Útero/metabolismo , Animais , Endométrio/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Fator de Transcrição GATA2/metabolismo , Infertilidade/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Gravidez , Proteínas Repressoras/genética , Complexos Ubiquitina-Proteína LigaseRESUMO
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.
Assuntos
Decídua/fisiologia , Endométrio/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Biologia Computacional , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Interferência de RNA , Células Estromais/citologia , Células Estromais/metabolismo , TranscriptomaRESUMO
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.
Assuntos
Endométrio/fisiologia , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Coativador 2 de Receptor Nuclear/fisiologia , Transcriptoma , Células Cultivadas , Feminino , Humanos , Receptores de Progesterona/metabolismo , Análise de Sequência de RNARESUMO
STUDY QUESTION: Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization? SUMMARY ANSWER: GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization. WHAT IS KNOWN ALREADY: Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined. STUDY DESIGN, SIZE, DURATION: Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression. PARTICIPANTS/MATERIALS, SETTING, METHODS: Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8-12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs. MAIN RESULTS AND THE ROLE OF CHANCE: Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P < 0.05, and 5.2 ± 0.26, P < 0.01, respectively) increase in GREB1 transcript levels. This increase was significantly reduced in the uteri of PR knock-out mice (P < 0.01), in HESCs treated with the PR antagonist RU486 (P < 0.01), or in HESCs in which PR expression was knocked down (P < 0.05). When GREB1 expression was knocked down, progesterone-driven decidualization markers in both immortalized and primary HESCs was significantly reduced (P < 0.05 and P < 0.01). Finally, GREB1 knock down signficantly reduced expression of the PR target genes WNT4 and FOXOA1 (P < 0.05 and P < 0.01, respectively). LARGE SCALE DATA: This study used the Nuclear Receptor Signaling Atlas. LIMITATIONS, REASONS FOR CAUTION: Although in vitro cell culture studies indicate that GREB1 is required for endoemtrial decidualization, the in vivo role of GREB1 in endometrial function and dysfunction should be assessed by using knock-out mouse models. WIDER IMPLICATIONS OF THE FINDINGS: Identification and functional analysis of GREB1 as a key molecular mediator of decidualization may lead to improved diagnosis and clinical management of women with peri-implantation loss due to inadequate endometrial decidualization. STUDY FUNDING AND COMPETING INTEREST(S): This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.
Assuntos
Decídua/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Neoplasias/genética , Progesterona/farmacologia , Receptores de Progesterona/genética , Células Estromais/efeitos dos fármacos , Animais , Diferenciação Celular , Decídua/citologia , Decídua/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Camundongos , Camundongos Knockout , Mifepristona/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Coativador 2 de Receptor Nuclear/antagonistas & inibidores , Coativador 2 de Receptor Nuclear/genética , Coativador 2 de Receptor Nuclear/metabolismo , Gravidez , Cultura Primária de Células , Prolactina/genética , Prolactina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/deficiência , Transdução de Sinais , Células Estromais/citologia , Células Estromais/metabolismo , Proteína Wnt4/genética , Proteína Wnt4/metabolismoRESUMO
Progesterone-RankL paracrine signaling has been proposed as a driver of stem cell expansion in the mammary gland, and Elf5 is essential for the differentiation of mammary epithelial progenitor cells. We demonstrate that Elf5 expression is induced by progesterone and that Elf5 and progesterone cooperate to promote alveolar development. The progesterone receptor and Elf5 are expressed in a mutually exclusive pattern, and we identify RankL as the paracrine mediator of the effects of progesterone on Elf5 expression in CD61+ progenitor cells and their consequent differentiation. Blockade of RankL action prevented progesterone-induced side branching and the expansion of Elf5(+) mature luminal cells. These findings describe a mechanism by which steroid hormones can produce the expansion of steroid hormone receptor-negative mammary epithelial cells.
Assuntos
Proteínas de Ligação a DNA/genética , Glândulas Mamárias Animais/efeitos dos fármacos , Progesterona/farmacologia , Ligante RANK/farmacologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Ligante RANK/metabolismo , Ligante RANK/fisiologia , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Progesterona/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
Early embryo miscarriage is linked to inadequate endometrial decidualization, a cellular transformation process that enables deep blastocyst invasion into the maternal compartment. Although much of the cellular events that underpin endometrial stromal cell (ESC) decidualization are well recognized, the individual gene(s) and molecular pathways that drive the initiation and progression of this process remain elusive. Using a genetic mouse model and a primary human ESC culture model, we demonstrate that steroid receptor coactivator-2 (SRC-2) is indispensable for rapid steroid hormone-dependent proliferation of ESCs, a critical cell-division step which precedes ESC terminal differentiation into decidual cells. We reveal that SRC-2 is required for increasing the glycolytic flux in human ESCs, which enables rapid proliferation to occur during the early stages of the decidualization program. Specifically, SRC-2 increases the glycolytic flux through induction of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3), a major rate-limiting glycolytic enzyme. Similarly, acute treatment of mice with a small molecule inhibitor of PFKFB3 significantly suppressed the ability of these animals to exhibit an endometrial decidual response. Together, these data strongly support a conserved mechanism of action by which SRC-2 accelerates the glycolytic flux through PFKFB3 induction to provide the necessary bioenergy and biomass to meet the demands of a high proliferation rate observed in ESCs prior to their differentiation into decidual cells. Because deregulation of endometrial SRC-2 expression has been associated with common gynecological disorders of reproductive-age women, this signaling pathway, involving SRC-2 and PFKFB3, promises to offer new clinical approaches in the diagnosis and/or treatment of a non-receptive uterus in patients presenting idiopathic infertility, recurrent early pregnancy loss, or increased time to pregnancy.
Assuntos
Aborto Espontâneo/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Coativador 2 de Receptor Nuclear/genética , Fosfofrutoquinase-2/biossíntese , Aborto Espontâneo/etiologia , Aborto Espontâneo/patologia , Animais , Células Cultivadas , Decídua/citologia , Decídua/metabolismo , Implantação do Embrião/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Coativador 2 de Receptor Nuclear/metabolismo , Fosfofrutoquinase-2/genética , Gravidez , Transdução de Sinais/genética , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
The p160/steroid receptor coactivator (SRC) family comprises three pleiotropic coregulators (SRC-1, SRC-2, and SRC-3; otherwise known as NCOA1, NCOA2, and NCOA3, respectively), which modulate a wide spectrum of physiological responses and clinicopathologies. Such pleiotropy is achieved through their inherent structural complexity, which allows this coregulator class to control both nuclear receptor and non-nuclear receptor signaling. As observed in other physiologic systems, members of the SRC family have recently been shown to play pivotal roles in uterine biology and pathobiology. In the murine uterus, SRC-1 is required to launch a full steroid hormone response, without which endometrial decidualization is markedly attenuated. From "dovetailing" clinical and mouse studies, an isoform of SRC-1 was recently identified which promotes endometriosis by reprogramming endometrial cells to evade apoptosis and to colonize as endometriotic lesions within the peritoneal cavity. The endometrium fails to decidualize without SRC-2, which accounts for the infertility phenotype exhibited by mice devoid of this coregulator. In related studies on human endometrial stromal cells, SRC-2 was shown to act as a molecular "pacemaker" of the glycolytic flux. This finding is significant because acceleration of the glycolytic flux provides the necessary bioenergy and biomolecules for endometrial stromal cells to switch from quiescence to a proliferative phenotype, a critical underpinning in the decidual progression program. Although studies on uterine SRC-3 function are in their early stages, clinical studies provide tantalizing support for the proposal that SRC-3 is causally linked to endometrial hyperplasia as well as with endometrial pathologies in patients diagnosed with polycystic ovary syndrome. This proposal is now driving the development and application of innovative technologies, particularly in the mouse, to further understand the functional role of this elusive uterine coregulator in normal and abnormal physiologic contexts. Because dysregulation of this coregulator triad potentially presents a triple threat for increased risk of subfecundity, infertility, or endometrial disease, a clearer understanding of the individual and combinatorial roles of these coregulators in uterine function is urgently required. This minireview summarizes our current understanding of uterine SRC function, with a particular emphasis on the next critical questions that need to be addressed to ensure significant expansion of our knowledge of this underexplored field of uterine biology.
Assuntos
Coativadores de Receptor Nuclear/fisiologia , Doenças Uterinas/genética , Útero/fisiologia , Animais , Decídua/metabolismo , Implantação do Embrião/genética , Feminino , Humanos , Camundongos , Família Multigênica , Gravidez , Transdução de Sinais/genética , Útero/fisiopatologiaRESUMO
Recent data from human and mouse studies strongly support an indispensable role for steroid receptor coactivator-2 (SRC-2)-a member of the p160/SRC family of coregulators-in progesterone-dependent endometrial stromal cell decidualization, an essential cellular transformation process that regulates invasion of the developing embryo into the maternal compartment. To identify the key progesterone-induced transcriptional changes that are dependent on SRC-2 and required for endometrial decidualization, we performed comparative genome-wide transcriptional profiling of endometrial tissue RNA from ovariectomized SRC-2(flox/flox) (SRC-2(f/f) [control]) and PR(cre/+)/SRC-2(flox/flox) (SRC-2(d/d) [SRC-2-depleted]) mice, acutely treated with vehicle or progesterone. Although data mining revealed that only a small subset of the total progesterone-dependent transcriptional changes is dependent on SRC-2 (â¼13%), key genes previously reported to mediate progesterone-driven endometrial stromal cell decidualization are present within this subset. Along with providing a more detailed molecular portrait of the decidual transcriptional program governed by SRC-2, the degree of functional diversity of these progesterone mediators underscores the pleiotropic regulatory role of SRC-2 in this tissue. To showcase the utility of this powerful informational resource to uncover novel signaling paradigms, we stratified the total SRC-2-dependent subset of progesterone-induced transcriptional changes in terms of novel gene expression and identified transcription factor 23 (Tcf23), a basic-helix-loop-helix transcription factor, as a new progesterone-induced target gene that requires SRC-2 for full induction. Importantly, using primary human endometrial stromal cells in culture, we demonstrate that TCF23 function is essential for progesterone-dependent decidualization, providing crucial translational support for this transcription factor as a new decidual mediator of progesterone action.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Decídua/citologia , Coativador 2 de Receptor Nuclear/genética , Células Estromais/citologia , Animais , Decídua/fisiologia , Feminino , Humanos , Camundongos , Camundongos Mutantes , Coativador 2 de Receptor Nuclear/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Progesterona/metabolismo , RNA Interferente Pequeno/genética , Células Estromais/fisiologia , Transcrição Gênica/fisiologia , Transcriptoma/fisiologia , Útero/citologia , Útero/fisiologiaRESUMO
The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues. Here, we investigated how different cellular states modify the p53 transcriptional program in human cells through a combination of computational analyses of publicly available large-scale datasets and in vitro studies using an isogenic system consisting of induced pluripotent stem cells (iPSCs) and two derived lineages. Analysis of publicly available mRNA expression and genetic dependency data demonstrated wide variation in terms of expression and function of a core p53 transcriptional program across various tissues and lineages. To monitor the impact of cell differentiation on the p53 transcriptome within an isogenic cell culture system, we activated p53 by pharmacological inhibition of its negative regulator MDM2. Using cell phenotyping assays and genome wide transcriptome analyses, we demonstrated that cell differentiation confines and modifies the p53 transcriptional network in a lineage-specific fashion. Although hundreds of p53 target genes are transactivated in iPSCs, only a small fraction is transactivated in each of the differentiated lineages. Mechanistic studies using small molecule inhibitors and genetic knockdowns revealed the presence of two major regulatory mechanisms contributing to this massive heterogeneity across cellular states: gene silencing by epigenetic regulatory complexes and constitutive transactivation by lineage-specific transcription factors. Altogether, these results illuminate the impact of cell differentiation on the p53 program, thus advancing our understanding of how this tumor suppressor functions in different contexts.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Camundongos , Animais , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ativação Transcricional/genética , Fatores de Transcrição/metabolismo , Diferenciação Celular/genética , Neoplasias/genética , Inativação GênicaRESUMO
Inactivation of the p53 tumor suppressor, either by mutations or through hyperactivation of repressors such as MDM2 and MDM4, is a hallmark of cancer. Although many inhibitors of the p53-MDM2/4 interaction have been developed, such as Nutlin, their therapeutic value is limited by highly heterogeneous cellular responses. We report here a multi-omics investigation of the cellular response to MDM2/4 inhibitors, leading to identification of FAM193A as a widespread regulator of p53 function. CRISPR screening identified FAM193A as necessary for the response to Nutlin. FAM193A expression correlates with Nutlin sensitivity across hundreds of cell lines. Furthermore, genetic codependency data highlight FAM193A as a component of the p53 pathway across diverse tumor types. Mechanistically, FAM193A interacts with MDM4, and FAM193A depletion stabilizes MDM4 and inhibits the p53 transcriptional program. Last, FAM193A expression is associated with better prognosis in multiple malignancies. Altogether, these results identify FAM193A as a positive regulator of p53.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/farmacologia , Apoptose , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Neoplasias/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Steroid receptor coactivator-3 (SRC-3; also known as NCOA3 or AIB1) is a member of the multifunctional p160/SRC family of coactivators, which also includes SRC-1 and SRC-2. Clinical and cell-based studies as well as investigations on mice have demonstrated pivotal roles for each SRC in numerous physiological and pathophysiological contexts, underscoring their functional pleiotropy. We previously demonstrated the critical involvement of SRC-2 in murine embryo implantation as well as in human endometrial stromal cell (HESC) decidualization, a cellular transformation process required for trophoblast invasion and ultimately placentation. We show here that, like SRC-2, SRC-3 is expressed in the epithelial and stromal cellular compartments of the human endometrium during the proliferative and secretory phase of the menstrual cycle as well as in cultured HESCs. We also found that SRC-3 depletion in cultured HESCs results in a significant attenuation in the induction of a wide-range of established biomarkers of decidualization, despite exposure of these cells to a deciduogenic stimulus and normal progesterone receptor expression. These molecular findings are supported at the cellular level by the inability of HESCs to morphologically transform from a stromal fibroblastoid cell to an epithelioid decidual cell when endogenous SRC-3 levels are markedly reduced. To identify genes, signaling pathways and networks that are controlled by SRC-3 and potentially important for hormone-dependent decidualization, we performed RNA-sequencing on HESCs in which SRC-3 levels were significantly reduced at the time of administering the deciduogenic stimulus. Comparing HESC controls with HESCs deficient in SRC-3, gene enrichment analysis of the differentially expressed gene set revealed an overrepresentation of genes involved in chromatin remodeling, cell proliferation/motility, and programmed cell death. These predictive bioanalytic results were confirmed by the demonstration that SRC-3 is required for the expansion, migratory and invasive activities of the HESC population, cellular properties that are required in vivo in the formation or functioning of the decidua. Collectively, our results support SRC-3 as an important coregulator in HESC decidualization. Since perturbation of normal homeostatic levels of SRC-3 is linked with common gynecological disorders diagnosed in reproductive age women, this endometrial coregulator-along with its new molecular targets described here-may open novel clinical avenues in the diagnosis and/or treatment of a non-receptive endometrium, particularly in patients presenting non-aneuploid early pregnancy loss.
RESUMO
Although constitutive murine transgenic models have provided important insights into ß-catenin signaling in tissue morphogenesis and tumorigenesis, these models are unable to express activated ß-catenin in a temporally controlled manner. Therefore, to enable the induction (and subsequent de-induction) of ß-catenin signaling during a predetermined time-period or developmental stage, we have generated and characterized a TETO-ΔN89ß-catenin responder transgenic mouse. Crossed with the MTB transgenic effector mouse, which targets the expression of the reverse tetracycline transactivator (rtTA) to the mammary epithelium, we demonstrate that the stabilized (and activated) form of ß-catenin (ΔN89ß-catenin) is expressed only in the presence doxycycline-activated rtTA in the mammary epithelial compartment. Furthermore, we show that transgene-derived ΔN89ß-catenin elicits significant mammary epithelial proliferation and precocious alveologenesis in the virgin doxycycline-treated MTB/TETO-ΔN89ß-catenin bitransgenic. Remarkably, deinduction of TETO-ΔN89ß-catenin transgene expression (through doxycycline withdrawal) results in the reversal of these morphological changes. Importantly, continued activation of the TETO-ΔN89ß-catenin transgene results in palpable mammary tumors (within 7-9 months) in the doxycycline-treated virgin MTB/TETO-ΔN89ß-catenin bigenic but not in the same bitransgenic without doxycycline administration. Collectively, these mammary epithelial responses to ΔN89ß-catenin expression agree with previous reports using conventional transgenesis and therefore confirm that ΔN89ß-catenin functions as expected in this doxycycline-responsive bigenic system. In sum, our mammary gland studies demonstrate "proof-of-principle" for using the TETO-ΔN89ß-catenin transgenic responder to activate (and then de-activate) ß-catenin signaling in any tissue of interest in a spatiotemporal specific fashion.