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1.
PLoS Biol ; 21(10): e3002334, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37856394

RESUMO

Tissue development entails genetically programmed differentiation of immature cell types to mature, fully differentiated cells. Exposure during development to non-mutagenic environmental factors can contribute to cancer risk, but the underlying mechanisms are not understood. We used a mouse model of endometrial adenocarcinoma that results from brief developmental exposure to an estrogenic chemical, diethylstilbestrol (DES), to determine causative factors. Single-cell RNA sequencing (scRNAseq) and spatial transcriptomics of adult control uteri revealed novel markers of uterine epithelial stem cells (EpSCs), identified distinct luminal and glandular progenitor cell (PC) populations, and defined glandular and luminal epithelium (LE) cell differentiation trajectories. Neonatal DES exposure disrupted uterine epithelial cell differentiation, resulting in a failure to generate an EpSC population or distinguishable glandular and luminal progenitors or mature cells. Instead, the DES-exposed epithelial cells were characterized by a single proliferating PC population and widespread activation of Wnt/ß-catenin signaling. The underlying endometrial stromal cells had dramatic increases in inflammatory signaling pathways and oxidative stress. Together, these changes activated phosphoinositide 3-kinase/AKT serine-threonine kinase signaling and malignant transformation of cells that were marked by phospho-AKT and the cancer-associated protein olfactomedin 4. Here, we defined a mechanistic pathway from developmental exposure to an endocrine disrupting chemical to the development of adult-onset cancer. These findings provide an explanation for how human cancers, which are often associated with abnormal activation of PI3K/AKT signaling, could result from exposure to environmental insults during development.


Assuntos
Adenocarcinoma , Fosfatidilinositol 3-Quinases , Animais , Feminino , Camundongos , Adenocarcinoma/induzido quimicamente , beta Catenina/genética , beta Catenina/metabolismo , Diferenciação Celular , Estrogênios , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Útero
2.
Biochem Pharmacol ; 195: 114848, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34801523

RESUMO

Exposure to naturally derived estrogen receptor activators, such as the phytoestrogen genistein, can occur at physiologically relevant concentrations in the human diet. Soy-based infant formulas are of particular concern because infants consuming these products have serum genistein levels almost 20 times greater than those seen in vegetarian adults. Comparable exposures in animal studies have adverse physiologic effects. The timing of exposure is particularly concerning because infants undergo a steroid hormone-sensitive period termed "minipuberty" during which estrogenic chemical exposure may alter normal reproductive tissue patterning and function. The delay between genistein exposure and reproductive outcomes poses a unique challenge to collecting epidemiological data. In 2010, the U.S. National Toxicology Program monograph on the safety of the use of soy formula stated that the use of soy-based infant formula posed minimal concern and emphasized a lack of data from human subjects. Since then, several new human and animal studies have advanced our epidemiological and mechanistic understanding of the risks and benefits of phytoestrogen exposure. Here we aim to identify clinically relevant findings regarding phytoestrogen exposure and female reproductive outcomes from the past 10 years, with a focus on the phytoestrogen genistein, and explore the implications of these findings for soy infant formula recommendations. Research presented in this review will inform clinical practice and dietary recommendations for infants based on evidence from both clinical epidemiology and basic research advances in endocrinology and developmental biology from mechanistic in vitro and animal studies.


Assuntos
Desenvolvimento Infantil/efeitos dos fármacos , Genisteína/farmacologia , Fórmulas Infantis/análise , Fitoestrógenos/farmacologia , Alimentos de Soja/análise , Animais , Desenvolvimento Infantil/fisiologia , Feminino , Genisteína/administração & dosagem , Humanos , Lactente , Fitoestrógenos/administração & dosagem , Reprodução/efeitos dos fármacos , Reprodução/fisiologia
3.
Environ Health Perspect ; 128(3): 37001, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32186404

RESUMO

BACKGROUND: Embryo implantation relies on precise hormonal regulation, associated gene expression changes, and appropriate female reproductive tract tissue architecture. Female mice exposed neonatally to the phytoestrogen genistein (GEN) at doses similar to those in infants consuming soy-based infant formulas are infertile due in part to uterine implantation defects. OBJECTIVES: Our goal was to determine the mechanisms by which neonatal GEN exposure causes implantation defects. METHODS: Female mice were exposed to GEN on postnatal days (PND)1-5 and uterine tissues collected on PND5, PND22-26, and during pregnancy. Analysis of tissue weights, morphology, and gene expression was performed using standard histology, confocal imaging with three-dimensional analysis, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and microarrays. The response of ovariectomized adults to 17ß-estradiol (E2) and artificial decidualization were measured. Leukemia inhibitory factor (LIF) injections were given intraperitoneally and implantation sites visualized. Gene expression patterns were compared with curated data sets to identify upstream regulators. RESULTS: GEN-exposed mice exhibited reduced uterine weight gain in response to E2 treatment or artificial decidualization compared with controls; however, expression of select hormone responsive genes remained similar between the two groups. Uteri from pregnant GEN-exposed mice were posteriorized and had reduced glandular epithelium. Implantation failure was not rescued by LIF administration. Microarray analysis of GEN-exposed uteri during early pregnancy revealed significant overlap with several conditional uterine knockout mouse models, including Foxa2, Wnt4, and Sox17. These models exhibit reduced endometrial glands, features of posteriorization and implantation failure. Expression of Foxa2, Wnt4, and Sox17, as well as genes important for neonatal uterine differentiation (Wnt7a, Hoxa10, and Msx2), were severely disrupted on PND5 in GEN-exposed mice. DISCUSSION: Our findings suggest that neonatal GEN exposure in mice disrupts expression of genes important for uterine development, causing posteriorization and diminished gland function during pregnancy that contribute to implantation failure. These findings could have implications for women who consumed soy-based formulas as infants. https://doi.org/10.1289/EHP6336.


Assuntos
Implantação do Embrião/efeitos dos fármacos , Genisteína/efeitos adversos , Fitoestrógenos/efeitos adversos , Útero/efeitos dos fármacos , Animais , Feminino , Camundongos , Gravidez , Útero/crescimento & desenvolvimento , Útero/fisiopatologia
4.
Toxicol Pathol ; 47(8): 1049-1071, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31833458

RESUMO

During the past 20 years, investigations involving endocrine active substances (EAS) and reproductive toxicity have dominated the landscape of ecotoxicological research. This has occurred in concert with heightened awareness in the scientific community, general public, and governmental entities of the potential consequences of chemical perturbation in humans and wildlife. The exponential growth of experimentation in this field is fueled by our expanding knowledge into the complex nature of endocrine systems and the intricacy of their interactions with xenobiotic agents. Complicating factors include the ever-increasing number of novel receptors and alternate mechanistic pathways that have come to light, effects of chemical mixtures in the environment versus those of single EAS laboratory exposures, the challenge of differentiating endocrine disruption from direct cytotoxicity, and the potential for transgenerational effects. Although initially concerned with EAS effects chiefly in the thyroid glands and reproductive organs, it is now recognized that anthropomorphic substances may also adversely affect the nervous and immune systems via hormonal mechanisms and play substantial roles in metabolic diseases, such as type 2 diabetes and obesity.


Assuntos
Disruptores Endócrinos/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Reprodução/efeitos dos fármacos , Animais , Congressos como Assunto , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/embriologia , Humanos , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Especificidade da Espécie , Testículo/efeitos dos fármacos , Testículo/embriologia , Testículo/patologia , Útero/efeitos dos fármacos , Útero/embriologia , Útero/patologia
5.
Mol Cancer Res ; 17(12): 2369-2382, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597742

RESUMO

Early-life exposure to estrogenic chemicals can increase cancer risk, likely by disrupting normal patterns of cellular differentiation. Female mice exposed neonatally to the synthetic estrogen diethylstilbestrol (DES) develop metaplastic and neoplastic uterine changes as adults. Abnormal endometrial glands express the oncofetal protein sine oculis homeobox 1 (SIX1) and contain cells with basal [cytokeratin (CK)14+/18-] and poorly differentiated features (CK14+/18+), strongly associating SIX1 with aberrant differentiation and cancer. Here, we tested whether SIX1 expression is necessary for abnormal endometrial differentiation and DES-induced carcinogenesis by using Pgr-cre to generate conditional knockout mice lacking uterine Six1 (Six1 d/d). Interestingly, corn oil (CO) vehicle-treated Six1 d/d mice develop focal endometrial glandular dysplasia and features of carcinoma in situ as compared with CO wild-type Six1 (Six1 +/+) mice. Furthermore, Six1 d/d mice neonatally exposed to DES had a 42% higher incidence of endometrial cancer relative to DES Six1 +/+ mice. Although DES Six1 d/d mice had >10-fold fewer CK14+/18- basal cells within the uterine horns as compared with DES Six1 +/+ mice, the appearance of CK14+/18+ cells remained a feature of neoplastic lesions. These findings suggest that SIX1 is required for normal endometrial epithelial differentiation, CK14+/18+ cells act as a cancer progenitor population, and SIX1 delays DES-induced endometrial carcinogenesis by promoting basal differentiation of CK14+/18+ cells. In human endometrial biopsies, 35% of malignancies showed CK14+/18+ expression, which positively correlated with tumor stage and grade and was not present in normal endometrium. IMPLICATIONS: Aberrant epithelial differentiation is a key feature in both the DES mouse model of endometrial cancer and human endometrial cancer. The association of CK14+/18+ cells with human endometrial cancer provides a novel cancer biomarker and could lead to new therapeutic strategies.


Assuntos
Dietilestilbestrol/toxicidade , Hiperplasia Endometrial/genética , Neoplasias do Endométrio/genética , Estrogênios/toxicidade , Proteínas de Homeodomínio/genética , Animais , Animais Recém-Nascidos , Carcinogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Óleo de Milho/farmacologia , Dietilestilbestrol/farmacologia , Modelos Animais de Doenças , Hiperplasia Endometrial/induzido quimicamente , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/induzido quimicamente , Neoplasias do Endométrio/patologia , Endométrio/efeitos dos fármacos , Endométrio/patologia , Células Epiteliais/efeitos dos fármacos , Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Queratina-14/genética , Camundongos
6.
Toxicol Pathol ; 46(5): 574-596, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29895210

RESUMO

Developmental exposure to estrogenic chemicals is an established risk factor for cancer of the female reproductive tract. This increase in risk has been associated with disruption of normal patterns of cellular differentiation during critical stages of morphogenesis. The goal of this study was to document uterine epithelial phenotypes over time following neonatal treatment with the synthetic estrogen diethylstilbestrol (DES) or the soy phytoestrogen genistein (GEN) in female CD-1 mice. Both DES and GEN induced three distinct populations of abnormal endometrial epithelial cells: luminal (SIX1+/P63-/CK14-/CK18+), basal (SIX1+/P63+/CK14+/CK18-), and mixed/bipotential (SIX1+/P63-/CK14+/CK18+), which were all established by early adulthood. In older animals, DES and GEN resulted in uterine carcinomas with mixed glandular, basal, and squamous cell elements. All carcinomas were composed largely of the three abnormal cell types. These findings identify novel epithelial differentiation patterns in the uterus and support the idea that disruption of cellular programming in early development can influence cancer risk later in life.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Neoplasias do Endométrio/induzido quimicamente , Endométrio/efeitos dos fármacos , Estrogênios/toxicidade , Morfogênese/efeitos dos fármacos , Lesões Pré-Cancerosas/induzido quimicamente , Animais , Animais Recém-Nascidos , Dietilestilbestrol/toxicidade , Neoplasias do Endométrio/patologia , Endométrio/crescimento & desenvolvimento , Endométrio/patologia , Feminino , Genisteína/toxicidade , Imuno-Histoquímica , Camundongos Endogâmicos , Lesões Pré-Cancerosas/patologia
7.
Nucleic Acids Res ; 46(11): 5487-5503, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29648668

RESUMO

Little is known regarding how steroid hormone exposures impact the epigenetic landscape in a living organism. Here, we took a global approach to understanding how exposure to the estrogenic chemical, diethylstilbestrol (DES), affects the neonatal mouse uterine epigenome. Integration of RNA- and ChIP-sequencing data demonstrated that ∼80% of DES-altered genes had higher H3K4me1/H3K27ac signal in close proximity. Active enhancers, of which ∼3% were super-enhancers, had a high density of estrogen receptor alpha (ERα) binding sites and were correlated with alterations in nearby gene expression. Conditional uterine deletion of ERα, but not the pioneer transcription factors FOXA2 or FOXO1, prevented the majority of DES-mediated changes in gene expression and H3K27ac signal at target enhancers. An ERα dependent super-enhancer was located at the Padi gene locus and a topological connection to the Padi1 TSS was documented using 3C-PCR. Chromosome looping at this site was independent of ERα and DES exposure, indicating that the interaction is established prior to ligand signaling. However, enrichment of H3K27ac and transcriptional activation at this locus was both DES and ERα-dependent. These data suggest that DES alters uterine development and consequently adult reproductive function by modifying the enhancer landscape at ERα binding sites near estrogen-regulated genes.


Assuntos
Dietilestilbestrol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios não Esteroides/farmacologia , Regulação da Expressão Gênica/genética , Sequências Reguladoras de Ácido Nucleico/genética , Útero/embriologia , Animais , Sítios de Ligação/genética , Receptor alfa de Estrogênio/genética , Estrogênios não Esteroides/metabolismo , Feminino , Proteína Forkhead Box O1/genética , Fator 3-beta Nuclear de Hepatócito/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Metilação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Regiões Promotoras Genéticas/genética
8.
Reprod Toxicol ; 68: 85-104, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27421580

RESUMO

Sex-specific differentiation, development, and function of the reproductive system are largely dependent on steroid hormones. For this reason, developmental exposure to estrogenic and anti-androgenic endocrine disrupting chemicals (EDCs) is associated with reproductive dysfunction in adulthood. Human data in support of "Developmental Origins of Health and Disease" (DOHaD) comes from multigenerational studies on offspring of diethylstilbestrol-exposed mothers/grandmothers. Animal data indicate that ovarian reserve, female cycling, adult uterine abnormalities, sperm quality, prostate disease, and mating behavior are susceptible to DOHaD effects induced by EDCs such as bisphenol A, genistein, diethylstilbestrol, p,p'-dichlorodiphenyl-dichloroethylene, phthalates, and polyaromatic hydrocarbons. Mechanisms underlying these EDC effects include direct mimicry of sex steroids or morphogens and interference with epigenomic sculpting during cell and tissue differentiation. Exposure to EDCs is associated with abnormal DNA methylation and other epigenetic modifications, as well as altered expression of genes important for development and function of reproductive tissues. Here we review the literature exploring the connections between developmental exposure to EDCs and adult reproductive dysfunction, and the mechanisms underlying these effects.


Assuntos
Disruptores Endócrinos/toxicidade , Exposição Ambiental/efeitos adversos , Epigênese Genética/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Reprodução/efeitos dos fármacos , Animais , Feminino , Idade Gestacional , Humanos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Reprodução/genética
9.
Mol Cancer Res ; 14(9): 849-58, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27259717

RESUMO

UNLABELLED: The oncofetal protein sine oculis-related homeobox 1 (SIX1) is a developmental transcription factor associated with carcinogenesis in several human cancer types but has not been investigated in human endometrial cancer. In a model of hormonal carcinogenesis, mice neonatally exposed to the soy phytoestrogen genistein (GEN) or the synthetic estrogen diethylstilbestrol (DES) develop endometrial cancer as adults. Previously, we demonstrated that SIX1 becomes aberrantly expressed in the uteri of these mice. Here, we used this mouse model to investigate the role of SIX1 expression in endometrial carcinoma development and used human tissue microarrays to explore the utility of SIX1 as a biomarker in human endometrial cancer. In mice neonatally exposed to GEN or DES, the Six1 transcript level increased dramatically over time in uteri at 6, 12, and 18 months of age and was associated with development of endometrial carcinoma. SIX1 protein localized within abnormal basal cells and all atypical hyperplastic and neoplastic lesions. These findings indicate that developmental estrogenic chemical exposure induces persistent endometrial SIX1 expression that is strongly associated with abnormal cell differentiation and cancer development. In human endometrial tissue specimens, SIX1 was not present in normal endometrium but was expressed in a subset of endometrial cancers in patients who were also more likely to have late-stage disease. These findings identify SIX1 as a disease biomarker in a model of hormonal carcinogenesis and suggest that SIX1 plays a role in endometrial cancer development in both mice and women. IMPLICATIONS: The SIX1 oncoprotein is aberrantly expressed in the endometrium following developmental exposure to estrogenic chemicals, correlates with uterine cancer, and is a biomarker in human endometrial cancers. Mol Cancer Res; 14(9); 849-58. ©2016 AACR.


Assuntos
Biomarcadores Tumorais/biossíntese , Carcinogênese/metabolismo , Neoplasias do Endométrio/metabolismo , Proteínas de Homeodomínio/biossíntese , Animais , Animais Recém-Nascidos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Diferenciação Celular/fisiologia , Dietilestilbestrol , Modelos Animais de Doenças , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Genisteína , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Fatores de Transcrição/metabolismo , Útero/metabolismo , Útero/patologia
10.
Nat Commun ; 4: 2910, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24335996

RESUMO

Although hypertension is a worldwide health issue, an incomplete understanding of its aetiology has hindered our ability to treat this complex disease. Here we identify arhgap42 (also known as GRAF3) as a Rho-specific GAP expressed specifically in smooth muscle cells (SMCs) in mice and humans. We show that GRAF3-deficient mice exhibit significant hypertension and increased pressor responses to angiotensin II and endothelin-1; these effects are prevented by treatment with the Rho-kinase inhibitor, Y27632. RhoA activity and myosin light chain phosphorylation are elevated in GRAF3-depleted SMCs in vitro and in vivo, and isolated vessel segments from GRAF3-deficient mice show increased contractility. Taken together, our data indicate that GRAF3-mediated inhibition of RhoA activity in vascular SMCs is necessary for maintaining normal blood pressure homoeostasis. Moreover, these findings provide a potential mechanism for a hypertensive locus recently identified within arhgap42 and provide a foundation for the future development of innovative hypertension therapies.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Hipertensão/fisiopatologia , Músculo Liso Vascular/metabolismo , Sequência de Aminoácidos , Angiotensina II/farmacologia , Animais , Aorta/metabolismo , Pressão Sanguínea/genética , Vasos Sanguíneos/fisiopatologia , Células Cultivadas , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/etiologia , Hipertensão/genética , Técnicas In Vitro , Masculino , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Ratos , Proteína rhoA de Ligação ao GTP/metabolismo
11.
J Mol Biol ; 425(8): 1378-89, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23396064

RESUMO

Identification of residues responsible for functional specificity in enzymes is a challenging and important problem in protein chemistry. Active-site residues are generally easy to identify, but residues outside the active site are also important to catalysis and their identities and roles are more difficult to determine. We report a method based on analysis of multiple sequence alignments, embodied in our program Janus, for predicting mutations required to interconvert structurally related but functionally distinct enzymes. Conversion of aspartate aminotransferase into tyrosine aminotransferase is demonstrated and compared to previous efforts. Incorporation of 35 predicted mutations resulted in an enzyme with the desired substrate specificity but low catalytic activity. A single round of DNA back-shuffling with wild-type aspartate aminotransferase on this variant generated mutants with tyrosine aminotransferase activities better than those previously realized from rational design or directed evolution. Methods such as this, coupled with computational modeling, may prove invaluable in furthering our understanding of enzyme catalysis and engineering.


Assuntos
Biologia Computacional/métodos , Análise Mutacional de DNA , Escherichia coli/enzimologia , Mutação de Sentido Incorreto , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Especificidade por Substrato , Tirosina Transaminase/genética , Tirosina Transaminase/metabolismo
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