scholarly journals Estrogen suppresses SOX9 and activates markers of female development in a human testis-derived cell line

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Melanie K. Stewart ◽  
Deidre M. Mattiske ◽  
Andrew J. Pask
Keyword(s):  
Reproductive Health ◽  
Cell Line ◽  
Cell Fate ◽  
Sertoli Cell ◽  
Endocrine Disruptors ◽  
Estrogen Receptor Alpha ◽  
Human Testis ◽  
Mrna Levels ◽  
Cell Fate Decisions ◽  
Estrogen Exposure

Abstract Background The increasing incidence of reproductive disorders in humans has been attributed to in utero exposure to estrogenic endocrine disruptors. In particular, exposure of the developing testis to exogenous estrogen can negatively impact male reproductive health. To determine how estrogens impact human gonad function, we treated the human testis-derived cell line NT2/D1 with estrogen and examined its impact on SOX9 and the expression of key markers of granulosa (ovarian) and Sertoli (testicular) cell development. Results Estrogen successfully activated its cognate receptor (estrogen receptor alpha; ESR1) in NT2/D1 cells. We observed a significant increase in cytoplasmic SOX9 following estrogen treatment. After 48 h of estrogen exposure, mRNA levels of the key Sertoli cell genes SOX9, SRY, AMH, FGF9 and PTGDS were significantly reduced. This was followed by a significant increase in mRNA levels for the key granulosa cell genes FOXL2 and WNT4 after 96 h of estrogen exposure. Conclusions These results are consistent with estrogen's effects on marsupial gonads and show that estrogen has a highly conserved impact on gonadal cell fate decisions that has existed in mammals for over 160 million years. This effect of estrogen presents as a potential mechanism contributing to the significant decrease in male fertility and reproductive health reported over recent decades. Given our widespread exposure to estrogenic endocrine disruptors, their effects on SOX9 and Sertoli cell determination could have considerable impact on the adult testis.

scholarly journals The research progress of p53 tumour suppressor activity controlled by Numb in triple-negative breast cancer

2018 ◽  
Author(s):  
Jie Xian ◽  
Yu Cheng ◽  
Xue Qin ◽  
Yequn Luo ◽  
Yijia Cao ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cell Fate ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Research Progress ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Suppressor Activity ◽  
Cell Fractions

AbstractNumb is known as a cell fate determinant as it determines the direction of cell differentiation by asymmetrically partitioning at mitosis. It has been shown to be a tumor suppressor, and there is frequent loss of Numb expression in breast cancer. Numb enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. The aim of this study was to investigate the expression and migration of Numb, HDM2 and p53 proteins in the membrane, cytoplasmic and nuclear fractions of MCF-10A cells and MDA-MB-231 cells. We extracted the cell fractions to detect changes of these three protein levels after re-expression of NUMB in the basal-like triple-negative cell line MDA-MB-231 and knocking down NUMB in the normal mammary epithelial cell line MCF-10A. Our results show that Numb protein can migrate from cytoplasm to nucleus of the MDA-MB-231 cells. Numb protein regulates p53 levels in nucleus of MCF-10A cells and MDA-MB-231 cells, and the Numb levels is positively correlated with p53 levels. We have a finding on HDM2 protein that after knocking down NUMB in MCF-10A cells, it was remarkably reduced in the membrane fraction of NUMB knockdown cells, but its mRNA levels was significantly increased. We also examined the Numb expression in 125 patients with triple-negative breast cancer, 61(48.8%) displayed deficient or reduced expression of Numb. The percent of Ki67>14% in retained Numb group was significantly lower than that in the reduced and deficient Numb group (86.00% vs. 98.40%, P=0.0171).

SOX9 and SF1 are involved in cyclic AMP-mediated upregulation of anti-Müllerian gene expression in the testicular prepubertal Sertoli cell line SMAT1

2011 ◽  
Vol 301 (3) ◽  
pp. E539-E547 ◽  
Author(s):  
Celina Lasala ◽  
Helena F. Schteingart ◽  
Nassim Arouche ◽  
Patricia Bedecarrás ◽  
Romina P. Grinspon ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cell Line ◽  
Sertoli Cell ◽  
Nuclear Translocation ◽  
Targeted Mutagenesis ◽  
Signaling Cascades ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Alternative Pathways ◽  
Sertoli Cell Line

In Sertoli cells, anti-Müllerian hormone (AMH) expression is upregulated by FSH via cyclic AMP (cAMP), although no classical cAMP response elements exist in the AMH promoter. The response to cAMP involves NF-κB and AP2; however, targeted mutagenesis of their binding sites in the AMH promoter do not completely abolish the response. In this work we assessed whether SOX9, SF1, GATA4, and AP1 might represent alternative pathways involved in cAMP-mediated AMH upregulation, using real-time RT-PCR (qPCR), targeted mutagenesis, luciferase assays, and immunocytochemistry in the Sertoli cell line SMAT1. We also explored the signaling cascades potentially involved. In qPCR experiments, Amh, Sox9, Sf1, and Gata4 mRNA levels increased after SMAT1 cells were incubated with cAMP. Blocking PKA abolished the effect of cAMP on Sox9, Sf1, and Gata4 expression, inhibiting PI3K/PKB impaired the effect on Sf1 and Gata4, and reducing MEK1/2 and p38 MAPK activities curtailed Gata4 increase. SOX9 and SF1 translocated to the nucleus after incubation with cAMP. Mutations of the SOX9 or SF1 sites, but not of GAT4 or AP1 sites, precluded the response of a 3,063-bp AMH promoter to cAMP. In conclusion, in the Sertoli cell line SMAT1 cAMP upregulates SOX9, SF1, and GATA4 expression and induces SOX9 and SF1 nuclear translocation mainly through PKA, although other kinases may also participate. SOX9 and SF1 binding to the AMH promoter is essential to increase the activity of the AMH promoter in response to cAMP.

scholarly journals Antisense oligonucleotides targeting Notch2 ameliorate the osteopenic phenotype in a mouse model of Hajdu-Cheney syndrome

2020 ◽  
Vol 295 (12) ◽  
pp. 3952-3964 ◽  
Author(s):  
Ernesto Canalis ◽  
Tamar R. Grossman ◽  
Michele Carrer ◽  
Lauren Schilling ◽  
Jungeun Yu
Keyword(s):  
Mouse Model ◽  
Cell Fate ◽  
Antisense Oligonucleotides ◽  
Target Genes ◽  
Skeletal Development ◽  
Subcutaneous Administration ◽  
Mrna Levels ◽  
Developmental Abnormalities ◽  
Cell Fate Decisions ◽  
Related Family

Notch receptors play critical roles in cell-fate decisions and in the regulation of skeletal development and bone remodeling. Gain–of–function NOTCH2 mutations can cause Hajdu-Cheney syndrome, an untreatable disease characterized by osteoporosis and fractures, craniofacial developmental abnormalities, and acro-osteolysis. We have previously created a mouse model harboring a point 6955C→T mutation in the Notch2 locus upstream of the PEST domain, and we termed this model Notch2tm1.1Ecan. Heterozygous Notch2tm1.1Ecan mutant mice exhibit severe cancellous and cortical bone osteopenia due to increased bone resorption. In this work, we demonstrate that the subcutaneous administration of Notch2 antisense oligonucleotides (ASO) down-regulates Notch2 and the Notch target genes Hes-related family basic helix–loop–helix transcription factor with YRPW motif 1 (Hey1), Hey2, and HeyL in skeletal tissue from Notch2tm1.1Ecan mice. Results of microcomputed tomography experiments indicated that the administration of Notch2 ASOs ameliorates the cancellous osteopenia of Notch2tm1.1Ecan mice, and bone histomorphometry analysis revealed decreased osteoclast numbers in Notch2 ASO-treated Notch2tm1.1Ecan mice. Notch2 ASOs decreased the induction of mRNA levels of TNF superfamily member 11 (Tnfsf11, encoding the osteoclastogenic protein RANKL) in cultured osteoblasts and osteocytes from Notch2tm1.1Ecan mice. Bone marrow-derived macrophage cultures from the Notch2tm1.1Ecan mice displayed enhanced osteoclastogenesis, which was suppressed by Notch2 ASOs. In conclusion, Notch2tm1.1Ecan mice exhibit cancellous bone osteopenia that can be ameliorated by systemic administration of Notch2 ASOs.

scholarly journals Epstein-Barr Virus Nuclear Antigen 2 trans-Activates the Cellular Antiapoptotic bfl-1 Gene by a CBF1/RBPJκ-Dependent Pathway

2006 ◽  
Vol 80 (16) ◽  
pp. 8133-8144 ◽  
Author(s):  
Pamela M. Pegman ◽  
Sinéad M. Smith ◽  
Brendan N. D'Souza ◽  
Sinéad T. Loughran ◽  
Sabine Maier ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Mrna Levels ◽  
Term Survival ◽  
Enhancer Element ◽  
Cell Fate Decisions ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The human herpesvirus Epstein-Barr virus (EBV) establishes latency and promotes the long-term survival of its host B cell by targeting the molecular machinery controlling cell fate decisions. The cellular antiapoptotic bfl-1 gene confers protection from apoptosis under conditions of growth factor deprivation when expressed ectopically in an EBV-negative Burkitt's lymphoma-derived cell line (B. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000), and the EBV latent membrane protein 1 (LMP1) and its cellular functional homologue CD40 can both drive bfl-1 via an NF-κB-dependent enhancer element in the bfl-1 promoter (B. N. D'Souza, L. C. Edelstein, P. M. Pegman, S. M. Smith, S. T. Loughran, A. Clarke, A. Mehl, M. Rowe, C. Gélinas, and D. Walls, J. Virol. 78:1800-1816, 2004). Here we show that the EBV nuclear antigen 2 (EBNA2) also upregulates bfl-1. EBNA2 trans-activation of bfl-1 requires CBF1 (or RBP-Jκ), a nuclear component of the Notch signaling pathway, and there is an essential role for a core consensus CBF1-binding site on the bfl-1 promoter. trans-activation is dependent on the EBNA2-CBF1 interaction, is modulated by other EBV gene products known to interact with the CBF1 corepressor complex, and does not involve activation of NF-κB. bfl-1 expression is induced and maintained at high levels by the EBV growth program in a lymphoblastoid cell line, and withdrawal of either EBNA2 or LMP1 does not lead to a reduction in bfl-1 mRNA levels in this context, whereas the simultaneous loss of both EBV proteins results in a major decrease in bfl-1 expression. These findings are relevant to our understanding of EBV persistence, its role in malignant disease, and the B-cell developmental process.

Estrogens and falling sperm counts

2001 ◽  
Vol 13 (4) ◽  
pp. 317 ◽  
Author(s):  
D. J. Handelsman
Keyword(s):  
Reproductive Health ◽  
Male Fertility ◽  
Human Sperm ◽  
Population Based ◽  
Human Testis ◽  
Estrogen Exposure ◽  
Surrogate Variables ◽  
Sperm Counts ◽  
Sperm Output ◽  
Male Reproductive Health

Extravagant claims have been made repeatedly in recent years that human sperm counts are falling and that global exposure to environmental estrogens are responsible. The basis for these two distinct claims is reviewed. The claims of falling human sperm output, reviving an old debate, are prompted by a paper by Carlsen et al. (1992). This meta-analysis, however, is marred by numerous flaws that invalidate its claims. Major defects include severe heterogeneity of component studies, rendering them unsuitable for aggregation, and defective data analysis based on arithmetic mean rather than median, which showed no significant changes over time. This debate is likely to remain unresolved until valid, representative population-based studies of human sperm output can be achieved. None have been reported, or seem feasible in the near future, and so alternative strategies, based on surrogate variables for human male fertility not requiring sperm counts, need to be developed and validated. The plausible hypothesis that prenatal estrogen exposure might influence development of the human testis through effects on Sertoli cell replication and sperm carrying capacity has, however, been conclusively refuted by studies of boys born to women exposed to high doses of oral diethylstilbestrol during pregnancy. Neither fertility nor sperm output were adversely influenced by massive maternal estrogen exposure during pregnancy, although minor urogenital malformations did occur. The still wider claims of deteriorating male reproductive health, notably changes in prevalence or incidence of hypospadias or cryptorchidism, also lack convincing population-based evidence, although cancer registry data indicate a gradual increase in testis cancer in some countries. In summary, the available evidence does not support claims of falling sperm counts or any general deterioration in male reproductive health. Population-based studies of valid surrogate variables for male fertility not requiring semen analysis are needed. If population-based evidence regarding male fertility or sperm output could be generated, it is highly unlikely that prenatal estrogen exposure could be a valid explanation of any deterioration as massive maternal exposure to oral estrogen has negligible effects on male fertility or sperm output.

scholarly journals Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation

2020 ◽  
Vol 29 (15) ◽  
pp. 2535-2550
Author(s):  
Judith C Birkhoff ◽  
Rutger W W Brouwer ◽  
Petros Kolovos ◽  
Anne L Korporaal ◽  
Ana Bermejo-Santos ◽  
...  
Keyword(s):  
Cell Fate ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Regulatory Elements ◽  
Specific Cell ◽  
Mrna Levels ◽  
Gene Desert ◽  
Protein Coding ◽  
Cell Fate Decisions ◽  
In Cis

Abstract The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat–Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2’s gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.

C/EBPβ (CCAAT/Enhancer Binding Protein) Controls Cell Fate Determination during Mammary Gland Development

2000 ◽  
Vol 14 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Tiffany N. Seagroves ◽  
John P. Lydon ◽  
Russell C. Hovey ◽  
Barbara K. Vonderhaar ◽  
Jeffrey M. Rosen
Keyword(s):  
Mammary Gland ◽  
Cell Fate ◽  
Binding Protein ◽  
Mammary Glands ◽  
Mammary Development ◽  
Mrna Levels ◽  
Wild Type ◽  
Cell Fate Decisions ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Abstract Deletion of the transcription factor CCAAT/enhancer binding protein (C/EBP)β results in a severe inhibition of lobuloalveolar development in the mouse mammary gland. Because progesterone receptor (PR) is requisite for alveolar development, the expression of PR was investigated in C/EBPβ−/− mice. Unexpectedly, the number of PR-positive cells, as well as the levels of PR mRNA, were elevated 3-fold in the mammary glands of C/EBPβ−/− mice. Furthermore, in contrast to wild-type nulliparous mice, in which PR distribution shifted from a uniform to nonuniform pattern between 8–12 weeks of age, C/EBPβ−/− mice exhibited uniform PR distribution throughout all stages of mammary development analyzed. No change in C/EBPβ mRNA levels was observed in the mammary glands of PR−/− mice, suggesting that PR acts in a pathway either in parallel to or downstream of C/EBPβ. The overexpression and disrupted cellular distribution of PR in C/EBPβ−/− mice were coincident with a striking 10-fold decrease in cell proliferation after acute steroid hormone treatment, assayed by incorporation of bromodeoxyuridine. In wild-type mice, PR and bromodeoxyuridine-positive cells were adjacent to each other and rarely colocalized. No differences in the level or pattern of PR expression were observed in the uterus, suggesting that C/EBPβ influences PR in a mam-mary-specific fashion. Together, these data suggest that C/EBPβ may control cell fate decisions in the mammary gland through the appropriate temporal and spatial expression of molecular markers, such as PR, that induce the proliferation of alveolar progenitor cells via juxtacrine mechanisms.

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