211. Expression and cellular localization of HTRA3 protease during placental development in mice

2005 ◽  
Vol 17 (9) ◽  
pp. 81
Author(s):  
Y. Li ◽  
L. A. Salamonsen ◽  
G. Nie
Keyword(s):  
Blot Analysis ◽  
Cellular Localization ◽  
Giant Cells ◽  
Binding Domain ◽  
Placental Development ◽  
Mouse Uterus ◽  
Decidual Cells ◽  
Igf Binding ◽  
Near Term ◽  
And Function

Placental development in mice involves highly regulated interactions between fetal- and maternal-derived cells. We have previously cloned a novel serine protease (HtrA3) containing an insulin-like growth factor (IGF) binding domain, which was upregulated during pregnancy, especially post-implantation in the mouse uterus.1 The present study examined HtrA3 regulation during placental development in mice, in particular, its expression in the different compartments of the placenta. Expression of mRNA was determined by Northern blot analysis in implantation units containing the decidua, placenta and fetus (day 8.5 to near-term). A specific HtrA3 antibody was generated, affinity-purified and used for Western blot analysis and immunohistochemistry. Both mRNA and protein of HtrA3 were identified specifically in the maternal decidua. In contrast, HtrA3 expression was below detection in trophoblasts, including the giant cells that are in direct contact with the decidua. This pattern persisted from the early stages of placentation to near term. The level of decidual HtrA3 mRNA and its protein gradually decreased as the placenta matured. In the decidua, only the maternal decidual cells, but not blood vessels or uterine NK cells that are present in large numbers, were positive for HtrA3. The specific localization of HtrA3, a protease possessing an IGF binding domain at the maternal–fetal interface, suggests that this protein plays an important role in mediating maternal decidual remodelling and maintenance, probably in association with the IGF system, in placental development and function. (1)Nie et al. (2003). Mol. Hum. Reprod.

scholarly journals Studying the Role of ETV6 in Megakaryopoiesis and Thrombopoiesis Using a Novel CRISPR-Cas9 Halotag Genome Editing Strategy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2468-2468 ◽  
Author(s):  
Marlie H Fisher ◽  
Leila J. Noetzli ◽  
Michael Callaghan ◽  
Walter H. Kahr ◽  
Jesse W Rowley ◽  
...  
Keyword(s):  
Dna Binding ◽  
Research Funding ◽  
Cellular Localization ◽  
Binding Domain ◽  
Clinical Approach ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Transcriptional Targets ◽  
And Function

Emerging data indicate that germline mutations in transcription factors involved in hematopoiesis can lead to a cascade of downstream molecular alterations that modify the function of megakaryocytes (MK) and platelets. Our group and others have found that mutations in ETV6 lead to mild thrombocytopenia with a bleeding diathesis, red cell macrocytosis, and predisposition to lymphoblastic leukemia. The mechanisms responsible for thrombocytopenia and propensity for bleeding in patients with ETV6 mutations are unknown. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.Pro214Leu mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. We hypothesize that normal regulation and function of ETV6 is essential for the transcriptional machinery that controls megakaryocyte differentiation and formation of platelets that function normally under homeostatic conditions. We have successfully generated a CRISPR-Cas9 model to edit the genome of ETV6-expressing iPSC derived megakaryocyte cell line (imMKCL) to characterize the role of wild-type ETV6 in megakaryocyte development and elucidate the molecular mechanism driving mutant ETV6 mislocalization, transcriptional dysregulation, and subsequent dysmegakaryopoiesis and thrombocytopenia. In this imMKCL model, we have genetically engineered the cells to express wild-type, P214L, and the DNA binding domain mutations R418G and R369Q ETV6 fused to HALOtag, a reporter protein that can react with ligands carrying a variety of functionalities, including fluorescent labels, affinity handles, and attachment to solid phase, making this novel reporter conducive to immunofluorescence imaging, biochemical pulldown, and ChIPSeq. This system allows us to express wild type and mutant forms of ETV6 in appropriate allele ratios in imMKCL cells and various hematopoietic-relevant cell lines. Using this approach, we detected nuclear localization of wild-type ETV6 and altered cytoplasmic localization of both P214L and R418G ETV6 mutants. We have also demonstrated dimerization between both wild-type and mutant ETV6 in this cell model. Importantly, we have used HALOtag protein immunoprecipitation to demonstrate ETV6 binding to FLI1, another ETS family member and key transcriptional regulator of megakaryocyte development, suggesting that ETV6 and FLI1 cooperate to regulate megakaryopoiesis under homeostatic conditions. Altogether, these data suggest that mutant ETV6 functions as a dominant negative, sequestering wild type ETV6 in the cytoplasm, de-regulating key transcriptional targets for homeostatic megakaryocyte development. Ongoing studies will define the full repertoire of protein interactions and transcriptional targets of wild-type and mutant ETV6. Discoveries from this novel tool will further advance our understanding of normal megakaryocyte and platelet biology, and will provide potential therapeutic targets for disorders of platelet number and function to optimize the clinical approach to these patients. Disclosures Callaghan: Bayer: Consultancy, Speakers Bureau; Alnylum: Equity Ownership; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.

scholarly journals IGFBP-1 forms associated with placental cell membranes

2009 ◽  
Vol 74 (7) ◽  
pp. 707-716 ◽  
Author(s):  
Romana Masnikosa ◽  
Bogovid Zivkovic ◽  
Olgica Nedic
Keyword(s):  
Molecular Mass ◽  
Cell Membranes ◽  
High Molecular Mass ◽  
Matrix Metalloproteases ◽  
Placental Development ◽  
Placental Cell ◽  
Igf Receptors ◽  
Igf Binding ◽  
And Function ◽  
Igf Binding Protein

Fetal growth in utero depends on the proper development and function of the placenta. Insulin-like growth factors (IGFs) are critically involved in placental development. During pregnancy, an IGF-binding protein, IGFBP-1, which is produced by maternal decidua, plays an important role in the control of the bioavailability of IGFs. It has recently been proposed that cleavage of decidual IGFBP-1 by matrix metalloproteases is a novel mechanism in the control of placental development. The presence of IGFBP-1 in solubilized placental cell membranes, i.e. its association with the membranes, was detected in an earlier work. Herein, it is shown that IGFBP-1 from the solubilized membranes forms dimers, as well as high molecular mass complexes. IGFBP-1 dimers preferably contain the non-phosphorylated form of IGFBP-1. The high molecular mass forms are polymers of IGFBP-1 or its complexes with other membrane proteins. Dimerization of IGFBP-1, together with its association with the placental cell membrane, could serve as an additional mechanism of the regulation of IGF availability to the type 1 IGF receptors.

scholarly journals Transforming Growth Factor-β (TGFβ) Receptors I/II Differentially Regulate TGFβ1 and IGF-Binding Protein-3 Mitogenic Effects in the Human Placenta

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1723-1731 ◽  
Author(s):  
Karen Forbes ◽  
Benoit Souquet ◽  
Rebecca Garside ◽  
John D. Aplin ◽  
Melissa Westwood
Keyword(s):  
Transforming Growth Factor ◽  
Immunohistochemical Analysis ◽  
First Trimester ◽  
Signaling Molecules ◽  
Placental Development ◽  
Igf Binding Proteins ◽  
Tgfβ Receptors ◽  
Igf Binding ◽  
And Function ◽  
Igfbp 3

Maternal IGFs regulate cytotrophoblast proliferation and, thereby, placental growth and function. IGF bioavailability is controlled by IGF-binding proteins (IGFBPs); in placenta, IGFBP-3 is particularly abundant. In other systems, IGFBP-3 can regulate cellular events independently of IGFs; these effects are thought to be mediated by TGFβ receptors (TβR). We have examined IGFBP-3 regulation of IGF-dependent and -independent cytotrophoblast proliferation in first-trimester placental explants and the role of TβRII in mediating these effects. In the presence of IGFBP-3 (50 nm), IGF-induced (10 nm) proliferation (monitored by immunohistochemical analysis of Ki67 expression and bromodeoxyuridine incorporation) was significantly reduced (P < 0.05). IGFBP-3 also reduced basal proliferation independently of IGF receptor signaling. Immunohistochemical analysis demonstrated that TGFβ signaling molecules [TGFβ receptor I (TβRI), TβRII, TβRV, Smad-2, and ERK] are expressed in syncytium and/or cytotrophoblast. TGFβ1 (10 ng/ml) enhanced cytotrophoblast proliferation and activated both Smad-2 and ERK-1/2, whereas IGFBP-3 activated only Smad-2. The function of both TGFβ1 and IGFBP-3 was attenuated by a TβRII function-blocking antibody and by small interfering RNA-mediated knockdown of TβRII (P < 0.05); this was accompanied by a reduction in Smad-2 activation. This study demonstrates that both TGFβ1 and IGFBP-3 signal through TβRI/II to influence human cytotrophoblast proliferation. However, downstream pathways are distinct, because IGFBP-3 acts only through Smad-2, whereas TGFβ1 also phosphorylates ERK, resulting in opposite effects on cytotrophoblast proliferation. The effects of maternal growth signals on placental growth and function therefore depend on the balance of ligands, receptors, and signaling molecules at the syncytiotrophoblast surface. Therapeutic manipulation of this balance might offer a strategy to optimize placental development and pregnancy outcome.

scholarly journals Development of ovine chorionic somatomammotropin hormone-deficient pregnancies

2016 ◽  
Vol 310 (9) ◽  
pp. R837-R846 ◽  
Author(s):  
Callie M. Baker ◽  
Lindsey N. Goetzmann ◽  
Jeremy D. Cantlon ◽  
Kimberly M. Jeckel ◽  
Quinton A. Winger ◽  
...  
Keyword(s):  
Intrauterine Growth Restriction ◽  
Fetal Liver ◽  
Secretory Product ◽  
Placental Development ◽  
Mortality And Morbidity ◽  
Chorionic Somatomammotropin ◽  
Near Term ◽  
High Concentrations ◽  
And Function

Intrauterine growth restriction (IUGR) is a leading cause of neonatal mortality and morbidity. Chorionic somatomammotropin hormone (CSH), a placenta-specific secretory product found at high concentrations in maternal and fetal circulation throughout gestation, is significantly reduced in human and sheep IUGR pregnancies. The objective of this study was to knock down ovine CSH ( oCSH) expression in vivo using lentiviral-mediated short-hairpin RNA to test the hypothesis that oCSH deficiency would result in IUGR of near-term fetal lambs. Three different lentiviral oCSH-targeting constructs were used and compared with pregnancies ( n = 8) generated with a scrambled control (SC) lentiviral construct. Pregnancies were harvested at 135 days of gestation. The most effective targeting sequence, “target 6” (tg6; n = 8), yielded pregnancies with significant reductions ( P ≤ 0.05) in oCSH mRNA (50%) and protein (38%) concentrations, as well as significant reductions ( P ≤ 0.05) in placental (52%) and fetal (32%) weights compared with the SC pregnancies. Fetal liver weights were reduced 41% ( P ≤ 0.05), yet fetal liver insulin-like growth factor-I ( oIGF1) and -II mRNA concentrations were reduced ( P ≤ 0.05) 82 and 71%, respectively, and umbilical artery oIGF1 concentrations were reduced 62% ( P ≤ 0.05) in tg6 pregnancies. Additionally, fetal liver oIGF-binding protein ( oIGFBP) 2 and oIGFBP3 mRNA concentrations were reduced ( P ≤ 0.05), whereas fetal liver oIGFBP1 mRNA concentration was not impacted nor was maternal liver o IGF and o IGFBP mRNA concentrations or uterine artery oIGF1 concentrations ( P ≥ 0.10). Based on our results, it appears that oCSH deficiency does result in IUGR, by impacting placental development as well as fetal liver development and function.

scholarly journals Mouse Model of Abortion Induced by Brucella abortus Infection

2012 ◽  
Vol 7 (3) ◽  
pp. 313-318
Author(s):  
Masahisa Watarai ◽  
Keyword(s):  
Bacterial Infection ◽  
Brucella Abortus ◽  
Bacterial Colonization ◽  
Giant Cells ◽  
Placental Development ◽  
Pregnant Mice ◽  
Ifn Γ ◽  
Bacterial Replication ◽  
And Function ◽  
Trophoblast Giant Cells

The mechanisms of abortion induced by bacterial infection are largely unknown. We found that Brucella abortus, a causative agent of brucellosis and a facultative intracellular pathogen, caused abortion in pregnant mice. High rates of abortion are observed for bacterial infection on day 4.5 of gestation, but not for other days. Regardless of whether fetuses are aborted or not, the transmission of bacteria to the fetus and bacterial replication in the placenta are observed. There is a higher degree of bacterial colonization in the placenta than in other organs and many bacteria are detected in trophoblast giant cells in the placenta. The intracellular growth-defective virB4 mutant and attenuated vaccine strain S19 do not induce abortion. In the case of abortion, the induction of IFN-γ and RANTES production is observed at day 7.5 of gestation – the placental development period – for infection by the wild type strain but not by the virB4 mutant or S19. B. abortus-infected pregnant IFN-γ knockoutmice die within 15 days of infection, but nonpregnant IFN-γ knockout mice remain alive. The neutralization of IFN-γ or RANTES, in which production is induced by infection with B. abortus serves to prevent abortion. These results indicate that abortion induced by B. abortus infection is regulated by IFN-γ during the period of placental development, and the production and function of RANTES are correlated with IFN-γ.

scholarly journals 016.Role of cited genes in placental morphogenesis: studies in null mutant mice

2004 ◽  
Vol 16 (9) ◽  
pp. 16
Author(s):  
S. L. Dunwoodie ◽  
S. L. Withington ◽  
D. B. Sparrow ◽  
A. N. Scott ◽  
J. I. Preis ◽  
...  
Keyword(s):  
Hypoxia Inducible Factor ◽  
Giant Cells ◽  
Maternal Blood ◽  
Postnatal Period ◽  
Null Mutant ◽  
Placental Development ◽  
Null Mutant Mice ◽  
And Function ◽  
Trophoblast Giant Cells ◽  
Null Mutants

Cited1 and Cited2 interact with CBP and p300. CBP/p300 bind numerous proteins and evidence exists, for Cited2 at least, that Cited binding prevents the binding of other proteins to CBP/p300. Since CBP/p300 interact with many proteins, can acetylate protein and DNA, and act as a ubiquitin ligase, it is likely that Cited1 and Cited2 function at a number of sites during development. We have generated mice that carry a null mutant allele for each of these genes. Analysis of null mutant embryos demonstrates that both Cited1 and Cited2 are required for normal embryonic development and survival. Although both Cited1 and Cited2 are expressed in the developing embryo and placenta, it appears that abnormal placental development and function is the cause of embryonic death. The defect that develops in the placentas of Cited1 null mutants is not apparent until late in gestation (16.5dpc). Cited1 null mutants are smaller than controls at birth and die during the early postnatal period. The placentas of these mutants are disorganised, with spongiotrophoblasts projecting in to the labyrinthine layer. In addition, resin casts of the maternal blood spaces within these placentas revealed extremely enlarged blood sinuses. We are searching for factors that could result in the increased size of the maternal blood sinuses. Cited2 null placentas and embryos are significantly smaller than controls; mutants die 3/4 the way through gestation (15.5dpc). The null mutant placentas have proportionally fewer spongiotrophoblasts, trophoblast giant cells and invasive trophoblasts. In addition, resin casts of fetal vasculature of the placenta reveal that the capillary network is underdeveloped. Through the isolation of trophoblast stem (TS) cells we are exploring the possibility that TS cell proliferation and/or differentiation is impaired due to a lack of Cited2. We suspect that the development of the phenotype may relate to the Hypoxia Inducible Factor-1a (HIF1a) transcription factor as Cited2 expression is induced by HIF1 and it acts to negatively regulate its activity.

scholarly journals Alpha-1 Antitrypsin-Induced Endoplasmic Reticulum Stress Promotes Invasion by Extravillous Trophoblasts

2021 ◽  
Vol 22 (7) ◽  
pp. 3683
Author(s):  
Kanoko Yoshida ◽  
Kazuya Kusama ◽  
Yuta Fukushima ◽  
Takako Ohmaru-Nakanishi ◽  
Kiyoko Kato ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Invasion ◽  
Trophoblast Invasion ◽  
Placental Development ◽  
Functional Link ◽  
Inflammatory Conditions ◽  
Decidual Cells ◽  
Alpha 1 Antitrypsin ◽  
And Function

Alpha-1 antitrypsin (A1AT) is a glycoprotein that has been shown to protect tissues from proteolytic damage under various inflammatory conditions. Several studies show that A1AT may be associated with pre-eclampsia. However, the role of A1AT expression in placental physiology is not fully understood. In the present study, we aim to characterize the expression and function of placental A1AT. A1AT knockdown is found to reduce the expression of the serine protease HTRA1 in a trophoblast cell line. In addition, A1AT overexpression (A1AT-OE) increases the expression of HTRA1, IL6, CXCL8, and several markers of endoplasmic reticulum (ER) stress. Treatment with tunicamycin or thapsigargin, which induces ER stress, increases HTRA1 expression. Furthermore, immunohistochemistry reveals that HTRA1 is expressed in trophoblasts and the endometrial decidual cells of human placentas. An invasion assay shows that A1AT and HTRA1 stimulate cell invasion, but treatment with the ER stress inhibitors reduces the expression of HTRA1 and ER stress markers and prevents cell invasion in A1AT-OE trophoblasts. These results suggest that endogenous A1AT regulates inflammatory cytokine expression and HTRA1-induced trophoblast invasion via the induction of ER stress. It is concluded that an imbalance in the functional link between A1AT and ER stress at the maternal–fetal interface might cause abnormal placental development.

Inhibition of TMEM16A impedes embryo implantation and decidualization in mice

Reproduction ◽  
2018 ◽  
Author(s):  
Qianrong Qi ◽  
Yifan Yang ◽  
Kailin Wu ◽  
Qingzhen Xie
Keyword(s):  
Progesterone Receptor ◽  
Stromal Cells ◽  
Embryo Implantation ◽  
Mouse Uterus ◽  
Endometrial Stromal Cells ◽  
Uterine Endometrium ◽  
Molecule Inhibitor ◽  
Pathway Inhibition ◽  
And Function ◽  
Reproductive Processes

Recent studies revealed that TMEM16A is involved in several reproductive processes, including ovarian estrogen secretion and ovulation, sperm motility and acrosome reaction, fertilization, and myometrium contraction. However, little is known about the expression and function of TMEM16A in embryo implantation and decidualization. In this study, we focused on the expression and regulation of TMEM16A in mouse uterus during early pregnancy. We found that TMEM16A is up-regulated in uterine endometrium in response to embryo implantation and decidualization. Progesterone treatment could induce TMEM16A expression in endometrial stromal cells through progesterone receptor/c-Myc pathway, which is blocked by progesterone receptor antagonist or the inhibitor of c-Myc signaling pathway. Inhibition of TMEM16A by small molecule inhibitor (T16Ainh-A01) resulted in impaired embryo implantation and decidualization in mice. Treatment with either specific siRNA of Tmem16a or T16Ainh-A01 inhibited the decidualization and proliferation of mouse endometrial stromal cells. In conclusion, our results revealed that TMEM16A is involved in embryo implantation and decidualization in mice, compromised function of TMEM16A may lead to impaired embryo implantation and decidualization.

Effects of Maternal Obesity and Gestational Diabetes Mellitus on the Placenta: Current Knowledge and Targets for Therapeutic Interventions

2020 ◽  
Vol 19 (2) ◽  
pp. 176-192
Author(s):  
Samantha Bedell ◽  
Janine Hutson ◽  
Barbra de Vrijer ◽  
Genevieve Eastabrook
Keyword(s):  
Diabetes Mellitus ◽  
Gestational Diabetes ◽  
Gestational Diabetes Mellitus ◽  
Maternal Obesity ◽  
Environmental Changes ◽  
Current Knowledge ◽  
Therapeutic Interventions ◽  
Placental Development ◽  
Exchange Site ◽  
And Function

: Obesity and gestational diabetes mellitus (GDM) are becoming more common among pregnant women worldwide and are individually associated with a number of placenta-mediated obstetric complications, including preeclampsia, macrosomia, intrauterine growth restriction and stillbirth. The placenta serves several functions throughout pregnancy and is the main exchange site for the transfer of nutrients and gas from mother to fetus. In pregnancies complicated by maternal obesity or GDM, the placenta is exposed to environmental changes, such as increased inflammation and oxidative stress, dyslipidemia, and altered hormone levels. These changes can affect placental development and function and lead to abnormal fetal growth and development as well as metabolic and cardiovascular abnormalities in the offspring. This review aims to summarize current knowledge on the effects of obesity and GDM on placental development and function. Understanding these processes is key in developing therapeutic interventions with the goal of mitigating these effects and preventing future cardiovascular and metabolic pathology in subsequent generations.

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