201. Homeobox gene DLX3 regulates forskolin induced trophoblast differentiation

2008 ◽  
Vol 20 (9) ◽  
pp. 1
Author(s):  
A. Chui ◽  
B. Kalionis ◽  
S. Brennecke ◽  
P. Murthi
Keyword(s):  
Transcription Factors ◽  
Real Time ◽  
Fetal Growth ◽  
Real Time Pcr ◽  
Fetal Growth Restriction ◽  
Homeobox Gene ◽  
Trophoblast Cell ◽  
Trophoblast Differentiation ◽  
Human Trophoblast ◽  
Β Hcg

Trophoblast cells carry out important functions required for the development of the normal placenta. Disruption of these functions is associated with significant pregnancy disorders such as fetal growth restriction and pre-eclampsia. Transcription factors regulate trophoblast functions. We are interested in one such class of transcription factors known as homeobox genes. The homeobox gene Distal-less 3 (DLX3) plays a vital role in the development of the mouse placenta (Morasso, Grinberg et al. 1999) and increased levels of DLX3 have been found in placentae affected by human fetal growth restriction (Murthi and Chui, unpubl. data). However, the function of DLX3 in the human placenta is not well established. Here, we investigated whether DLX3 regulates trophoblast differentiation using a plasmid construct to overexpress DLX3 in the human trophoblast cell line, BeWo. Real-time PCR showed a significant increase in DLX3 mRNA (3.1 ± 0.1 v. 1.0 ± 0.2 control, P < 0.05, n = 3), as well as the mRNA of two known markers of differentiation 3-β-hydroxysteroid dehydrogenase (3β-HSD) (8.1 ± 1.8 v. 1.2 ± 0.1 control, P < 0.05, n = 3) and β-human chorionic gonadotropin (β-hCG) (54.9 ± 0.9 v. 49.2 ± 1.6 control, P < 0.05, n = 3). Furthermore, forskolin mediated trophoblast differentiation was verified in BeWo cells. Following forskolin induction, real-time PCR showed a significant increase in the expression of DLX3 mRNA (12.9 ± 1.2 v. 3.8 ± 0.9 control, P < 0.05, n = 4), as well as a significant increase in the mRNA expression of the differentiation markers previously tested, 3β-HSD (28.3 ± 2.4 v. 1.0 ± 0.08 control, P < 0.05, n = 4) and β-hCG (2.3 ± 1.9 v. 30.9 ± 0.08 control, P < 0.001, n = 3). The expression of an additional differentiation marker, syncytin, was also significantly increased (4.0 ± 1.9 v. 1.0 ± 0.08 control, P < 0.05, n = 4). Thus, we have shown that DLX3 is a regulator of human trophoblast cell differentiation, and that forskolin acts through DLX3 to induce trophoblast differentiation. (1) Morasso, M. I., A. Grinberg et al. (1999).

scholarly journals The role of insulin-like growth factor 2 receptor-mediated homeobox gene expression in human placental apoptosis, and its implications in idiopathic fetal growth restriction

2019 ◽  
Vol 25 (9) ◽  
pp. 572-585 ◽  
Author(s):  
Lynda K Harris ◽  
Priyadarshini Pantham ◽  
Hannah E J Yong ◽  
Anita Pratt ◽  
Anthony J Borg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Line ◽  
Gene Transcription ◽  
Fetal Growth ◽  
Fetal Growth Restriction ◽  
Homeobox Gene ◽  
Trophoblast Cell

Abstract Fetal growth restriction (FGR) is caused by poor placental development and function early in gestation. It is well known that placentas from women with FGR exhibit reduced cell growth, elevated levels of apoptosis and perturbed expression of the growth factors, cytokines and the homeobox gene family of transcription factors. Previous studies have reported that insulin-like growth factor-2 (IGF2) interacts with its receptor-2 (IGF2R) to regulate villous trophoblast survival and apoptosis. In this study, we hypothesized that human placental IGF2R-mediated homeobox gene expression is altered in FGR and contributes to abnormal trophoblast function. This study was designed to determine the association between IGF2R, homeobox gene expression and cell survival in pregnancies affected by FGR. Third trimester placentas were collected from FGR-affected pregnancies (n = 29) and gestation matched with control pregnancies (n = 30). Functional analyses were then performed in vitro using term placental explants (n = 4) and BeWo trophoblast cells. mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence IGF2R expression in placental explants and the BeWo cell-line. cDNA arrays were used to screen for downstream targets of IGF2R, specifically homeobox gene transcription factors and apoptosis-related genes. Functional effects of silencing IGF2R were then verified by β-hCG ELISA, caspase activity assays and a real-time electrical cell-impedance assay for differentiation, apoptosis and cell growth potential, respectively. IGF2R expression was significantly decreased in placentas from pregnancies complicated by idiopathic FGR (P < 0.05 versus control). siRNA-mediated IGF2R knockdown in term placental explants and the trophoblast cell line BeWo resulted in altered expression of homeobox gene transcription factors, including increased expression of distal-less homeobox gene 5 (DLX5), and decreased expression of H2.0-Like Homeobox 1 (HLX) (P < 0.05 versus control). Knockdown of IGF2R transcription increased the expression and activity of caspase-6 and caspase-8 in placental explants, decreased BeWo proliferation and increased BeWo differentiation (all P < 0.05 compared to respective controls). This is the first study linking IGF2R placental expression with changes in the expression of homeobox genes that control cellular signalling pathways responsible for increased trophoblast cell apoptosis, which is a characteristic feature of FGR.

220. Homeobox gene HLX is a regulator of HGF/c-met mediated trophoblast migration

2008 ◽  
Vol 20 (9) ◽  
pp. 20
Author(s):  
G. Rajaraman ◽  
P. Murthi ◽  
S. P. Brennecke ◽  
B. Kalionis
Keyword(s):  
Cell Migration ◽  
Mrna Expression ◽  
Fetal Growth ◽  
Fetal Growth Restriction ◽  
Homeobox Gene ◽  
Trophoblast Cell ◽  
Growth Restriction ◽  
Trophoblast Cells ◽  
Cytotrophoblast Cell ◽  
Trophoblast Migration

Homeobox gene transcription factors play critical roles in normal placental development and are expressed in specialised trophoblast cells. Abnormal trophoblast cell function is associated with pregnancy disorders including fetal growth restriction. Our studies show homeobox gene HLX expression in trophoblast cells (1) and that HLX is significantly decreased in fetal growth restriction (2). HLX gene inactivation in cultured trophoblast cells shows that HLX is a regulator of cytokine-dependent trophoblast proliferation (3). Hepatocyte growth factor (HGF) activates trophoblast cell migration in a paracrine fashion and its receptor, c-met, is expressed in trophoblast cells. This study investigates the regulation of HGF/c-met mediated trophoblast migration by HLX, in two human trophoblast cell lines SGHPL-4 and HTR-8/SVNeo. HGF stimulation significantly increased HLX mRNA expression (e.g. 43.2 ± 2.5, HGF v. 18.4 ± 1.7 control, densitometric units, P < 0.001, n = 3). siRNA-mediated inactivation of HLX resulted in significantly decreased trophoblast migration (e.g. 32 ± 4, siRNA v. 127 ± 12 control, migrated cells, P < 0.05, n = 4). When HLX was inactivated in the presence of HGF stimulation, migration remained significantly decreased (e.g. 112 ± 15, siRNA + HGF v. 368 ± 44 HGF, migrated cells, P < 0.05, n = 4). In order to determine if HGF is acting via the c-met receptor, the Met tyrosine kinase inhibitor, SU11274, was employed to inhibit c-met activity. c-met inhibition resulted in significantly reduced HLX mRNA expression (e.g. 2.1 ± 0.32, SU11274 v. 12.3 ± 1.4 control, densitometric units, P < 0.05, n = 3). HLX expression remained significantly reduced with HGF stimulation and SU11274 mediated c-met inhibition (e.g. 8.02 ± 1.3, SU11274 v. 38.3 ± 5.4 HGF, densitometric units, P < 0.05, n = 3). This is the first study to show that homeobox gene HLX is a downstream effector gene of HGF, that HLX regulates trophoblast migration and that HGF, via its receptor c-met, acts through HLX to control cell migration. (1) Rajaraman G, Murthi P, Quinn L, Brennecke SP, Kalionis B. Homeodomain protein HLX is expressed primarily in cytotrophoblast cell types in the early human placenta. (2008) Reproduction, Fertility a (2) Murthi P, Doherty V, Said J, Donath S, Brennecke SP, Kalionis B. Homeobox gene HLX1 expression is decreased in idiopathic human fetal growth restriction. (2006) Am J Pathol. 2006 Feb;168(2):511–8. (3) Rajaraman G, Murthi P, Leo B, Brennecke SP, Kalionis B. Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent cell proliferation. (2007) Placenta Volume 28, Issue 10, October

Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor

2000 ◽  
Vol 278 (5) ◽  
pp. C982-C988 ◽  
Author(s):  
Roni Levy ◽  
Steven D. Smith ◽  
Kala Chandler ◽  
Yoel Sadovsky ◽  
D. Michael Nelson
Keyword(s):  
Cell Differentiation ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Fetal Growth ◽  
Fetal Growth Restriction ◽  
Caspase Inhibitor ◽  
Trophoblast Differentiation ◽  
Epidermal Growth ◽  
Induced Apoptosis

Preeclampsia and fetal growth restriction are associated with placental hypoperfusion and villous hypoxia. The villous response to this environment includes diminished trophoblast differentiation and enhanced apoptosis. We tested the hypothesis that hypoxia induces apoptosis in cultured trophoblasts, and that epidermal growth factor (EGF), an enhancer of trophoblast differentiation, diminishes hypoxia-induced apoptosis. Trophoblasts isolated from placentas of term-uncomplicated human pregnancies were cultured up to 72 h in standard ([Formula: see text]= 120 mmHg) or hypoxic ([Formula: see text] < 15 mmHg) conditions. Exposure to hypoxia for 24 h markedly enhanced trophoblast apoptosis as determined by DNA laddering, internucleosomal in situ DNA fragmentation, and histomorphology, as well as by the reversibility of the apoptotic process with a caspase inhibitor. Apoptosis was accompanied by increased expression of p53 and Bax and decreased expression of Bcl-2. Addition of EGF to cultured trophoblasts or exposure of more differentiated trophoblasts to hypoxia significantly lowered the level of apoptosis. We conclude that hypoxia enhances apoptosis in cultured trophoblasts by a mechanism that involves an increase in p53 and Bax expression. EGF and enhancement of cell differentiation protect against hypoxic-induced apoptosis.

scholarly journals Verbascoside-Rich Abeliophyllum distichum Nakai Leaf Extracts Prevent LPS-Induced Preterm Birth Through Inhibiting the Expression of Proinflammatory Cytokines from Macrophages and the Cell Death of Trophoblasts Induced by TNF-α

Molecules ◽  
2020 ◽  
Vol 25 (19) ◽  
pp. 4579
Author(s):  
Ho Won Kim ◽  
A-Reum Yu ◽  
Minji Kang ◽  
Nak-Yun Sung ◽  
Byung Soo Lee ◽  
...  
Keyword(s):  
Cell Death ◽  
Preterm Birth ◽  
Conditioned Medium ◽  
Fetal Growth ◽  
Fetal Growth Restriction ◽  
Fetal Loss ◽  
Trophoblast Cell ◽  
Tnf Α ◽  
Abeliophyllum Distichum ◽  
Cell Conditioned Medium

Background: Preterm birth is a known leading cause of neonatal mortality and morbidity. The underlying causes of pregnancy-associated complications are numerous, but infection and inflammation are the essential high-risk factors. However, there are no safe and effective preventive drugs that can be applied to pregnant women. Objective: The objectives of the study were to investigate a natural product, Abeliophyllum distichum leaf (ADL) extract, to examine the possibility of preventing preterm birth caused by inflammation. Methods: We used a mouse preterm birth model by intraperitoneally injecting lipopolysaccharides (LPS). ELISA, Western blot, real-time PCR and immunofluorescence staining analyses were performed to confirm the anti-inflammatory efficacy and related mechanisms of the ADL extracts. Cytotoxicity and cell death were measured using Cell Counting Kit-8 (CCK-8) analysis and flow cytometer. Results: A daily administration of ADL extract significantly reduced preterm birth, fetal loss, and fetal growth restriction after an intraperitoneal injection of LPS in mice. The ADL extract prevented the LPS-induced expression of TNF-α in maternal serum and amniotic fluid and attenuated the LPS-induced upregulation of placental proinflammatory genes, including IL-1β, IL-6, IL-12p40, and TNF-α and the chemokine gene CXCL-1, CCL-2, CCL3, and CCL-4. LPS-treated THP-1 cell-conditioned medium accelerated trophoblast cell death, and TNF-α played an essential role in this effect. The ADL extract reduced LPS-treated THP-1 cell-conditioned medium-induced trophoblast cell death by inhibiting MAPKs and the NF-κB pathway in macrophages. ADL extract prevented exogenous TNF-α-induced increased trophoblast cell death and decreased cell viability. Conclusions: We have demonstrated that the inhibition of LPS-induced inflammation by ADL extract can prevent preterm birth, fetal loss, and fetal growth restriction.

46 EXPRESSION OF TRANSCRIPTION FACTORS SPECIFIC TO THE TROPHOBLAST LINEAGE IN MOUSE SOMATIC NUCLEAR TRANSFER EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 103
Author(s):  
T. Mitani ◽  
M. Nishiwaki ◽  
M. Anzai ◽  
H. Kato ◽  
Y. Hosoi ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Real Time ◽  
Real Time Pcr ◽  
Nuclear Transfer ◽  
Ectopic Expression ◽  
Expression Level ◽  
Pcr Analysis ◽  
Cell Stage ◽  
Cdx2 Expression ◽  
Morula Stage

Somatic cell nuclear transfer (SCNT) embryos can develop at relatively high rates during the preimplantation period; however, most of these fail after implantation. Development of extraembryonic tissue is indispensable for normal embryonic development. Hence, an abnormality of trophoblast development might be a significant factor in post-implantation lethality of SCNT embryos. A transcription factor, caudal-related homeobox 2 (Cdx2), appears to be involved in the segregation of ICM and trophectoderm (TE) in preimplantation embryos (Niwa et al. 2005 Cell 123, 917–929). Both Cdx2 and Oct3/4 are expressed in all cells at the morula stage, and then Cdx2 expression becomes restricted to the TE and Oct3/4 to the ICM as the blastocyst develops. Mouse embryos deficient in Cdx2 are able to develop to normal blastocysts but die soon after implantation, probably because of defects in the TE lineage. Moreover, dysplasia of the spongiotrophoblast layer might attribute to an abnormality of Tpbpa expression in mouse SCNT embryos (Wakisaka-Saito et al. 2006 Biochem. Biophys. Res. Commun. 349, 106–114). In this study, we examined the expression profiles of transcription factors implicated in trophoblast development in mouse SCNT embryos and intracytoplasmic sperm injection (ICSI) embryos by immunohistochemistry and real-time PCR analysis. SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998 Nature 394, 369–374). In brief, B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. After nuclear transfer, the oocytes were activated and cultured in KSOM to the morula and blastocyst stages. Immunohistochemical analysis demonstrated that in ICSI embryos Cdx2 was only partially expressed at the 8-cell stage but completely in early morulae. In contrast, in SCNT embryos, it was absent at the 8-cell stage and appeared partially at the early morula stage. Thereafter, Cdx2 expression became restricted to the TE cells in both the ICSI and the SCNT blastocysts. However, ectopic expression of Oct3/4 was observed in the TE cells of SCNT, but not in ICSI blastocysts. Real-time PCR analysis showed that at the 8-cell stage, Cdx2 was expressed in ICSI but not in SCNT embryos. In addition, the expression level of Cdx2 in SCNT embryos at the blastocyst stage was only half that in ICSI embryos (P < 0.05). However, there was no significant difference in expression level of Oct3/4 between ICSI and SCNT embryos. Eomesodermin (Eomes) is also implicated in trophoblast development and its expression depends on Cdx2, BMP4, and FGF4. In SCNT embryos, the expression level of Eomes was also only half that in ICSI embryos. These results indicate that the delayed expression of Cdx2 in SCNT embryos may lead to the ectopic expression of Oct3/4 in blastocysts and, along with the limited expression of Cdx2 and Eomes, may contribute to disorders in the function of the trophoblast lineage for normal placental development. This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

scholarly journals Homeobox Gene HLX1Expression Is Decreased in Idiopathic Human Fetal Growth Restriction

2006 ◽  
Vol 168 (2) ◽  
pp. 511-518 ◽  
Author(s):  
Padma Murthi ◽  
Vicki Doherty ◽  
Joanne Said ◽  
Susan Donath ◽  
Shaun P. Brennecke ◽  
...  
Keyword(s):  
Fetal Growth ◽  
Fetal Growth Restriction ◽  
Homeobox Gene ◽  
Growth Restriction
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