Rcho-1 Cells Research Articles

Galectin-4 expression is down-regulated in response to autophagy during differentiation of rat trophoblast cells

Placental development and trophoblast invasion of the maternal endometrium establish the maternal-fetal interface, which is critical for the developing embryo and fetus. Herein we show that overexpression of Galectin-4 (Gal-4) during trophoblast differentiation inhibited the enlargement of Rcho-1 cells (a model for rat trophoblast differentiation) and promoted cell-cell adhesion, whereas trophoblast specific markers and MMP-9 activity were not affected. In the rat placenta, microtubule associated protein 1 light chain 3 alpha (LC3) protein, an autophagy marker, is highly expressed on the maternal side of the decidua where Gal-4 expression is weak. In vitro assays showed that the expression of trophoblast-specific differentiation markers was reduced by 3-Methyladenine (3-MA) and Bafilomycin A1, known as autophagy inhibitors, compared to control cells. Furthermore, Gal-4 expression in Rcho-1 cells, which is normally down-regulated during differentiation, was not attenuated in the presence of autophagy inhibitors, suggesting that autophagy is upstream of Gal-4 expression. We herein describe a possible mechanism by which autophagy regulates trophoblast differentiation via regulation of Gal-4 expression in order to establish the maternal-fetal interface.

Expression pattern of Galectin 4 in rat placentation

Galectin 4 (Gal4) is abundantly expressed in the epithelium of the gastrointestinal tract, and functional analysis has concentrated on its roles associated with polarized membrane trafficking. This study aimed to investigate the expression of Gal4 in placentation. The expression level of Gal4 was revealed to be lower in differentiated Rcho-1 cells (a model system of rat trophoblast differentiation) than in proliferative cells. In the rat placenta, immunohistochemical analysis showed that Gal4 is preferentially located in the maternal–fetal junctional zone. These results suggest that down-regulation of Gal4 may be involved in the promotion of trophoblast cell differentiation.

Transforming Growth Factor Beta Regulates Proliferation and Invasion of Rat Placental Cell Lines1

Implantation of an embryo in the endometrium is a critical step for continuation of pregnancy, and implantation failure is a major cause of infertility. In rats, the implantation process involves invasion of the endometrial epithelial lining by the trophoblastic cells in order to reach the underlying stromal cells. Transforming growth factor beta (TGFB) is a multifunctional cytokine that regulates proliferation, differentiation, and invasiveness of multiple cell lineages. We used rat HRP-1 and RCHO-1 placental cell lines to perform this study. HRP-1 cells were derived from midgestation chorioallantoic placental explants of the outbred Holtzman rat, whereas RCHO-1 cells were established from a rat choriocarcinoma. MTT proliferation assays revealed that each TGFB isoform decreased HRP-1 cell growth in a dose-dependent manner, whereas RCHO-1 cells were resistant to the growth-suppressive effect of TGFB1 and TGFB3. Only TGFB2 reduced RCHO-1 cell proliferation. Activation of ERK, MAPK14 (p38 MAPK), or SMAD pathways is known to play a role in cell proliferation, and we found that TGFB activates these pathways in both HRP-1 and RCHO-1 cells in an isoform-specific manner. MTT proliferation assays revealed that ERK pathway is partially implicated in TGFB3-reduced HRP-1 cell proliferation. Hoechst nuclear staining and caspase-3 cleavage demonstrated that TGFB isoforms failed to induce apoptosis in both cell lines. Matrigel invasion assays showed that both HRP-1 and RCHO-1 cells exhibit intrinsic invasive ability under untreated conditions. The capacity of HRP-1 cells to invade the Matrigel was selectively increased by TGFB2 and TGFB3, whereas all TGFB isoforms could increase the invasiveness of RCHO-1 cells. These important functional studies progressively reveal a key role for TGFB in regulating proliferation and invasiveness of placental cells.

DNA methylation contributes to the tissue-specific expression of the rPL-Iv gene

To understand the tissue-specific expression of the rat placental lactogen-I variant (rPL-Iv) gene, we investigated the methylation pattern of the 5′-flanking region of this gene in various rat tissues. We report that the 5′-flanking region of the rPL-Iv gene was hypomethylated in placenta that expressed the gene and hypermethylated in those tissues that did not express the gene. Moreover, the intron region of the rPL-Iv gene was hypomethylated in the placenta, but hypermethylated in the liver, kidney and pituitary. Although there are 5 CpG sites and the density of CpG dinucleotide is lower within 2 kb of the rPL-Iv 5′-flanking region, the methylated promoter reporter gene produced strong repression in the transcriptional activity of the gene. In addition, the 5′-flanking and intron regions of the rPL-Iv gene were hypomethylated on day 12 of gestation, and the methylation pattern in the placenta remained unchanged from mid-pregnancy until term. The entire genomic region of the rPL-Iv gene might be hypermethylated in tissues other than the placenta, within which its methylated status repress expression of the placenta-specific rPL-Iv gene. Interestingly, the methylation status of the intron region of the rPL-Iv in proliferating Rcho-1 cells was changed to the unmethylated status on day 8 and 12 of differentiation of Rcho-1 cells. These results demonstrate that demethylation in the rPL-Iv upstream region was induced at an early stage of placental development, and once the 5′-flanking region of the rPL-Iv had been demethylated, its status on the rPL-Iv genomic region was continued during pregnancy. Taken together, these results suggest that DNA methylation is responsible for the silencing of tissue-specific genes in non-expressing cells, while defined combinations of trophoblast factors dictate the expression of unmethylated rPL-Iv gene in placenta trophoblast cells.

Identification of trophoblast-specific binding sites for GATA-2 that are essential for rat placental lactogen-I gene expression

We identified a 3.4-kb 5'-flanking region of the rPL-I gene and examined its promoter activity using rat trophoblast Rcho-1 cells. A regulatory element between base pairs (bp) -2,487 and -2,310 in the 5'-flanking region was essential for maximum promoter activity of the rPL-I gene. This regulatory element was further characterized between bp -2,443 to -2,415 and -2,374 to -2,345. Electrophoretic mobility shift analysis showed that the interaction of nuclear extract proteins from differentiated Rcho-1 cells was inhibited by competition with a GATA-like sequence in the promoter, but not by a mutated GATA sequence. Moreover, the promoter activity of 2487 eLuc containing two novel GATA sites was significantly elevated by co-transfection of a GATA-2 expression vector in proliferating Rcho-1 cells. Our results demonstrate that GATA-2 is involved in multiple promoter regions to activate the specific expression of the rPL-I gene in placental tissue.

Protein processing by the placental protease, cathepsin P

Cathepsin P is a member of a family of placentally expressed cathepsins (PECs). The closest human homolog of cathepsin P is cathepsin L, a broad specificity enzyme that has functions in many tissues in addition to placenta. The gene duplications that gave rise to the PECs provide a rare opportunity to define proteolytic functions in placenta, a transient organ unique to mammals. Peptidyl substrate and inhibitor libraries have shown that cathepsin P has evolved an unusually restricted preference for substrates containing hydrophobic amino acids. Proteomic techniques were used to probe for substrates of this enzyme. Recombinant cathepsin P was incubated with rat choriocarcinoma (Rcho-1) cell proteins to identify substrates using two-dimensional difference gel electrophoresis. Substrate proteins were excised from gels and characterized by trypsin digestion and MALDI MS/MS. Two endoplasmic reticulum (ER) proteins, gp96 and calreticulin, emerged as potential substrates, and western blotting showed that these proteins are processed by cathepsin P from their C-terminus, removing the KDEL ER retention signal. Immunohistochemistry showed that a portion of cathepsin P co-localizes with calreticulin in Rcho-1 cells. Extracellular calreticulin induces differentiation of Rcho-1 cells, indicating a potential role of cathepsin P in processing and secretion of calreticulin during differentiation of trophoblast giant cells.

The nucleosomal binding protein NSBP1 is highly expressed in the placenta and modulates the expression of differentiation markers in placental Rcho‐1 cells

We report that NSBP1, a nucleosome binding protein that affects the structure of chromatin, is highly expressed in mouse placenta. In Rcho-1 cells, which recapitulate the differentiation of trophoblast giant cells of living placenta, NSBP1 expression is linked to differentiation. Disregulation of NSBP1 protein levels, by either siRNA treatment or by overexpression, alters the expression of several members of the prolactin gene family without affecting the levels of several transcription factors involved in placental differentiation. Our studies identify NSBP1 as a nucleosome binding protein that modulates the expression of prolactin gene family members most likely by inducing changes in chromatin structure.

Transforming Growth Factor β Regulates Proliferation and Invasion of Rat Trophoblastic Cells.

Implantation of embryo in the endometrium is a critical step for continuation of pregnancy and implantation failure is a major cause of infertility. In the rat, the implantation process involves invasion of the endometrial epithelial lining by the trophoblastic cells in order to reach the underneath stromal cells. Transforming Growth factor-β (TGF-β) is a multifunctional cytokine which regulates proliferation, differentiation and invasiveness of multiple cell lineages. One or more TGF-β isoforms (TGF-β1, TGF-β2 and TGF-β3) are secreted by the uterus during pregnancy, including at the time of implantation. Recently, we have showed that exposure to TGF-β3 increased the invasiveness of endometrial cells and the aim of the present study was to determine if TGF-β isoforms might be involved in the regulation of trophoblast invasiveness. We used the rat HRP-1 and RCHO-1 trophoblastic cell lines to perform this study. HRP-1 cells were derived from a normal midgestation chorioallantoic placenta, whereas RCHO-1 cells were established from a choriocarcinoma. MTT proliferation assays revealed that each TGF-β isoform decreased HRP-1 cell growth in a dose-dependent manner, whereas RCHO-1 cells were resistant to the growth-suppressive effect of TGF-β. Matrigel invasion assays showed that both HRP-1 and RCHO-1 cells exhibited intrinsic invasive ability under untreated conditions, and preliminary experiments have demonstrated that the capacity of HRP-1 cells to invade the Matrigel was selectively increased by TGF-β2 and TGF-β3, whereas the three TGF-β isoforms could increase the invasiveness of RCHO-1 cells. Activation of ERK, PI 3-K or Smad pathways is known to increase cellular motility and/or invasiveness, and we found that TGF-β activates these pathways in both HRP-1 and RCHO-1 cells in an isoform-specific manner. We thus recently initiated the characterization of the role of each of these pathways in TGF-β-induced invasiveness of trophoblastic cells. Preliminary studies showed that pharmacological inhibitor LY294002 could block TGF-β2-induced Akt phosphorylation and HRP-1 invasiveness, suggesting a role for PI 3-K activity in TGF-β2-induced invasiveness in this cell line. These important functional studies progressively reveal a key role for TGF-β in regulating the invasiveness of trophoblastic cells. Supported by NSERC (238501-06).

Leukemia Inhibitory Factor Regulates Trophoblast Giant Cell Differentiation via Janus Kinase 1-Signal Transducer and Activator of Transcription 3-Suppressor of Cytokine Signaling 3 Pathway

Suppressor of cytokine signaling 3 (SOCS3) inhibits leukemia-inhibitory factor (LIF) signaling and acts as a negative regulator. Deletion of SOCS3 causes embryonic lethality because of placental failure, and genetic reduction of LIF or the LIF receptor (LIFR) in SOCS3-deficient mice rescues placental defects and embryonic lethality; this indicates that SOCS3 is an essential inhibitor of LIFR signaling. However, the downstream signaling molecule that acts as a link between the LIFR and SOCS3 has not been identified. In this study we explored the downstream signaling of LIFR. The administration of LIF to SOCS3-heterozygous pregnant mice promotes trophoblast giant cell differentiation and accelerates placental failure in SOCS3-deficient mice. SOCS3-deficient trophoblast stem cells show enhanced and prolonged signal transducer and activator of transcription 3 (Stat3) activation by LIF stimulation. Further, in the trophoblasts of SOCS3-deficient placenta and differentiating cells from the choriocarcinoma-derived cell line Rcho-1 cells, constitutive activation of Stat3 is observed. The forced expression of SOCS3, dominant-negative Stat3, and dominant-negative Janus kinase 1 (JAK1) in Rcho-1 cells significantly suppressed the trophoblast giant cell differentiation of these cells. In addition, the number of trophoblast giant cells is significantly reduced concomitant with an increased number of precursor trophoblasts in JAK1-deficient placentas. Finally, JAK1 deficiency rescues placental defects and embryonic lethality in SOCS3-deficient mice. These results indicate that the LIFR signaling is finely coordinated by JAK1, Stat3, and SOCS3 and regulates trophoblast giant cell differentiation. In addition, these data establish that LIFR-JAK1-Stat3-SOCS3 signaling is an essential pathway for the regulation of trophoblast giant cell differentiation.

MammalianSox15Gene: Promoter Analysis and Implications for Placental Evolution

Sox15 belongs to the Sox (Sry-type HMG box) protein family, which is involved in placental development and muscle regeneration. Previously, we showed that the Sox15 gene is highly expressed in the trophoblast giant cells of the mouse placenta. To elucidate the molecular mechanisms of the tissue-dependent transcription of the gene, we isolated approximately 2.2 kb of the 5'-flanking sequence upstream of the transcription initiation site and used it to construct luciferase reporter plasmids. A variety of cell lines, including trophoblast stem (TS) cells, placenta-derived Rcho-1 cells, and myoblast C2C12 cells, required the same 5'-flanking sequence, from -109 to -8, for basal promoter activity. In contrast, the sequences from -297 to -149 and from -148 to -110 were required for cell-type-specific promoter activity in myoblast-derived C2C12 cells and placenta-derived Rcho-1 and TS cells, respectively. These results suggest that the region from -297 to -8 of the Sox15 gene contains three distinct cis-elements that respectively control placenta-specific, myoblast-specific, and common basal expression. We also searched for Sox15 ortholog(s) in the genome databases of various vertebrate species. The results indicated that the three regulatory promoter sequences of the Sox15 genes were conserved among eutherian mammals during vertebrate evolution. Interestingly, the marsupial opossum gene that is closest to Sox15 appeared to be a pseudogene. These findings indicate that Sox15 may have been involved in placental evolution.

Nucleolar release of Hand1 acts as a molecular switch to determine cell fate

The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis in the developing embryo. Here we implicate Hand1 as a molecular switch that determines whether a trophoblast stem cell continues to proliferate or commits to differentiation. We identify a novel interaction of Hand1 with a protein that contains an I-mfa (inhibitor of myogenic factor) domain that anchors Hand1 in the nucleolus where it negatively regulates Hand1 activity. In the trophoblast stem-cell line Rcho-1, nucleolar sequestration of Hand1 accompanies sustained cell proliferation and renewal, whereas release of Hand1 into the nucleus leads to its activation, thus committing cells to a differentiated giant-cell fate. Site-specific phosphorylation is required for nucleolar release of Hand1, for its dimerization and biological function, and this is mediated by the non-canonical polo-like kinase Plk4 (Sak). Sak is co-expressed in Rcho-1 cells, localizes to the nucleolus during G2 and phosphorylates Hand1 as a requirement for trophoblast stem-cell commitment to a giant-cell fate. This study defines a novel cellular mechanism for regulating Hand1 that is a crucial step in the stem-cell differentiation pathway.

Involvement of RelA-Associated Inhibitor in Regulation of Trophoblast Differentiation via Interaction with Transcriptional Factor Specificity Protein-1

Glucose transporter-1 (GLUT1), one of the key functional indicators of placental differentiation, has an important role in placental glucose transport. We previously showed that the protein levels of GLUT1 and nuclear transcription factor specificity protein-1 (Sp1) in rat choriocarcinoma cells (Rcho-1 cells) decreased during the differentiation of these cells to giant cells. We also showed that Sp1 was involved in the regulation of GLUT1 gene expression during this process. RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor-kappaB that was identified by a yeast two-hybrid screen and is preferably expressed in human placenta and heart. RAI was also found to interact with Sp1 and exert an inhibitory effect against the DNA-binding activity of Sp1. We first show here that RAI mRNA expression increased as gestation proceeded and that RAI was localized mainly in the syncytiotrophoblast throughout pregnancy. The chloramphenicol acetyltransferase activity assay in Rcho-1 cells revealed that cotransfection of RAI expression vector resulted in decreased activity of the rat GLUT1 promoter but not in that of a mutated rat GLUT1 promoter lacking the Sp1 binding site. Furthermore, the protein level of RAI increased during differentiation. In addition, transfection of RAI expression vector promoted the morphological differentiation of Rcho-1 cells, and RAI knockdown using RAI-specific small interfering RNA reveals inhibitory effects on the morphological differentiation, as assessed by photomicroscopy. Taken together, these findings suggest that RAI may be involved in the regulation of trophoblast differentiation via interaction with Sp1.

Hypoxia Represses the Differentiation of Rcho-1 Rat Trophoblast Giant Cells

Background: A hypoxic environment is known to be essential for early placentation. A low oxygen tension induces hypoxia-inducible factor-1 (HIF-1α) which may play an important role as a transcription factor in maintaining the proliferative and undifferentiated phenotype in human trophoblasts. Methods: We analyzed the effect of a low oxygen tension on the rat trophoblast giant cell differentiation pathway using Rcho-1 cells which were derived from rat choriocarcinomas and consist of trophoblast stem cells. Results: We found that a low oxygen tension suppressed the morphological changes and steroidogenesis during differentiation. The anticipated downregulation of the Id-1 transcription factor, a negative regulator of trophoblast giant cell differentiation, was not observed in the hypoxic environment. On the other hand, deferoxamine, which mimics hypoxia and induces HIF-1α, caused downregulation of Id-1 transcription factor and trophoblast giant cell differentiation. Conclusion: These results indicate that hypoxia represses rat trophoblast giant cell differentiation via an HIF-1α-independent pathway.

Involvement of Sp-1 in the Regulation of the Id-1 Gene during Trophoblast Cell Differentiation

Id-1, a member of the helix-loop-helix transcription factor family, inhibits the differentiation of Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells. Id-1 is expressed at a high level in undifferentiated trophoblast stem cells and then down-regulated during early differentiation, and is thought to be a key regulator in the trophoblast giant-cell differentiation pathway. In this study, we analyzed the signaling mechanism regulating the high expression levels of Id-1 in undifferentiated Rcho-1 cells. Promoter deletion analysis revealed that a 31-bp sequence (Box-2 region), located between −200 and −169 bp in the Id-1 promoter is necessary for the promoter activity. Electrophoretic mobility shift assays and DNA affinity precipitation assays showed that Box-2-binding activity was decreased during differentiation and that Sp-1 protein bound to this sequence. The protein level of Sp-1 was decreased during the differentiation. These results suggest that the Sp-1 protein level may regulate the Box-2-binding activity and the trophoblast giant-cell differentiation.

Differentiation of Trophoblast Giant Cells and Their Metabolic Functions Are Dependent on Peroxisome Proliferator-Activated Receptor β/δ

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

DNA Methylation-dependent Epigenetic Regulation of Dimethylarginine Dimethylaminohydrolase 2 Gene in Trophoblast Cell Lineage

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

Hypoxia Inhibits Differentiation of Lineage-Specific Rcho-1 Trophoblast Giant Cells1

Defects in placental development lead to pregnancies at risk for miscarriage and intrauterine growth retardation and are associated with preeclampsia, a leading cause of maternal death and premature birth. In preeclampsia, impaired placental formation has been associated with alterations in a specific trophoblast lineage, the invasive trophoblast cells. In this study, an RT-PCR Trophoblast Gene Expression Profile previously developed by our laboratory was utilized to examine the lineage-specific gene expression of the rat Rcho-1 trophoblast cell line. Our results demonstrated that Rcho-1 cells represent an isolated, trophoblast population committed to the giant cell lineage. RT-PCR analysis revealed that undifferentiated Rcho-1 cells expressed trophoblast stem cell marker, Id2, and trophoblast giant cell markers. On differentiation, Rcho-1 cells downregulated Id2 and upregulated Csh1, a marker of the trophoblast giant cell lineage. Neither undifferentiated nor differentiated Rcho-1 cells expressed spongiotrophoblast marker Tpbpa or labyrinthine markers Esx1 and Tec. Differentiating Rcho-1 cells in hypoxia did not alter the expression of lineage-specific markers; however, hypoxia did inhibit the downregulation of the trophoblast stem cell marker Id2. Differentiation in hypoxia also blocked the induction of CSH1 protein. In addition, hypoxia inhibited stress fiber formation and abolished the induction of palladin, a protein associated with stress fiber formation and focal adhesions. Thus, Rcho-1 cells can be maintained as a proliferative, lineage-specific cell line that is committed to the trophoblast giant cell lineage on differentiation in both normoxic and hypoxic conditions; however, hypoxia does inhibit aspects of trophoblast giant cell differentiation at the molecular, morphological, and functional levels.

STAT3-mediated Constitutive Expression of SOCS3 in an Undifferentiated Rat Trophoblast-like Cell Line

In the trophoblast, constitutive expression of SOCS3 is important for the negative regulation of trophoblast giant cell differentiation. In this study, we analyzed the signaling pathway regulating the constitutive SOCS3 expression in undifferentiated Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells that are capable of differentiating to trophoblast giant cells in vitro. PD98059, an MEK inhibitor, repressed the SOCS3 expression but AG490, a JAK2 inhibitor, did not. Promoter deletion analysis revealed that the STAT response element (SRE) in the SOCS3 promoter is necessary for the promoter activity. Overexpression of STAT3 increased the SOCS3 promoter activity, whereas expression of dominant-negative STAT3 reduced it. Constitutive STAT3 tyrosine phosphorylation that was not inhibited by either AG490 or PD98059 was demonstrated. Electrophoretic mobility shift assays showed the existence of a protein that bound to SRE and was supershifted with STAT3 antibody. This binding reaction was inhibited by neither AG490 nor PD98059. These findings imply that the ERK/MAPK pathway and STAT3 are involved in the constitutive activation of SOCS3 in undifferentiated Rcho-1 cells. Moreover, they indicate that the constitutive STAT3 tyrosine phosphorylation and the DNA binding activity of STAT3 do not depend on the ERK/MAPK or JAK kinase pathway. These results suggest that a trophoblast-specific STAT3 activation pathway is important for the regulation of giant cell differentiation.

Developmental expression of the homeobox protein Distal-less 3 and its relationship to progesterone production in mouse placenta

Distal-less 3 (Dlx3) is a homeobox factor that functions as a placental-specific transcriptional regulator. Dlx3 null mice (-/-) have compromised placental development and do not survive in utero past embryonic day (E) 9.5. The current studies were undertaken to examine the expression of Dlx3 in mouse placenta during gestation, and to determine whether Dlx3 was involved in placental progesterone production. Dlx3 was not detectable at E8.5 but was detected in E9.5 placenta with continuing but diminished expression through E15.5. Dlx3 immuno-localization was restricted to the labyrinth, was nuclear and was found in cytokeratin-positive cells. Previous studies in choriocarcinoma cell lines support the conclusion that Dlx3 is required for expression of 3'-hydroxysteroid dehydrogenase VI (3betaHSD VI), an obligate enzyme in the production of progesterone by trophoblast giant cells. In a rat trophoblast stem cell line (Rcho-1), Dlx3 expression was non-detectable in Rcho-1 cells induced to differ-entiate using mitogen withdrawal. In vitro progesterone production in placental cultures and 3betaHSD VI mRNA from Dlx3 (+/+), (+/-) and (-/-) mice were equivalent. In situ hybridization for 3betaHSD VI revealed mRNA expression restricted to trophoblast giants cells with no detectable expression in the labyrinth suggesting that Dlx3 and 3betaHSD VI were not colocalized within the placenta. These studies support the conclusion that Dlx3 protein expression is restricted to the labyrinth region of the murine placenta into late gestation and that Dlx3 does not appear to be expressed in trophoblast giant cells. Further, loss of Dlx3 was not correlated with synthesis of progesterone from E9.5 mouse placentas.

Induction of glucose transporter 1 expression through hypoxia-inducible factor 1α under hypoxic conditions in trophoblast-derived cells

Glucose transporter 1 (GLUT1) plays an important role in the transport of glucose in the placenta. During early pregnancy, placentation occurs in a relatively hypoxic environment that is essential for appropriate embryonic development, and GLUT1 expression is enhanced in response to oxygen deficiency in the placenta. Hypoxia-inducible factor-1 (HIF-1)alpha is involved in the induction of GLUT1 expression in other cells. The present study was designed to test whether HIF-1alpha is involved in hypoxia-induced activation of GLUT1 expression using trophoblast-derived human BeWo and rat Rcho-1 cells as models. GLUT1 mRNA and protein expression were elevated under 5% O2 or in the presence of cobalt chloride, which has been shown to mimic hypoxia. Using rat GLUT1 (rGLUT1) promoter-luciferase constructs, we showed that this up-regulation was mediated at the transcriptional level. Deletion mutant analysis of the rGLUT1 promoter indicated that a 184 bp hypoxia-responsive element (HRE) of the promoter was essential to increase GLUT1 reporter gene expression in response to low-oxygen conditions. BeWo and Rcho-1 cells cultured under 5% O2 or with CoCl2 showed increased expression of HIF-1alpha protein compared with those cultured under 20% O2. To test whether this factor is directly involved in hypoxia-induced GLUT1 promoter activation, BeWo and Rcho-1 cells were transiently transfected with an HIF-1alpha expression vector. Exogeneous HIF-1alpha markedly increased the GLUT1 promoter activity from constructs containing the HRE site, while the GLUT1 promoter constructs lacking the HRE site were not activated by exogenous HIF-1alpha These data demonstrate that GLUT1 is up-regulated under 5% O2 or in the presence of CoCl2 in the placental cell lines through HIF-1alpha interaction with a consensus HRE site of the GLUT1 promoter.