Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes

BACKGROUND: Krüppel-like factor-6 (KLF6) is a widely expressed member of the Sp1/KLF family of transcriptional regulators involved in differentiation, cell cycle control and proliferation in several cell systems. Even though the highest expression level of KLF6 has been detected in human and mice placenta, its function in trophoblast physiology is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance. Confocal immunofluorescence microscopy demonstrated that KLF6 is expressed throughout human cytotrophoblast differentiation showing no evident modifications in its nuclear and cytoplasmic localization pattern. KLF6 transcript and protein peaked early during the syncytialization process as determined by qRT-PCR and western blot assays. Overexpression of KLF6 in trophoblast-derived JEG-3 cells showed a preferential nuclear signal correlating with enhanced expression of human β-chorionic gonadotropin (βhCG) and pregnancy-specific glycoprotein (PSG) genes. Moreover, KLF6 transactivated βhCG5, PSG5 and PSG3 gene promoters. Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter. CONCLUSIONS/SIGNIFICANCE: Results are consistent with KLF6 playing a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG, in agreement with an early and transient increase of KLF6 expression during trophoblast syncytialization

Absence of the 9-bp Deletion of Mitochondrial DNA in Pre-Hispanic Inhabitants of Argentina

We investigated the incidence of the Region V mitochondrial DNA 9-base-pair (bp) deletion from human remains recovered from several archaeological sites and contexts throughout Argentina. Of the 34 samples analyzed, 24 yielded DNA extractions that gave clear amplification results. All of the individuals carried two repeats of the 9 bp, one of which has been shown to be deleted in some individuals of Asian origin and defines mitochondrial lineage B. Although most of the modern Amerindian groups in the region exhibit the deletion in high frequencies, the absence of the 9-bp deletion among ancient populations of South America seems to be the rule rather than the exception, as was reported by several studies involving extinct populations. The evidence gathered until now suggests that the earliest settlers of this region of South America did not carry mitochondrial lineage B

StarD7 knockdown modulates ABCG2 expression, cell migration, proliferation, and differentiation of human choriocarcinoma JEG-3 cells.

StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs.Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (-26.4% and -41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and (3)H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7.Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model

PSG gene expression is up-regulated by lysine acetylation involving histone and nonhistone proteins.

BackgroundLysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG) are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored.Methodology/principal findingsHere, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs) up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140), was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA). This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT) function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6.Conclusions/significanceResults are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization

The <i>de novo</i> biosynthesis of total glycerophospholipids in StarD7 siRNA-treated JEG-3 cells.

<p><b>A</b>- Relative ³H-glycerophospholipid synthesis in StarD7.1 (siD7) siRNA-treated JEG-3 cells compared to scrambled (siC) siRNA-treated cells defined as 1. Data are median and 25^th–75^th% percentiles (<i>n</i> = 4 independent experiments). *<i>p</i><0.05 compared to scrambled siRNA-transfected cells. <b>B</b>- Percentage of distribution of major individual ³H-phospholipids in StarD7 siRNA-treated JEG-3 cells or in scrambled siRNA cells. Data are mean ± SEM (<i>n</i> = 2 independent experiments). Phosphatidylcholine (PC) is the species with the highest precursor incorporation in both cell conditions, followed by phosphatidylserine (PS) and after phosphatidylinositol (PI) and phosphatidylethanolamine (PE) with a minor percentage.</p

Effect of StarD7 silencing on JEG-3 cell migration.

<p><b>A</b>- Wound healing assay in JEG-3 cells treated with StarD7.1 (siD7) or scrambled (siC) siRNAs. An open furrow was generated by scratching confluent cells using a pipette tip. Confluency was restored in controls after 24 h. However, in cells treated with StarD7 siRNA, confluency was not restored after 24 h. <b>B</b>- The distance between furrow edges in the scrambled or StarD7 siRNA-treated cells of three independent experiments was measured and presented graphically as percentage of the initial distance (0 h);*<i>p</i><0.05 compared to scrambled siRNA-transfected cells. <b>C</b>- Transwell <i>In vitro</i> migration assays. Left panels: representative images show cells migrated to the lower chamber after 48 hours (×200). Right panels: Bar graph represents the percentage of cell migration in seven fields of duplicate wells containing StarD7 siRNA-treated cells relative to scrambled siRNA ones (median and 25^th–75^th% percentiles, n = 3); *<i>p</i><0.05 compared to scrambled siRNA-transfected cells. <b>D-</b>Cell proliferation was determined by BrdU (red) staining of JEG-3 cells treated with scrambled (left panel) or StarD7.1 (right panel) siRNAs. The nuclei were labelled with Hoescht (blue, middle panels) and merge images are shown on the bottom panels. Bar  = 50 µm (×200). The images are representative of three experiments with consistent results.</p