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

Cutaneous Leishmaniasis and Sand Fly Fluctuations Are Associated with El Nino in Panama

BackgroundCutaneous Leishmaniasis (CL) is a neglected tropical vector-borne disease. Sand fly vectors (SF) and Leishmania spp parasites are sensitive to changes in weather conditions, rendering disease transmission susceptible to changes in local and global scale climatic patterns. Nevertheless, it is unclear how SF abundance is impacted by El Nino Southern Oscillation (ENSO) and how these changes might relate to changes in CL transmission.Methodology and FindingsWe studied association patterns between monthly time series, from January 2000 to December 2010, of: CL cases, rainfall and temperature from Panama, and an ENSO index. We employed autoregressive models and cross wavelet coherence, to quantify the seasonal and interannual impact of local climate and ENSO on CL dynamics. We employed Poisson Rate Generalized Linear Mixed Models to study SF abundance patterns across ENSO phases, seasons and eco-epidemiological settings, employing records from 640 night-trap sampling collections spanning 2000?2011. We found that ENSO, rainfall and temperature were associated with CL cycles at interannual scales, while seasonal patterns were mainly associated with rainfall and temperature. Sand fly (SF) vector abundance, on average, decreased during the hot and cold ENSO phases, when compared with the normal ENSO phase, yet variability in vector abundance was largest during the cold ENSO phase. Our results showed a three month lagged association between SF vector abundance and CL cases.ConclusionAssociation patterns of CL with ENSO and local climatic factors in Panama indicate that interannual CL cycles might be driven by ENSO, while the CL seasonality was mainly associated with temperature and rainfall variability. CL cases and SF abundance were associated in a fashion suggesting that sudden extraordinary changes in vector abundance might increase the potential for CL epidemic outbreaks, given that CL epidemics occur during the cold ENSO phase, a time when SF abundance shows its highest fluctuations

Transcriptional Control of the Human Pregnancy-specific Glycoprotein 5 Gene is Dependent on Two GT-boxes Recognized by the Ubiquitous Specificity Protein 1 (Sp1) Transcription Factor

International audiencePregnancy-specific glycoprotein 5 gene (PSG-5) belongs to the human pregnancy-specific glycoprotein family, encoded by eleven highly similar and transcriptionally active genes. High levels of PSG biosynthesis are restricted to the placenta syncytiotrophoblast and are essential for the maintenance of normal gestation in mammalian species. We have investigated here the nature of the transcription factors that recognize the FP1 (-455/-433) and the CPE (-147/-140) regulatory sequences that significantly contribute to basal PSG-5 promoter activity. Both elements bear a similar GT-box motif; and DNA-protein complex formation, as well as promoter activity, is largely dependent on the integrity of these GT-box sequences. Gel shift, super gel shift and UV-crosslinking experiments clearly demonstrate that the ubiquitous specificity protein 1 (Sp1) is the major transcription factor involved in complex formation with both cis-acting elements in normal term placenta tissue and in PSG-non-expressing COS-7 cells. Furthermore, transfection experiments indicate that Sp1 activates PSG-5 promoter constructs. In addition, we show that Sp1 is indeed co-expressed with PSG genes in the syncytiotrophoblast cells, stressing its potential role in the in vivo regulation of PSG expression

Hexosamine pathway regulates StarD7 expression in JEG-3 cells

StarD7 is a lipid binding protein involved in the delivery of phosphatidylcholine to the mitochondria whose promoter is activated by Wnt/β-catenin signaling. Although the majority of glucose enters glycolysis, ~ 2–5% of it can be metabolized via the hexosamine biosynthetic pathway (HBP). Considering that HBP has been implicated in the regulation of β-catenin we explored if changes in glucose levels modulate StarD7 expression by the HBP in trophoblast cells. We found an increase in StarD7 as well as in β-catenin expression following high-glucose (25 mM) treatment in JEG-3 cells; these effects were abolished in the presence of HBP inhibitors. Moreover, since HBP is able to promote unfolded protein response (UPR) the protein levels of GRP78, Ire1α, calnexin, p-eIF2α and total eIF2α as well as XBP1 mRNA was measured. Our results indicate that a diminution in glucose concentration leads to a decrease in StarD7 expression and an increase in the UPR markers: GRP78 and Ire1α. Conversely, an increase in glucose is associated to high StarD7 levels and low GRP78 expression, phospho-eIF2α and XBP1 splicing, although Ire1α remains high when cells are restored to high glucose. Taken together these findings indicate that glucose modulates StarD7 and β-catenin expression through the HBP associated to UPR, suggesting the existence of a link between UPR and HBP in trophoblast cells. This is the first study reporting the effects of glucose on StarD7 in trophoblast cells. These data highlight the importance to explore the role of StarD7 in placenta disorders related to nutrient availability.Fil: Flores Martín, Jésica Belén. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Reyna, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Cruz del Puerto, Mariano Matias Arzud. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Rojas, María L.. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Panzetta-Dutari, Graciela Maria del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Genti de Raimondi, Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Low oxygen tension induces Krüppel-Like Factor 6 expression in trophoblast cells

The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia.Fil: Racca, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ridano, Magali Evelin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bandeira, C. L.. Universidade de Sao Paulo; BrasilFil: Avvad Portari, E.. Universidade Federal do Rio de Janeiro; BrasilFil: Genti de Raimondi, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Graham, C. H.. Queens University; CanadáFil: Panzetta-Dutari, Graciela Maria del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Primer sequences and concentrations used in RT-PCR assays.

<p>Primer sequences and concentrations used in RT-PCR assays.</p

KLF6 protein expression throughout trophoblast cell differentiation.

<p><b>A-</b> Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). <b>B-</b> Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. <b>C-</b> Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. <b>D-</b> Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.</p

KLF6 transcript and protein levels increase during trophoblast cell differentiation.

<p><b>A-</b> KLF6 mRNA expression was quantified by qRT-PCR (ABI 7500, Applied Biosystems) in CTB cells isolated from three to eight normal term placentas, and cultured in differentiating medium during the indicated times. Results were normalized to cyclophilin A and expressed according to the 2^−ΔΔCt method using as calibrator the expression level at 0 h. Results are depicted as boxplot graphs representing the medians (horizontal bars), the 25–75th percentile interquartile range (box limits), and the lowest and highest values (whiskers) of three to eight experiments performed in triplicates. Inter-group comparisons were made using the Kruskal-Wallis one-way Analysis of variance (ANOVA) with the Dunn's multiple comparisons post-hoc test of statistical significance. *p<0.05 <i>vs</i> 0 h, #p<0.05 <i>vs</i> 2 h. <b>B-</b> Protein extracts (60 µg) prepared from CTB cells cultured for the indicated hours were subjected to western blot analysis using anti-KLF6 and α-tubulin antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022438#s4" target="_blank"><i>Materials and Methods</i></a>. A representative blot is shown. The bar graph represents the densitometric quantification of KLF6/α-tubulin ratio of three independent experiments expressed as mean ±SEM. *p<0.05 <i>vs</i> 0 h, ‡p<0.05 <i>vs</i> 16 h. (Kruskal-Wallis, Dunn's).</p