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

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

c-Jun Proto-Oncoprotein Plays a Protective Role in Lung Epithelial Cells Exposed to Staphylococcal α-Toxin

c-Jun is a member of the early mammalian transcriptional regulators belonging to the AP-1 family, which participates in a wide range of cellular processes such as proliferation, apoptosis, tumorigenesis, and differentiation. Despite its established role in cell survival upon stress, its participation in the stress response induced by bacterial infections has been poorly investigated. To study the potential role of c-Jun in this context we choose the widely studied α-toxin produced by Staphylococcus aureus, a pore-forming toxin that is a critical virulence factor in the pathogenesis of these bacteria. We analyzed the effect of α-toxin treatment in the activation, expression, and protein levels of c-Jun in A549 lung epithelial cells. Furthermore, we explored the role of c-Jun in the cellular fate after exposure to α-toxin. Our results show that staphylococcal α-toxin per se is able to activate c-Jun by inducing phosphorylation of its Serine 73 residue. Silencing of the JNK (c-Jun N-terminal Kinase) signaling pathway abrogated most of this activation. On the contrary, silencing of the ERK (Extracellular Signal-Regulated Kinase) pathway exacerbated this response. Intriguingly, while the exposure to α-toxin induced a marked increase in the levels of c-Jun transcripts, c-Jun protein levels noticeably decreased in the same time-frame as a consequence of active proteolytic degradation through the proteasome-dependent pathway. In addition, we established that c-Jun promoted cell survival when cells were challenged with α-toxin. Similarly, c-Jun phosphorylation was also induced in cells upon intoxication with the cytolysin produced by Vibrio cholerae in a JNK-dependent manner, suggesting that c-Jun-JNK axis would be a conserved responsive cellular pathway to pore-forming toxins. This study contributes to understanding the role of the multifaceted c-Jun proto-oncoprotein in cell response to bacterial pore-forming toxins, positioning it as a relevant component of the complex early machinery mounted to deal with staphylococcal infections

Analyzes In Silico Indicate the lncRNAs MIR31HG and LINC00939 as Possible Epigenetic Inhibitors of the Osteogenic Differentiation in PDLCs

Chromatin conformation, DNA methylation pattern, transcriptional profile, and non-coding RNAs (ncRNAs) interactions constitute an epigenetic pattern that influences the cellular phenotypic commitment and impacts the clinical outcomes in regenerative therapies. Here, we investigated the epigenetic landscape of the SP7 transcriptor factor (SP7) and Distal-Less Homeobox 4 (DLX4) osteoblastic transcription factors (TFs), in human periodontal ligament mesenchymal cells (PDLCs) with low (l-PDLCs) and high (h-PDLCs) osteogenic potential. Chromatin accessibility (ATAC-seq), genome DNA methylation (Methylome), and RNA sequencing (RNA-seq) assays were performed in l- and h-PDLCs, cultured at 10 days in non-induced (DMEM) and osteogenic (OM) medium in vitro. Data were processed in HOMER, Genome Studio, and edgeR programs, and metadata was analyzed by online bioinformatics tools and in R and Python environments. ATAC-seq analyses showed the TFs genomic regions are more accessible in l-PDLCs than in h-PDLCs. In Methylome analyses, the TFs presented similar average methylation intensities (AMIs), without differently methylated probes (DMPs) between l- and h-PDLCs; in addition, there were no differences in the expression profiles of TFs signaling pathways. Interestingly, we identified the long non-coding RNAs (lncRNAs), MIR31HG and LINC00939, as upregulated in l-PDLCs, in both DMEM and OM. In the following analysis, the web-based prediction tool LncRRIsearch predicted RNA:RNA base-pairing interactions between SP7, DLX4, MIR31HG, and LINC00939 transcripts. The machine learning program TriplexFPP predicted DNA:RNA triplex-forming potential for the SP7 DNA site and for one of the LINC00939 transcripts (ENST00000502479). PCR data confirmed the upregulation of MIR31HG and LINC00939 transcripts in l-PDLCs (× h-PDLCs) in both DMEM and OM (p SP7 and DLX4 were downregulated, confirming those results observed in the RNA-Seq analysis. Together, these results indicate the lncRNAs MIR31HG and LINC00939 as possible epigenetic inhibitors of the osteogenic differentiation in PDLCs by (post)transcriptional and translational repression of the SP7 and DLX4 TFs

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 used in conventional PCR for ChIP assays and their corresponding amplification product sizes.

<p>Primer sequences used in conventional PCR for ChIP assays and their corresponding amplification product sizes.</p

Induction of <i>PSG3</i> and <i>PSG5</i> promoter activity in TSA-exposed JEG-3 cells.

<p>Scheme of reporter constructs bearing the 5′ regulatory region of <i>PSG3</i> (A) and <i>PSG5</i> (C) genes. Circles depict the CPE regulatory element. Luciferase activity was determined in JEG-3 cells incubated with 150 nM TSA (grey bars) or DMSO (white bars). Results are expressed as relative luciferase activities (RLA) referred to the promoter activity of NB3luc (B) or UB5luc (D) construct in DMSO control cultures, arbitrarily set as 1. Data are shown as mean ± SEM of four independent experiments performed in triplicates. Italic bold numbers represent fold increase in luciferase activity of each construct in TSA-exposed cells relative to the control ones. Data were subjected to analysis of variance (one-way ANOVA) followed by Fisher's test to determine significant statistical differences (p≤0.05). Different letters on the right side of each bar indicate statistically different values.</p

TSA treatment increases acetylation of histone H3 associated with <i>PSG</i> gene promoters.

<p>JEG-3 cells were exposed to 150 nM TSA or 0 nM TSA (DMSO 0,015%, non-treated controls) for 18 h. A) Epifluorescence immune detection of acetyl H3 (Ac-H3) protein (green, left panels) and Höechst counterstaining (blue, middle panels). Merged images are shown on the right panels. Scale bar: 10 µm. Original magnification: 1500X. B) Western blot assay to detect whole cell content of acetyl H3 (Ac-H3) in protein extracts from JEG-3 cells treated with DMSO or TSA. β-actin protein was used for normalization of protein loading. A representative experiment of three independent assays is shown. C) ChIP analyses. TSA- and DMSO-treated JEG-3 cells were fixed with formaldehyde. DNA-protein complexes were sonicated and immunoprecipitated using anti-acetyl-H3 (Ac-H3), anti-PSG (as non related antibody, NR) or without antibodies (No Ab). Input and recovered DNA samples were amplified by conventional PCR employing primer pairs flanking positions −178/−49, −970/−551, and −1463/−977 of the regulatory region of <i>PSG</i> genes (positions are indicated relative to the <i>PSG5</i> gene). A <i>PSG5</i> coding sequence and a transcriptionally active euchromatin region of the <i>GAPDH</i> promoter were used as negative and positive immunoprecipitation controls, respectively. Results are representative of duplicated PCR reactions from two independent ChIP assays. D) Densitometric quantification of amplified products of the <i>PSG</i> gene regulatory regions normalized to the corresponding input is shown after subtraction of No Ab and NR amplification. Results are presented as mean ± SD and referred as fold change respect to DMSO control condition (arbitrarily defined as 1).</p

Effect of HAT co-activators on <i>PSG5</i> promoter activation mediated by Sp1 and KLF6.

<p>JEG-3 cells were transfected with the UB5luc plasmid and the expression vectors coding for Sp1 (A) or KLF6 (B) alone (lane 1) or together with either wild-type (lanes 2, 4, and 6) or HAT-deficient (lanes 3, 5, and 7) versions of p300 (light grey bars), CBP (dark grey bars) or PCAF (black bars). Data are shown as relative luciferase activity (RLA) to that of JEG-3 cultures co-transfected with the corresponding empty vectors defined as 1 (dashed line). Results are presented as mean ± SD of one representative experiment of two independent assays performed in triplicates, with consistent results. Statistical differences (p≤0.05) were identified using two-sided Student’s t-test. * depicts statistically different values compared to that of the culture co-transfected with the corresponding empty vectors. † represents statistically different values compared to that of the culture co-transfected with the wild type version of the corresponding co-activator. ‡ shows statistically different values compared to that of the culture co-transfected with the Sp1 or KLF6 expression vector alone.</p