Amphiregulin Downregulates E-cadherin Expression by Activating YAP/Egr-1/Slug Signaling in SKOV3 Human Ovarian Cancer Cells.

Amphiregulin (AREG) stimulates human epithelial ovarian cancer (EOC) cell invasion by downregulating E-cadherin expression. YAP is a transcriptional cofactor that has been shown to regulate tumorigenesis. This study aimed to examine whether AREG activates YAP in EOC cells and explore the roles of YAP in AREG-induced downregulation of E-cadherin and cell invasion. Analysis of the Cancer Genome Atlas (TCGA) showed that upregulation of AREG and EGFR were associated with poor survival in human EOC. Treatment of SKOV3 human EOC cells with AREG induced the activation of YAP. In addition, AREG downregulated E-cadherin, upregulated Egr-1 and Slug, and stimulated cell invasion. Using gain- and loss-of-function approaches, we showed that YAP was required for the AREG-upregulated Egr-1 and Slug expression. Furthermore, YAP was also involved in AREG-induced downregulation of E-cadherin and cell invasion. This study provides evidence that AREG stimulates human EOC cell invasion by downregulating E-cadherin expression through the YAP/Egr-1/Slug signaling.

HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements.

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

Cell diversity and plasticity during atrioventricular heart valve EMTs.

Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation, but the cellular complexity and small scale of this tissue have restricted analyses. To circumvent these issues, we analyzed over 50,000 murine single-cell transcriptomes from embryonic day (E)7.75 hearts to E12.5 atrioventricular canals. We delineate mesenchymal and endocardial bifurcation during endocardial EMT, identify a distinct, transdifferentiating epicardial population during epicardial EMT, and reveal the activation of epithelial-mesenchymal plasticity during both processes. In Sox9-deficient valves, we observe increased epithelial-mesenchymal plasticity, indicating a role for SOX9 in promoting endothelial and mesenchymal cell fate decisions. Lastly, we deconvolve cell interactions guiding the initiation and progression of cardiac valve EMTs. Overall, these data reveal mechanisms of emergence of mesenchyme from endocardium or epicardium at single-cell resolution and will serve as an atlas of EMT initiation and progression with broad implications in regenerative medicine and cancer biology.

Synthetic Multivalent Disulfide-Constrained Peptide Agonists Potentiate Wnt1/ß-Catenin Signaling via LRP6 Coreceptor Clustering.

Wnt ligands are critical for tissue homeostasis and form a complex with LRP6 and frizzled coreceptors to initiate Wnt/ß-catenin signaling. Yet, how different Wnts achieve various levels of signaling activation through distinct domains on LRP6 remains elusive. Developing tool ligands that target individual LRP6 domains could help elucidate the mechanism of Wnt signaling regulation and uncover pharmacological approaches for pathway modulation. We employed directed evolution of a disulfide constrained peptide (DCP) to identify molecules that bind to the third ß-propeller domain of LRP6. The DCPs antagonize Wnt3a while sparing Wnt1 signaling. Using PEG linkers with different geometries, we converted the Wnt3a antagonist DCPs to multivalent molecules that potentiated Wnt1 signaling by clustering the LRP6 coreceptor. The mechanism of potentiation is unique as it occurred only in the presence of extracellular secreted Wnt1 ligand. While all DCPs recognized a similar binding interface on LRP6, they displayed different spatial orientations that influenced their cellular activities. Moreover, structural analyses revealed that the DCPs exhibited new folds that were distinct from the parent DCP framework they were evolved from. The multivalent ligand design principles highlighted in this study provide a path for developing peptide agonists that modulate different branches of cellular Wnt signaling.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

G protein-coupled estrogen receptor stimulates human trophoblast cell invasion via YAP-mediated ANGPTL4 expression.

Insufficient invasion of trophoblast cells into the uterine decidua is associated with preeclampsia (PE). G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. GPER is expressed in human trophoblast cells and downregulated GPER levels are noted in PE. However, to date, the role of GPER in trophoblast cells remains largely unknown. Here, we applied RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to G1, an agonist of GPER, and identified angiopoietin-like 4 (ANGPTL4) as a target gene of GPER. Treatment of trophoblast cells with G1 or 17ß-estradiol (E2) activated Yes-associated protein (YAP), the major downstream effector of the Hippo pathway, via GPER but in a mammalian STE20-like protein kinase 1 (MST1)-independent manner. Using pharmacological inhibitors as well as loss- and gain-of-function approaches, our results revealed that YAP activation was required for GPER-stimulated ANGPTL4 expression. Transwell invasion assays demonstrated that activation of GPER-induced ANGPTL4 promoted cell invasion. In addition, the expression levels of GPER, YAP, and ANGPTL4 were downregulated in the placenta of patients with PE. Our findings reveal a mechanism by which GPER exerts its stimulatory effect on human trophoblast cell invasion by upregulating YAP-mediated ANGPTL4 expression.

Association of circulating monocyte chemoattractant protein-1 levels with polycystic ovary syndrome: A meta-analysis.

PROBLEM: Polycystic ovary syndrome (PCOS) is a common hormonal disorder that has a huge impact on the human infertility. Increased levels of various circulating inflammatory cytokines have been observed in PCOS patients, which can contribute to the pathogenesis of PCOS. Monocyte chemoattractant protein-1 (MCP-1), a secretory chemokine, is a potent chemotactic factor that recruits monocytes/macrophages to inflammatory foci. Several previous studies comparing the circulating MCP-1 levels between non-PCOS and PCOS patients have yielded contradictory results. Therefore, the aim of this meta-analysis was to investigate whether circulating MCP-1 levels vary between non-PCOS and PCOS patients. METHODS: Research articles published before November 11, 2020, were screened to identify eligible studies. Heterogeneity, sensitivity, and publication bias were analyzed using STATA software. Standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated by the STATA software using a random-effects model. RESULTS: 11 studies were included in this meta-analysis involving 897 individuals: 368 non-PCOS patient and 529 PCOS patients. Our pooled meta-analysis results show that circulating MCP-1 levels were significantly higher in PCOS patients than in non-PCOS patients (SMD = 0.84, 95% CI = [0.37, 1.31], Z = 3.50, p < 0.01). However, due to the limited number of studies included in this meta-analysis, subgroup analysis determined that circulating MCP-1 levels were not significantly varied between obese non-PCOS and obese PCOS patients (SMD = 0.42, 95% CI = [-0.65, 1.49], Z = 0.77, p = 0.442) as well as between non-PCOS and PCOS patients without obesity (SMD = 2.04, 95% CI = [-0.84, 4.93], Z = 1.39, p = 0.166). In addition, circulating MCP-1 levels were also not significantly different between obese and non-obese PCOS patients (SMD = -0.04, 95% CI = [-0.68, 0.60], Z = 0.11, p = 0.909). CONCLUSION: Our findings reveal that circulating MCP-1 levels are upregulated in women with PCOS and are associated with an increased risk of PCOS.

Protein Ligand-Induced Amplification in the 1/f Noise of a Protein-Selective Nanopore.

Previous studies of transmembrane protein channels have employed noise analysis to examine their statistical current fluctuations. In general, these explorations determined a substrate-induced amplification in the Gaussian white noise of these systems at a low-frequency regime. This outcome implies a lack of slowly appearing fluctuations in the number and local mobility of diffusing charges in the presence of channel substrates. Such parameters are among the key factors in generating a low-frequency 1/f noise. Here, we show that a protein-selective biological nanopore exhibits a substrate-induced amplification in the 1/f noise. The modular composition of this biological nanopore includes a hydrophilic transmembrane protein pore fused to a water-soluble binding protein on its extramembranous side. In addition, this protein nanopore shows an open substate populated by a high-frequency current noise because of the flickering of an engineered polypeptide adaptor at the tip of the pore. However, the physical association of the protein ligand with the binding domain reversibly switches the protein nanopore from a high-frequency noise substate into a quiet substate. In the absence of the protein ligand, our nanopore shows a low-frequency white noise. Remarkably, in the presence of the protein ligand, an amplified low-frequency 1/f noise was detected in a ligand concentration-dependent fashion. This finding suggests slowly occurring equilibrium fluctuations in the density and local mobility of charge carriers under these conditions. Furthermore, we report that the excess in 1/f noise is generated by reversible switches between the noisy ligand-released substate and the quiet ligand-captured substate. Finally, quantitative aspects of the low-frequency 1/f noise are in accord with theoretical predictions of the current noise analysis of protein channel-ligand interactions.

TGF-ß1 induces VEGF expression in human granulosa-lutein cells: a potential mechanism for the pathogenesis of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is one of the most serious and iatrogenic complications that can occur during in vitro fertilization treatment. Although the pathogenesis of OHSS is not fully understood, vascular endothelial growth factor (VEGF) has been recognized as an important mediator of the development of OHSS. Transforming growth factor-beta-1 (TGF-ß1) is known to regulate various ovarian functions. However, whether VEGF can be regulated by TGF-ß1 in human granulosa cells has not been determined. In addition, the role of TGF-ß1 in the pathogenesis of OHSS remains unknown. In the present study, we demonstrate that TGF-ß1 stimulates VEGF expression in and secretion from both immortalized human granulosa-lutein (hGL) cells and primary hGL cells. Our results demonstrate that the SMAD2/3, ERK1/2, and p38 MAPK signaling pathways are involved in TGF-ß1-induced VEGF expression and secretion. Using a mouse OHSS model, we show that the expression levels of TGF-ß1 and VEGF are increased in the ovaries of OHSS mice. Blocking TGF-ß1 signaling inhibits the development of OHSS by attenuating VEGF expression. Moreover, clinical results reveal that the protein levels of TGF-ß1 and VEGF are increased in the follicular fluid of patients with OHSS, and that the levels of these two proteins in the follicular fluid are positively correlated. The results of this study help to elucidate the mechanisms by which VEGF expression is regulated in hGL cells, which could lead to the development of alternative therapeutic approaches for treating OHSS.

Factors Associated with the Development of Secondary Multidrug-resistant Tuberculosis.

BACKGROUND: Spread of multidrug-resistant tuberculosis (TB) is a threat to India's TB control program. We conducted this study with the objective to determine the risk factors for the development of secondary multidrug-resistant TB. METHODS: We conducted an unmatched case-control study involving 247 multidrug-resistant TB patients as "cases" and 494 individuals who were declared as "cured" after category I DOTS treatment as "controls." Data were collected through face-to-face interviews and review of treatment records. Multivariable logistic regressions were used to analyze the collected data. RESULTS: The mean duration for which cases took first-line anti-TB drug was 19.7 months. The mean duration between initial diagnosis of TB and diagnosis of multi-drug resistant TB (MDR-TB) was 28.3 months. In our study, 26.7%, 50.2%, and 23.1% of MDR-TB cases had one, two, or more previous episodes of TB before being diagnosed as MDR-TB. In multivariable analysis, low or no formal education (album-oriented rock [AOR] =1.63 [confidence interval (CI) = 1.03-3.11]), labor occupation (AOR = 2.15 [CI = 1.18-3.90]), smoking (AOR = 2.56 [CI = 1.19-3.26]), having HIV (AOR = 9.45 [CI = 6.80-15.9]), migration for job (AOR = 3.70 [CI = 1.96-5.67]), stopping TB treatment due to comorbid conditions (AOR = 8.86 [CI = 5.45-11.2]), and having type 2 diabetes (AOR = 3.4 [CI = 1.96-5.16]) were associated with MDR-TB. CONCLUSIONS: Government of India should devise strategy to prevent interruption of treatment to stop the emergence and spread of MDR-TB. We need to better integrate TB control activities with diabetes and tobacco control programs for better health outcome among patients.

YAP transcriptionally regulates ErbB2 to promote liver cell proliferation.

The Hippo signaling pathway is implicated in regulation of liver size and dysregulation of this pathway contributes to tumorigenesis. The transcriptional targets and downstream pathways of the Hippo pathway effector YAP that contribute to liver growth have yet to be well-characterized. We examined the liver transcriptome in response to YAP overexpression and identify the ErbB signaling pathway as a mediator of cell growth downstream of YAP. ErbB2 is transcriptionally regulated by YAP in both the mouse liver and in HepG2 human hepatoma cells. Knockdown of YAP or pharmacological inhibition with verteporfin reduced ERBB2 levels in HepG2 cells. Analysis of ChIP-seq data revealed enrichment of the transcription factor TEAD4 at the ERBB2 promoter. Using luciferase reporter and chromatin immunoprecipitation assays, we show that YAP and TEAD4 directly bind to and activate a regulatory element in the ErbB2 promoter in both the mouse liver and HepG2 cells. Functionally, knockdown of YAP reduced EGF-induced ERBB2-mediated HepG2 cell proliferation and PI3K/AKT activation. Our findings highlight a mechanism by which YAP exerts its effects on liver cell proliferation through the ErbB signaling pathway by directly regulating the transcription of ErbB2.

S1P Stimulates Proliferation by Upregulating CTGF Expression through S1PR2-Mediated YAP Activation.

Dysregulation of the Hippo pathway in the liver results in overgrowth and eventually tumorigenesis. To date, several upstream mechanisms have been identified that affect the Hippo pathway, which ultimately regulate YAP, the major downstream effector of the pathway. However, upstream regulators of the Hippo pathway in the liver remain poorly defined. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been shown to stimulate hepatocellular carcinoma (HCC) cell proliferation, but whether the Hippo pathway is involved in S1P-stimulated HCC cell proliferation remains to be determined. Here it is demonstrated that S1P activates YAP and that the S1P receptor 2 (S1PR2/S1P2) mediates S1P-induced YAP activation in both human and mouse HCC cells. S1P promotes YAP-mediated upregulation of cysteine-rich protein 61 and connective tissue growth factor (CTGF), and stimulates HCC cell proliferation. By using siRNA-mediated knockdown approaches, only CTGF was required for S1P-stimulated cell proliferation. Of note, S1P activates YAP in a MST1/2-independent manner suggesting that the canonical Hippo kinase is not required for S1P-mediated proliferation in liver. The upregulation of CTGF and S1P2 were also observed in liver-specific YAP overexpression transgenic mouse hepatocytes. Moreover, YAP regulated liver differentiation-dependent gene expression by influencing the chromatin binding of HNF4α based on ChIP-seq analysis. Finally, results using gain- and loss-of-function approaches demonstrate that HNF4α negatively regulated S1P-induced CTGF expression.Implications: These findings reveal a role for S1P in stimulating HCC cell proliferation by upregulating CTGF expression through S1P2-mediated YAP activation. Mol Cancer Res; 16(10); 1543-55. ©2018 AACR.

Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression.

BACKGROUND: DNA methylation plays a vital role in the cell, but loss-of-function mutations of the maintenance methyltransferase DNMT1 in normal human cells are lethal, precluding target identification, and existing hypomorphic lines are tumour cells. We generated instead a hypomorphic series in normal hTERT-immortalised fibroblasts using stably integrated short hairpin RNA. RESULTS: Approximately two-thirds of sites showed demethylation as expected, with one-third showing hypermethylation, and targets were shared between the three independently derived lines. Enrichment analysis indicated significant losses at promoters and gene bodies with four gene classes most affected: (1) protocadherins, which are key to neural cell identity; (2) genes involved in fat homoeostasis/body mass determination; (3) olfactory receptors and (4) cancer/testis antigen (CTA) genes. Overall effects on transcription were relatively small in these fibroblasts, but CTA genes showed robust derepression. Comparison with siRNA-treated cells indicated that shRNA lines show substantial remethylation over time. Regions showing persistent hypomethylation in the shRNA lines were associated with polycomb repression and were derepressed on addition of an EZH2 inhibitor. Persistent hypermethylation in shRNA lines was, in contrast, associated with poised promoters. CONCLUSIONS: We have assessed for the first time the effects of chronic depletion of DNMT1 in an untransformed, differentiated human cell type. Our results suggest polycomb marking blocks remethylation and indicate the sensitivity of key neural, adipose and cancer-associated genes to loss of maintenance methylation activity.

Global redesign of a native ß-barrel scaffold.

One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded ß-barrel protein into a large-conductance protein pore. Here, we show that this redesigned ß-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric ß-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.

Synthesis of CNS active thyrotropin-releasing hormone (TRH)-like peptides: Biological evaluation and effect on cognitive impairment induced by cerebral ischemia in mice.

Thyrotropin-releasing hormone (TRH)-like peptides were synthesized by replacing critical histidine and pGlu residues in the native peptide. The peptides were evaluated in vitro for receptor binding activity assay and in the cell functional assay; the peptides exhibit selective basal signaling agonist behavior toward TRH-R2. For example, peptides 8a, 8b, 8c, 8 f, 8 h, 8 l and 12 d activated TRH-R2 with potency (EC50) of 0.53 µM, 0.048 µM, 0.05 µM, 0.006 µM, 0.31 µM, 0.034 µM and 0.004 µM, respectively. In contrast for signaling activation of TRH-R1, the same peptide required higher concentration of 19.35 µM, 3.98 µM, 2.54 µM, 0.287 µM, 11.28 µM, 0.986 µM and 0.944 µM, respectively. The results showed that peptides were 36.5, 82.9, 50.8, 47.8, 36.3, 32.6 and 235-fold selective to TRH-R2 receptor subtype. The peptides were investigated for CNS activity at 10 µmol/kg in pentobarbital-induced sleep assay study. Peptides 8c (16.5 ± 1.4 min) and 8l (16.5 ± 2.1 min) displayed excellent CNS activity. In an in vivo study, peptide 8c did not cause significant change in the rat plasma TSH levels. The peptide 8c was further investigated for neuroprotective potential, and significantly reduced infracts volume and neurological score in the focal cerebral ischemia model in mice. Peptide 8c also significantly lowered MDA levels, indicating reduction of oxidative and enhanced percentage cell survival in CA1 region, when compared to ischemic brain.

Isolating globose Basal stem cells from albino wistar rats using a highly specific monoclonal antibody.

INTRODUCTION: Olfactory mucosa which is situated in the roof of the nasal cavity possesses an extremely peculiar and exceptional type of pluripotent stem cells called Globose Basal Cells (GBCs) which help in lifelong regeneration of the olfactory mucosa. Previous literature doesn't provide much knowledge on the cytological, histochemical and electrophysiological properties of these cells, as they have never been isolated in pure form. MATERIAL AND METHODS: Olfactory mucosa was obtained from six Albino Wistar rats by using standardized surgical and chemical separation procedures. GBCs were isolated by using different chemical, surgical and fluorescent techniques. RESULTS: In this research work, we standardized the techniques for isolating these stem cells in pure form from rat olfactory mucosa by tagging them with GBC-III antibody and separating them from other epithelial cells by using Fluorescence Activated Cell Sorter (FACS). GBC-III antibody is a mouse monoclonal IgM antibody which recognizes a 40 kDa surface antigen, which is a laminin receptor surface protein present on the GBCs. It is a highly specific marker for GBCs, unlike the earlier antibodies used, like GBC-I, which were nonspecific markers for GBCs and showed positive reactions, even with Horizontal Basal Cells (HBCs), sustentacular cells (Sus) and duct cells. This study also standardized the techniques for surgically excising the olfactory mucosa from the nasal septum and chemically separating the olfactory epithelium from the lamina propria. CONCLUSION: GBCs are an important group of cells which can be exploited in future to study and treat neuro-degenerative disorders like multiple sclerosis, brain ischaemia, etc. and spinal cord trauma, as they reside in a niche similar to the microenvironment in the central nervous system and have the similar ectodermal development as the neuronal and non-neuronal cells of the CNS. Moreover, olfactory epithelium is easily accessible for autologous transplantation of GBCs for different CNS disorders.