Absence Of Exogenous Ligands Research Articles

The modern human aryl hydrocarbon receptor is more active when ancestralized by genome editing

The aryl hydrocarbon receptor (AHR) is a transcription factor that has many functions in mammals. Its best known function is that it binds aromatic hydrocarbons and induces the expression of cytochrome P450 genes, which encode enzymes that metabolize aromatic hydrocarbons and other substrates. All present-day humans carry an amino acid substitution at position 381 in the AHR that occurred after the divergence of modern humans from Neandertals and Denisovans. Previous studies that have expressed the ancestral and modern versions of AHR from expression vectors have yielded conflicting results with regard to their activities. Here, we use genome editing to modify the endogenous AHR gene so that it encodes to the ancestral, Neandertal-like AHR protein in human cells. In the absence of exogenous ligands, the expression of AHR target genes is higher in cells expressing the ancestral AHR than in cells expressing the modern AHR, and similar to the expression in chimpanzee cells. Furthermore, the modern human AHR needs higher doses of three ligands than the ancestral AHR to induce the expression of target genes. Thus, the ability of AHR to induce the expression of many of its target genes is reduced in modern humans.

Ectopic Expression of Human, Mouse, and Fruit Fly Aryl Hydrocarbon Receptor in <i>Drosophila</i> <i>melanogaster</i> Tissues

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor and its target genes play a fundamental role in detoxification, regulation of developmental processes, maintenance of homeostasis, as well as in the occurrence of oncological and autoimmune diseases and drug metabolism. The high conservatism of vertebrate AHR allowed us to study its functions in vivo using transformed Drosophila melanogaster fruit flies with human or mouse AHR gene and compare the ectopic effect of their expression with the expression of spineless gene, Drosophila AHR homologue. This work demonstrates for the first time that vertebrate AHR exhibits its functional activity in Drosophila embryogenesis, in leg imaginal discs and in somatic cells of female reproductive system in the absence of exogenous ligands.

Interactions between semaphorins and plexin–neuropilin receptor complexes in the membranes of live cells

Signaling of semaphorin ligands via their plexin–neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein–protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer–dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.

Distal lysine (de)coordination in the algal hemoglobin THB1: A combined computer simulation and experimental study

THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe–Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein–propionate interactions and strongly suggested that cleavage of the Fe–Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.

Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC

In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array.

CSIG-06. PLATELET DERIVED GROWTH FACTOR RECEPTOR ALPHA AS AN ONCOGENIC DRIVER IN GLIOBLASTOMA

Abstract Activated platelet derived growth factor receptor alpha (PDGFRα) is essential for the maintenance of oligodendrocyte progenitors. Differential signaling results from phosphorylation of specific tyrosine residues in the kinase insert (KI) domain present in most RTKs in variable length. Our goal is to identify the molecular contexts in which PDGFRA activation is an essential driver for maintenance of the malignant phenotype in glioblastoma. RNA sequencing was conducted on 13 glioblastoma mouse orthotopic PDXs (n=3/line). PDGFRA mRNA overexpression was observed for 3 glioblastomas: one carrying PDGFRA extrachromosomal (ecDNA) amplification (HF3253) and two non-amplified (Tukey p=0.41, 0.19). Corresponding PDGFRα KI activation (pY754) was observed only for HF3253. To assess activation as an adaptation to microenvironmental pressures, pY754 was quantified by reverse phase protein array on 4 growth conditions (normal, -growth factors, two serum concentrations) for 8 neurospheres. PDGFRɑ activation was highest in growth factor-depleted conditions in the absence of exogenous ligand (Tukey p=0.02). Taking advantage of genomic heterogeneity in HF3253, we isolated cellular lineages with divergent frequencies of PDGFRA ecDNA. PDGFRA copy number (TaqMan) decreased longitudinally. PDX tumors from higher passage had slower tumor growth due to strong selection for initial low frequency PDGFRA ecDNA-amplified clones. IHC showed that the tumors had comparably high total and activated PDGFRα to low passage tumors. Further exploiting intra-tumoral heterogeneity, we have isolated single cell clones. Tumor growth was reduced in 2 ecDNA(-) tumors compared to parental ecDNA(+) (log-rank test p= 3e-3, 1e-3). In contrast to parental, ecDNA(-) tumors demonstrated diffuse tumor morphology and weak PDGFRα activation. The staining index (mean intensity x area) for the ecDNA(-) clones was significantly lower than the ecDNA(+) (1-wayANOVA p=2e-16). We established dependency on PDGFRα signaling in a patient-derived glioblastoma model. Our data demonstrates that detection of ecDNA-amplified PDGFRA has the potential to be a predictive biomarker of future PDGFRɑ-targeted therapies.

Chronic and acute arsenic exposure enhance EGFR expression via distinct molecular mechanisms

The impacts of acute arsenic exposure (i.e. vomiting, diarrhea, and renal failure) are distinct from those brought about by sustained, low level exposure from environmental sources or drinking of contaminated well water. Chronic arsenic exposure is a risk factor for the development of pulmonary diseases, including lung cancer. How arsenic exposure leads to pulmonary disease is not fully understood. Both acute versus chronic arsenic exposure increase EGFR expression, but do so via distinct molecular mechanisms. BEAS-2B cells were exposed to either acute sodium arsenite (5 μM for 24 h) or chronic sodium arsenite (100 nM for 24 weeks). Cells treated with acute arsenic exhibited a decrease in viability, changes in morphology, and increased mRNA level of BTC. In contrast, during 24 weeks of arsenic exposure, the cells had increased EGFR expression and activity, and increased mRNA and protein levels of TGFα. Further, chronic arsenic treatment caused an increase in cell migration in the absence of exogenous ligand. Elevated TGFα and EGFR expression are features of many non-small cell lung cancers. We propose that lung epithelial cells chronically exposed to low level arsenic increases EGFR signaling via TGFα production to enhance ligand-independent cell migration.

Human colon organoids reveal distinct physiologic and oncogenic Wnt responses.

Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.

PPARγ is a nexus controlling alternative activation of macrophages via glutamine metabolism.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is known to regulate lipid metabolism in many tissues, including macrophages. Here we report that peritoneal macrophage respiration is enhanced by rosiglitazone, an activating PPARγ ligand, in a PPARγ-dependent manner. Moreover, PPARγ is required for macrophage respiration even in the absence of exogenous ligand. Unexpectedly, the absence of PPARγ dramatically affects the oxidation of glutamine. Both glutamine and PPARγ have been implicated in alternative activation (AA) of macrophages, and PPARγ was required for interleukin 4 (IL4)-dependent gene expression and stimulation of macrophage respiration. Indeed, unstimulated macrophages lacking PPARγ contained elevated levels of the inflammation-associated metabolite itaconate and express a proinflammatory transcriptome that, remarkably, phenocopied that of macrophages depleted of glutamine. Thus, PPARγ functions as a checkpoint, guarding against inflammation, and is permissive for AA by facilitating glutamine metabolism. However, PPARγ expression is itself markedly increased by IL4. This suggests that PPARγ functions at the center of a feed-forward loop that is central to AA of macrophages.

Aryl hydrocarbon receptor (AHR) is a potential tumour suppressor in pituitary adenomas.

Pituitary adenomas (PA) represent the largest group of intracranial neoplasms and yet the molecular mechanisms driving this disease remain largely unknown. The aim of this study was to use a high-throughput screening method to identify molecular pathways that may be playing a significant and consistent role in PA. RNA profiling using microarrays on eight local PAs identified the aryl hydrocarbon receptor (AHR) signalling pathway as a key canonical pathway downregulated in all PA types. This was confirmed by real-time PCR in 31 tumours. The AHR has been shown to regulate cell cycle progression in various cell types; however, its role in pituitary tissue has never been investigated. In order to validate the role of AHR in PA behaviour, further functional studies were undertaken. Over-expression of AHR in GH3 cells revealed a tumour suppressor potential independent of exogenous ligand activation by benzo α-pyrene (BαP). Cell cycle analysis and quantitative PCR of cell cycle regulator genes revealed that both unstimulated and BαP-stimulated AHR reduced E2F-driven transcription and altered expression of cell cycle regulator genes, thus increasing the percentage of cells in G0/G1 phase and slowing the proliferation rate of GH3 cells. Co-immunoprecipitation confirmed the interaction between AHR and retinoblastoma (Rb1) protein supporting this as a functional mechanism for the observed reduction. Endogenous Ahr reduction using silencing RNA confirmed the tumour suppressive function of the Ahr. These data support a mechanistic pathway for the putative tumour suppressive role of AHR specifically in PA, possibly through its role as a cell cycle co-regulator, even in the absence of exogenous ligands.

The Complex Biology of the Aryl Hydrocarbon Receptor and Its Role in the Pituitary Gland.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor best known for its ability to mediate the effects of environmental toxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), polycyclic aromatic hydrocarbons (PAHs), benzene, and polychlorinated biphenyls (PCBs) through the initiation of transcription of a number of metabolically active enzymes. Therefore, the AHR has been studied mostly in the context of xenobiotic signaling. However, several studies have shown that the AHR is constitutively active and plays an important role in general cell physiology, independently of its activity as a xenobiotic receptor and in the absence of exogenous ligands. Within the pituitary, activation of the AHR by environmental toxins has been implicated in disruption of gonadal development and fertility. Studies carried out predominantly in mouse models have revealed the detrimental influence of several environmental toxins on specific cell lineages of the pituitary tissue mediated by activation of AHR and its downstream effectors. Activation of AHR during fetal development adversely affected pituitary development while adult models exposed to AHR ligands demonstrated varying degrees of pituitary dysfunction. Such dysfunction may arise as a result of direct effects on pituitary cells or indirect effects on the hypothalamic-pituitary-gonadal axis. This review offers in-depth analysis of all aspects of AHR biology, with a particular focus on its role and activity within the adenohypophysis and specifically in pituitary tumorigenesis. A novel mechanism by which the AHR may play a direct role in pituitary cell proliferation and tumor formation is postulated. This review therefore attempts to cover all aspects of the AHR's role in the pituitary tissue, from fetal development to adult physiology and the pathophysiology underlying endocrine disruption and pituitary tumorigenesis.

Aryl hydrocarbon receptor (AhR)-dependent regulation of pulmonary miRNA by chronic cigarette smoke exposure

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor historically known for its toxic responses to man-made pollutants such as dioxin. More recently, the AhR has emerged as a suppressor of inflammation, oxidative stress and apoptosis from cigarette smoke by mechanisms that may involve the regulation of microRNA. However, little is known about the AhR regulation of miRNA expression in the lung in response to inhaled toxicants. Therefore, we exposed Ahr−/− and Ahr+/− mice to cigarette smoke for 4 weeks and evaluated lung miRNA expression by PCR array. There was a dramatic regulation of lung miRNA by the AhR in the absence of exogenous ligand. In response to cigarette smoke, there were more up-regulated miRNA in Ahr−/− mice compared to Ahr+/− mice, including the cancer-associated miRNA miR-96. There was no significant change in the expression of the AhR regulated proteins HuR and cyclooxygenase-2 (COX-2). There were significant increases in the anti-oxidant gene sulfiredoxin 1 (Srxn1) and FOXO3a- predicted targets of miR-96. Collectively, these data support a prominent role for the AhR in regulating lung miRNA expression. Further studies to elucidate a role for these miRNA may further uncover novel biological function for the AhR in respiratory health and disease.

Aryl hydrocarbon receptor negatively regulates lipid synthesis and involves in cell differentiation of SZ95 sebocytes in vitro

The aryl hydrocarbon receptor (AhR) is the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene (BaP) and other exogenous compounds. In human sebocytes, TCDD and BaP were found to activate the expression of multiple genes, including cytochrome P450 1A1 (CYP1A1), and inhibit lipid synthesis via AhR, while little is known about endogenous functions of the AhR. In order to expand this knowledge, we analyzed the impact of AhR knockdown on lipid synthesis as well as on cell differentiation of SZ95 sebocytes in vitro and observed that lipid synthesis was significantly induced in AhR silenced SZ95 sebocytes. In line with this result, expression of lipogenesis-associated genes, such as peroxisome proliferator activated receptor (PPAR) δ and PPARγ, was increased. Morphological changes with smaller cells in size but more abundant cytoplasmic lipids were observed in AhR silenced SZ95 sebocytes compared with the AhR activated cells. Besides, the expression of keratin 7, an early sebaceous differentiation marker, was increased, while the expression of the terminal sebocyte differentiation marker epithelial membrane antigen (EMA) was reduced. Moreover, the terminal keratinocyte differentiation markers keratin 10 and involucrin, and the AhR downstream protein CYP1A1 were reduced after AhR silencing. To the best of our knowledge, we provide evidence that in the absence of exogenous ligands, the AhR inhibits lipid synthesis and involves in cell differentiation of human SZ95 sebocytes, which indicates the physiological function of this receptor in human sebocytes.

Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells

Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10–20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs.

Cross-species transcriptomic analysis elucidates constitutive aryl hydrocarbon receptor activity.

BackgroundResearch on the aryl hydrocarbon receptor (AHR) has largely focused on variations in toxic outcomes resulting from its activation by halogenated aromatic hydrocarbons. But the AHR also plays key roles in regulating pathways critical for development, and after decades of research the mechanisms underlying physiological regulation by the AHR remain poorly characterized. Previous studies identified several core genes that respond to xenobiotic AHR ligands across a broad range of species and tissues. However, only limited inferences have been made regarding its role in regulating constitutive gene activity, i.e. in the absence of exogenous ligands. To address this, we profiled transcriptomic variations between AHR-active and AHR-less-active animals in the absence of an exogenous agonist across five tissues, three of which came from rats (hypothalamus, white adipose and liver) and two of which came from mice (kidney and liver). Because AHR status alone has been shown sufficient to alter transcriptomic responses, we reason that by contrasting profiles amongst AHR-variant animals, we may elucidate effects of the AHR on constitutive mRNA abundances.ResultsWe found significantly more overlap in constitutive mRNA abundances amongst tissues within the same species than from tissues between species and identified 13 genes (Agt, Car3, Creg1, Ctsc, E2f6, Enpp1, Gatm, Gstm4, Kcnj8, Me1, Pdk1, Slc35a3, and Sqrdl) that are affected by AHR-status in four of five tissues. One gene, Creg1, was significantly up-regulated in all AHR-less-active animals. We also find greater overlap between tissues at the pathway level than at the gene level, suggesting coherency to the AHR signalling response within these processes. Analysis of regulatory motifs suggests that the AHR mostly mediates transcriptional regulation via direct binding to response elements.ConclusionsThese findings, though preliminary, present a platform for further evaluating the role of the AHR in regulation of constitutive mRNA levels and physiologic function.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1053) contains supplementary material, which is available to authorized users.

Engineered chimeras reveal the structural basis of hexacoordination in globins: A case study of neuroglobin and myoglobin

BackgroundMyoglobin (Mb) and neuroglobin (Ngb) are representative members of pentacoordinated and bis-histidyl, hexacoordinated globins. In spite of their low sequence identity, they show surprisingly similar three-dimensional folds. The ability of Ngb to form a hexacoordinated bis-histidyl complex with the distal HisE7 has a strong impact on ligand affinity. The factors governing such different behaviors have not been completely understood yet, even though they are extremely relevant to establish structure–function relationships within the globin superfamily. MethodsIn this work we generated chimeric proteins by swapping a previously identified regulatory segment – the CD region – and evaluated comparatively the structural and functional properties of the resulting proteins by molecular dynamics simulations, and spectroscopic and kinetic investigations. ResultsOur results show that chimeric proteins display heme coordination properties displaced towards those expected for the corresponding CD region. In particular, in the absence of exogenous ligands, chimeric Mb is found as a partially hexacoordinated bis-histidyl species, whereas chimeric Ngb shows a lower equilibrium constant for forming a hexacoordinated bis-histidyl species. ConclusionsWhile these results confirm the regulatory role of the CD region for bis-histidyl hexacoordination, they also suggest that additional sources contribute to fine tune the equilibrium.General significanceGlobins constitute a ubiquitous group of heme proteins widely found in all kingdoms of life. These findings raise challenging questions regarding the structure–function relationships in these proteins, as bis-histidyl hexacoordination emerges as a novel regulatory mechanism of the physiological function of globins.

CD1d-mediated Presentation of Endogenous Lipid Antigens by Adipocytes Requires Microsomal Triglyceride Transfer Protein

Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and -δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis.

Autocrine signaling via release of ATP and activation of P2X7 receptor influences motile activity of human lung cancer cells

Extracellular nucleotides, such as ATP, are released from cells and play roles in various physiological and pathological processes through activation of P2 receptors. Here, we show that autocrine signaling through release of ATP and activation of P2X7 receptor influences migration of human lung cancer cells. Release of ATP was induced by stimulation with TGF-β1, which is a potent inducer of cell migration, in human lung cancer H292 cells, but not in noncancerous BEAS-2B cells. Treatment of H292 cells with a specific antagonist of P2X7 receptor resulted in suppression of TGF-β1-induced migration. PC-9 human lung cancer cells released a large amount of ATP under standard cell culture conditions, and P2X7 receptor-dependent dye uptake was observed even in the absence of exogenous ligand, suggesting constitutive activation of P2X7 receptor in this cell line. PC-9 cells showed high motile activity, which was inhibited by treatment with ecto-nucleotidase and P2X7 receptor antagonists, whereas a P2X7 receptor agonist enhanced migration. PC-9 cells also harbor a constitutively active mutation in epidermal growth factor receptor (EGFR). Treatment with EGFR tyrosine kinase inhibitor AG1478 suppressed both cell migration and P2X7 receptor expression in PC-9 cells. Compared to control PC-9 cells, cells treated with P2X7 antagonist exhibited broadened lamellipodia around the cell periphery, while AG1478-treated cells lacked lamellipodia. These results indicate that P2X7-mediated signaling and EGFR signaling may regulate migration of PC-9 cells through distinct mechanisms. We propose that autocrine ATP-P2X7 signaling is involved in migration of human lung cancer cells through regulation of actin cytoskeleton rearrangement.

Single Nucleotide Polymorphisms of Human STING Can Affect Innate Immune Response to Cyclic Dinucleotides

The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3’5’-3’5’ cyclic GMP-AMP (3’3’ cGAMP) produced by Vibrio cholerae and metazoan second messenger 2’5’-3’5’ Cyclic GMP-AMP (2’3’ cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-β and NF-κB promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-βsignaling, while it was defective for the response to c-di-AMP and 3’3’ cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-β signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides.

Bowl versus Funnel Supramolecular Concept for Cu I Complexes within the Biomimetic Tris(imidazole) Core

The coordination of CuI to a poly(imidazole) core is of the most importance in biology as it is present in most enzymes as well as in peptides involved in pathological processes. Here, we report a detailed study of the binding of CuI to a tris(imidazole) core constructed on a resorcinarene scaffold. NMR and IR spectroscopy as well as cyclic voltammetry have been used to characterize the different species obtained in solution as a function of the presence or absence of exogenous ligands, mainly CO and MeCN, but also Cl–. Within the resorcinarene scaffold, the metal ion can switch from a dynamic two-coordinate to a four-coordinate environment upon binding to a π acceptor. A comparison with previously reported calix[6]arene-based tris(imidazole) Cu complexes highlights several key points: (1) the importance of the shape and size of the macrocycle that forms the pocket in which the metal center is partly embedded, (2) the importance of the flexibility of the system as it undergoes induced-fit processes (calixarene) or not (resorcinarene), (3) the consequences on the affinity of the metal center for CO and MeCN in relation to its π-backbonding capabilities, and finally (4), the redox behavior of the system.