Messenger Activity Research Articles

Ca2+-dependent contractile signaling induced by dsDNA-cGAS ligation mediates type 1 interferon generation and vascular injury

Acute Lung injury (ALI) is an uncontrolled systemic inflammatory response from bacterial or viral infection. The cyclic GMP-AMP synthase (cGAS) detects cytosolic double stranded DNA (dsDNA) release from bacteria, viruses, or apoptotic cells and induces innate immunity by generating the second messenger cGAMP and activation of stimulator of interferon genes (STING). However, the mechanisms regulating cGAS activity upon binding dsDNA remain elusive. Here, we show for the first in-depth analysis of mechanisms inducing cGAS activation in using lung endothelial cells (EC) and establish its translational relevance in causing ALI using a mouse model. We show that within 15 min, dsDNA ligation with cGAS mediates its interaction with a motor protein, myosin light chain 6 (MYL6), leading to phosphorylation of MLC and actin stress fiber formation. cGAS-MYL6-mediated MLC phosphorylation required calcium release and organization of an ER-Ca2+ sensor, Stromal Interaction Molecule 1 (STIM1) as puncta. Activated STIM1 also induced cGAS tyrosine phosphorylation at Y215/242 residues via cSrc. Interfering with MYL6 or cSrc expression impaired cGAS condensation and cGAMP generation. Liposome delivery of phosphodeficient cGAS (Y215/Y242àF215/F242) in EC of cGAS null mice blocked type1-IFN production during bacterial pneumonia. Our finding suggests that STIM1 is crucial in inducing cGAS activity and type1-IFN production. cGAS thus represents a novel target for inhibiting STING activation, thereby suppressing tissue injury and inflammation. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Is the Neuropeptide PEN a Ligand of GPR83?

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.

Urtica Dioica Leaf Extract Attenuates Oxidative Stress Induced by Environment Pollutant Benzo[a] Pyrene in Mouse Testicular Tissues

Background: Epidemiological studies have shown that elevated levels of air pollutants are associated with various adverse health effects, including infertility. Objective: We aimed to assess the protective effects of aqueous Urtica dioica leaf extract against benzo[a]pyrene -induced oxidative damage in mouse testis. Methods: Mice exposed to benzo[a]pyrene were treated with or without aqueous Urtica dioica extract for five weeks, and changes in body and testes weights, messenger RNA levels and activities of antioxidant enzymes, plasma testosterone levels, sperm characteristics, and testicular tissue histology were determined. Results: Exposure to benzo[a]pyrene remarkably reduced testis and body weights, the expression and activity of antioxidant enzymes, increased lipid peroxidation, decreased plasma testosterone levels and sperm count and motility, affected sperm morphology and viability, and damaged the seminiferous tubules. Treatment with aqueous Urtica dioica leaf extract attenuated benzo[a]pyrene -induced oxidative stress in the testicular tissue by increasing the expression of antioxidant genes. Further, Urtica dioica leaf extract reduced lipid peroxidation, increased antioxidative enzyme activity, enhanced sperm characteristics, increased plasma testosterone levels, and improved the morphology of the seminiferous tubules. Conclusion: Aqueous Urtica dioica leaf extract protects testicular tissue from benzo[a]pyrene -induced oxidative damage and could potentially reverse benzo[a]pyrene -induced infertility.

Selenium Ameliorated Oxidized Fish Oil-Induced Lipotoxicity via the Inhibition of Mitochondrial Oxidative Stress, Remodeling of Usp4-Mediated Deubiquitination, and Stabilization of Pparα.

Aims: Studies demonstrated that oxidized fish oil (OFO) promoted oxidative stress and induced mitochondrial dysfunction and lipotoxicity, which attenuated beneficial effects of fish oil supplements in the treatment of nonalcoholic fatty liver disease (NAFLD). The current study was performed on yellow catfish, a good model to study NAFLD, and its hepatocytes to explore whether selenium (Se) could alleviate OFO-induced lipotoxicity via the inhibition of oxidative stress and determine its potential mechanism. Results: The analysis of triglycerides content, oxidative stress parameters, and histological and transmission electronic microscopy observation showed that high dietary Se supplementation alleviated OFO-induced lipotoxicity, oxidative stress, and mitochondrial injury and dysfunction. RNA-sequencing and immunoblotting analysis indicated that high dietary Se reduced OFO-induced decline of peroxisome-proliferator-activated receptor alpha (Pparα) and ubiquitin-specific protease 4 (Usp4) protein expression. High Se supplementation also alleviated OFO-induced reduction of thioredoxin reductase 2 (txnrd2) messenger RNA (mRNA) expression level and activity. The txnrd2 knockdown experiments revealed that txnrd2 mediated Se- and oxidized eicosapentaenoic acid (oxEPA)-induced changes of mitochondrial reactive oxygen species (mtROS) and further altered Usp4 mediated-deubiquitination and stabilization of Pparα, which, in turn, modulated mitochondrial fatty acid β-oxidation and metabolism. Mechanistically, Usp4 deubiquitinated Pparα and ubiquitin-proteasome-mediated Pparα degradation contributed to oxidative stress-induced mitochondrial dysfunction. Innovation: These findings uncovered a previously unknown mechanism by which Se and OFO interacted to affect lipid metabolism via the Txnrd2-mtROS-Usp4-Pparα pathway, which provides the new target for NAFLD prevention and treatment. Conclusion: Se ameliorated OFO-induced lipotoxicity via the inhibition of mitochondrial oxidative stress, remodeling of Usp4-mediated deubiquitination, and stabilization of Pparα. Antioxid. Redox Signal. 40, 433-452.

Repentance and Compassion in 4Q400–405 Songs of the Sabbath Sacrifice. Are the Deputy Princes in Song Eight Angelic or Human Beings?

The pervasive ambiguity in 4QSongs of the Sabbath Sacrifice (SOSS) presents a major problem: When are the activities described in this text performed by angels and when by humans? This paper builds on research on Songs One, Seven, Twelve, Thirteen, and Knowledge in SOSS that hypothesised that as the liturgy proceeds through the thirteen songs of SOSS a gradual transition takes place in the human participants. In Song Six, seven chief princes initially bless God and then proceed to bless various other participants in the liturgy, concluding with “those who wait for Him … for a return of His gracious compassion.” Song Seven, in the middle of the liturgy, describes angelic messenger activity arising from the throne of God, based on Ezekiel’s merkebah chapters. After Song Seven, a dramatic change takes place. In Song Eight, those who do the blessing are no longer “chief” princes, but “deputy” princes. The hypothesis of this article is that these deputy princes are the “people of discernment” introduced in Song One, who, having expressed repentance and persevered in joint exaltation of God with the “holiest of holy ones,” have been transformed into “god-like” messengers of God, to convey God’s compassion to those “who wait for Him.” This paper refers to recent cognitive neuroscience insights in order to, to some extent, support Fletcher-Louis’s proposal of a fluid identity achieved by the participants in the liturgy as a “community of human priests as God’s angels.” In its focus on repentance, compassion, and messenger activity, SOSS may be a witness to the beginnings of a new way of achieving atonement.

Liquid Chromatographic-Electrospray Ionization-Mass Spectrometry Method for the Simultaneous Determination of Gramicidin, Neomycin & Triamcinolone Acetonide and Characterization of Novel Forced Degradation Products

The dosage form iotrim (gramicidin, neomycin and triamcinolone) is a combination of two antibiotics (gramicidin and neomycin) and a steroid (triamcinolone). The antibiotics work by killing the bacteria that cause infections. The steroid blocks the action of chemical messengers (prostaglandins) that make the affected area red, swollen and itchy. Consequently, there was still a need to develop a simple, less time consuming and economical method for the simultaneous determination of gramicidin, neomycin and triamcinolone acetonide. The current work is an effort to develop a fast and reproducible LC-MS technique for the simultaneous estimation of gramicidin, neomycin and triamcinolone acetonide. The objective of the present procedure was to validate and develop a precise and accurate liquid chromatography-mass spectrometry (LC-MS) technique for the simultaneous quantification of gramicidin, neomycin and triamcinolone acetonide. Gramicidin, neomycin and triamcinolone acetonide were monitored on Shimadzu-8045 mass spectrometer equipped with electro spray ionization interface. The retention times of gramicidin, neomycin and triamcinolone acetonide were found at 9.145 min, 7.273 min and 2.435 min, respectively. The limit of detection (LOD) results for gramicidin, neomycin and triamcinolone acetonide were observed to be 0.15, 1.5 and 0.6 μg/mL, respectively while the limit of quantification (LOQ) results were observed to be 0.5, 5, 2 μg/mL concentration, respectively. The linear range for gramicidin, neomycin and triamcinolone acetonide were found in the concentration ranges from 1.25-7.5 μg/mL, 12.5-75 μg/mL and 5-30 μg/mL with regression coefficient of 0.9991, 0.9996, 0.9999, respectively. Accuracy values for gramicidin, neomycin and triamcinolone acetonide were found to be in the range of 98.64%, 99.4%, 99.5% respectively. The % RSD for six replicates in precision was less than 2%. According to ICH Q2(R1) recommendations, this method was successfully tested with LC-MS to confirm the chemical structures of newly produced degradation products of triamcinolone acetonide and neomycin. The developed process was validated efficaciously as per ICH guidelines.

Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additionally, dissecting cell type-specific responses is challenging when the same GPCR is expressed on different cells within a tissue. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that bind clozapine-N-oxide and mimic a GPCR-of-interest. We show that chimeric DREADD-β2AR triggers responses comparable to β2AR on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate β2AR-mediated filopodia formation in microglia, an immune cell capable of driving central nervous system inflammation. When dissecting microglial inflammation, we included two additional DREADD-based chimeras mimicking microglia-enriched GPR65 and GPR109A. DREADD-β2AR and DREADD-GPR65 modulate the inflammatory response with high similarity to endogenous β2AR, while DREADD-GPR109A shows no impact. Our DREADD-based approach allows investigation of cell type-dependent pathways without known endogenous ligands.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells

AbstractPlatelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbβ3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2–infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

A qualitative exploratory interview study on birth companion support actions for women during childbirth

BackgroundThe World Health Organization recommends that women are supported continuously throughout labor by a companion of their choice. And, that companions have clearly designated roles and responsibilities to ensure that their presence is beneficial to both the woman and her health care providers. Presently, there is lack of strong evidence regarding specific support actions in relation to women’s needs of care. Thus, we aimed to explore birth companion support actions for women during childbirth.MethodsThis was an exploratory descriptive qualitative study conducted between August 2019 and December 2019; at a referral hospital in the Eastern part of Uganda. Ten women were purposively selected: those who were admitted in early labor, expecting a normal delivery, and had fulltime birth companion. Nonparticipant direct observation and in-depth interviews were used to collect data. Latent content analysis was used.ResultsThree themes were identified: “Support actions aiding a good childbirth experience”, “Support actions hindering coping with labor”, and “Women’s needs and expectations of care”. Support actions aiding a good experience described were; emotional presence, motivation, providing nourishments, messenger activities, body massage for pain relief, assisting in ambulation and coaching. Companion fearful behaviors and disrespectful care in form of unacknowledged needs and hostility from birth companions were reported to hinder coping. The women desired thoughtful communication, trust, for birth companions to anticipate their needs and recognize non perceptive phases of labor to allow them focus on themselves.ConclusionBirth companions from this study largely supported women emotionally, and attended to their physical needs. The greater part of support actions provided were esteemed by the women. Presence of birth companion will be of benefit when individual needs of women are put into consideration. Also, more guidance for birth companions is necessary to boost their role and mitigate shortcomings of their presence during childbirth.

Nonradioactive In Situ Hybridization of MT1 Melatonin Receptor for the Identification of Melatonin Target Cells.

Identifying and phenotyping the target cells of a neuroendocrine messenger is one of the key steps to understand neuroendocrine networks and the physiological action of such messengers. In the absence of reliable antibodies directed against the receptor of a neuroendocrine messenger, detecting the expression of the messenger RNA of this receptor is an important tool to identify the target cells of a neuroendocrine messenger such as melatonin. While radioactive in situ hybridization has a higher sensitivity, nonradioactive in situ hybridization has a much better cellular resolution than radioactive in situ hybridization and is therefore better suited for phenotyping the target cells of melatonin. Here we describe a nonradioactive in situ hybridization protocol with its adaptations to various types of histological preparations. This protocol allowed the phenotyping of melatonin target cells in the pars tuberalis of the adenohypophysis, leading to the discovery of photoperiodic melatonin signaling from the pars tuberalis to the hypothalamus.

Glycogen Synthase Kinase-3 (GSK-3) Inhibitors as a New Lead for Treating Breast and Ovarian Cancer.

A serine/threonine-protein kinase, recognized as Glycogen Synthase Kinase-3 (GSK-3), is documented as a regulator of assorted cellular roles. GSK-3 activates by phosphorylation and thereby controls the action of many physiological, messenger, and membrane-bound structures. GSK-3α and GSK-3β are two vastly homologous forms of GSK-3 in mammals. Recent information has recommended that GSK-3β is a constructive controller of cancer cell proliferation and a promising key target against cancer cells. GSK-3 is overexpressed in various tumor types, including ovarian tumors. In human breast carcinoma, it has been revealed that the overexpression of GSK-3β was linked with breast cancer patients. The inhibition of GSK-3 or inhibitors of GSK-3 is a promising therapeutic tactic to overcome breast and ovarian cancer. This article features an important aspect of inhibitors of Glycogen Synthase Kinase-3 as a new lead for treating breast and ovarian Cancer.

Genome-wide characterization and expression profiling of the Phospholipase C (PLC) gene family in three orchids of economic importance

BackgroundPhospholipases hydrolyze glycerophospholipids and generate diverse lipid-derived molecules with secondary messenger activity. Out of these, phospholipase C (PLC) specifically cleaves the phospholipids at ester linkages and yields diacylglycerol (DAG) and phosphorylated head groups. PLCs are classified further as phosphatidylinositol-specific PLCs (PI-PLCs) and non-specific PLCs with biased specificity for phosphatidylcholine (NPC/PC-PLC). ResultsIn the present report, we identified and characterized PLC genes in the genomes of three orchids, Phalaenopsis equestris (seven PePLCs), Dendrobium catenatum (eight DcPLCs), and Apostasia shenzhenica (seven AsPLCs). Multiple sequence alignment analysis confirmed the presence of conserved X and Y catalytic domains, calcium/lipid-binding domain (C2 domain) at the C terminal region, and EF-hand at the N-terminal region in PI-PLC proteins and esterase domain in PC-PLC. Systematic phylogenetic analysis established the relationship of the PLC protein sequences and clustered them into two groups (PI-PLC and PC-PLC) along with those of Arabidopsis thaliana and Oryza sativa. Gene architecture studies showed the presence of nine exons in all PI-PLC genes while the number varied from one to five in PC-PLCs. RNA-seq-based spatio-temporal expression profile for PLC genes was generated, which showed that PePC-PLC1, PePC-PLC2A, DcPC-PLC1A, DcPC-PLC1B, DcPC-PLC2, DcPC-PLC1B, and AsPC-PLC1 had significant expression in all reproductive and vegetative tissues. The expression profile is matched to their upstream cis-regulatory promoter elements, which indicates that PLC genes have a role in various growth and development processes and during stress responses. ConclusionsThe present study unwrapped the opportunity for functional characterization of selected PLC genes in planta for plant improvement.

Biochemical Activity Architectures Visualized-Using Genetically Encoded Fluorescent Biosensors to Map the Spatial Boundaries of Signaling Compartments.

All biological processes arise through the coordinated actions of biochemical pathways. How such functional diversity is achieved by a finite cast of molecular players remains a central mystery in biology. Spatial compartmentation-the idea that biochemical activities are organized around discrete spatial domains within cells-was first proposed nearly 40 years ago and has become firmly rooted in our understanding of how biochemical pathways are regulated to ensure specificity. However, directly interrogating spatial compartmentation and its mechanistic origins has only really become possible in the last 20 or so years, following technological advances such as the development of genetically encoded fluorescent biosensors. These powerful molecular tools permit a direct, real-time visualization of dynamic biochemical processes in native biological contexts, and they are essential for probing the spatial regulation of biochemical activities. In this Account, we review our lab's efforts in developing and using biosensors to map the spatial compartmentation of intracellular signaling pathways and illuminate key mechanisms that establish the boundaries of an intricate biochemical activity architecture. We first discuss the role of regulatory fences, wherein the dynamic activation and deactivation of diffusible messengers produce diverse signaling compartments. For example, we used biosensors for the Ca2+ effector calmodulin and its downstream target calcineurin to reveal a spatial gradient of calmodulin that controls the temporal dynamics of calcineurin signaling. Our studies using cyclic adenosine monophosphate (cAMP) biosensors have similarly elucidated fenced cAMP domains generated by competing production and degradation pathways, ranging in size from cell-spanning gradients to nanoscale hotspots. Second, we describe the role played by intracellular membranes in creating unique signaling platforms with distinctive pathway regulation, as revealed through studies using subcellularly targeted fluorescent biosensors. Using biosensors to visualize subcellular extracellular response kinase (ERK) pathway activity, for example, led us to discover a local signaling circuit that mediates distinct plasma membrane ERK dynamics versus global ERK signaling. Similarly, our work developing biosensors to monitor the subcellular mechanistic target of rapamycin complex 1 (mTORC1) signaling allowed us to not only clarify the presence of mTORC1 activity in the nucleus but also identify a novel mechanism governing the activation of mTORC1 in this location. Finally, we detail how molecular assemblies enable the precise spatial tuning of biochemical activity, through investigations enabled by cutting-edge advances in biosensor design. We recently identified liquid-liquid phase separation as a major factor in cAMP compartmentation aided by a new strategy for targeting biosensors to endogenously expressed proteins via genome editing, for instance, and have also been able to directly visualize nanometer-scale protein kinase signalosomes using an entirely new class of biosensors specifically developed for the dynamic super-resolution imaging of live-cell biochemical activities. Our work provides key insights into the molecular logic of spatially regulated signaling and lays the foundation for a broader exploration of biochemical activity architectures across multiple spatial scales.

The Princes in 4QSongs of the Sabbath Sacrifice, Songs Six and Eight: Are They Angelic or Human?

The pervasive ambiguity in Songs of the Sabbath Sacrifice (SOSS) presents a major problem: When are the activities described in this text performed by angels and when by humans? This article builds on previous research on Songs One, Seven, Twelve, Thirteen, and knowledge in SOSS that hypothesized that as the liturgy proceeds through the thirteen songs of 4QSOSS a gradual transition takes place in the human participants. In Song Six the seven chief princes initially bless God and then proceed to bless “all councils in the sanctuary, and those who have knowledge of eternal things,” “those whose way is perfect,” “all who wait for Him … for a return of His gracious compassion.” The following song, significantly the middle of the liturgy, describes angelic messenger activity arising from the throne of God, based on Ezekiel’s merkebah chapters. After Song Seven, a dramatic change takes place. In Song Eight those who do the blessing are no longer “chief” princes, but “deputy” princes. The hypothesis of this article is that these deputy princes are the “people of discernment” introduced in Song One, who, having persevered in joint exaltation of God with the “holiest of holy ones,” are enabled to become “god-like” messengers of God, to convey God’s compassion to those “who wait for Him.”

How the biomimetic assembly of membrane receptors into multivalent domains is regulated by a small ligand.

In living cells, communication requires the action of membrane receptors that are activated following very small environmental changes. A binary all-or-nothing behavior follows, making the organism extremely efficient at responding to specific stimuli. Using a minimal system composed of lipid vesicles, chemical models of a membrane receptor and their ligands, we show that bio-mimetic ON/OFF assembly of high avidity, multivalent domains is triggered by small temperature changes. Moreover, the intensity of the ON signal at the onset of the switch is modulated by the presence of small, weakly binding divalent ligands, reminiscent of the action of primary messengers in biological systems. Based on the analysis of spectroscopic data, we develop a mathematical model that rigorously describes the temperature-dependent switching of the membrane receptor assembly and ligand binding. From this we derive an equation that predicts the intensity of the modulation of the ON signal by the ligand-messenger as a function of the pairwise binding parameters, the number of binding sites that it features and the concentration. The behavior of our system, and the model derived, highlight the usefulness of weakly binding ligands in the regulation of membrane receptors and the pitfalls inherent to their binding promiscuity, such as non-specific binding to the membrane. Our model, and the equations derived from it, offer a valuable tool for the study of membrane receptors in both biological and biomimetic settings. The latter can be exploited to program membrane receptor avidity on sensing vesicles, create hierarchical protocell tissues or develop highly specific drug delivery vehicles.

Transcriptional activation of urokinase by the Krüppel-like factor Zf9/COPEB activates latent TGF-β1 in vascular endothelial cells

Understanding the regulation of genes controlling fibrinolysis and matrix homeostasis is essential for elucidating the basis of tissue repair. A recently described novel Krüppel-like factor, Zf9, is up-regulated in acute liver injury in activated hepatic stellate cells. Because Zf9 can be induced widely, its activity was examined in vascular endothelium, a key cell in vascular injury. Zf9 is induced as an immediate-early response gene in bovine aortic endothelial cells (BAECs) following treatment with serum or phorbol ester. Zf9 transcriptionally activates urokinase plasminogen activator (uPA). Recombinant Zf9-GST binds to wild-type but not mutated 'GC-box' motifs within the human uPA promoter (-63 to -32), with greatest affinity to the middle of 3 contiguous GC boxes. Transient transfection of Zf9 drives transactivation of a full-length uPA promoter- and GC box-construct, but not a uPA promoter-construct devoid of GC boxes. Transactivation of uPA by Zf9 is also supported in Drosophila S2 cells. Most importantly, transiently transfected Zf9 up-regulates endogenous uPA messenger RNA and activity in BAECs, resulting in increased bioactive transforming growth factor-beta (TGF-beta) via enhancement of proteolytic activation of the latent molecule. Furthermore, concomitant expression of Zf9 and uPA proteins was observed in arterial endothelial cells after balloon injury in rats, suggesting a potential role of Zf9 in uPA expression not only in vitro but also in vivo. These findings suggest a role of Zf9 in the injury response by enhancing uPA synthesis and subsequent activation of latent TGF-beta. (Blood. 2000;95:1309-1316)

Abstract A20: Gαq controls the Hippo pathway through MOB1 tyrosine phosphorylation by FAK

Abstract YAP overactivation is an essential molecular event for cancer initiation and growth of most solid tumors, but pharmacologic targeting of the YAP or Hippo pathway has been proven to be challenging. In this regard, YAP activity is also required for stem and progenitor cell maintenance and function in multiple tissues, and as such, YAP is necessary for normal tissue homeostasis and regeneration. Thus, targeting YAP directly may result in normal cell growth arrest and tissue damage. The elucidation of the signaling networks sustaining YAP pervasive activation in each cancer type may afford the opportunity to identify novel suitable precision therapeutic targets for cancer treatment. GNAQ and GNA11 activate mutations (encoding GTPase deficient and constitutively active Gαq proteins) occurs in ~90% of uveal melanoma (UM) and 4% of skin cutaneous melanoma (SKCM), respectively, firmly established UM, and a subset of SKCM as Gαq-driven human malignancies. Approximately 50% of UM patients develop liver metastasis within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. To date, there are no effective treatment options for metastatic UM disease. Through the bioinformatics analysis of the TCGA UM dataset (n=80) using skin melanoma samples that do not harbor Gαq genomic alteration (n=209) as control, (1) we first identified the genes that are highly overexpressed in UM. (2) We then filtered the genes for those whose reduced expression leads to better patient survival in UM. (3) We finally used large datasets of gene essentiality and drug response screenings in cancer cells to identify genes that are predicted to reduce cell viability when targeted in Gαq-gain cells. (4) Finally, 7 top predicted drug targets were found from a set of genes that can be targeted by approved and experimental drugs (n=756). The top predicted gene target was PTK2, which encodes the nonreceptor tyrosine kinase FAK (focal adhesion kinase). We found that Gαq activates pY379-FAK (reflecting FAK activation) though a Trio-RhoA signaling pathway, independent of PLC-β regulated second messenger activation. Interestingly, genome-wide transcriptional analysis (RNA-seq) of UM cells treated with a FAK inhibitor (VS-4718) revealed that YAP signatures were highly inhibited by FAK blockade. This suggested that FAK signaling actives YAP downstream of Gαq in UM, in addition to our previously found Trio/Rho GTPase/F-actin polymerization regulation of YAP activation mechanism. Further signaling analysis uncovered that FAK stimulates YAP through direct YAP tyrosine phosphorylation (enhancing YAP protein stability) and, unexpectedly, MOB1 tyrosine phosphorylation on Y26. The latter disrupts MOB1/LATS interaction, causing Hippo signaling inhibition and YAP activation. Interestingly, FAK inhibitors are already under clinical evaluation, and showed strong inhibition of YAP activity and UM growth in vitro and in vivo, thus representing a potential precision therapeutic option for UM treatment. Citation Format: Xiaodong Feng, Nadia Arang, Damiano Cosimo Rigiracciolo, Joo Sang Lee, Huwate Yeerna, Zhiyong Wang, Simone Lubrano, Ayush Kishore, Jonathan A. Pachter, Gabriele M. König, Marcello Maggiolini, Evi Kostenis, David D. Schlaepfer, Pablo Tamayo, Qianming Chen, Eytan Ruppin, J. Silvio Gutkind. Gαq controls the Hippo pathway through MOB1 tyrosine phosphorylation by FAK [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A20.

Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels.

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

Cocaine-induced changes in behaviour and DNA methylation in rats are influenced by inter-individual differences in spontaneous exploratory activity.

Individual differences in behavioural traits influence susceptibility to addictive disorders. Drug addiction involves changes in gene expression, proposed to occur via DNA methylation (DNAm). To investigate DNAm changes in reward-related brain structures (nucleus accumbens (NAc), lateral habenula (LHb)) in response to cocaine exposure in rats differing in spontaneous exploratory activity. Rats were observed in the exploration box and categorised as high- (HE) or low explorers (LE). Rats were administered vehicle or cocaine (12 mg/kg, i.p.) for 7 days, followed by a 14-day withdrawal period and cocaine challenge (7 mg/kg); horizontal locomotor activity was recorded. Brain tissue was dissected after 24 h; we analysed messenger RNA (mRNA) and activity levels of epigenetic DNA modifiers (DNMTs and TETs) as well as mRNA and promoter methylation levels at selected genes previously linked to addictive behaviours. The cocaine challenge dose stimulated locomotor activity in both LE- and HE rats only when administered after a repeated cocaine schedule, suggesting development of behavioural sensitisation. Quantitative polymerase chain reaction analyses demonstrated higher basal expression of Dnmt3a, Tet2 and Tet3 in the LHb of HE- vs. LE rats, and we observed differential effects of cocaine exposure on the expression and activity of epigenetic DNA modifiers in the NAc and LHb of HE- and LE rats. Furthermore, cocaine exposure differentially altered promoter methylation levels of A2AR, Ppp1cc, and Taar7b in the NAc and LHb of HE- and LE rats. DNAm might play a role in the HE- and LE phenotypes as well as mediate behavioural effects of LE- and HE rats in response to drugs of abuse.

Myristoylated Protein Kinase C Beta II Peptide Inhibitor Improves Cell Survival in Cultured Human Umbilical Vein Endothelial Cells Subjected to Hypoxia/Reoxygenation

Endothelial dysfunction as a result of ischemia/reperfusion (I/R) injury is known to contribute to damage in myocardial infarction and organ transplant patients. In vascular cells, NADPH oxidase (NOX) and the mitochondrial electron transport chain are primary sources of reactive oxygen species (ROS) during I/R injury resulting in reduced nitric oxide bioavailability. Protein kinase C beta II (PKCβII) is an attractive therapeutic target due to its regulation of these downstream mediators of ROS production. PKCβII phosphorylates p66Shc to enhance mitochondrial‐derived ROS production and p47 phox to promote ROS release from NOX. We have previously shown that myristoylated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) improved post‐reperfusion cardiac function and reduced infarct size in rat myocardial I/R injury compared to non‐treated controls. The decrease in myocardial I/R injury with myr‐PKCβII‐treatment may be attributed to improved vascular endothelial function. The myristoylated peptide sequence (cargo sequence) targets a highly conserved peptide sequence among mammalian species and inhibits PKCβII translocation to protein substrates (e.g. NOX and p66Shc) after second messenger activation. We hypothesize that myr‐PKCβII‐ will confer protection by directly inhibiting ROS production from NOX and mitochondria in human umbilical vein endothelial cells (HUVECs) subjected to hypoxia/reoxygenation (H/R) injury. HUVECs were cultured in gelatin‐coated 96‐well plates and subjected to 24h hypoxia and 24h reoxygenation in Billups‐Rothenburg hypoxia chamber with 1% O2, 5% CO2, and balanced nitrogen. Myr‐PKCβII‐ (20 mM) treatment was administered at the beginning of the 24h reoxygenation period. Cell viability was assessed using tetrazolium‐salt (WST‐8) colorimetric assay with a microplate reader (450 nm) and normalized against the normoxia control group. Data were analyzed using Student‐Newman‐Keuls post‐hoc analysis. At the end of the 24h reoxygenation period, cell viability (%) was significantly reduced to 78±2% (p<0.05; n=5) in the non‐treated H/R group compared to normoxia controls (n=5). Myr‐PKCβII‐significantly improved HUVEC survival (95±4%; p<0.01; n=5) compared to non‐treated H/R controls and was not significantly changed compared to normoxia controls. The data suggests that PKCβII inhibition promotes cell survival subjected to H/R possibly due to directly attenuating NOX and mitochondrial derived ROS. Further studies are needed to control for the myristoylated acid conjugation of the peptide cargo.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at the Philadelphia College of Osteopathic Medicine. Current license is supported by Young Therapeutics, LLC.