Extracellular Signaling Molecules Research Articles

Characterizing purinergic receptors in the gastrointestinal epithelium and immune cells

Background: Extracellular release of nucleotides (e.g., ATP, ADP, UTP, UDP) are well established as potent intercellular signaling molecules during homeostasis but are also released from cells during pathogenic microbial infection and tissue injury to serve as danger signals. These important extracellular signaling molecules activate purinergic receptors, of which there are 19 different purinergic receptor subtypes that are classified into P1 and P2 receptors. P1 receptor subtypes are activated by adenosine, while P2 receptors are differentially activated by ATP, ADP, UTP, UDP, and UP-glucose. P2 receptors are further divided into P2Y receptors (P2YRs) and P2X receptors (P2XRs) based on their structures and distinct signal-transduction mechanisms. Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, yet the majority of work to date has focused on purinergic signaling in immune cells. Our group has previously identified a role for P2Y1 in mediating ADP-driven calcium waves during rotavirus infection. While purinergic signaling is a well-established mode of signaling in the enteric nervous system, the functional roles of purinergic signaling in the gastrointestinal tract remain incompletely understood. Hypothesis: Since epithelial cells serve as the first barrier against irritants and infection, we hypothesized that gut epithelium express multiple purinergic receptors that regulate cellular responses to extracellular nucleotide signals. Methods & Results: Using the Human Protein Atlas, we queried single cell RNA sequencing data for the P2 purinergic receptors. We examined these receptors along the length of the gastrointestinal tract, including esophagus, small intestine, colon, and rectum as well as in flow-sorted immune cells, peripheral blood mononuclear cells and and bone marrow derived immune cells. In silico analysis revealed high expression of P2Y1, P2Y2, P2Y11 and P2X4 among the purinergic receptors throughout the gastrointestinal tract. Interestingly, P2Y1 showed highest expression in esophageal epithelial cells, small intestinal enterocytes, and goblet cells. P2Y6 was also observed in promixal enterocytes of the small intestine, but was largely absent from the colon and rectum. In contrast, we found that immune cells from bone marrow, peripheral blood and flow sorted cells had relatively high expression of P2X1 and P2X4. We noted P2X5 expression in plasma cells and B -cells in all sets. Pathway analysis from the Reactome database revealed P2Y receptors are associated with signal transduction, metabolism and hemostasis pathways, while P2X receptors were primarily involved in immune response and infectious disease pathways. Conclusions: These findings confirm that the gastrointestinal tract possess robust P2Y1, P2Y2, P2Y11 and P2X4 receptor expression and can participate in purinergic signaling pathways. R01 DK115507 (Hyser); R01 AI158683 (Hyser); APS Postdoctoral Fellowship (Engevik); F32 DK130288 (Engevik) This is the full abstract presented at the American Physiology Summit 2023 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.

Extracellular HSPA5 is autocrinally involved in the regulation of neuronal process elongation

The molecular mechanisms by which neuronal processes grow are extremely complicated, involving fine-tuned regulation of extracellular and intracellular signals. It remains to be elucidated which molecules are contained in the regulation. Herein, we report for the first time that heat shock protein family A member 5 (HSPA5, also called immunoglobulin heavy chain binding endoplasmic reticulum [ER] protein [BiP]) is secreted from mouse primary dorsal neuronal ganglion (DRG) cells or neuronal cell line N1E-115, a frequently used neuronal differentiation model. Supporting these results, HSPA5 protein was co-localized not only with ER antigen KDEL but also with intracellular vesicles such as Rab11-positive secretory vesicles. Unexpectedly, addition of HSPA5 inhibited elongation of neuronal processes, whereas neutralization of extracellular HSPA5 with the antibodies elongated processes, characterizing extracellular HSPA5 as a negative regulator of neuronal differentiation. Treatment of cells with neutralizing antibodies for low-density lipoprotein receptor (LDLR) did not have significant effects on process elongation, whereas LDLR-related protein 1 (LRP1) antibodies promoted differentiation, implying that LRP1 may act as a receptor candidate for HSPA5. Interestingly, extracellular HSPA5 was greatly decreased following treatment with tunicamycin, an ER stress inducer, illustrating that the ability to form neuronal processes could be preserved, even under stress. These results suggest that neuronal HSPA5 itself is secreted to contribute to inhibitory effects on neuronal cell morphological differentiation and can be included on the list of extracellular signaling molecules negatively controlling differentiation.

Purinergic signaling and purine base metabolism at the crossroads between immunity, metabolism, and cancer: A review

AbstractIn addition to its universally known role in transferring genetic material, DNA nucleotides and nucleosides are regarded as the most ancient form of extracellular signaling molecules. This unique signaling pathway was first reported by Dr. Albert Szent‐Györgyi in 1937 and established by Dr. Geoffrey Burnstock in 1972, who coined the term “purinergic signaling.” The significance of purinergic signaling is now recognized in various cellular processes, including immune responses. With an increased understanding of how changes in immunity impact cancer progression, the groundbreaking successes of immunotherapies, and emerging challenges facing patients receiving these treatments, in this review we revisit the history of purinergic signaling, provide a comprehensive summary of its impact on immune cells, and discuss the therapeutic potential of targeting this pathway for cancer treatment.

The Wheel of p53 Helps to Drive the Immune System.

The p53 tumor suppressor protein is best known as an inhibitor of the cell cycle and an inducer of apoptosis. Unexpectedly, these functions of p53 are not required for its tumor suppressive activity in animal models. High-throughput transcriptomic investigations as well as individual studies have demonstrated that p53 stimulates expression of many genes involved in immunity. Probably to interfere with its immunostimulatory role, many viruses code for proteins that inactivate p53. Judging by the activities of immunity-related p53-regulated genes it can be concluded that p53 is involved in detection of danger signals, inflammasome formation and activation, antigen presentation, activation of natural killer cells and other effectors of immunity, stimulation of interferon production, direct inhibition of virus replication, secretion of extracellular signaling molecules, production of antibacterial proteins, negative feedback loops in immunity-related signaling pathways, and immunologic tolerance. Many of these p53 functions have barely been studied and require further, more detailed investigations. Some of them appear to be cell-type specific. The results of transcriptomic studies have generated many new hypotheses on the mechanisms utilized by p53 to impact on the immune system. In the future, these mechanisms may be harnessed to fight cancer and infectious diseases.

The arabinose transporter MtLat-1 is involved in hemicellulase repression as a pentose transceptor in Myceliophthora thermophila

BackgroundFilamentous fungi possess an array of secreted enzymes to depolymerize the structural polysaccharide components of plant biomass. Sugar transporters play an essential role in nutrient uptake and sensing of extracellular signal molecules to inhibit or trigger the induction of lignocellulolytic enzymes. However, the identities and functions of transceptors associated with the induction of hemicellulase genes remain elusive.ResultsIn this study, we reveal that the l-arabinose transporter MtLat-1 is associated with repression of hemicellulase gene expression in the filamentous fungus Myceliophthora thermophila. The absence of Mtlat-1 caused a decrease in l-arabinose uptake and consumption rates. However, mycelium growth, protein production, and hemicellulolytic activities were markedly increased in a ΔMtlat-1 mutant compared with the wild-type (WT) when grown on arabinan. Comparative transcriptomic analysis showed a different expression profile in the ΔMtlat-1 strain from that in the WT in response to arabinan, and demonstrated that MtLat-1 was involved in the repression of the main hemicellulase-encoding genes. A point mutation that abolished the l-arabinose transport activity of MtLat-1 did not impact the repression of hemicellulase gene expression when the mutant protein was expressed in the ΔMtlat-1 strain. Thus, the involvement of MtLat-1 in the expression of hemicellulase genes is independent of its transport activity. The data suggested that MtLat-1 is a transceptor that senses and transduces the molecular signal, resulting in downstream repression of hemicellulolytic gene expression. MtAra-1 protein directly regulated the expression of Mtlat-1 by binding to its promoter region. Transcriptomic profiling indicated that the transcription factor MtAra-1 also plays an important role in expression of arabinanolytic enzyme genes and l-arabinose catabolism.ConclusionsM. thermophila MtLat-1 functions as a transceptor that is involved in l-arabinose transport and signal transduction associated with suppression of the expression of hemicellulolytic enzyme-encoding genes. The data presented in this study add to the models of the regulation of hemicellulases in filamentous fungi.

Oleic acid stimulates cell proliferation and BRD4–L-MYC-dependent glucose transporter transcription through PPARα activation in ovarian cancer cells

Fatty acids (FAs) play important roles in cell membrane structure maintenance, energy production via β-oxidation, and as extracellular signaling molecules. Prior studies have demonstrated that exposure of cancer cells to FAs affects cell survival, cell proliferation, and cell motility. Oleic acid (OA) has somewhat controversial effects in cancer cells, with both pro- and anti-cancer effects, depending on cell type. Our prior findings suggested that OA enhances cell survival in serum starved HNOA ovarian cancer cells by activating glycolysis, but not β-oxidation. Here, we pharmacologically examined the cellular mechanisms by which OA stimulates glycolysis in HNOA cells. OA induced cell cycle progression, leading to increase in cell number through peroxisome proliferator activated receptor (PPAR) α activation. OA-induced glycolysis was mediated by increased GLUT expression, and increases in GLUT expression were mediated by increased L-MYC expression. Furthermore, L-MYC expression was due to BRD4 activation. These findings suggested involvement of the BRD4–L-MYC–GLUT axis in OA-stimulated glycolysis. These results suggested that OA could activate PPARα to stimulate two pathways: glycolysis and cell cycle progression, and provided insight into the role of OA in ovarian cancer cell growth.

Genome-Wide Investigation and Co-Expression Network Analysis of SBT Family Gene in Gossypium.

Subtilases (SBTs), which belong to the serine peptidases, control plant development by regulating cell wall properties and the activity of extracellular signaling molecules, and affect all stages of the life cycle, such as seed development and germination, and responses to biotic and abiotic environments. In this study, 146 Gossypium hirsutum, 138 Gossypium barbadense, 89 Gossypium arboreum and 84 Gossypium raimondii SBTs were identified and divided into six subfamilies. Cotton SBTs are unevenly distributed on chromosomes. Synteny analysis showed that the members of SBT1 and SBT4 were expanded in cotton compared to Arabidopsis thaliana. Co-expression network analysis showed that six Gossypium arboreum SBT gene family members were in a network, among which five SBT1 genes and their Gossypium hirsutum and Arabidopsis thaliana direct homologues were down-regulated by salt treatment, indicating that the co-expression network might share conserved functions. Through co-expression network and annotation analysis, these SBTs may be involved in the biological processes of auxin transport, ABA signal transduction, cell wall repair and root tissue development. In summary, this study provides valuable information for the study of SBT genes in cotton and excavates SBT genes in response to salt stress, which provides ideas for cotton breeding for salinity resistance.

Abstract P2-23-19: Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform

Abstract Tumors are multicellular ecosystems that communicate through the exchange of extracellular signaling molecules. In breast cancer, this multicellular communication can enable tumor cells to cooperate in a range of contexts, including tumor invasion and the outgrowth of distal metastases. Illuminating the mechanisms by which tumor cells communicate may reveal new therapies. To this end, three-dimensional tumor organoid models have emerged as versatile platforms for modeling multicellular behavior ex vivo. However, organoid culture typically requires the use of poorly defined, animal-derived extracellular matrices, such as Matrigel. These exogenous matrices contain thousands of proteins that dominate over and conceal cell-secreted factors in conventional proteomics approaches. Revealing low abundance cell-secreted factors from this complex exogenous background presents a formidable challenge. To surmount this challenge, we develop a new method to isolate the pericellular proteome in 3D organotypic culture models. This technology harnesses biorthogonal click chemistry to bypass exogenous factors, infiltrate intercellular spaces, and retrieve the endogenous intercellular proteome. This approach requires no genetic manipulation, requires only 1 million cells, and can be adapted to diverse organotypic models. These capabilities enable isolation of intercellular signaling factors in models that yield precious amounts of material and that can sustain only limited manipulation ex vivo, which we demonstrate using organoids established from patient-derived xenografts. To establish the generalizability of our approach, we apply this technology to a panel of breast cancer and small-cell lung cancer organoid models. Furthermore, we demonstrate that this method can be readily adapted to different extracellular matrix environments that are widely employed in organoid research, including basement membrane matrices and collagen gels. Taken together, our results establish this technology as a scalable and generalizable platform that may open opportunities for researchers investigating diverse questions in organoid models of cancer. Moving forward, we are leveraging these capabilities to investigate changes in the intercellular proteome that emerge after the acquisition of therapy resistance. We hope to uncover new mechanisms of collective signaling that may be targeted to overcome therapy resistance. Citation Format: Brad A. Krajina, Ami Yamamoto, Kevin J. Cheung, Samuel Madasu. Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-19.

Stochastic effects in bacterial communication mediated by extracellular vesicles.

Quorum sensing (QS) allows bacterial cells to sense changes in local cell density and, hence, to regulate multicellular processes, including biofilm formation, regulation of virulence, and horizontal gene transfer. While, traditionally, QS was thought to involve the exchange of extracellular signal molecules free in solution, recent experiments have shown that for some bacterial systems a substantial fraction of signal molecules are packaged and delivered in extracellular vesicles. How the packaging of signal molecules in extracellular vesicles influences the ability of cells to communicate and coordinate multicellular behaviors remains largely unknown. We present here a stochastic reaction-diffusion model of QS that accounts for the exchange of both freely diffusing and vesicle-associated signal molecules. We find that the delivery of signal molecules via extracellular vesicles amplifies local fluctuations in the signal concentration, which can strongly affect the dynamics and spatial range of bacterial communication. For systems with multiple bacterial colonies, extracellular vesicles provide an alternate pathway for signal transport between colonies, and may be crucial for long-distance signal exchange in environments with strong degradation of free signal molecules.

Role of senescent tumor cells in building a cytokine shield in the tumor microenvironment: mathematical modeling.

Cellular senescence can induce dual effects (promotion or inhibition) on cancer progression. While immune cells naturally respond and migrate toward various chemotactic sources from the tumor mass, various factors including senescent tumor cells (STCs) in the tumor microenvironment may affect this chemotactic movement. In this work, we investigate the mutual interactions between the tumor cells and the immune cells that either inhibit or facilitate tumor growth by developing a mathematical model that consists of taxis-reaction-diffusion equations and receptor kinetics for the key players in the interaction network. We apply a mathematical model to a transwell Boyden chamber invasion assay used in the experiments to illustrate that STCs can play a pivotal role in negating immune attack through tight regulation of intra- and extra-cellular signaling molecules. In particular, we show that senescent tumor cells in cell cycle arrest can block intratumoral infiltration of CD8+ T cells by secreting a high level of CXCL12, which leads to significant reduction its receptors, CXCR4, on T cells, and thus impaired chemotaxis. The predictions of nonlinear responses to CXCL12 were in good agreement with experimental data. We tested several hypotheses on immune-tumor interactions under various biochemical conditions in the tumor microenvironment and developed new concepts for anti-tumor strategies targeting senescence induced immune impairment.

P2-type purinergic signaling in the regulation of pancreatic β-cell functional plasticity as a promising novel therapeutic approach for the treatment of type 2 diabetes?

Diabetes Mellitus is a metabolic disorder characterized by a chronic hyperglycemia due to an impaired insulin secretion and a decreased in peripheral insulin sensitivity. This disease is a major public health problem due to it sharp prevalence. Therefore, it is crucial to readapt therapeutic approaches for the treatment of this pathology. One of the strategies would be through P2-type purinergic receptors pathway via ATP binding. In addition to its well-known role as an intracellular energy intermediary in numerous biochemical and physiological processes, ATP is also an important extracellular signaling molecule. ATP mediates its effects by binding and activating two classes of P2 purinoreceptors: P2X receptors that are ligand-gated ion channel receptors, existing in seven isoforms (P2X 1 to 7) and P2Y receptors that are G-protein coupled receptors, existing in eight isoforms (P2Y 1/2/4/6/11/12/13/14). These receptors are ubiquitously distributed and involved in numerous physiological processes in several tissues. The concept of purinergic signaling, originally formulated by Geoffrey Burnstock (1929-2020), was also found to mediate various responses in the pancreas. Several studies have shown that P2 receptors are expressed in the endocrine pancreas, notably in β cells, where ATP could modulate their function but also their plasticity and thus play a physiological role in stimulating insulin secretion to face some metabolic demands. In this review, we provide a historical perspective and summarize current knowledge on P2-type purinergic signaling in the regulation of pancreatic β-cell functional plasticity, which would be a promising novel therapeutic approach for the treatment of type 2 diabetes.

Proteins secreted by brain arteriolar smooth muscle cells are instructive for neural development

Intercellular communication between vascular and nerve cells mediated by diffusible proteins has recently emerged as a critical intrinsic program for neural development. However, whether the vascular smooth muscle cell (VSMC) secretome regulates the connectivity of neural circuits remains unknown. Here, we show that conditioned medium from brain VSMC cultures enhances multiple neuronal functions, such as neuritogenesis, neuronal maturation, and survival, thereby improving circuit connectivity. However, protein denaturation by heating compromised these effects. Combined omics analyses of donor VSMC secretomes and recipient neuron transcriptomes revealed that overlapping pathways of extracellular matrix receptor signaling and adhesion molecule integrin binding mediate VSMC-dependent neuronal development. Furthermore, we found that human arterial VSMCs promote neuronal development in multiple ways, including expanding the time window for nascent neurite initiation, increasing neuronal density, and promoting synchronized firing, whereas human umbilical vein VSMCs lack this capability. These in vitro data indicate that brain arteriolar VSMCs may carry direct instructive information for neural development through intercellular communication in vivo.

Quorum Sensing Based Drug Screening Against Vibrio Cholerae

The QS method is a means of bacterial cell-to-cell communication, which uses extracellular signal molecules called autoinducers to transmit information between cells.Bacteria can use QS to collaborate on tasks. The pathogen Vibrio cholerae uses QS to inhibit the development of virulence factors and the formation of biofilms. Cholera is caused by the Gram-negative, curved bacteria Vibrio cholerae (Clemens et al., 2017). There are also a number of virulence components produced by this disease, including cholera hemolysin (CH), toxin-co-regulated pilus (TCP), flagellum, etc. By constraining the target protein, HapR, with adequate bioactive compounds, the pathogenic activity of in vibrio cholerae can be suppressed.Bioactive substances from various natural food sources were chosen and analysed for their quorum quenching effect against HapR protein utilising bioinformatics methods. The in-silico analysis produced notable results for thirteen of the 25 substances evaluated, with the best docking score. These chemicals could be employed for QSI-based therapeutics against vibrio cholerae infections and could be suggested for in vitro and in vivo investigations.

Microbial Communication via Pyrazine Signaling: A New Class of Signaling Molecules Identified in Vibrio cholerae

AbstractQuorum sensing is a microbial signaling process that relies on the production, release, and response to extracellular signaling molecules called autoinducers. This Review covers quorum‐sensing systems controlled by a pyrazine‐type autoinducer, 3,5‐dimethyl‐pyrazin‐2‐ol (DPO). The systems we cover span the human pathogen Vibrio cholerae, where DPO regulates pathogenicity and biofilm formation, and phage VP882, where DPO controls lytic development and viral dissemination. In both cases, detection of DPO occurs via a dedicated receptor‐transcription factor called VqmA. In V. cholerae, DPO activates the production of the VqmR regulatory RNA, and in the phage, DPO triggers production of the small protein Qtip. We present our current understanding of DPO biosynthesis, ongoing efforts to resolve the mechanisms underlying DPO‐mediated signal transduction, and a forward‐facing perspective on the role of DPO in human health and undiscovered pyrazine‐based signals in biology, more broadly.

Addressing the Enzyme-independent tumor-promoting function of NAMPT via PROTAC-mediated degradation

Aberrant overexpression of nicotinamide phosphoribosyltransferase (NAMPT) has been reported in a variety of tumor cells and is a poor prognosis factor for patient survival. It plays an important role in tumor cell proliferation, acting concurrently as an nicotinamide adenine dinucleotide (NAD+) synthase and, unexpectedly, as an extracellular signaling molecule for several tumor-promoting pathways. Although previous efforts to modulate NAMPT activity were limited to enzymatic inhibitors with low success in clinical studies, protein degradation offers the possibility to simultaneously disrupt NAMPT's enzyme activity and ligand capabilities. Here we report the development of two highly selective proteolysis-targeting chimeras (PROTACs) that promote NAMPT degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a clinical candidate, FK866, in killing effect on hematological tumor cells. These results emphasize the importance and feasibility of applying PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions like NAMPT, which is not easily achieved by conventional enzymatic inhibitors.

Protocol: a simple method for biosensor visualization of bacterial quorum sensing and quorum quenching interaction on Medicago roots

BackgroundDefining interactions of bacteria in the rhizosphere (encompassing the area near and on the plant root) is important to understand how they affect plant health. Some rhizosphere bacteria, including plant growth promoting rhizobacteria (PGPR) engage in the intraspecies communication known as quorum sensing (QS). Many species of Gram-negative bacteria use extracellular autoinducer signal molecules called N-acyl homoserine lactones (AHLs) for QS. Other rhizobacteria species, including PGPRs, can interfere with or disrupt QS through quorum quenching (QQ). Current AHL biosensor assays used for screening and identifying QS and QQ bacteria interactions fail to account for the role of the plant root.MethodsMedicago spp. seedlings germinated on Lullien agar were transferred to soft-agar plates containing the broad-range AHL biosensor Agrobacterium tumefaciens KYC55 and X-gal substrate. Cultures of QS and QQ bacteria as well as pure AHLs and a QQ enzyme were applied to the plant roots and incubated for 3 days.ResultsWe show that this expanded use of an AHL biosensor successfully allowed for visualization of QS/QQ interactions localized at the plant root. KYC55 detected pure AHLs as well as AHLs from live bacteria cultures grown directly on the media. We also showed clear detection of QQ interactions occurring in the presence of the plant root.ConclusionsOur novel tri-trophic system using an AHL biosensor is useful to study QS interspecies interactions in the rhizosphere.

A 2A ADENOSINE RECEPTORS REGULATE MULTIPLE ORGAN FAILURE AFTER HEMORRHAGIC SHOCK IN MICE.

Trauma hemorrhagic shock (T/HS) is a clinical condition that causes multiple organ failure that needs rapid intervention. Restricted oxygen at the cellular level causes inflammation and subsequent cell death. Adenosine triphosphate is the universal intracellular energy currency and an important extracellular inflammatory signaling molecule. Adenosine, an endogenous nucleotide formed as a result of the breakdown of adenosine triphosphate, is also released during T/HS. Adenosine binds to four G protein-coupled receptors (A 1R , A 2a , A 2b , A 3R ) called adenosine receptors or P1 receptors. In the present study, we evaluated the effect of activation, inactivation, and genetic absence of A2aR (A2aR -/- mice) on T/HS-induced multiple organ failure. Wild-type mice were pretreated (30 min before shock induction) with an agonist or antagonist and then subjected to T/HS by withdrawing arterial blood and maintaining the blood pressure between 28 and 32 mm Hg. A2aR -/- mice were subjected to T/HS in the absence of pharmacologic treatment. Neutrophil sequestration was assessed by detecting myeloperoxidase, and Evans blue dye (EBD) method was used to analyze lung permeability. Blood and lung inflammatory cytokine levels were determined by sandwich enzyme-linked immunosorbent assay. The liver enzymes aspartate aminotransferase and alanine aminotransferase were determined spectrophotometrically from plasma. Activation of the apoptotic cascade was evaluated using a mouse apoptosis array. Our results demonstrate that the selective A2aR agonist CGS21680 decreases lung neutrophil sequestration, lung proinflammatory cytokines IL-6 and TNF-α, and bronchoalveolar lavage EBD. Pretreatment with the selective antagonist ZM241385 and genetic blockade in A2aR -/- mice increased neutrophil sequestration, proinflammatory cytokine levels, and bronchoalveolar lavage fluid EBD. The myeloperoxidase level in the lung was also increased in A2aR -/- mice. We observed that antiapoptotic markers decreased significantly with the absence of A2aR in the lung and spleen after T/HS. In conclusion, our data demonstrate that activation of A2aR regulates organ injury and apoptosis in the setting of T/HS.

Tyrosine hydroxylase gene promoter activity is upregulated in female catecholaminergic neuroblastoma cells following activation of a Gαq-coupled designer receptor

Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis that catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine. The tyrosine hydroxylase gene is regulated by extracellular signaling molecules such as epidermal growth factor, nerve growth factor and steroids. Here, we investigated whether the activity of the tyrosine hydroxylase gene promoter is upregulated by activation of G protein-coupled receptors, the largest group of plasma membrane receptors. We used catecholaminergic neuroblastoma cells as a cellular model and chromatin-integrated tyrosine hydroxylase promoter-luciferase reporter genes. The results show that stimulation of Rαq, a Gαq-coupled designer receptor, triggered transcription of a reporter gene driven by the tyrosine hydroxylase promoter. Transcription was attenuated by overexpression of regulator of G-protein signaling-2, which activates the GTPase activity of the G protein α-subunit, and by a truncated, dominant-negative mutant of phospholipase Cβ3. Extracellular signal-regulated protein kinase was identified as the signal transducer. At the transcriptional level, tyrosine hydroxylase promoter activity was found to be controlled by the transcription factor CREB. Expression experiments with the adenoviral regulator protein E1A, an inhibitor of CBP/p300 histone acetyltransferases, showed that transcription of the reporter gene controlled by the tyrosine hydroxylase is under epigenetic control. We identified the protein phosphatases MAP kinase phosphatase-1 and calcineurin as part of a shutdown device of the signaling cascade linking Rαq designer receptor activation to tyrosine hydroxylase gene transcription. We conclude that tyrosine hydroxylase promoter activity is controlled by Gαq-coupled receptors.

Exploring the mechanism of berberine-mediated Tfh cell immunosuppression

BackgroundOur previous research revealed a novel function of berberine (BBR), a clinically relevant plant-derived alkaloid, as a suppressor of follicular T helper (Tfh) cell proliferation in secondary lymphoid organs of BBR-treated mice that underwent immunization for collagen-induced arthritis (CIA) in DBA1/J mice. Due to the importance of Tfh cell and B cell interactions in the generation of T cell-dependent humoral responses, the suppression of Tfh cell activity may have implications for the general safety of BBR as a prophylactic dietary supplement, and its potential use in antibody-driven autoimmune and hypersensitivity disorders. PurposeThis research aims to characterize BBR's impact on the activation, differentiation, and proliferation of Tfh cells by examining the expression of key extracellular signaling molecules, as well as the activity of intracellular signaling molecules involved in the Ca2+-calcineurin-NFAT pathway and STAT3 phosphorylation, following activation. Study designIn vitro experimental study using primary tissues. MethodsTo explore the direct effects of BBR on the proliferation and differentiation of Tfh cells, isolated naïve CD4+ T cells (>95% pure) were activated and differentiated into pre- Tfh cells in the presence or absence of BBR. The resulting Tfh cell populations and the expression of the key extracellular molecules CXCR5, ICOS, and PD-1 were measured. In addition, we examined the impact of BBR treatment on the activity of key intracellular signaling molecules involved in Tfh cell activation and differentiation following TCR ligation and/or CD28 signaling (p-ZAP-70, p-Lck, p-PLCγ1, NFATc1 and intracellular calcium, Ca2+, concentrations), as well as IL-6 signaling (p-STAT3). ResultsTreatment with BBR significantly reduced the expression of both CXCR5 (p < 0.01) and ICOS (p < 0.005), but not PD-1, and reduced the percentage of Tfh cells within the total CD4+ T cell population. BBR treatment also led to a reduction in intracellular Ca2+ flux, activation of p-STAT3, and IL-21 production. ConclusionOur observations provide insight into the mechanism of BBR-mediated Tfh cell suppression and suggest that BBR treatment can directly inhibit Tfh cell activity, perhaps through interfering with cytokine receptor or downstream signaling.

Nutritional and metabolic signalling through GPCRs.

Deregulated metabolism is a well-known feature of several challenging diseases, including diabetes, obesity and cancer. Besides their important role as intracellular bioenergetic molecules, dietary nutrients and metabolic intermediates are released in the extracellular environment. As such, they may achieve unconventional roles as hormone-like molecules by activating cell surface G-protein-coupled receptors (GPCRs) that regulate several pathophysiological processes. In this review, we provide an insight into the role of lactate, succinate, fatty acids, amino acids as well as ketogenesis-derived and β-oxidation-derived intermediates as extracellular signalling molecules. Moreover, the mechanisms by which their cognate metabolite-sensing GPCRs integrate nutritional and metabolic signals with specific intracellular pathways will be described. A better comprehension of these aspects is of fundamental importance to identify GPCRs as novel druggable targets.