Increase In Intracellular cAMP Research Articles

Evidence for the Type IV Pilus Retraction Motor PilT as a Component of the Surface Sensing System in Pseudomonas aeruginosa.

Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3',5'-cyclic AMP (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the type IV pilus retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations in PilT, and in particular those impacting the ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its PilT retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current T4P-dependent surface sensing models for P. aeruginosa. IMPORTANCE T4P are cellular appendages that allow P. aeruginosa to sense a surface, leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.

OR2AT4 and OR1A2 counterregulate molecular pathophysiological processes of steroid-resistant inflammatory lung diseases in human alveolar macrophages

BackgroundTherapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are lacking. Alveolar macrophages are central in the progression of these diseases by releasing proinflammatory cytokines, making them promising targets for new therapeutic approaches. Extra nasal expressed olfactory receptors (ORs) mediate various cellular processes, but clinical data are lacking. This work investigates whether ORs in human primary alveolar macrophages could impact pathophysiological processes and could be considered as therapeutic targets.MethodsHuman primary alveolar macrophages were isolated from bronchoalveolar lavages of 50 patients with pulmonary diseases. The expression of ORs was validated using RT-PCR, immunocytochemical staining, and Western blot. Changes in intracellular calcium levels were analyzed in real-time by calcium imaging. A luminescent assay was used to measure the cAMP concentration after OR stimulation. Cytokine secretion was measured in cell supernatants 24 h after stimulation by ELISA. Phagocytic ability was measured by the uptake of fluorescent-labeled beads by flow cytometry.ResultsWe demonstrated the expression of functional OR2AT4 and OR1A2 on mRNA and protein levels. Both ORs were primarily located in the plasma membrane. Stimulation with Sandalore, the ligand of OR2AT4, and Citronellal, the ligand of OR1A2, triggered a transient increase of intracellular calcium and cAMP. In the case of Sandalore, this calcium increase was based on a cAMP-dependent signaling pathway. Stimulation of alveolar macrophages with Sandalore and Citronellal reduced phagocytic capacity and release of proinflammatory cytokines.ConclusionThese are the first indications for utilizing olfactory receptors as therapeutic target molecules in treating steroid-resistant lung diseases with non-type 2 inflammation.

Induced Human Regulatory T Cells Express the Glucagon-like Peptide-1 Receptor.

The glucagon-like peptide-1 receptor (GLP-1R) plays a key role in metabolism and is an important therapeutic target in diabetes and obesity. Recent studies in experimental animals have shown that certain subsets of T cells express functional GLP-1R, indicating an immune regulatory role of GLP-1. In contrast, less is known about the expression and function of the GLP-1R in human T cells. Here, we provide evidence that activated human T cells express GLP-1R. The expressed GLP-1R was functional, as stimulation with a GLP-1R agonist triggered an increase in intracellular cAMP, which was abrogated by a GLP-1R antagonist. Analysis of CD4+ T cells activated under T helper (Th) 1, Th2, Th17 and regulatory T (Treg) cell differentiation conditions indicated that GLP-1R expression was most pronounced in induced Treg (iTreg) cells. Through multimodal single-cell CITE- and TCR-sequencing, we detected GLP-1R expression in 29–34% of the FoxP3+CD25+CD127- iTreg cells. GLP-1R+ cells showed no difference in their TCR-gene usage nor CDR3 lengths. Finally, we demonstrated the presence of GLP-1R+CD4+ T cells in skin from patients with allergic contact dermatitis. Taken together, the present data demonstrate that T cell activation triggers the expression of functional GLP-1R in human CD4+ T cells. Given the high induction of GLP-1R in human iTreg cells, we hypothesize that GLP-1R+ iTreg cells play a key role in the anti-inflammatory effects ascribed to GLP-1R agonists in humans.

CAMP Response Element-Binding Protein Controls the Appearance of Neuron-Like Traits in Chorion Mesenchymal Cells.

Mesenchymal stromal cells (MSC) from bone marrow have been reported to undergo the initial phases of neural differentiation in response to an increase of intracellular cAMP. We investigated the possibility that a similar effect applies to chorion-derived MSC. The intracellular concentration of cAMP was increased either by forskolin, to promote its synthesis, or by inhibitors of its degradation. The consequent reduction in the expression of mesenchymal markers was associated with the appearance of neuron-like morphology in a subset of cells. The effect was measured and characterized using biomarkers and an inhibitor of cAMP response element-binding protein (CREB). The dramatic morphological change induced by all the treatments that promoted intracellular cAMP was transient and peaked on the third day. After that, cells returned to the typical fibroblast-like appearance within 24 hours. The distinctive morphology was associated to the expression of neuregulin 1, doublecortin, neuron-specific class III β-tubulin, and required cAMP response element-binding protein activity. Basic-fibroblast growth factor (b-FGF) treatment increased both the timeframe and number of cells undergoing the morphological change induced by the effect of forskolin. As opposite, arginine-vasopressin (AVP) and sphingosine-1-phosphate (S1P) reduced it. We conclude that cAMP and the ensuing CREB activation trigger a preliminary step towards neuronal differentiation of chorion-derived MSC. However, likewise other MSC, the stimulus is not sufficient to promote stable differentiation.

Effects of a Phosphodiesterase inhibitor on the Browning of Adipose Tissue in Mice.

Cilostazol is a selective inhibitor of phosphodiesterase type 3 (PDE3) that increases intracellular cyclic adenosine monophosphate (cAMP), which plays a critical role in the development of the beige phenotype and the activation of its thermogenic program in white adipose tissue (WAT). We investigated the metabolic effects of PDE3B inhibition with cilostazol treatment in the adipose tissue of high-fat diet (HFD)-fed mice. Seven-week-old male C57BL/6J mice were randomly assigned to either the cilostazol or control group. The control group was divided into two groups: the chow diet and HFD. The expression of uncoupling Protein 1 (UCP1) and other brown adipocyte markers was compared. In the HFD-fed cilostazol group, C57BL/6J mice displayed improvements in systemic metabolism, including improved glucose tolerance and lipid profile, but only modest effects on body weight were observed. In the visceral WAT of HFD-fed cilostazol-treated mice, cAMP/protein kinase A (PKA) signaling pathways were activated, resulting in the “browning” phenotype, smaller fat deposits, and enhanced mRNA expression of UCP1 and other brown adipocyte markers. PDE3B appears to be an important regulator of lipid metabolism, insulin sensitivity, and thermogenic programs in adipose tissues. An increase in intracellular cAMP via PDE3B inhibition with cilostazol treatment promoted the browning of visceral WAT.

Expression of SLC26A9 in Airways and Its Potential Role in Asthma.

SLC26A9 is an epithelial anion transporter with a poorly defined function in airways. It is assumed to contribute to airway chloride secretion and airway surface hydration. However, immunohistochemistry showing precise localization of SLC26A9 in airways is missing. Some studies report localization near tight junctions, which is difficult to reconcile with a chloride secretory function of SLC26A9. We therefore performed immunocytochemistry of SLC26A9 in sections of human and porcine lungs. Obvious apical localization of SLC26A9 was detected in human and porcine superficial airway epithelia, whereas submucosal glands did not express SLC26A9. The anion transporter was located exclusively in ciliated epithelial cells. Highly differentiated BCi-NS1 human airway epithelial cells grown on permeable supports also expressed SLC26A9 in the apical membrane of ciliated epithelial cells. BCi-NS1 cells expressed the major Cl− transporting proteins CFTR, TMEM16A and SLC26A9 in about equal proportions and produced short-circuit currents activated by increases in intracellular cAMP or Ca2+. Both CFTR and SLC26A9 contribute to basal chloride currents in non-stimulated BCi-NS1 airway epithelia, with CFTR being the dominating Cl− conductance. In wtCFTR-expressing CFBE human airway epithelial cells, SLC26A9 was partially located in the plasma membrane, whereas CFBE cells expressing F508del-CFTR showed exclusive cytosolic localization of SLC26A9. Membrane localization of SLC26A9 and basal chloride currents were augmented by interleukin 13 in wild-type CFTR-expressing cells, but not in cells expressing the most common disease-causing mutant F508del-CFTR. The data suggest an upregulation of SLC26A9-dependent chloride secretion in asthma, but not in the presence of F508del-CFTR.

Disruption of CRTC1 and CRTC2 in Sim1 cells strongly increases high-fat diet intake in female mice but has a modest impact on male mice.

cAMP responsive element binding protein (CREB)-regulated transcription coactivators (CRTCs) regulate gene transcription in response to an increase in intracellular cAMP or Ca2+ levels. To date, three isoforms of CRTC have been identified in mammals. All CRTCs are widely expressed in various regions of the brain. Numerous studies have shown the importance of CREB and CRTC in energy homeostasis. In the brain, the paraventricular nucleus of the hypothalamus (PVH) plays a critical role in energy metabolism, and CRTC1 and CRTC2 are highly expressed in PVH neuronal cells. The single-minded homolog 1 gene (Sim1) is densely expressed in PVH neurons and in some areas of the amygdala neurons. To determine the role of CRTCs in PVH on energy metabolism, we generated mice that lacked CRTC1 and CRTC2 in Sim1 cells using Sim-1 cre mice. We found that Sim1 cell-specific CRTC1 and CRTC2 double-knockout mice were sensitive to high-fat diet (HFD)-induced obesity. Sim1 cell-specific CRTC1 and CRTC2 double knockout mice showed hyperphagia specifically for the HFD, but not for the normal chow diet, increased fat mass, and no change in energy expenditure. Interestingly, these phenotypes were stronger in female mice than in male mice, and a weak phenotype was observed in the normal chow diet. The lack of CRTC1 and CRTC2 in Sim1 cells changed the mRNA levels of some neuropeptides that regulate energy metabolism in female mice fed an HFD. Taken together, our findings suggest that CRTCs in Sim1 cells regulate gene expression and suppress excessive fat intake, especially in female mice.

Phosphodiesterase (PDE) 5 inhibitors sildenafil, tadalafil and vardenafil impact cAMP-specific PDE8 isoforms-linked second messengers and steroid production in a mouse Leydig tumor cell line

Type 5 phosphodiesterase (PDE5) blockade by inhibitors (PDE5i) results in intracellular cyclic guanosine monophosphate (cGMP) increase and smooth muscle relaxation and are used for the treatment of men erectile dysfunction. Although they have high specificity for PDE5, these inhibitors are suspected to cross-interact also with cyclic adenosine monophosphate (cAMP)-specific PDEs, inducing the intracellular accumulation of this cyclic nucleotide and related testosterone increase, positively impacting male reproductive parameters. However, the link between the use of PDE5i and the activation of cAMP-mediated steroidogenesis is still unclear. We have investigated whether three PDE5i, sildenafil, tadalafil and vardenafil, cross-interacts with the high affinity cAMP-specific enzymes type 8A and 8B PDEs (PDE8A and PDE8B), in live, transfected mouse Leydig tumor (mLTC1) and human embryonic kidney (HEK293) cell lines in vitro. The PDE5i-induced production of cAMP-dependent testosterone and its precursor progesterone was evaluated as well. We have developed PDE8A/B biosensors and modified cyclic nucleotides confirming enzyme binding to cAMP, but not to cGMP, in our cell models. cAMP binding to PDE8A/B was displaced upon cell treatment with PDE5i, revealing that sildenafil, tadalafil and vardenafil have similar effectiveness in live cells, in vitro. The cross-interaction between PDE5i and PDE8A/B supports the gonadotropin-enhanced intracellular cAMP increase, occurring together with cGMP increase, as well as steroid synthesis. Indeed, we found that Leydig cell treatment by PDE5i increases progesterone and testosterone production triggered by gonadotropins. We demonstrated that PDE5i may interact with the cAMP-specific PDE8A and PDE8B, possibly inducing intracellular cAMP and sex steroid hormone increase. These findings support clinical data suggesting that PDE5i might increase testosterone levels in men.

Angiotensin(1–7) activates MAS-1 and upregulates CFTR to promote insulin secretion in pancreatic β-cells: the association with type 2 diabetes

ObjectiveThe beneficial effect of angiotensin(1–7) (Ang(1–7)), via the activation of its receptor, MAS-1, has been noted in diabetes treatment; however, how Ang(1–7) or MAS-1 affects insulin secretion remains elusive and whether the endogenous level of Ang(1–7) or MAS-1 is altered in diabetic individuals remains unexplored. We recently identified an important role of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl− channel, in the regulation of insulin secretion. Here, we tested the possible involvement of CFTR in mediating Ang(1–7)’s effect on insulin secretion and measured the level of Ang(1–7), MAS-1 as well as CFTR in the blood of individuals with or without type 2 diabetes.MethodsAng(1–7)/MAS-1/CFTR pathway was determined by specific inhibitors, gene manipulation, Western blotting as well as insulin ELISA in a pancreatic β-cell line, RINm5F. Human blood samples were collected from 333 individuals with (n = 197) and without (n = 136) type 2 diabetes. Ang(1–7), MAS-1 and CFTR levels in the human blood were determined by ELISA.ResultsIn RINm5F cells, Ang(1–7) induced intracellular cAMP increase, cAMP-response element binding protein (CREB) activation, enhanced CFTR expression and potentiated glucose-stimulated insulin secretion, which were abolished by a selective CFTR inhibitor, RNAi-knockdown of CFTR, or inhibition of MAS-1. In human subjects, the blood levels of MAS-1 and CFTR, but not Ang(1–7), were significantly higher in individuals with type 2 diabetes as compared to those in non-diabetic healthy subjects. In addition, blood levels of MAS-1 and CFTR were in significant positive correlation in type-2 diabetic but not non-diabetic subjects.ConclusionThese results suggested that MAS-1 and CFTR as key players in mediating Ang(1–7)-promoted insulin secretion in pancreatic β-cells; MAS-1 and CFTR are positively correlated and both upregulated in type 2 diabetes.

Extracellular cAMP: The Past and Visiting the Future in cAMP-Enriched Extracellular Vesicles.

It is recently discovered that the cyclic nucleotide, cyclic adenosine monophosphate (cAMP) can be enriched in the extracellular vesicles (EVs) isolated from endothelial cells. In the current perspective a historical context for the discovery of the extracellular cAMP is provided. The story of extracellular cAMP through investigations addressing the molecule's role in the adenosine pathway is followed, which is widespread in mammalian physiology. The adenosine pathway mediates normal physiological conditions such as renin release, phosphate transport, etc., and participates in pathological conditions such as bronchoconstriction of the airways. Furthermore, adenosine mediated biological pathways are regulated via the receptor mediated intracellular cAMP pathway in mammalian cells. It then speculates on the question of whether cAMP enriched EVs could bypass typical receptor mediated cell signaling and directly activate cAMP signaling cascade in target cells. Preliminary studies to suggest cAMP enriched EVs are provided, added to naïve endothelial cells, results in an increase in intracellular cAMP. An alternate mechanism is proposed, apart from the traditional adenosine pathway, that extracellular cAMP may exert its effects and put into perspective how it might consider circulating cAMP moving forward.

β3-Adrenergic receptors regulate human brown/beige adipocyte lipolysis and thermogenesis.

β3-Adrenergic receptors (β3-ARs) are the predominant regulators of rodent brown adipose tissue (BAT) thermogenesis. However, in humans, the physiological relevance of BAT and β3-AR remains controversial. Herein, using primary human adipocytes from supraclavicular neck fat and immortalized brown/beige adipocytes from deep neck fat from 2 subjects, we demonstrate that the β3-AR plays a critical role in regulating lipolysis, glycolysis, and thermogenesis. Silencing of the β3-AR compromised genes essential for thermogenesis, fatty acid metabolism, and mitochondrial mass. Functionally, reduction of β3-AR lowered agonist-mediated increases in intracellular cAMP, lipolysis, and lipolysis-activated, uncoupling protein 1–mediated thermogenic capacity. Furthermore, mirabegron, a selective human β3-AR agonist, stimulated BAT lipolysis and thermogenesis, and both processes were lost after silencing β3-AR expression. This study highlights that β3-ARs in human brown/beige adipocytes are required to maintain multiple components of the lipolytic and thermogenic cellular machinery and that β3-AR agonists could be used to achieve metabolic benefit in humans.

The homotetramerization of a GPCR transmits the 20-hydroxyecdysone signal and increases its entry into cells for insect metamorphosis.

Animal steroid hormones initiate signaling by passive diffusion into cells and binding to their nuclear receptors to regulate gene expression. Animal steroid hormones can initiate signaling via G protein-coupled receptors (GPCRs); however, the underlying mechanisms are unclear. Here, we show that a newly discovered ecdysone-responsive GPCR, ErGPCR-3, transmits the steroid hormone 20-hydroxyecdysone (20E) signal by binding 20E and promoting its entry into cells in the lepidopteran insect Helicoverpa armigera Knockdown of ErGPCR-3 in larvae caused delayed and abnormal pupation, inhibited remodeling of the larval midgut and fat body, and repressed 20E-induced gene expression. Also, 20E induced both the interaction of ErGPCR-3 with G proteins and rapid intracellular increase in calcium, cAMP and protein phosphorylation. ErGPCR-3 was endocytosed by GPCR kinase 2-mediated phosphorylation, and interacted with β-arrestin-1 and clathrin, to terminate 20E signaling under 20E induction. We found that 20E bound to ErGPCR-3 and induced the ErGPCR-3 homodimer to form a homotetramer, which increased 20E entry into cells. Our study revealed that homotetrameric ErGPCR-3 functions as a cell membrane receptor and increases 20E diffusion into cells to transmit the 20E signal and promote metamorphosis.

Dibutyryl cAMP down-regulates gene expression of endothelin receptor A subtype in rat astrocytoma C6 cells

In this study, we have investigated the gene expression of endothelin receptors in rat astrocytoma C6 cells regulated by the increase of intracellular cAMP. Following treatment with 1 mM dibutyryl cAMP, the specific binding of [125I]-endothelin-1 to the C6 cells was down-regulated to about 40 % of the original level. Radioligand binding assay and Northern blot analysis indicated that endothelin receptors on C6 cells, which were either treated or untreated with 1 mM dibutyryl cAMP, belong to subtype A. Analysis of RNA by Northern blotting and slot hybridization demonstrated that the presence of dibutyryl cAMP induced the down regulation of ET(A) endothelin receptor gene in C6 cells. The level of ET(A) mRNA was rapidly reduced to about 10 % of its initial level after 4 hour treatment with dibutyryl cAMP and then was slightly elevated to about 40 % after one day treatment. All the findings suggest that the down regulation of ET(A) receptor in C6 cells by the treatment of dibutyryl cAMP occurs not only at the receptor binding level but also at the mRNA level.

Astrocytes with TDP-43 inclusions exhibit reduced noradrenergic cAMP and Ca2+ signaling and dysregulated cell metabolism

Most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons and non-neuronal cells, including astrocytes, which metabolically support neurons with nutrients. Neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline. Activation of astroglial adrenergic receptors with noradrenaline triggers cAMP and Ca2+ signaling and augments aerobic glycolysis with production of lactate, an important neuronal energy fuel. Astrocytes with cytoplasmic TDP-43 inclusions can cause motor neuron death, however, whether astroglial metabolism and metabolic support of neurons is altered in astrocytes with TDP-43 inclusions, is unclear. We measured lipid droplet and glucose metabolisms in astrocytes expressing the inclusion-forming C-terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited a 3-fold increase in the accumulation of lipid droplets versus astrocytes expressing wild-type TDP-43, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increases in intracellular cAMP and Ca2+ levels were reduced by 35% and 31%, respectively, likely due to the downregulation of β2-adrenergic receptors. Although noradrenaline triggered a similar increase in intracellular lactate levels in astrocytes with and without TDP-43 inclusions, the probability of activating aerobic glycolysis was facilitated by 1.6-fold in astrocytes with TDP-43 inclusions and lactate MCT1 transporters were downregulated. Thus, while in astrocytes with TDP-43 inclusions noradrenergic signaling is reduced, aerobic glycolysis and lipid droplet accumulation are facilitated, suggesting dysregulated astroglial metabolism and metabolic support of neurons in TDP-43-associated ALS and FTD.

Characterization of the first insect prostaglandin (PGE2) receptor: MansePGE2R is expressed in oenocytoids and lipoteichoic acid (LTA) increases transcript expression

In arthropods, eicosanoids derived from the oxygenated metabolism of arachidonic acid are significant in mediating immune responses. However, the lack of information about insect eicosanoid receptors is an obstacle to completely decipher immune mechanisms underlying both eicosanoid downstream signal cascades and their relationship to immune pathogen-associated molecular patterns (PAMPs). Here, we cloned and sequenced a G protein-coupled receptor (MW 46.16 kDa) from the model lepidopteran, Manduca sexta (Sphingidae). The receptor shares similarity of amino acid motifs to human prostaglandin E2 (PGE2) receptors, and phylogenetic analysis supports its classification as a prostaglandin receptor. In agreement, the recombinant receptor was activated by PGE2 resulting in intracellular cAMP increase, and therefore designated MansePGE2R. Expression of MansePGE2R in Sf9 cells in which the endogenous orthologous receptor had been silenced showed similar cAMP increase upon PGE2 challenge. Receptor transcript expression was identified in various tissues in larvae and female adults, including Malpighian tubules, fat body, gut and hemocytes, and in female ovaries. In addition to the cDNA cloned that encodes the functional receptor, an mRNA was found featuring the poly-A tail but lacking the predicted transmembrane (TM) regions 2 and 3, suggesting the possibility that internally deleted receptor proteins exist in insects. Immunocytochemistry and in situ hybridization revealed that among hemocytes, the receptor was exclusively localized in the oenocytoids. Larval immune challenges injecting bacterial components showed that lipoteichoic acid (LTA) increased MansePGE2R expression in hemocytes. In contrast, injection of LPS or peptidoglycan did not increase MansePGE2R transcript levels in hemocytes, suggesting the LTA-associated increase in receptor transcript is regulated through a distinct pathway. This study provides the first characterization of an eicosanoid receptor in insects, and paves the way for establishing the hierarchy in signaling steps required for establishing insect immune responses to infections.

Intracellular cyclic AMP levels modulate differential adaptive responses on epimastigotes and cell culture trypomastigotes of Trypanosoma cruzi

Among the many environmental challenges the parasite Trypanosoma cruzi has to overcome to complete its life cycle through different hosts, oxidative stress plays a central role. Different stages of this parasite encounter distinct sources of oxidative stress, such as the oxidative burst of the immune system, or the Heme released from hemoglobin degradation in the triatomine's midgut. Also, the redox status of the surroundings functions as a signal to the parasite, triggering processes coupled to differentiation or proliferation. Intracellular second messengers, like cAMP, are responsible for the transduction of environmental queues and initiating cellular processes accordingly. In trypanosomatids cAMP is involved in a variety of processes, including proliferation, differentiation, osmoregulation and quorum sensing. Trypanosomatid phosphodiesterases (PDE) show atypical pharmacological properties and some have been involved in key processes for the survival of the parasites, which validates them as attractive therapeutic targets. Our work here shows that cAMP modulates different processes according to parasite stage. Epimastigotes become more resistant to oxidative stress when pre-treated with cAMP analogs, while in trypomastigotes an increase in intracellular cAMP doesn't seem to aid in this response, although it does increase the number of amastigotes obtained 48 h after infection, compared to the control group. Also, we show that TcrPDEA1, a functionally enigmatic phosphodiesterase with very high Km, is involved in the epimastigotes response to oxidative stress.

Cyclic-AMP Is an Actionable Novel Regulator of PD-L1 Expression in Untransformed Lymphocytes and Diffuse Large B Cell Lymphomas

Antigen-specific T lymphocytes can recognize and eliminate aberrant cells. Cancer cells halt this process by hijacking a system of immune checkpoints, the programmed cell death 1 (PD-1) and its ligands (PD-L1/2) pathway, which physiologically regulates the quantity and activity of T cells, establishing peripheral T cell tolerance and limiting tissue damage. PD-L1-expressing cancer cells interact with and inhibit PD-1 positive T cells, thus abrogating anti-cancer immunity, which can be restored by checkpoint inhibitors (CPI). Improved understanding of the regulation of PD-L1 expression will shed further light on how cancer cells escape immune surveillance, and it may help in the design of combinatorial therapeutic strategies that expand the activity of CPI. Oncogenes (e.g., MYC, STAT3, HIF1 and NF-KB) have been shown to directly induce PD-L1 transcription. In addition, pro-inflammatory cytokines, notably IFN-γ, via the JAK/STAT pathway, also increase PD-L1 expression, an intuitive counteracting regulatory axis that prevents unchecked inflammation and auto-immunity. The second messenger cyclic-AMP (cAMP) is a classical mediator of anti-inflammatory and immunosuppressive inputs. However, its putative role in PD-L1 regulation is unknown. Addressing this knowledge gap is especially relevant because this signaling node can be modulated with a class of FDA-approved agents, the phosphodiesterase 4 (PDE4) inhibitors. We have recently reviewed the pleiotropic roles that cAMP/PDE4 plays in diffuse large B-cell lymphoma (DLBCL) biology (BloodPMID: 27756749). Thus, to examine if cAMP modulates PD-L1 expression, we first used DLBCL cell lines (n=10). Raising the levels of intracellular cAMP readily induced PD-L1 expression (measured by WB and FACS) in ABC-DLBCLs but not in GCB-DLBCLs. This cAMP-mediated induction of PD-L1 occurred also at RNA level; however, using reporter assays we found that the canonical cAMP-PKA-CREB pathway does not directly activate the PD-L1 promoter. The immune modulatory activity of cAMP is mediated, at least in part, by transcriptional activation/secretion of cytokines. Thus, we considered that cAMP induction of PD-L1 in DLBCL may be driven by an autocrine loop. In agreement with this idea, cAMP promoted JAK/STAT activation and culturing DLBCL cell lines in conditioned media (CM) from cAMP-high models induced PD-L1 expression. These assays pointed to secreted factor(s) as intermediaries in the cAMP/PD-L1 axis. Therefore, we screened a panel of 105 cytokines to identify those secreted by DLBCL cell lines following cAMP up-modulation - in most models, we detected a significant cAMP-driven increase in IL-6, IL-8, IL-10 and IL-1α secretion. For validation, we focused on IL-10 because this was the most commonly cAMP-induced cytokine across the DLBCL models. We found that recombinant IL-10 induced PD-L1, albeit this induction was significantly less marked than that observed following an increase in intra-cellular cAMP. Concordantly, antibody-based blocking of the IL-10 signals, and pharmacologically inhibiting the JAK/STAT pathway, only partially abrogated the cAMP-mediated induction of PD-L1. We concluded that IL-10 and JAK/STAT signals relay part, but not all, of the cAMP effects on PD-L1 expression in DLBCL. Next, we utilized the Pde4b null mouse model to examine if these observations were present in an organismal level and in non-immortalized immune cells. In these assays, spleens of Pde4b WT, +/- and -/- mice (8-16 weeks old, male and female, n=8) were collected and analyzed by WB and FACS. Spleen cells from Pde4b deficient mice had markedly higher expression of PD-L1 (WB). By FACS, we found that the increase in PDL1 expression in Pde4b null mice derived from T cells, B cells, but from the smaller non-B/T cell population (CD19/CD3 negative). Finally, we found that the PDE4 inhibitor roflumilast used as a single agent in vitro robustly induced PD-L1 expression in DLBCL cell lines. In summary, we identified cAMP as an "actionable" novel regulator of PD-L1 expression in normal and malignant immune cells. Mechanistically, cAMP drives an autocrine loop enacted by cytokines and transduced in part by JAK/STAT. This finding supports the clinical testing of roflumilast to induce PD-L1 expression, a strategy that may improve the activity of checkpoint inhibitors in DLBCL and related tumor types. Disclosures No relevant conflicts of interest to declare.

The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation.

Holometabolous insects stop feeding at the final larval instar stage and then undergo metamorphosis; however, the mechanism is unclear. In the present study, using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20-hydroxyecdysone (20E) binds to the dopamine receptor (DopEcR), a G protein-coupled receptor, to stop larval feeding and promote pupation. DopEcR was expressed in various tissues and its level increased during metamorphic molting under 20E regulation. The 20E titer was low during larval feeding stages and high during wandering stages. By contrast, the dopamine (DA) titer was high during larval feeding stages and low during the wandering stages. Injection of 20E or blocking dopamine receptors using the inhibitor flupentixol decreased larval food consumption and body weight. Knockdown of DopEcR repressed larval feeding, growth, and pupation. 20E, via DopEcR, promoted apoptosis; and DA, via DopEcR, induced cell proliferation. 20E opposed DA function by repressing DA-induced cell proliferation and AKT phosphorylation. 20E, via DopEcR, induced gene expression and a rapid increase in intracellular calcium ions and cAMP. 20E induced the interaction of DopEcR with G proteins αs and αq. 20E, via DopEcR, induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element. DopEcR could bind 20E inside the cell membrane or after being isolated from the cell membrane. Mutation of DopEcR decreased 20E binding levels and related cellular responses. 20E competed with DA to bind to DopEcR. The results of the present study suggested that 20E, via binding to DopEcR, arrests larval feeding and promotes pupation.

Agonists activate different A2B adenosine receptor signaling pathways in MBA-MD-231 breast cancer cells with distinct potencies.

It is well established that some receptor types including G protein-coupled receptors may transduce effects through more than one signaling pathway. This holds also true for A2B adenosine receptors which were shown to trigger an increase in intracellular Ca2+ levels in addition to the canonical stimulation of adenylyl cyclase. We have recently shown that activation of A2B receptors in the breast cancer cell line MBA-MD-231 elicits a reduction in ERK1/2 phosphorylation, an effect that might be exploited in treatment of cancer cell growth and proliferation. In this study, we investigate whether structurally divers agonists show functional selectivity for any of the signaling pathways leading to an increase of intracellular cAMP or Ca2+, or the reduction of ERK1/2 phosphorylation. As agonists, adenosine derivatives were used bearing different substitutions in 2- and 6-position and, in addition, a ligand with a non-nucleoside structure was tested. It was found that all the tested ligands showed similar pharmacological profiles for the three responses investigated in MBA-MD-231 cells. However, the reduction of ERK1/2 phosphorylation occurred with 40-500-fold higher potency compared to stimulation of adenylyl cyclase or increasing intracellular Ca2+ levels. Based on these observations, it seems possible to utilize activation of A2B adenosine receptors expressed in certain cancers to limit cell growth and proliferation due to reduction of MAPK activity without activation of other signaling pathways potentially responsible for side effects.

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors

Several 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.