Role Of cAMP Signaling Research Articles

Methylxanthines Modulate Circadian Period Length Independently of the Action of Phosphodiesterase.

In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Δpkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive. IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.

CAMP/PKA signaling promotes AKT deactivation by reducing CIP2A expression, thereby facilitating decidualization

cAMP signaling is widely known to be indispensable for decidualization, but the details are not fully understood. Here, we show that cAMP signaling promotes AKT deactivation in endometrial stromal cells, which favors their decidualization. The deactivation of AKT is found to be a consequence of the reduced expression of several inhibitors of PP2A, the major phosphatase of AKT, with CIP2A being the most prominent. CIP2A reduction is obligatory for decidualization, as persistent CIP2A expression impairs chromatin remodeling and the expression of several decidualization markers (IGFBP1, PRL, MAOA, and IL-15). Furthermore, analyses of the responsiveness of the CIP2A promoter to cAMP signaling suggest the ETS family to be a bridge between cAMP signaling and CIP2A reduction. Our results provide novel insights into the role of cAMP signaling in decidualization and might benefit the development of novel therapies for decidualization deficiency, AKT-driven tumors, and the reverse, insulin resistance.

Role of A-Kinase Anchoring Proteins in Respiratory Disorder

Published on: October 2022Journal of Young Pharmacists, 2022; 14(4):359-365Review Article | doi:10.5530/jyp.2022.14.73 Authors:Mustakim M Mansuri1,*, Krupa D Patel1, Gaurang B Shah1, Avinash Khadela1, Manish P Patel21Department of Pharmacology, L. M. College of Pharmacy, Ahmedabad, Gujarat, INDIA.2Department of Pharmaceutics, L. M. College of Pharmacy, Ahmedabad, Gujarat, INDIA. Abstract:Protein kinase A, plays a crucial role in cAMP signaling and its further downstream regulation of received stimuli. A kinase anchoring protein (AKAP); a binding protein provides a platform to the substrates to perform signaling events. AKAP serve its role in cAMP signaling by interacting with GPCR, PKA, ACs, Epac or PDE. More than 70 AKAP have been identified from which role of some AKAPs have been identified. AKAPs are found in heart, brain, testis, skeletal muscle, pancreas, placenta, oocytes, thyroid, kidney, liver, lungs. Amongst which the regulatory role of some AKAPs like AKAP149, AKAP5, AKAP4 and AKAP18 has been reported in mitochondrial function, recycling of β1-adrenoceptors, sperm motility, and insulin secretion respectively; whereas AKAP149 interaction is involved in reverse transcriptase activity of HIV; mutation in AKAP9 isoform causes prolonged QT interval, AKAP10 causes arrhythmia, risk of colorectal cancer and familial breast cancer. Although its dominating role is majorly reported in heart and brain, few reports are available regarding its role in respiratory system. The probable action of AKAP is via IL-8 release; IL-10, IL-6 and iNOS synthesis; mitochondrial function mediation, ROS generation and apoptosis. In the current review, attempt has been made to explore the general role of AKAPs with special emphasis in respiratory system.Keywords: Anchoring protein, Cyclic adenosine monophosphate, Protein kinase A, QT interval, Inflammatory mediators.

Abstract 112: Epac signaling facilitates malignant transformation of melanocytes

Abstract Cyclic AMP (cAMP) signaling plays a critical role in melanocyte proliferation and differentiation. However, the role of cAMP signaling in malignant transformation of human melanocytes and initiation of melanoma is not fully understood. Previously, we demonstrated that cAMP signaling plays opposite roles in primary and metastatic melanoma cells, i.e., pro- and anti-proliferative roles, respectively. This switch in cAMP action is mediated by the alternative cAMP signaling axis involving EPAC-RAP1 (exchange protein directly activated by cyclic AMP-Ras-related protein 1). In this study, we tested the hypothesis that activation of EPAC signaling is an early event in malignant transformation of melanocytes and is required for survival and proliferation of malignant melanocytes. Here, we show that shRNA-mediated knockdown of PTEN alone or PTEN-KD with lentiviral transduction with BRAF(V600E) results in upregulation of EPAC expression and activation of RAP1 (i.e., RAP1-GTP) in human primary melanocytes. Interestingly, activation of phosphoinositide 3-kinase (PI3K) signaling alone is sufficient for stimulation of maximal EPAC expression as well as proliferation suggesting that EPAC activation is an early event associated with loss of PTEN and melanoma tumor initiation. Inhibition of EPAC activity by either EPAC 1/2 or EPAC2-selective inhibitors suppressed the growth of transformed melanocytes by inhibiting cell cycle progression as evident by downregulation of cyclins E, -A2 and -B1. Inhibition of EPAC signaling also increased cells expressing senescence-associate beta galactosidase activity suggesting that EPAC may contribute to the senescence escape mechanisms in BRAF(V600E) melanocytes. Our data suggest that activation of PI3K pathway alone or in combination with mitogen-activated protein kinase pathway activates EPAC signaling in melanocytes. Since primary but not metastatic melanoma cells remain dependent on EPAC signaling, understanding the mechanisms of action EPAC can open new avenues to prevent melanomagenesis and inhibit melanoma tumor progression. Citation Format: Mithalesh Kumar Singh, Sarah Altameemi, Marcos Lares, Shreyans Sadangi, Vijayasaradhi Setaluri. Epac signaling facilitates malignant transformation of melanocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 112.

The Role of POPDC Proteins in Cardiac Pacemaking and Conduction.

The Popeye domain-containing (POPDC) gene family, consisting of Popdc1 (also known as Bves), Popdc2, and Popdc3, encodes transmembrane proteins abundantly expressed in striated muscle. POPDC proteins have recently been identified as cAMP effector proteins and have been proposed to be part of the protein network involved in cAMP signaling. However, their exact biochemical activity is presently poorly understood. Loss-of-function mutations in animal models causes abnormalities in skeletal muscle regeneration, conduction, and heart rate adaptation after stress. Likewise, patients carrying missense or nonsense mutations in POPDC genes have been associated with cardiac arrhythmias and limb-girdle muscular dystrophy. In this review, we introduce the POPDC protein family, and describe their structure function, and role in cAMP signaling. Furthermore, the pathological phenotypes observed in zebrafish and mouse models and the clinical and molecular pathologies in patients carrying POPDC mutations are described.

Botrytis cinerea G Protein β Subunit Bcgb1 Controls Growth, Development and Virulence by Regulating cAMP Signaling and MAPK Signaling.

Botrytis cinerea is a necrotrophic phytopathogenic fungus that causes gray mold disease in many crops. To better understand the role of G protein signaling in the development and virulence of this fungus, the G protein β subunit gene Bcgb1 was knocked out in this study. The ΔBcgb1 mutants showed reduced mycelial growth rate, but increased aerial hyphae and mycelial biomass, lack of conidiation, failed to form sclerotia, increased resistance to cell wall and oxidative stresses, delayed formation of infection cushions, and decreased virulence. Deletion of Bcgb1 resulted in a significant reduction in the expression of several genes involved in cAMP signaling, and caused a notable increase in intracellular cAMP levels, suggesting that G protein β subunit Bcgb1 plays an important role in cAMP signaling. Furthermore, phosphorylation levels of MAP kinases (Bmp1 and Bmp3) were increased in the ΔBcgb1 mutants. Yeast two-hybrid assays showed that Bcgb1 interacts with MAPK (Bmp1 and Bmp3) cascade proteins (BcSte11, BcBck1, BcMkk1, and BcSte50), and the Bmp1-regulated gene Bcgas2 was up-regulated in the ΔBcgb1 mutant. These results indicated that Gβ protein Bcgb1 is involved in the MAPK signaling pathway in B. cinerea. In summary, our results revealed that Gβ protein Bcgb1 controls development and virulence through both the cAMP and MAPK (Bmp1 and Bmp3) signaling pathways in B. cinerea.

Coregulation of dimorphism and symbiosis by cyclic AMP signaling in the lichenized fungus Umbilicaria muhlenbergii

Umbilicaria muhlenbergii is the only known dimorphic lichenized fungus that grows in the hyphal form in lichen thalli but as yeast cells in axenic cultures. However, the regulation of yeast-to-hypha transition and its relationship to the establishment of symbiosis are not clear. In this study, we show that nutrient limitation and hyperosmotic stress trigger the dimorphic change in U. muhlenbergii Contact with algal cells of its photobiont Trebouxia jamesii induced pseudohyphal growth. Treatments with the cAMP diphosphoesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) induced pseudohyphal/hyphal growth and resulted in the differentiation of heavily melanized, lichen cortex-like structures in culture, indicating the role of cAMP signaling in regulating dimorphism. To confirm this observation, we identified and characterized two Gα subunits UmGPA2 and UmGPA3 Whereas deletion of UmGPA2 had only a minor effect on pseudohyphal growth, the ΔUmgpa3 mutant was defective in yeast-to-pseudohypha transition induced by hyperosmotic stress or T. jamesii cells. IBMX treatment suppressed the defect of ΔUmgpa3 in pseudohyphal growth. Transformants expressing the UmGPA3G45V or UmGPA3Q208L dominant active allele were enhanced in the yeast-to-pseudohypha transition and developed pseudohyphae under conditions noninducible to the wild type. Interestingly, T. jamesii cells in close contact with pseudohyphae of UmGPA3G45V and UmGPA3Q208L transformants often collapsed and died after coincubation for over 72 h, indicating that improperly regulated pseudohyphal growth due to dominant active mutations may disrupt the initial establishment of symbiotic interaction between the photobiont and mycobiont. Taken together, these results show that the cAMP-PKA pathway plays a critical role in regulating dimorphism and symbiosis in U. muhlenbergii.

Cyclooxygenase-2 dependent prostaglandin E2 signaling through the EP3 receptor is required for cell-mediated immunity to Listeria monocytogenes

Abstract The bacterial pathogen Listeria monocytogenes (Lm) is being developed as a cancer immunotherapeutic platform due to its ability to elicit robust CD8+ T cell responses. Though the role of cytokines in Lm-stimulated immunity is well studied, the role of eicosanoids, lipid modulators of inflammation, is unclear. We previously demonstrated that cyclooxygenase-2 (COX-2) dependent prostaglandin E2 (PGE2) is essential for optimal T-cell priming. Antigen specific T cells and protective immunity were decreased following either treatment with the COX-2 inhibitor celecoxib or immunization of mice deficient in microsomal PGE2 synthase-1. Analysis of splenic eicosanoid levels revealed an upregulation of PGE2 twelve hours post immunization and administration of exogenous PGE2 at this time restored immunity during COX-2 inhibition. To understand how PGE2 impacts T-cell priming we assessed the role of the receptors EP1–4 in mediating this response using small molecule agonists and antagonists. EP3 blockade impaired immune responses while the use of an EP3 specific agonist restored immunity during COX-2 inhibition, suggesting that PGE2 signaling through EP3 is critical for T-cell priming. Conversely, consistent with its predicted inverse role in cAMP signaling, EP4 blockade enhanced responses, providing a mechanism by which to improve Listeria-based immunotherapeutics. Ongoing studies with floxed COX-2, EP3 and EP4 mice will illuminate the cell types producing and responding to PGE2. In conclusion, PGE2 signaling through EP3 is necessary Lm-stimulated immunity while EP4 signaling is detrimental. Further understanding these signaling pathways and where they function will be key to optimizing Lm as therapeutic platform.

Cell-type-specific Modulation of Non-homologous End Joining of Gamma Ray-induced DNA Double-strand Breaks by cAMP Signaling in Human Cancer Cells.

BackgroundCyclic AMP (cAMP) signaling is activated by various hormones and neurotransmitters and regulates numerous physiological phenomena, including energy metabolism, gene expression, and proliferation. cAMP signaling plays a role in the repair of DNA damage, but its specific function is inconsistent in the literature. The present study aimed to investigate the mechanism of the different roles of cAMP signaling in DNA repair by analyzing the cell-type differences in the modulation of DNA repair by cAMP signaling following γ-ray irradiation.MethodscAMP signaling was activated in human malignant melanoma cells (SK-MEL-2 and SK-MEL-28), human uterine cervical cancer cells (HeLa and SiHa) and human non-small cell lung cancer cells (H1299 and A549) by expressing a constitutively active mutant of the long-form stimulatory α subunit of GTP-binding protein or by treating with isoproterenol and prostaglandin E2 before γ-ray irradiation. DNA damage was quantitated by western blot analysis of γ-H2AX, and non-homologous end joining (NHEJ) was assessed by fluorescent reporter plasmid repair assay and immunofluorescence of microscopic foci of XRCC4 and DNA-ligase IV.ResultscAMP signaling modulated DNA damage, apoptosis and the NHEJ repair following γ-ray irradiation differently depending upon the cell type. cAMP signaling regulated the phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at Ser2056 and Thr2609 in cell-type-specific manners following γ-ray irradiation, an activity that was mediated by protein kinase A.ConclusioncAMP signaling modulates the NHEJ repair of γ-ray-induced DNA damage in melanoma cells, uterine cervical cancer cells and lung cancer cells in a cell-type-specific manner, and the modulation is likely mediated by protein kinase A-dependent phosphorylation of DNA-PKcs. This study suggests that cell- and tissue-specific modulation of DNA damage repair by cAMP signaling may contribute to improve the therapeutic efficiency of radiation therapy.

Aspects of cAMP Signaling in Epileptogenesis and Seizures and Its Potential as Drug Target.

Epilepsy is one of the most common chronic neurological conditions. Today, close to 30 different medications to prevent epileptic seizures are in use; yet, far from all patients become seizure free upon medical treatment. Thus, there is a need for new pharmacological approaches including novel drug targets for the management of epilepsy. Despite the fact that a role for cAMP signaling in epileptogenesis and seizures was first suggested some four decades ago, none of the current medications target the cAMP signaling system. The reasons for this are probably many including limited knowledge of the underlying biology and pathology as well as difficulties in designing selective drugs for the different components of the cAMP signaling system. This review explores selected aspects of cAMP signaling in the context of epileptogenesis and seizures including cAMP response element binding (CREB)-mediated transcriptional regulation. We discuss the therapeutic potential of targeting cAMP signaling in epilepsy and point to an increased knowledge of the A-kinase anchoring protein-based signaling hubs as being of seminal importance for future drug discovery within the field. Further, in terms of targeting CREB, we argue that targeting upstream cAMP signals might be more fruitful than targeting CREB itself. Finally, we point to astrocytes as cellular targets in epilepsy since cAMP signals may regulate astrocytic K+ clearance affecting neuronal excitability.

The Astrocytic cAMP Pathway in Health and Disease.

Astrocytes are major glial cells that play critical roles in brain homeostasis. Abnormalities in astrocytic functions can lead to brain disorders. Astrocytes also respond to injury and disease through gliosis and immune activation, which can be both protective and detrimental. Thus, it is essential to elucidate the function of astrocytes in order to understand the physiology of the brain to develop therapeutic strategies against brain diseases. Cyclic adenosine monophosphate (cAMP) is a major second messenger that triggers various downstream cellular machinery in a wide variety of cells. The functions of astrocytes have also been suggested as being regulated by cAMP. Here, we summarize the possible roles of cAMP signaling in regulating the functions of astrocytes. Specifically, we introduce the ways in which cAMP pathways are involved in astrocyte functions, including (1) energy supply, (2) maintenance of the extracellular environment, (3) immune response, and (4) a potential role as a provider of trophic factors, and we discuss how these cAMP-regulated processes can affect brain functions in health and disease.

The role of cAMP signaling in the regulation of interleukin-33 production by RAW264.7 macrophages in response to various doses of lipopolysaccharide

The role of cAMP signaling in the regulation of interleukin-33 production by RAW264.7 macrophages in response to various doses of lipopolysaccharide

Cyclic nucleotide signaling is required during synaptic refinement at the Drosophila neuromuscular junction.

The removal of miswired synapses is a fundamental prerequisite for normal circuit development, leading to clinical problems when aberrant. However, the underlying activity-dependent molecular mechanisms involved in synaptic pruning remain incompletely resolved. Here the dynamic properties of intracellular calcium oscillations and a role for cAMP signaling during synaptic refinement in intact Drosophila embryos were examined using optogenetic tools. We provide In vivo evidence at the single gene level that the calcium-dependent adenylyl cyclase rutabaga, the phosphodiesterase dunce, the kinase PKA, and Protein Phosphatase 1 (PP1) all operate within a functional signaling pathway to modulate Sema2a-dependent chemorepulsion. It was found that presynaptic cAMP levels were required to be dynamically maintained at an optimal level to suppress connectivity defects. It was also proposed that PP1 may serve as a molecular link between cAMP signaling and CaMKII in the pathway underlying refinement. The results introduced an in vivo model where presynaptic cAMP levels, downstream of electrical activity and calcium influx, act via PKA and PP1 to modulate the neuron's response to chemorepulsion involved in the withdrawal of off-target synaptic contacts. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 39-60, 2017.

Editorial: Frontiers in the Pharmacological Manipulation of Intracellular cAMP Levels.

Cyclic adenosine monophosphate (cAMP) is a second messenger of paramount biological importance, involved in the regulation of a significant number of cellular functions via the cAMP-dependent intracellular signal transduction pathways. Being the first second messenger described (Rall and Sutherland, 1958), cAMP has been extensively studied throughout the years and has revealed mainstream mechanisms of cellular signaling (Beavo and Brunton, 2002; Baillie et al., 2005; McCormick and Baillie, 2014). The aim of this Research Topic was to attract contributions that highlight emerging ideas in the cAMP field that: (i) describe its role in cellular function and homeostasis, (ii) present the current approaches to its pharmacological manipulation, and (iii) clarify its central role in the development of more targeted therapeutic approaches toward a spectrum of diseases. To our belief, this aim has been successfully fulfilled. The present collection of articles highlights, in a representative (but certainly not exhaustive) way, the research activity and emerging concepts in the field, while it also reveals the therapeutic potential that targeted pharmacological manipulation of intracellular cAMP levels could exert on a number of pathological conditions. We organized the papers of this Research Topic/e-Book into three sections (A–C). The first section (A: “Frontiers in the role of cAMP in cellular function, homeostasis and disease”) opens with a very informative review article on the reciprocal regulation between cAMP signaling and the ubiquitin-proteasome system, as well as its relevance to certain human diseases (Rinaldi et al.). A more focused perspective on the importance of cAMP in cardiac physiology and pathophysiology is presented by the review article of Boularan and Gales and the mini review article of Froese and Nikolaev; both articles provide an up-to-date account of recent developments in the field and should be considered by readers as complementary to each other. Later in section A, readers will discover an elegant mini review on the role of cAMP signaling in neural plasticity, learning and memory (Lee), that summarizes findings generated by research on the fruit fly Drosophila melanogaster and briefly highlights implications of this signaling pathway to potential therapeutic applications. Section A concludes with an original article on the role of cAMP signaling in human trophoblast fusion (Gerbaud et al.), and with a perspective article on the spatiotemporal regulation of cAMP signaling in blood platelets (Raslan et al.). The second section (B: “Approaches to the study and pharmacological manipulation of intracellular cAMP levels”) includes an informative review article on state-of-the-art methodologies which can be utilized toward the study of cAMP-mediated signal transduction via G-protein coupled receptors (Wright et al.), as well as two excellent review articles on the pharmacological manipulation of intracellular cAMP levels through interference of the molecular interactions between the A-kinase anchoring proteins and other signaling protein intermediates (Calejo and Tasken; Nygren and Scott). This section also accommodates a contribution by Rock et al. with an original research article in which analysis of protein kinase A (PKA) dynamics following the integration of patient mutations into its catalytic (PKAc) and regulatory (RIa) subunits is presented. The section is completed with two analytical review articles that present recent developments in the utilization of genetically encoded tools for cAMP measurement in health and disease (Paramonov et al.; Patel and Gold). The third section (C: “Potential therapeutic applications”) is introduced with a perspective article on the role of patients' sex toward a more targeted (cAMP pathway-regulated) therapeutic approach to brain tumors (Warrington et al.), followed by two original research articles on the role of cAMP signaling in the context of hematological malignancies and the potential therapeutic applications that could arise through its pharmacological manipulation (Dong et al.; Fernandez-Araujo et al.). The current Research Topic / e-Book is concluded with a well-written review article on cAMP signaling in trypanosomatids that provides an overview of the complex functions of cAMP in these protozoan parasites, as well as a fine perspective on how potential targets for the trypanosomatid-specific cAMP pathway could provide efficient therapeutics for serious human diseases such as the sleeping sickness and other trypanosomatid-induced pathological entities (Tagoe et al.). The recruited articles cover multiple aspects of the ongoing research in the cAMP field and allow an appreciation of the difficult task ahead in fully-understanding the complexity of this ubiquitous signaling cascade and taming it to develop efficient therapeutic applications. Hopefully, readers will consider this collection of papers as a small step toward these goals.

Abstract 2680: Cyclic AMP signaling in melanoma: Paradoxical downregulation of pCREB upon activation of adenylate cyclase confers resistance to MAPK inhibition

Abstract Melanoma, which arises from malignant transformation of melanocytes, has proven to be difficult to treat due its intrinsic and acquired resistance for chemotherapy. Although polymorphisms in the melanocortin receptor 1 (MC1R), which result in impairment of cyclic AMP (cAMP) signaling, are associated with increased risk for melanoma development, the role of cAMP signaling in melanoma, specifically in drug resistance, is not fully understood. Given the importance of cAMP signaling in melanocytes proliferation and differentiation, we investigated the role of cAMP signaling in melanoma cell proliferation, survival and drug resistance. We found that upregulation of cAMP signaling, through activation of adenylate cyclase with forskolin (FSK), has both positive and negative effects on proliferation and survival of melanoma cells. The effect of FSK on melanoma proliferation and survival was not dependent on oncogenic (RAS vs. BRAF) mutation. Moreover, upregulation of cAMP conferred resistance to Selumetinib (AZD6244)-mediated MAPK inhibition by restoring the pERK levels. We also show that FSK-mediated resistance is paradoxically related to a downregulation of phosphorylated cAMP Responsive Element Binding Protein (pCREB), suggesting a CREB-independent mechanism of FSK-induced MAPK reactivation in acquired drug resistance. Citation Format: Carlos I. Rodriguez, Vijayasaradhi Setaluri. Cyclic AMP signaling in melanoma: Paradoxical downregulation of pCREB upon activation of adenylate cyclase confers resistance to MAPK inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2680. doi:10.1158/1538-7445.AM2015-2680

CAMP‐signaling via EPAC1 mediates vascular endothelial cell adaptation to fluid‐shear stress

Herein, we report a novel role for cAMP‐signaling in human aortic endothelial cell (HAEC) adaptation to fluid‐mediated shear stress (FSS). Our results showed that increases in cAMP enhanced elongation and alignment of HAECs cultured under LSS for 48hrs and that this effect was largely mediated by the cAMP‐effector molecule, exchange protein activated by cAMP (EPAC)‐1, but not Protein Kinase A (PKA). Additionally, major HAEC cAMP‐hydrolyzing enzyme, phosphodiesterase (PDE)4D, was also shown to play a crucial role in integrating FSS signals in these cells.Our results demonstrated that flow‐mediated increases in atheroprotective genes, Kruppel‐like factor, endothelial nitric oxide synthase and thrombomodulin mRNA expression were reduced in EPAC‐1 and PDE4D knockdown HAECs exposed to both LSS and HSS. Furthermore, EPAC‐1 knockdown increased leukocyte extravasation across HAEC monolayers in vitro and activation of EPAC significantly reduced LPS‐induced leukocyte rolling flux in mice.VECs have numerous mechanosensors which facilitate the integration of FSS signals from the flowing blood. One such sensor consists of platelet‐endothelial cell adhesion molecule (PECAM)‐1, vascular endothelial‐cadherin (VECAD) and vascular endothelial growth factor receptor (VEFGR)‐2. Our current data suggests that EPAC1 and PDE4D can additionally mediate flow‐sensing through this mechanosensory complex. All together, this data provides important mechanistic insight providing therapeutic targets in endothelial dysfunction and atherosclerosis.

VgrG2 of type VI secretion system 2 of Vibrio parahaemolyticus induces autophagy in macrophages.

Type VI secretion system (T6SS) is a macromolecular transenvelope machine encoded within the genomes of several proteobacteria species. Vibrio parahaemolyticus contains two putative T6SS systems, VpT6SS1 and VpT6SS2, both contributing to adherence to Caco-2 and/or HeLa cells. However, it remains unknown if these systems are involved in cellular responses. In order to exclude the effects of other virulence factors known to induce cytotoxicity or autophagy, a triple deletion mutant dTTT (with deletion of tdh, and T3SS1 and T3SS2 structural protein genes) was used as the parent strain to construct deletion mutants of T6SS genes. The mutant dTTT-ΔicmF2, but not dTTT-ΔicmF1, reduced autophagic response upon 4 h of infection of the macrophage. Further attempt was made to search for the possible effector proteins that might be responsible for direct induction of autophagy by deletion of the genes encoding Hcp2 and VgrG2, two putative translocons of T6SS2 of V. parahaemolyticus. Deletion of either hcp2 or vgrG2 did reduce the autophagic response. However, increased LC3-II lipidation was seen only in the macrophage cells transfected with pVgrG2, but not with pHcp2. Chloroquinine treatment increased accumulation of LC3-II, suggesting that VgrG2 enhanced autophagic flux. The fact that vgrG2 deletion led to reduced level of intracellular cAMP suggests a possible role of cAMP signaling in autophagic responses to the bacterium. We conclude that VgrG2 of V. parahaemolyticus induces autophagy in macrophages.

Cyclic Adenosine Monophosphate Signalling in Inflammatory Skin Disease

The second messenger cyclic adenosine monophosphate (cAMP) regulates numerous key pathways that impact the immune system. Distinct cellular cAMP signaling pathways can lead to both pro- and anti-inflammatory effects depending upon the cell type. When dysregulated, these cAMP pathways can influence the pathogenesis of inflammatory cutaneous diseases, such as atopic dermatitis and psoriasis. In psoriasis and atopic dermatitis, cAMP and/or its effector proteins (e.g., protein kinase A) are downregulated suggesting that elevation of cAMP might be a therapeutic option. cAMP levels are the result of balance between synthesis by adenylyl cyclases and degradation by phosphodiesterases (PDEs). Pharmacologically inhibiting PDEs represents one effective mechanism to raise intracellular cAMP levels perhaps leading to targeted immune suppression. Several drugs have been developed to target PDEs and while some toxicities (e.g., nausea and emesis) exist these drugs are generally well tolerated. Perhaps the best characterized is Apremilast, a PDE4 specific inhibitor, which has been FDA approved for the treatment of psoriasis and shows great promise as a safe and novel immunosuppressive medication. Following on the heels of Apremilast are numerous oral and topical PDE inhibitors in various stages of clinical trials. In this review, we examine the role of cAMP signaling in inflammatory cutaneous diseases and the development of PDE inhibitors as therapeutics.

Not all glucocorticoid-induced obesity is the same: differences in adiposity among various diagnostic groups of Cushing syndrome.

The cAMP signaling pathway is implicated in bilateral adrenocortical hyperplasias (BAHs), which are often associated with ACTH-independent Cushing syndrome (CS). Although CS is invariably associated with obesity and is frequently associated with PKA signaling defects, we recently reported that its different forms appear to also present with variable weight gain and adiposity. The present study was aimed at characterizing further the phenotypic and molecular differences in periadrenal adipose tissue (PAT) among patients with subtypes of CS, by anthropometric/biochemical analyses and quantification of PKA expression and activity in BAHs in comparison to a non-CS group with aldosterone producing adenomas (APAs). Glucocorticoid levels, serum parameters, and BMI were analyzed among a larger patient cohort including those with different forms of CS, APAs, and Cushing disease. Abdominal CT scans were available for a small subset of patients examined for fat distribution. PAT collected during adrenalectomy was assayed for PKA activity, cAMP, and PKA expression. BMI and BMI z-score were lower in adults with PPNAD with PRKAR1A mutations and in pediatric patients with PPNAD with and without PRKAR1A mutations, respectively. Patients with PPNAD had higher cAMP levels in PAT and different fat distribution. Thus, PKA activity in PAT differed between CS diagnostic groups. Increased cAMP and PKA activity may have contributed to phenotypic differences among subtypes of CS. In agreement with the known roles of cAMP signaling in the regulation of adiposity, patients with PPNAD were less obese than other patients with CS.

Role of cAMP signaling in mucin production in response to IL‐13 in human bronchial epithelial cells (660.12)

Asthma, a chronic airway inflammatory disease, is associated with mucus hypersecretion by airway epithelium. Accumulated mucus leads to airway obstruction and morbidity. We have shown that β2AR signaling has pro‐inflammatory effects in cultured normal human bronchial epithelial cells (NHBECs) in response to IL‐13(1). Our objective here was to elucidate the role of cAMP signaling in mucin production.NHBECs growing at air‐liquid interface (ALI) were subjected to various treatments, and levels of MUC5AC transcripts, intracellular mucin, cAMP levels, pCREB and pSTAT6 were measured on day 14 ALI.Treatment with forskolin+IBMX (to raise global intracellular cAMP levels) increased MUC5AC transcripts and intracellular mucin in the presence or absence of epinephrine in response to IL‐13 pCREB and pSTA6 were also elevated only in the presence of epinephrine, IL‐13 and forskolin+IBMX. A compartmentalized increase in cAMP caused by treatment with roflumilast, a PDE4 inhibitor, did not affect MUC5AC expression but reduced intracellular mucin only in the presence of epinephrine+IL‐13. Roflumilast did not affect the levels of cAMP, pCREB or pSTAT6.Our data provide evidence for the different influences of global and compartmentalized increases in cAMP on mucin production in human airway epithelium in response to IL‐13.1.Pokkunuri et al, BPS, 2011 Winter MeetingGrant Funding Source: Supported by NIH 1RO1A179236 awarded to RAB and BJK