Elevation Of Cyclic AMP Research Articles

Insulin Resistance Develops Due to an Imbalance in the Synthesis of Cyclic AMP and the Natural Cyclic AMP Antagonist Prostaglandylinositol Cyclic Phosphate (Cyclic PIP)

The reasons initiating insulin resistance are not identified. Various metabolic derailments have been characterized. These are the outcome and not the initiation of insulin resistance. In animal models of type 2 diabetes and hypertension, a decreased hormonal stimulation of the synthesis of the cyclic AMP antagonist prostaglandylinositol cyclic phosphate (cyclic PIP) was determined. The resultant imbalance of the action of cyclic AMP and cyclic PIP shifts metabolic regulation to the dominance of catabolism and a decrease in imperative anabolism. This dominance develops gradually since the more cyclic AMP dominates, the more the synthesis of cyclic PIP will be inhibited. Vanishing actions of cyclic PIP are its 10-fold activation of glucose uptake in adipocytes, its inhibition of insulin release from pancreatic β-cells, its inhibition of PKA and its 7-fold activation of protein ser/thr phosphatase. Reduced synthesis of cyclic PIP results from (a) decreased substrate availability, (b) long-time elevated cyclic AMP levels resulting from stress overloads and (c) aging and the gradual decrease in the synthesis of hormones which likely maintain mechanisms that stimulate cyclic PIP synthesis. The need is to discover which hormones, such as growth hormone, insulin-like growth factor-1, dehydroepiandrosterone, and testosterone, are involved in maintaining the stimulation of cyclic PIP synthesis.

A phenotypic screen for compounds that reverse cAMP-mediated suppression of T cell functions.

The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that suppresses T cell receptor (TCR) signaling. We developed a phenotypic assay to screen for small molecules that interfere with this pathway using TALL-104 human leukemic cytotoxic T lymphocytes pretreated with prostaglandin E2 to elevate cAMP. Beads coated with anti-CD3 antibodies stimulate lytic granule exocytosis, which is detected via binding of an antibody against lysosome associated membrane protein 1 (LAMP-1) measured with flow cytometry. Confirming that the assay can find compounds with desired activity, treating cells with a phorbol ester restores exocytosis. The assay behaves well in 96-well format and we screened a collection of compounds expected to have effects on epigenetic regulatory proteins. Compounds in this collection affected lytic granule exocytosis after 24-hour treatment, but none prevented cAMP from suppressing lytic granule exocytosis. We used a fully automated 384-well version of the assay to screen the Prestwick Compound Library but obtained no confirmed hits. Analyzing this assay's performance reveals two points of interest. First, cytometry offers multiple ways to quantify signals. Z' was higher using percent positive cells than mean fluorescence because the relationship between the two measures saturates, but using percent positive could make it harder to find hits in some assays. Second, variance was higher in positive controls than in negative controls in this assay, which degrades assay performance less than if variance was higher in negative controls.

Gs‐coupled GPCR‐mediated Activation of Rapgef2‐ and ERK‐dependent Gene Regulation and Neuritogenesis in NS‐1 Cells

Gs‐coupled GPCRs signal through the second messenger cAMP. In the neuroendocrine NS‐1 cell line, cAMP acts through three parcellated signal pathways using distinct cAMP sensors as third messenger: protein kinase A (PKA) is linked to neuroendocrine‐specific gene expression, the exchange protein (EPAC) to cell proliferation arrest, and the neuritogenic cAMP sensor NCS‐Rapgef2 to neuritogenesis (Xu et al., J. Neuroendocrinol. 33: e12974). NCS‐RapGEF2‐mediated neuritogenesis occurs via the Rap‐Braf‐MEK‐ERK pathway, which is activated by a variety of Gs‐coupled GPCRs, including the endogenously expressed PAC1 receptor, and receptors for GLP‐1 (GLP1R) and VIP (VIPR1, VIPR2) when exogenously expressed in NS‐1 cells, followed by exposure to exendin‐4 or VIP (Xu et al., ibid) or by ADRB2, after exposure to isoproterenol. We have noted previously that other Gs‐coupled GPCRs expressed in NS‐1 cells mediate measurable cAMP elevation, which however is apparently below the threshold required for triggering and maintaining neuritogenesis (Xu et al., ibid). Here, we identify changes in the NS‐1 cell transcriptome associated with PACAP activation of PAC1 receptor and the role of ERK phosphorylation in sustaining gene expression potentially associated with cAMP‐dependent neuritogenesis. Continuous ERK phosphorylation is required for neuritogenic signaling: inhibition of ERK phosphorylation with U0126 for up to 24 hours after PACAP treatment arrests neuritogenesis, while inhibition of PKA with H89 has no effect on neuritogenesis.NS‐1 cells were treated for 24 hours with PACAP in the presence and absence of either the MEK inhibitor U0126 or the PKA inhibitor H89, and subjected to transcriptomic analysis. We identified 25 genes significantly upregulated by PACAP but downregulated (P<0.001, >2 fold, n=3) by co‐treatment with U0126. DCLK1 is a serine/threonine protein kinase important for neuronal development. Dclk1 mRNA is upregulated ~six‐fold at 8 hr and four‐fold at 24 hr after PACAP treatment, and this was reduced to <two‐fold by U0126. Dlg2 encodes a postsynaptic scaffolding protein, PSD‐93, and Dlg2 mRNA is also upregulated significantly at 8 and 24 hr after PACAP (P<0.01, >2 fold compared to no treat) and its upregulation by PACAP is also blocked by U0126. The gene encoding the orphan GPCR, GPR50, is upregulated more than 100‐fold by 8 hours after PACAP, and induction of GPR50 mRNA is blocked more than 97% by U0126, with partial blockade (80%) by H89. We are currently investigating the impact of GPR50 on neuritogenesis using GPR50 knockdown with shRNA in NS‐1 cells. Forty‐five genes upregulated by PACAP and blocked by H89, but not U0126, include Slit2 and Gas1. Regulation of these genes by Gs‐coupled GPCR signaling through PKA may mediate NS‐1 cell responses separate from activation of neuritogenesis.Cyclic AMP elevation associated with Gs‐coupled GPCR signaling in NS‐1 cells results in upregulation of both RapGEF2/ERK‐dependent and PKA‐dependent gene induction. A threshold for cAMP elevation, either in amount or duration of elevation, or both, appears to be a requirement for Gs‐coupled GPCR‐mediated neuritogenesis. NS‐1 cells provide a model for investigating the functional importance of RapGEF2‐ and PKA‐dependent gene regulation initiated by cAMP in neuroendocrine cells.

Monocyte-derived SDF1 supports optic nerve regeneration and alters retinal ganglion cells’ response to Pten deletion

Although mammalian retinal ganglion cells (RGCs) normally cannot regenerate axons nor survive after optic nerve injury, this failure is partially reversed by inducing sterile inflammation in the eye. Infiltrative myeloid cells express the axogenic protein oncomodulin (Ocm) but additional, as-yet-unidentified, factors are also required. We show here that infiltrative macrophages express stromal cell–derived factor 1 (SDF1, CXCL12), which plays a central role in this regard. Among many growth factors tested in culture, only SDF1 enhances Ocm activity, an effect mediated through intracellular cyclic AMP (cAMP) elevation and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activation. SDF1 deficiency in myeloid cells (CXCL12flx/flxLysM-Cre−/+ mice) or deletion of the SDF1 receptor CXCR4 in RGCs (intraocular AAV2-Cre in CXCR4flx/flx mice) or SDF1 antagonist AMD3100 greatly suppresses inflammation-induced regeneration and decreases RGC survival to baseline levels. Conversely, SDF1 induces optic nerve regeneration and RGC survival, and, when combined with Ocm/cAMP, SDF1 increases axon regeneration to levels similar to those induced by intraocular inflammation. In contrast to deletion of phosphatase and tensin homolog (Pten), which promotes regeneration selectively from αRGCs, SDF1 promotes regeneration from non-αRGCs and enables the latter cells to respond robustly to Pten deletion; however, SDF1 surprisingly diminishes the response of αRGCs to Pten deletion. When combined with inflammation and Pten deletion, SDF1 enables many RGCs to regenerate axons the entire length of the optic nerve. Thus, SDF1 complements the effects of Ocm in mediating inflammation-induced regeneration and enables different RGC subtypes to respond to Pten deletion.

Dorsal Striatum Transcriptome Profile Profound Shift in Repeated Aggression Mouse Model Converged to Networks of 12 Transcription Factors after Fighting Deprivation.

Both aggressive and aggression-deprived (AD) species represent pathologic cases intensely addressed in psychiatry and substance abuse disciplines. Previously, we reported that AD mice displayed a higher aggressive behavior score than the aggressive group, implying the manifestation of a withdrawal effect. We employed an animal model of chronic social conflicts, curated in our lab for more than 30 years. In the study, we pursued the task of evaluating key events in the dorsal striatum transcriptome of aggression experienced mice and AD species compared to controls using RNA-Seq profiling. Aggressive species were subjected to repeated social conflict encounters (fights) with regular positive (winners) experience in the course of 20 consecutive days (A20 group). This led to a profoundly shifted transcriptome expression profile relative to the control group, outlined by more than 1000 differentially expressed genes (DEGs). RNA-Seq cluster analysis revealed that elevated cyclic AMP (cAMP) signaling cascade and associated genes comprising 170 differentially expressed genes (DEGs) in aggressive (A20) species were accompanied by a downturn in the majority of other metabolic/signaling gene networks (839 DEGs) via the activation of transcriptional repressor DEGs. Fourteen days of a consecutive fighting deprivation period (AD group) featured the basic restoration of the normal (control) transcriptome expression profile yielding only 62 DEGs against the control. Notably, we observed a network of 12 coordinated DEG Transcription Factor (TF) activators from 62 DEGs in total that were distinctly altered in AD compared to control group, underlining the distinct transcription programs featuring AD group, partly retained from the aggressive encounters and not restored to normal in 14 days. We found circadian clock TFs among them, reported previously as a withdrawal effect factor. We conclude that the aggressive phenotype selection with positive reward effect (winning) manifests an addiction model featuring a distinct opioid-related withdrawal effect in AD group. Along with reporting profound transcriptome alteration in A20 group and gaining some insight on its specifics, we outline specific TF activator gene networks associated with transcriptional repression in affected species compared to controls, outlining Nr1d1 as a primary candidate, thus offering putative therapeutic targets in opioid-induced withdrawal treatment.

Cyclic AMP links luteinizing hormone signaling to dephosphorylation and inactivation of the NPR2 guanylyl cyclase in ovarian follicles†.

Cyclic AMP elevation links luteinizing hormone receptor signaling to dephosphorylation and inactivation of the NPR2 guanylyl cyclase in preovulatory mouse follicles.

TBK1 regulates regeneration of pancreatic \u03b2-cells

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate β-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in β-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat β-cells. Conversely, TBK1 overexpression decreased sensitivity of β-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of β-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of β-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived β-cells and human islets. TBK1 expression was increased in β-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and β-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional β-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a β-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional β-cells.

Genetic and epigenetic regulation of osteopontin by cyclic adenosine 3' 5'-monophosphate in osteoblasts.

Osteopontin (OPN) is not only a marker of osteoblasts but it is also related to cancer progression and inflammation. The expression of OPN increases in response to inflammatory cytokines, hormones, and mechanical stress. Among them, cyclic-AMP (cAMP) elevating agents stimulate OPN expression in the presence of 1, 25-OH vitamin D3 (VD3). We aimed to clarify the mechanism by which cAMP enhances OPN expression in osteoblastic cells. The OPN promoter (-2335 to +76, OPNp2335) exerted a cell type specific response to forskolin (FK) and VD3. Sequential deletion analysis of OPNp revealed that the OPNp (-833 to +76) contained essential responsive regions to respond to cAMP signaling. In particular, both Vitamin D response element (VDRE, -758 to -743) and osteoblast-specific cis- acting element 2 (OSE2, -695 to -690) were essential for cAMP-mediated OPNp activity. The expression of vitamin D receptor (VDR), but not runt-related transcription factor 2 (Runx2), a nuclear receptor for OSE2, was induced by the treatment of the cells with FK. Although, VD3-induced OPNp activity was slightly enhanced in VDR-overexpressing osteoblasts, it reached the same level as that of osteoblasts induced by both VD3 and FK in the presence of histone deacetylase (HDAC) inhibitor. Moreover, we identified histone acetylation on the OPN promoter region by FK treatment. These results strongly suggest that OPNp activity is controlled by the cAMP signaling via genetic and epigenetic regulations.

Neurofibromatosis 1 - Mutant microglia exhibit sexually-dimorphic cyclic AMP-dependent purinergic defects

As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/−) mice, purinergic control of phagocytosis was only affected in male Nf1+/− mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/−, microglia. These defects resulted from Nf1+/− male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/− microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain.

Neurotrophic and neuroprotective effects of a monomeric GLP-1/GIP/Gcg receptor triagonist in cellular and rodent models of mild traumatic brain injury

A synthetic monomeric peptide triple receptor agonist, termed “Triagonist” that incorporates glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon (Gcg) actions, was previously developed to improve upon metabolic and glucose regulatory benefits of single and dual receptor agonists in rodent models of diet-induced obesity and type 2 diabetes. In the current study, the neurotrophic and neuroprotective actions of this Triagonist were probed in cellular and mouse models of mild traumatic brain injury (mTBI), a prevalent cause of neurodegeneration in both the young and elderly. Triagonist dose- and time-dependently elevated cyclic AMP levels in cultured human SH-SY5Y neuronal cells, and induced neurotrophic and neuroprotective actions, mitigating oxidative stress and glutamate excitotoxicity. These actions were inhibited only by the co-administration of antagonists for all three receptor types, indicating the balanced co-involvement of GLP-1, GIP and Gcg receptors. To evaluate physiological relevance, a clinically translatable dose of Triagonist was administered subcutaneously, once daily for 7 days, to mice following a 30 g weight drop close head injury. Triagonist fully mitigated mTBI-induced visual and spatial memory deficits, evaluated at 7 and 30 days post injury. These results establish Triagonist as a novel neurotrophic/protective agent worthy of further evaluation as a TBI treatment strategy.

Elevated cyclic-AMP represses expression of exchange protein activated by cAMP (EPAC1) by inhibiting YAP-TEAD activity and HDAC-mediated histone deacetylation

Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts.Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP.Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.

A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1A4V). Moreover, we found that the administration of TUDCA in an hSOD1G93A mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.

Mechanisms underlying vasorelaxation induced in the porcine coronary arteries by Thymus linearis, Benth

Ethnopharmacological relevanceThymus linearis, Benth indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. Aim of the studyPresent study aims to investigate vasorelaxant effect of Thymus linearis and its underlying vasorelaxation mechanisms in porcine coronary artery rings. Materials and methodsAqueous-methanolic extract of aerial parts of Thymus linearis was prepared by maceration process and then bio-guided fractionation was carried out using different solvents. The effects of extract and subsequent fractions were assessed on coronary artery rings with intact and denuded endothelium. The mechanisms of vasorelaxant effect were investigated using different pharmacological tools. The in-vitro inhibitory effects of the test fractions were also assessed on purified phophodiestrases using radioenzymatic assay. Phytochemical studies were carried out using GCMS. ResultsThe aqueous-methanolic extract elicited similar relaxations in coronary artery rings with and without endothelium in dose dependent fashion and removal of endothelium did not alter this response. Further, n-butanolic fraction of Thymus liniaris (TLB) was found to be the most potent among other derived fractions. TLB did not alter the relaxation produced by endothelium dependent vasodilators in rings with intact endothelium. However, TLB significantly potentiated the relaxation elicited by cyclic AMP and cyclic GMP elevating drugs but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41–2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with TLB inhibited in a concentration-dependent manner contractions to KCl, CaCl2 and U46619 in coronary artery rings without endothelium. Further, TLB was found to non-selectively inhibit the PDE activity in concentration manner. Conclusionn-Butanolic fraction of Thymus linearis possesses endothelium independent vasorelaxant effects in coronary artery by direct acting on the smooth muscles. These effects involve the elevation of the cyclic AMP and cyclic GMP possibly through the inhibition of various PDEs. GCMS analysis revel presence of thymole and carvacrol as major constituents. Furthermore, these investigations also support the folklore use of Thymus linearis in hypertension.

Elevated Cyclic AMP Inhibits Mycobacterium tuberculosis-Stimulated T-cell IFN-γ Secretion Through Type I Protein Kinase A.

Cyclic adenosine monophosphate (cAMP) is critical in immune regulation, and its role in tuberculosis infection remains unclear. We determined the levels of cAMP in peripheral blood mononuclear cells (PBMC) from tuberculosis patients and the mechanisms for cAMP suppression of IFN-γ production. PBMC from tuberculosis patients contained significantly elevated cAMP than latent tuberculosis infected subjects (LTBI), with an inverse correlation with IFN-γ production. Consistent with this, the expression of cAMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun were reduced in tuberculosis patients compared with LTBI. PKA type I specific cAMP analogs inhibited Mtb-stimulated IFN-g production by PBMC through suppression of Mtb-induced IFN-γ promoter binding activities of CREB, ATF-2, and c-Jun and also miR155, the target miRNA of these transcription factors. Neutralizing both IL-10 and TGF-β1 or supplementation of IL-12 restored cAMP-suppressed IFN-g production. We conclude that increased cAMP inhibits IFN-g production through PKA type I pathway in tuberculosis infection.

Altered GNAS imprinting due to folic acid deficiency contributes to poor embryo development and may lead to neural tube defects.

Disturbed epigenetic modifications have been linked to the pathogenesis of Neural Tube Defects (NTDs) in those with folate deficiency during pregnancy. However, evidence is lacking to delineate the critical region in epigenome regulated by parental folic acid and mechanisms by which folate deficiency affects normal embryogenesis. Our data from clinical samples revealed the presence of aberrant DNA methylation in GNAS imprinting cluster in NTD samples with low folate concentrations. Results from mouse models indicated that the establishment of GNAS imprinting was influenced by both maternal and paternal folate-deficient diets. Such aberrant GNAS imprinting was present prior to the gametogenesis period. Imprinting in Exon1A/GNAS gDMR was abolished in both spermatozoa and oocytes upon treating with a parental folate-deficient diet (3.6% in spermatozoa, 9.8% in oocytes). Interestingly, loss of imprinting in the GNAS gene cluster altered chromatin structure to an overwhelmingly open structure (58.48% in the folate-free medium group vs. 39.51% in the folate-normal medium group; P < 0.05), and led to a disturbed expression of genes in this region. Furthermore, an elevated cyclic AMP levels was observed in folate acid deficiency group. Our results imply that GNAS imprinting plays major roles in folic acid metabolism regulation during embryogenesis. Aberrant GNAS imprinting is an attribute to NTDs, providing a new perspective for explaining the molecular mechanisms by which folate supplementation in human pregnancy provides protection from NTDs.

Elevated cyclic AMP levels promote BRAFCA/Pten−/− mouse melanoma growth but pCREB is negatively correlated with human melanoma progression

Melanocyte development and differentiation are regulated by cAMP, which is produced by the adenylate cyclase (AC) enzyme upon activation of the melanocortin-1-receptor (MC1R). Individuals carrying single amino acid substitution variants of MC1R have impaired cAMP signaling and higher risk of melanoma. However, the contribution of AC to this risk is not clear. Downstream of AC, the phosphorylated transcription factor, cyclic AMP Responsive Element Binding Protein (pCREB), which is activated by protein kinase A, regulates the expression of several genes including the melanocyte master regulator MITF. The roles of AC and CREB in melanoma development and growth are not well understood. Here, we investigated the effect of topical application of AC inhibitor on BrafCA/Pten−/− mouse melanoma development. We show that AC inhibitor delays melanoma growth independent of MAPK pathway activity and melanin content. Next, employing a primary melanoma tissue microarray and quantitative immunohistochemistry, we show that pCREB levels are positively correlated with the proliferative status of melanoma, but low pCREB expression is associated with tumor aggressiveness and metastatic recurrence. These data suggest that low cAMP signaling inhibits tumor growth but is a predictor of melanoma aggressiveness.

Misoprostol Inhibits Lipopolysaccharide-Induced Pro-inflammatory Cytokine Production by Equine Leukocytes.

Pro-inflammatory cytokines including tumor necrosis factor α (TNFα), IL-1β, IL-6, and IL-8 are potent immune mediators that exacerbate multiple equine diseases such as sepsis and laminitis. Unfortunately, safe and effective cytokine-targeting therapies are lacking in horses; therefore, novel mechanisms of inhibiting cytokine production are critically needed. One potential mechanism for inhibiting cytokine synthesis is elevation of intracellular cyclic AMP (cAMP). In human leukocytes, intracellular cAMP production is induced by activation of E-prostanoid (EP) receptors 2 and 4. These receptors can be targeted by the EP2/4 agonist and prostaglandin E1 analog, misoprostol. Misoprostol is currently used as a gastroprotectant in horses but has not been evaluated as a cytokine-targeting therapeutic. Thus, we hypothesized that misoprostol treatment would inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-stimulated equine leukocytes in an in vitro inflammation model. To test this hypothesis, equine leukocyte-rich plasma (LRP) was collected from 12 healthy adult horses and used to model LPS-mediated inflammatory signaling. LRP was treated with varying concentrations of misoprostol either before (pretreated) or following (posttreated) LPS stimulation. LRP supernatants were assayed for 23 cytokines using an equine-specific multiplex bead immunoassay. Leukocytes were isolated from LRP, and leukocyte mRNA levels of four important cytokines were evaluated via RT-PCR. Statistical differences between treatments were determined using one-way RM ANOVA (Holm–Sidak post hoc testing) or Friedman’s RM ANOVA on Ranks (SNK post hoc testing), where appropriate (p < 0.05, n = 3–6 horses). These studies revealed that misoprostol pre- and posttreatment inhibited LPS-induced TNFα and IL-6 protein production in equine leukocytes but had no effect on IL-8 protein. Interestingly, misoprostol pretreatment enhanced IL-1β protein synthesis following 6 h of LPS stimulation, while misoprostol posttreatment inhibited IL-1β protein production after 24 h of LPS stimulation. At the mRNA level, misoprostol pre- and posttreatment inhibited LPS-induced TNFα, IL-1β, and IL-6 mRNA production but did not affect IL-8 mRNA. These results indicate that misoprostol exerts anti-inflammatory effects on equine leukocytes when applied before or after a pro-inflammatory stimulus. However, the effects we observed were cytokine-specific and sometimes differed at the mRNA and protein levels. Further studies are warranted to establish the inhibitory effects of misoprostol on equine cytokine production in vivo.

Gastric Acid Secretion from Parietal Cells Is Mediated by a Ca2+ Efflux Channel in the Tubulovesicle

Gastric acid secretion by parietal cells requires trafficking and exocytosis of H/K-ATPase-rich tubulovesicles (TVs) toward apical membranes in response to histamine stimulation via cyclic AMP elevation. Here, we found that TRPML1 (ML1), a protein that is mutated in type IV mucolipidosis (ML-IV), is a tubulovesicular channel essential for TV exocytosis and acid secretion. Whereas ML-IV patients are reportedly achlorhydric, transgenic overexpression of ML1 in mouse parietal cells induced constitutive acid secretion. Gastric acid secretion was blocked and stimulated by ML1 inhibitors and agonists, respectively. Organelle-targeted Ca2+ imaging and direct patch-clamping of apical vacuolar membranes revealed that ML1 mediates a PKA-activated conductance on TV membranes that is required for histamine-induced Ca2+ release from TV stores. Hence, we demonstrated that ML1, acting as a Ca2+ channel in TVs, links transmitter-initiated cyclic nucleotide signaling with Ca2+-dependent TV exocytosis in parietal cells, providing a regulatory mechanism that could be targeted to manage acid-related gastric diseases.

Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates

Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons.

Protein kinase A regulates C-terminally truncated CaV 1.2 in Xenopus oocytes: roles of N- and C-termini of the α1C subunit.

β-Adrenergic stimulation enhances Ca2+ entry via L-type CaV 1.2 channels, causing stronger contraction of cardiac muscle cells. The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of CaV 1.2 remain controversial despite extensive research. We show that PKA regulation of CaV 1.2 can be reconstituted in Xenopus oocytes when the distal C-terminus (dCT) of the main subunit, α1C , is truncated. The PKA upregulation of CaV 1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase-anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the β subunit of CaV 1.2. The gating element within the initial segment of the N-terminus of the cardiac isoform of α1C is essential for the PKA effect. We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of CaV 1.2 in the heart. β-Adrenergic stimulation enhances Ca2+ currents via L-type, voltage-gated CaV 1.2 channels, strengthening cardiac contraction. The signalling via β-adrenergic receptors (β-ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post-translational proteolytic truncation of the distal C-terminus (dCT) of the main (α1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated CaV 1.2 in Xenopus oocytes, which previously failed with the non-truncated α1C . cAMP and the purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced CaV 1.2 currents by ∼40% (rabbit α1C ) to ∼130% (mouse α1C ). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase-anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The CaV β2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long-N-terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV 1.2 in the heart. We hypothesize that PKA is acting on CaV 1.2, in part, by affecting a structural 'scaffold' comprising the interacting cytosolic N- and C-termini of α1C .