cAMP-PKA Pathway Research Articles

Chenodeoxycholic acid stimulates Cl− secretion via cAMP signaling and increases cystic fibrosis transmembrane conductance regulator phosphorylation in T84 cells

High levels of chenodeoxycholic acid (CDCA) and deoxycholic acid stimulate Cl(-) secretion in mammalian colonic epithelia. While different second messengers have been implicated in this action, the specific signaling pathway has not been fully delineated. Using human colon carcinoma T84 cells, we elucidated this cascade assessing Cl(-) transport by measuring I(-) efflux and short-circuit current (Isc). CDCA (500 μM) rapidly increases I(-) efflux, and we confirmed by Isc that it elicits a larger response when added to the basolateral vs. apical surface. However, preincubation with cytokines increases the monolayer responsiveness to apical addition by 55%. Nystatin permeabilization studies demonstrate that CDCA stimulates an eletrogenic apical Cl(-) but not a basolateral K(+) current. Furthermore, CDCA-induced Isc was inhibited (≥67%) by bumetanide, BaCl2, and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172. CDCA-stimulated Isc was decreased 43% by the adenylate cyclase inhibitor MDL12330A and CDCA increases intracellular cAMP concentration. The protein kinase A inhibitor H89 and the microtubule disrupting agent nocodazole, respectively, cause 94 and 47% reductions in CDCA-stimulated Isc. Immunoprecipitation with CFTR antibodies, followed by sequential immunoblotting with Pan-phospho and CFTR antibodies, shows that CDCA increases CFTR phosphorylation by approximately twofold. The rapidity and side specificity of the response to CDCA imply a membrane-mediated process. While CDCA effects are not blocked by the muscarinic receptor antagonist atropine, T84 cells possess transcript and protein for the bile acid G protein-coupled receptor TGR5. These results demonstrate for the first time that CDCA activates CFTR via a cAMP-PKA pathway involving microtubules and imply that this occurs via a basolateral membrane receptor.

Modeling mutant phenotypes and oscillatory dynamics in the Saccharomyces cerevisiae cAMP-PKA pathway

Background The cyclic AMP-Protein Kinase A (cAMP-PKA) pathway is an evolutionarily conserved signal transduction mechanism that regulates cellular growth and differentiation in animals and fungi. We present a mathematical model that recapitulates the short-term and long-term dynamics of this pathway in the budding yeast, Saccharomyces cerevisiae. Our model is aimed at recapitulating the dynamics of cAMP signaling for wild-type cells as well as single (pde1Δ and pde2Δ) and double (pde1Δpde2Δ) phosphodiesterase mutants.Results Our model focuses on PKA-mediated negative feedback on the activity of phosphodiesterases and the Ras branch of the cAMP-PKA pathway. We show that both of these types of negative feedback are required to reproduce the wild-type signaling behavior that occurs on both short and long time scales, as well as the the observed responses of phosphodiesterase mutants. A novel feature of our model is that, for a wide range of parameters, it predicts that intracellular cAMP concentrations should exhibit decaying oscillatory dynamics in their approach to steady state following glucose stimulation. Experimental measurements of cAMP levels in two genetic backgrounds of S. cerevisiae confirmed the presence of decaying cAMP oscillations as predicted by the model.Conclusions Our model of the cAMP-PKA pathway provides new insights into how yeast respond to alterations in their nutrient environment. Because the model has both predictive and explanatory power it will serve as a foundation for future mathematical and experimental studies of this important signaling network.

Dopaminergic modulation of axonal potassium channels and action potential waveform in pyramidal neurons of prefrontal cortex

Voltage-gated K(+) (KV) channels play critical roles in shaping neuronal signals. KV channels distributed in the perisomatic regions and thick dendrites of cortical pyramidal neurons have been extensively studied. However, the properties and regulation of KV channels distributed in the thin axons remain unknown. In this study, by performing somatic and axonal patch-clamp recordings from layer 5 pyramidal neurons of prefrontal cortical slices, we showed that the rapidly inactivating A-currents mediated the transient K(+) currents evoked by action potential (AP) waveform command (KAP) at the soma, whereas the rapidly activating but slowly inactivating KV1-mediated D-currents dominated the KAP at the axon. In addition, activation of D1-like receptors for dopamine decreased the axonal K(+) currents, as a result of an increase in the activity of cAMP-PKA pathway. In contrast, activation of D2-like receptors showed an opposite effect on the axonal K(+) currents. Further experiments demonstrated that functional D1-like receptors were expressed at the main axon trunk and their activation could broaden the waveforms of axonal APs. Together, these results show that axonal KV channels were subjected to dopamine modulation, and this modulation could regulate the waveforms of propagating APs at the axon, suggesting an important role of dopaminergic modulation of axonal KV channels in regulating neuronal signalling.

Evidence for Gating Roles of Protein Kinase A and Protein Kinase C in Estradiol-Induced Luteinizing Hormone Receptor (lhcgr) Expression in Zebrafish Ovarian Follicle Cells

Estradiol (E2) stimulates luteinizing hormone receptor (lhcgr) expression in zebrafish follicle cells via nuclear estrogen receptors (nERs) that are likely expressed on the membrane, and lhcgr responds to E2 in a biphasic manner during 24-h treatment. These observations raise an interesting question on the signaling mechanism underlying E2 regulation, in particular the biphasic response of lhcgr expression. In the present study, we demonstrated that E2 regulation of lhcgr was significantly influenced by the activity of cAMP-PKA pathway. Activation of cAMP-PKA pathway by forskolin or db-cAMP suppressed E2-stimulated lhcgr expression in short-term (3 h) but enhanced its effect in long-term (24 h), suggesting differential roles of PKA at these two phases of lhcgr response. PKA inhibitor H89 showed reversed effects. In contrast, PKC pathway had consistent permissive effect on E2-induced lhcgr expression as evidenced by strong inhibition of E2 effect by PKC inhibitors GF109203X and Ro-31-8220 at both 3 and 24 h. One of the mechanisms by which PKA and PKC gated E2 effect might be through regulating nERs, particularly esr2a. Despite the strong influence of PKA and PKC, our data did not suggest direct mediating roles for these two pathways in E2 stimulation of lhcgr expression; yet they likely play critical gating roles in E2 signal transduction. As a follow-up study to our previous report on E2 regulation of gonadotropin receptors in the zebrafish ovary, the present study provides further evidence for the involvement of classical intracellular signal transduction pathways in E2 stimulation of lhcgr expression in the follicle cells.

Prostaglandin E2 promotes Th1 differentiation via synergistic amplification of IL-12 signalling by cAMP and PI3-kinase

T helper 1 (Th1) cells have critical roles in various autoimmune and proinflammatory diseases. cAMP has long been believed to act as a suppressor of IFN-γ production and Th1 cell-mediated immune inflammation. Here we show that cAMP actively promotes Th1 differentiation by inducing gene expression of cytokine receptors involved in this process. PGE2 signalling through EP2/EP4 receptors mobilizes the cAMP-PKA pathway, which induces CREB- and its co-activator CRTC2-mediated transcription of IL-12Rβ2 and IFN-γR1. Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28. Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo. These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea

BackgroundThe transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development.ResultsWe evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom.ConclusionsWe implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea.

Dysfunctional mitochondria modulate cAMP-PKA signaling and filamentous and invasive growth of Saccharomyces cerevisiae.

Mitochondrial metabolism is targeted by conserved signaling pathways that mediate external information to the cell. However, less is known about whether mitochondrial dysfunction interferes with signaling and thereby modulates the cellular response to environmental changes. In this study, we analyzed defective filamentous and invasive growth of the yeast Saccharomyces cerevisiae strains that have a dysfunctional mitochondrial genome (rho mutants). We found that the morphogenetic defect of rho mutants was caused by specific downregulation of FLO11, the adhesin essential for invasive and filamentous growth, and did not result from general metabolic changes brought about by interorganellar retrograde signaling. Transcription of FLO11 is known to be regulated by several signaling pathways, including the filamentous-growth-specific MAPK and cAMP-activated protein kinase A (cAMP-PKA) pathways. Our analysis showed that the filamentous-growth-specific MAPK pathway retained functionality in respiratory-deficient yeast cells. In contrast, the cAMP-PKA pathway was downregulated, explaining also various phenotypic traits observed in rho mutants. Thus, our results indicate that dysfunctional mitochondria modulate the output of the conserved cAMP-PKA signaling pathway.

Group II metabotropic glutamate receptors modify N-methyl-D-aspartate receptors via Src kinase

Group II metabotropic glutamate receptors (mGluR2/3) have emerged as important targets for the treatment of schizophrenia. Since hypofunction of N-methyl-D-aspartate receptors (NMDARs) has also been implicated in the etiology of schizophrenia, we examined whether postsynaptic mGluR2/3 regulate NMDAR function. Activation of mGluR2/3 significantly decreased the ratio of AMPA-to-NMDA excitatory postsynaptic currents at Schaffer Collateral-CA1 synapses and enhanced the peak of NMDA-evoked currents in acutely isolated CA1 neurons. The mGluR2/3-mediated potentiation of NMDAR currents was selective for GluN2A-containing NMDARs and was mediated by the Src family kinase Src. Activation of mGluR2/3 inhibited the adenylyl cyclase-cAMP-PKA pathway and thereby activated Src by inhibiting its regulatory C-terminal Src kinase (Csk). We suggest a novel model of regulation of NMDARs by Gi/o-coupled receptors whereby inhibition of the cAMP-PKA pathway via mGluR2/3 activates Src kinase and potentiates GluN2A-containing NMDAR currents. This represents a potentially novel mechanism to correct the hypoglutamatergic state found in schizophrenia.

Neuropeptide PACAP in mouse liver ischemia and reperfusion injury: Immunomodulation by the cAMP-PKA pathway

Hepatic ischemia and reperfusion injury (IRI), an exogenous antigen-independent local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The immune system and the nervous system maintain extensive communication and mount a variety of integrated responses to danger signals through intricate chemical messengers. This study examined the function and potential therapeutic potential of neuropeptide pituitary adenylate cyclase-activating polypeptides (PACAP) in a murine model of partial liver "warm" ischemia (90 minutes) followed by reperfusion. Liver IRI readily triggered the expression of intrinsic PACAP and its receptors, whereas the hepatocellular damage was exacerbated in PACAP-deficient mice. Conversely, PACAP27, or PACAP38 peptide monotherapy, which elevates intracellular cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) signaling, protected livers from IRI, as evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. The liver protection rendered by PACAP peptides was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and selectively augmented hepatic interleukin (IL)-10 expression. Strikingly, PKA inhibition readily restored liver damage in otherwise IR-resistant, PACAP-conditioned mice. In vitro, PACAP treatment not only diminished macrophage tumor necrosis factor alpha/IL-6/IL-12 levels in a PKA-dependent manner, but also prevented necrosis and apoptosis in primary mouse hepatocyte cultures. Our novel findings document the importance of PACAP-mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to manage liver inflammation and IRI in transplant patients.

Evaluation of Pathogenicity of Candida albicans in Germination- ready States Using a Silkworm Infection Model

We previously developed an N-acetyl-D-glucosamine (GlcNAc) medium which induces Candida albicans to undergo a yeast-to-hyphal transition through a cAMP-PKA pathway. Microarray analysis demonstrated that 18 genes, including ALS3 that encodes a cell wall adhesion, were upregulated by 30-min incubation of yeast cells at 37°C in the GlcNAc medium. To investigate the differences between morphological transition and morphotype in C. albicans as a consequence of infection, this study utilized a silkworm infection model as an invertebrate mini-host. We prepared 3 different conditions of C. albicans cells in vitro by changing the incubation times in the GlcNAc medium: yeast-form cells at 0 min (Y0 cells), yeast-form cells in germination-ready state at 60 min (Y60 cells), and hyphal cells at 120 min (H120 cells), and compared their pathogenicities. We performed the infection study at various temperatures to find temperature-dependent virulence factors in vivo. Y60 cells in germination-ready state in the GlcNAc medium showed higher pathogenicity in vivo compared to Y0 and H120 cells at 30°C. Y60 cells proliferated in silkworms 24 h post-injection at 30°C, whereas the other 2 cell types did not. In vitro analysis demonstrated that Y60 cells, but not Y0 cells, germinated in the silkworm hemolymph at 30°C. However, Y0 and Y60 cells showed a similar degree of germination in the silkworm hemolymph at 37°C, although no significant difference in silkworm survival after infection with each cell type was observed at 37°C. These results suggested that the germination-ready state induced by the GlcNAc medium contributed to virulence in the silkworm.

Phosphodiesterase activity is regulated by CC2D1A that is implicated in non-syndromic intellectual disability

BackgroundCyclic adenosine 3′,5′-monophosphate (cAMP) is a key regulator of many cellular processes, including in the neuronal system, and its activity is tuned by Phosphodiesterase (PDE) activation. Further, the CC2D1A protein, consisting of N-Terminal containing four DM14 domains and C-terminal containing C2 domain, was shown to regulate the cAMP-PKA pathway. A human deletion mutation lacking the fourth DM14 and the adjacent C2 domain results in Non Syndromic Intellectual Disability (NSID) also referred to as Non Syndromic Mental Retardation (NSMR).FindingsHere we demonstrate that in Mouse Embryonic Fibroblasts (MEF) CC2D1A co-localizes with PDE4D in the cytosol before cAMP stimulation and on the periphery after stimulation, and that the movement to the periphery requires the full-length CC2D1A. In CC2D1A mouse mutant cells, the absence of three of the four DM14 domains abolishes migration of the complex to the periphery and causes constitutive phosphorylation of PDE4D Serine 126 (S126) via the cAMP-dependent protein kinase A (PKA) resulting in PDE4D hyperactivity. Suppressing PDE4D activity with Rolipram in turn restores the down-stream phosphorylation of the “cAMP response element-binding protein” (CREB) that is defective in mouse mutant cells.ConclusionOur findings suggest that CC2D1A is a novel regulator of PDE4D. CC2D1A interacts directly with PDE4D regulating its activity and thereby fine-tuning cAMP-dependent downstream signaling. Based on our in vitro evidence we propose a model which links CC2D1A structure and function to cAMP homeostasis thereby affecting CREB phosphorylation. We speculate that CC2D1A and/or PDE4D may be promising targets for therapeutic interventions in many disorders with impaired PDE4D function such as NSID.

ECM17-dependent methionine/cysteine biosynthesis contributes to biofilm formation in Candida albicans

Candida albicans has become the fourth leading pathogen of nosocomial bloodstream infections largely due to biofilm formation on implanted medical devices. Previous microarray data indicated that almost all genes in methionine (Met)/cysteine (Cys) biosynthesis pathway were up-regulated during biofilm formation, especially during the adherence period. In this work, we studied the role of Met/Cys biosynthesis pathway by disrupting ECM17, a gene encoding sulfite reductase in C. albicans. It was found that the ecm17Δ/Δ mutant failed to catalyze the biochemical reaction from sulfite to H2S and hardly grew in media lacking Met and Cys. NaSH, the donor of H2S, dose-dependently improved the growth of ecm17Δ/Δ in media lacking a sulfur source. Sufficient Met/Cys supply inhibited the expression of ECM17 in a dose-dependent manner. These results validated the important role of ECM17 in Met/Cys biosynthesis. Interestingly, the ecm17Δ/Δ mutant showed diminished ability to form biofilm, attenuated adhesion on abiotic substrate and decreased filamentation on solid SLD medium, especially under conditions lacking Met/Cys. Further results indicated that ECM17 affected the expressions of ALS3, CSH1, HWP1 and ECE1, and that the cAMP–protein kinase A (PKA) pathway was associated with ECM17 and Met/Cys biosynthesis pathway. These results provide new insights into the role of Met/Cys biosynthesis pathway in regulating cAMP-PKA pathway and benefiting biofilm formation.

The inhibitory activity of linalool against the filamentous growth and biofilm formation inCandida albicans

Candida spp. are part of the natural human microbiota, but they also represent important opportunistic human pathogens. Biofilm-associated Candida albicans infections are clinically relevant due to their high levels of resistance to traditional antifungal agents. In this study, we investigated the ability of linalool to inhibit the formation of C. albicans biofilms and reduce existing C. albicans biofilms. Linalool exhibited antifungal activity against C. albicans ATCC 14053, with a minimum inhibitory concentration (MIC) of 8 mM. Sub-MIC concentrations of linalool also inhibited the formation of germ tubes and biofilms in that strain. The defective architecture composition of C. albicans biofilms exposed to linalool was characterized by scanning electron microscopy. The expression levels of the adhesin genes HWP1 and ALS3 were downregulated by linalool, as assessed by real-time RT-PCR. The expression levels of CYR1 and CPH1, which encode components of the cAMP-PKA and MAPK hyphal formation regulatory pathways, respectively, were also suppressed by linalool, as was the gene encoding their upstream regulator, Ras1. The expression levels of long-term hyphae maintenance associated genes, including UME6, HGC1, and EED1, were all suppressed by linalool. These results indicate that linalool may have therapeutic potential in the treatment of candidiasis associated with medical devices because it interferes with the morphological switch and biofilm formation of C. albicans.

Glucagon-like peptide 1 potentiates glucotoxicity-diminished insulin secretion via stimulation of cAMP-PKA signaling in INS-1E cells and mouse islets

Glucagon-like peptide-1 (GLP-1)-enhanced insulin secretion is mainly mediated by cAMP-PKA and cAMP-Epac2 signaling pathways at physiological glucose concentrations. However the cellular mechanisms underlying the insulinotropic action of GLP-1 at glucotoxicity remain largely unknown. In the present study, we examined the effects of GLP-1 on glucotoxicity-diminished insulin secretion and explored the roles of these two cAMP-linked pathways in mediating the effects of GLP-1 under glucotoxic conditions. Consistent with the previous reports, exposure of INS-1E cells and mouse islets to 30mM glucose for 72h almost abolished glucose-stimulated insulin secretion. Addition of 10nM GLP-1 significantly increased glucose-stimulated insulin secretion. This was not due to a protective effect of GLP-1 against glucotoxicity-induced apoptosis but instead improvement of the secretory capacity of the insulin-secreting β-cells. It is of note that GLP-1 preferentially increased the expression and activity of PKA, whereas had no effects on Epac2 at high glucose. In correlation with the observations, treatment of INS-1E cells with the specific PKA inhibitor Rp-cAMPS completely abolished the insulinotropic action of GLP-1, whereas knock-down of Epac2 did not interfere the effects of GLP-1. Moreover, GLP-1 did not increase further insulin secretion in the presence of the PKA agonist 6-Bnz-cAMP-AM. By contrast, it produced additional enhancement of insulin secretion when Epac2 was maximally stimulated by its selective agonist 8-pCPT-2′-O-Me-cAMP-AM. Taken together, our results suggest that GLP-1 potentiates glucotoxicity-diminished insulin secretion mainly through cAMP-PKA signaling pathway.

PDE4B Modulates Glucocorticoid Sensitivity in Childhood Acute Lymphoblastic Leukemia

AbstractAbstract 530Although cure rates of childhood acute lymphoblastic leukemia (ALL) have improved dramatically, a substantial portion of children still relapse and the prognosis of relapsed ALL is extremely poor. Therefore, a better understanding of molecular determinants of drug resistance in ALL is imperative for the development of more efficacious and individualized therapy, particularly in the context of relapsed disease. In a recent genome-wide association study of 2,534 children with ALL, we identified that genetic variation in PDE4B– phosphodiesterase 4B–strongly influenced the risk of ALL relapse across various ALL treatment regimens (Nat Genet 2011: 43:237). While PDE4B is the predominant phosphodiesterase in lymphoid tissue and a major regulator of cyclic AMP, its role in ALL pathobiology is largely unknown. To this end, we sought to characterize the molecular mechanisms by which PDE4B modulates antileukemic drug sensitivity in ALL. We first characterized PDE4B expression in ALL blasts at diagnosis and its relationship with drug response in vivo in 3 independent cohorts of children with ALL. In 191 children with newly diagnosed ALL enrolled on the COG P9906 protocol, PDE4B expression in ALL blasts was positively correlated with minimal residual disease status at the end of remission induction (P=0.0096). Higher PDE4B expression was also associated with slower early response to induction therapy in COG 1961 (N=82, P=0.019). In 275 children with newly-diagnosed ALL enrolled on the Shanghai Children’s Medical Center ALL05 study, we determined that PDE4B2 was the predominant isoform of PDE4B in ALL blasts (P<0.0001); there was also a trend that children with poor in vivo response to the upfront single-agent prednisone treatment had higher PDE4B2 expression in the diagnostic blasts (P=0.042). In parallel, shRNA-mediated knock-down of PDE4B in a glucocorticoid-sensitive (i.e., Nalm6) and a glucocorticoid-resistant (i.e., UOCB1) ALL cell line significantly potentiated cytotoxic effects of prednisolone, whereas Nalm6 and CEM ALL cells over-expressing PDE4B2 were significantly more resistant to prednisolone compared to cells transduced with empty vectors. Sensitization to glucocorticoid was further amplified by forskolin, a stimulator of cAMP synthesis, and was concomitant with activation of PKA as determined by CREB phosphorylation, suggesting that the effects of PDE4B on glucocorticoid sensitivity involve signaling of the cAMP-PKA cascade. Importantly, PDE4B knockdown by shRNA and pharmacologic inhibition by rolipram in Nalm6 and UOCB1 cells consistently led to upregulation of BIM, a key apoptosis regulator and a critical mediator of glucocorticoid sensitivity in lymphoid cells. In both cells lines, BIM upregulation following PDE4B inhibition was enhanced by forskolin but suppressed by PKA inhibitor H89, indicating that BIM might act as a downstream effector of cAMP-PKA signaling in ALL. Finally, we evaluated effects of small molecule regulators of the PDE-cAMP pathway (namely, forskolin and rolipram) on glucocorticoid response in primary ALL cells (N=23) in vitro. Measuring IC50 by MTT assay, increased cytotoxicity was observed in 16 (70%), 8 (35%), and 17 (74%) cases, when forskolin, rolipram, or both were added to prednisolone, respectively. In conclusion, tumor expression of PDE4B at diagnosis was associated with poorer early treatment response in ALL, particularly resistance to glucocorticoids in vivo. Inhibition of PDE4B sensitized both cultured and primary ALL cells to glucocorticoids via activating the cAMP-PKA pathway and subsequent upregulation of BIM. Disclosures:No relevant conflicts of interest to declare.

Angiotensin-(1–7) in Paraventricular Nucleus Modulates Sympathetic Activity and Cardiac Sympathetic Afferent Reflex in Renovascular Hypertensive Rats

BackgroundExcessive sympathetic activity contributes to the pathogenesis and progression of hypertension. Enhanced cardiac sympathetic afferent reflex (CSAR) is involved in sympathetic activation. This study was designed to determine the roles of angiotensin (Ang)-(1–7) in paraventricular nucleus (PVN) in modulating sympathetic activity and CSAR and its signal pathway in renovascular hypertension.Methodology/Principal FindingsRenovascular hypertension was induced with two-kidney, one-clip method. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in sinoaortic-denervated and cervical-vagotomized rats with anesthesia. CSAR was evaluated with the RSNA and MAP responses to epicardial application of capsaicin. PVN microinjection of Ang-(1–7) and cAMP analogue db-cAMP caused greater increases in RSNA and MAP, and enhancement in CSAR in hypertensive rats than in sham-operated rats, while Mas receptor antagonist A-779 produced opposite effects. There was no significant difference in the angiotensin-converting enzyme 2 (ACE2) activity and Ang-(1–7) level in the PVN between sham-operated rats and hypertensive rats, but the Mas receptor protein expression in the PVN was increased in hypertensive rats. The effects of Ang-(1–7) were abolished by A-779, adenylyl cyclase inhibitor SQ22536 or protein kinase A (PKA) inhibitor Rp-cAMP. SQ22536 or Rp-cAMP reduced RSNA and MAP in hypertensive rats, and attenuated the CSAR in both sham-operated and hypertensive rats.ConclusionsAng-(1–7) in the PVN increases RSNA and MAP and enhances the CSAR, which is mediated by Mas receptors. Endogenous Ang-(1–7) and Mas receptors contribute to the enhanced sympathetic outflow and CSAR in renovascular hypertension. A cAMP-PKA pathway is involved in the effects of Ang-(1–7) in the PVN.

Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure-function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs-cAMP-PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure-function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

MyRIP interaction with MyoVa on secretory granules is controlled by the cAMP-PKA pathway.

Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)-anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited by small interfering RNA knockdown of MyRIP, which also reduced cAMP-mediated hormone secretion. Demonstrating the importance of this phosphorylation, nonphosphorylatable and phosphomimic Rph-3A mutants significantly altered hormone release when PKA was activated. These data suggest that MyRIP only forms a functional protein complex with BR-MyoVa on SGs when cAMP is elevated and under this condition facilitates phosphorylation of SG-associated proteins, which in turn can enhance secretion.

Group I mGluR-Mediated Inhibition of Kir Channels Contributes to Retinal Müller Cell Gliosis in a Rat Chronic Ocular Hypertension Model

Müller cell gliosis, which is characterized by upregulated expression of glial fibrillary acidic protein (GFAP), is a universal response in many retinal pathological conditions. Whether down-regulation of inward rectifying K+ (Kir) channels, which commonly accompanies the enhanced GFAP expression, could contribute to Müller cell gliosis is poorly understood. We investigated changes of Kir currents, GFAP and Kir4.1 protein expression in Müller cells in a rat chronic ocular hypertension (COH) model, and explored the mechanisms underlying Müller cell gliosis. We show that Kir currents and Kir4.1 protein expression in Müller cells were reduced significantly, while GFAP expression was increased in COH rats, and these changes were eliminated by MPEP, a group I metabotropic glutamate receptors (mGluR I) subtype mGluR5 antagonist. In normal isolated Müller cells, the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) suppressed the Kir currents and the suppression was blocked by MPEP. The DHPG effect was mediated by the intracellular Ca2+ -dependent PLC/IP3-ryanodine/PKC signaling pathway, but the cAMP-PKA pathway was not involved. Moreover, intravitreal injection of DHPG in normal rats induced changes in Müller cells, similar to those observed in COH rats. The DHPG-induced increase of GFAP expression in Müller cells was obstructed by Ba2+, suggesting the involvement of Kir channels. We conclude that overactivation of mGluR5 by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir channels.

Adipose‐derived factor CTRP9 attenuates vascular smooth muscle cell proliferation and neointimal formation

Obesity is closely associated with the progression of vascular disorders, including atherosclerosis and postangioplasty restenosis. C1q/TNF-related protein (CTRP) 9 is an adipocytokine that is down-regulated in obese mice. Here we investigated whether CTRP9 modulates neointimal hyperplasia and vascular smooth muscle cell (VSMC) proliferation in vivo and in vitro. Left femoral arteries of wild-type (WT) mice were injured by a steel wire. An adenoviral vector expressing CTRP9 (Ad-CTRP9) or β-galactosidase as a control was intravenously injected into WT mice 3 d before vascular injury. Delivery of Ad-CTRP9 significantly attenuated the neointimal thickening and the number of bromodeoxyuridine-positive proliferating cells in the injured arteries compared with that of control. Treatment of VSMCs with CTRP9 protein attenuated the proliferative and chemotactic activities induced by growth factors including platelet-derived growth factor (PDGF)-BB, and suppressed PDGF-BB-stimulated phosphorylation of ERK. CTRP9 treatment dose-dependently increased cAMP levels in VSMCs. Blockade of cAMP-PKA pathway reversed the inhibitory effect of CTRP9 on DNA synthesis and ERK phosphorylation in response to PDGF-BB. The present data indicate that CTRP9 functions to attenuate neointimal formation following vascular injury through its ability to inhibit VSMC growth via cAMP-dependent mechanism, suggesting that the therapeutic approaches to enhance CTRP9 production could be valuable for prevention of vascular restenosis after angioplasty.