Induced cAMP Accumulation Research Articles

A2B adenosine receptor activation and modulation by protein kinase C

Protein kinase C (PKC) isoforms regulate many important signaling pathways. Here, we report that PKC activation by phorbol 12-myristate 13-acetate (PMA) enhanced A2B adenosine receptor (AR)-mediated, but not β2-adrenergic receptor-mediated, cAMP accumulation, in H9C2 cardiomyocyte-like and HEK293 cells. In addition to enhancement, PKC (PMA-treatment) also activated A2BAR with low Emax (H9C2 and NIH3T3 cells endogenously expressing A2BAR), or with high Emax (A2BAR-overexpressing HEK293 cells) to induce cAMP accumulation. A2BAR activation induced by PKC was inhibited by A2BAR and PKC inhibitors but enhanced by A2BAR overexpression. Gαi isoforms and PKCγ isoform were found to be involved in both enhancement of A2BAR function and A2BAR activation. Thus,we establish PKC as an endogenous modulator and activator of A2BAR, involving Giα and PKCγ. Depending on signaling pathway, PKC could activate and enhance,or alternatively inhibit A2BAR activity. These findings are relevant to common functions of A2BAR and PKC, e.g. cardioprotection and cancer progression/treatment.

Artesunate inhibits PDE4 leading to intracellular cAMP accumulation, reduced ERK/MAPK signaling, and blockade of influenza A virus vRNP nuclear export

Influenza A viruses (IAV) have been a major cause of mortality. Given the potential for future deadly pandemics, effective drugs are needed for the treatment of severe influenzas, such as those caused by H5N1 IAV. The anti-malaria drugs artemisinin and its derivates, including artesunate (AS), have been reported to have broad antiviral activities. Here, we showed AS's antiviral activity against H5N1, H1N1, H3N2 and oseltamivir-resistant influenza A(H1N1)virus in vitro. Moreover, we showed that AS treatment significantly protected mice from lethal challenges with H1N1 and H5N1 IAV. Strikingly, the combination of AS and peramivir treatment significantly improved survival outcomes compared to their monotherapy with either AS or peramivir. Furthermore, we demonstrated mechanistically that AS affected the later stages of IAV replication and limited nuclear export of viral ribonucleoprotein (vRNP) complexes. In A549 cells, we demonstrated for the first time that AS treatment induced cAMP accumulation via inhibiting PDE4, and consequently reduced ERK phosphorylation and blocked IAV vRNP export, and thus suppressed IAV replication. These AS's effects were reversed by the pre-treatment with a cAMP inhibitor SQ22536. Our findings suggest that AS could serve as a novel IAV inhibitor by interfering vRNP nuclear export to prevent and treat IAV infection.

Calofolic Acid-A from Calophyllum scriblitifolium Bark Has Vasorelaxant Activity via Indirect PKA Activation Caused by PI-3 Kinase Inhibition in Rat Vascular Smooth Muscle Cells.

Previously, we isolated 2R,3S,15R-calofolic acids (CAs) from Calophyllum scriblitifolium bark, which showed vasorelaxant activity on phenylephrine (PE)-precontracted rat aortic rings. Although the effect was suggested to be induced via an extracellular Ca2+-independent manner and mainly acts on vascular smooth muscle, the exact mechanism of action of CAs remained unclear. Thus, this study investigated the detailed mechanism of calofolic acid-A (CA-A) induced vasorelaxation in an aortic ring specimen using rat vascular smooth muscle cells (VSMCs). The levels of PE-induced phosphorylation on MLC Ser19 decreased in VSMCs pretreated with CA-A. CA-A also decreased the phosphorylation of MYPT1 Thr696 and MYPT1 Thr853. On the other hand, CA-A increased the PE-induced phosphorylation of MYPT1 Ser695 and MYPT1 Ser668, which are reported to be phosphorylated by a cAMP-dependent protein kinase (PKA). CA-A slightly increased PKA substrate phosphorylation in a concentration-dependent manner. Furthermore, CA-A enhanced isoproterenol (ISO)-induced cAMP accumulation and PKA substrate phosphorylation. Treatment with PI-3 kinase (PI3K) inhibitor, LY294002, enhanced ISO-induced cAMP accumulation and PKA substrate phosphorylation in the same manner as CA-A treatment. Furthermore, CA-A was found to directly inhibit PI3K enzyme activity in a dose-dependent manner. Taken together, the present study indicated that CA-A induces vasorelaxation through an indirectly activated PKA-MYPT1 pathway caused by inhibition of PI3K activity.

Signaling profiles in HEK 293T cells co-expressing GLP-1 and GIP receptors.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are regarded as 'incretins' working closely to regulate glucose homeostasis. Unimolecular dual and triple agonists of GLP-1R and GIPR have shown remarkable clinical benefits in treating type 2 diabetes. However, their pharmacological characterization is usually carried out in a single receptor-expressing system. In the present study we constructed a co-expression system of both GLP-1R and GIPR to study the signaling profiles elicited by mono, dual and triple agonists. We show that when the two receptors were co-expressed in HEK 293T cells with comparable receptor ratio to pancreatic cancer cells, GIP predominately induced cAMP accumulation while GLP-1 was biased towards β-arrestin 2 recruitment. The presence of GIPR negatively impacted GLP-1R-mediated cAMP and β-arrestin 2 responses. While sharing some common modulating features, dual agonists (peptide 19 and LY3298176) and a triple agonist displayed differentiated signaling profiles as well as negative impact on the heteromerization that may help interpret their superior clinical efficacies.

Synthesis, in vitro biological investigation, and molecular dynamics simulations of thiazolopyrimidine based compounds as corticotrophin releasing factor receptor-1 antagonists

Corticotrophin releasing factor receptor-1 (CRFR1) is a potential target for treatment of depression and anxiety through modifying stress response. A series of new thiazolo[4,5-d]pyrimidine derivatives were designed, prepared and biologically evaluated as potential CRFR1 antagonists. Four compounds produced more than fifty percent inhibition in the [125I]-Tyr0-sauvagine specific binding assay. Assessment of binding affinities revealed that compound (3-(2,4-dimethoxyphenyl)-7-(dipropylamino)-5-methylthiazolo[4,5-d]pyrimidin-2(3H)-one) 8c was the best candidate with highest binding affinity (Ki = 32.1 nM). Further evaluation showed the ability of compound 8c to inhibit CRF induced cAMP accumulation in a dose response manner. Docking and molecular dynamics simulations were used to investigate potential binding modes of synthesized compounds as well as the stability of 8c-CRFR1 complex. These studies suggest similar allosteric binding of 8c compared to that of the co-crystalized ligand CP-376395 in 4K5Y pdb file.

β-Arrestin-2-Dependent Mechanism of GPR52 Signaling in Frontal Cortical Neurons.

The orphan Gαs-coupled receptor GPR52 is expressed exclusively in the brain, predominantly in circuitry relating to symptoms of neuropsychiatric and cognitive disorders such as schizophrenia. While GPR52 agonists have displayed antipsychotic and procognitive efficacy in murine models, there remains limited evidence delineating the molecular mechanisms of these effects. Indeed, previous studies have solely reported canonical cAMP signaling and CREB phosphorylation downstream of GPR52 activation. In the present study, we demonstrated that the synthetic GPR52 agonist, 3-BTBZ, equipotently induces cAMP accumulation, ERK1/2 phosphorylation, and β-arrestin-1 and -2 recruitment in transfected HEK293T cells. In cultured frontal cortical neurons, however, 3-BTBZ-induced ERK1/2 phosphorylation was significantly more potent than cAMP signaling, with a more prolonged signaling profile than that in HEK293T cells. Furthermore, knock down of β-arrestin-2 in frontal cortical neurons abolished 3-BTBZ-induced ERK1/2 phosphorylation, but not cAMP accumulation. These results suggest a β-arrestin-2-dependent mechanism for GPR52-mediated ERK1/2 signaling, which may link to cognitive function in vivo. Finally, these findings highlight the context-dependence of GPCR signaling in recombinant cells and neurons, offering new insights into translationally relevant GPR52 signaling mechanisms.

Astragaloside IV Inhibits Adipose Lipolysis and Reduces Hepatic Glucose Production via Akt Dependent PDE3B Expression in HFD-Fed Mice.

Objective: This study aims to investigate the effect of astragaloside IV on adipose lipolysis and hepatic gluconeogenesis.Methods: High-fat diet (HFD) feeding induced adipose dysfunction with enhanced endogenous glucose production in mice. The effects of Astragaloside IV on lipolysis and hepatic glucose production were investigated.Results: HFD feeding induced cAMP accumulation through reducing PDE3B expression and activity in adipose tissue. As a result, HFD feeding increased adipose lipolysis in mice. Astragaloside IV enhanced Akt phosphorylation and promoted Akt binding to PDE3B to preserve PDE3B content, resultantly reducing adipose cAMP accumulation. Knockdown of Akt1/2 diminished the effect of astragaloside IV on PDE3B induction, indicative of the role of Akt in astragaloside IV action. As a result from blocking of cAMP/PKA signaling, astragaloside IV suppressed hormone-sensitive lipase (HSL) activation and inhibited inflammation-associated adipose lipolysis. Moreover, astragaloside IV reduced ectopic fat deposition in the liver and inhibited FoxO1 activation via regulation of Akt, resultantly restraining excess hepatic glucose production.Conclusion: We showed that preserving PDE3B content by Akt is a key regulation to prevent lipolysis. Astragaloside IV inhibited lipolysis by reducing cAMP accumulation via regulation of Akt/PDE3B, contributing to limiting hepatic lipid deposition and restraining excessive hepatic glucose production.

Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor.

19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists.

Abstract LB-259: Bone targeted parathyroid hormone antagonist for prevention of breast cancer bone metastases

Abstract Breast cancer metastasis to bone is a major source of morbidity and mortality in patients. Local erosion of bone increases risk of fracture and decreases mean survival time. Currently patients are treated with bisphosphonates and denosumab. Breast cancer bone metastases secrete high local concentrations of a parathyroid hormone (PTH) analog termed parathyroid hormone-related peptide (PTHrP). PTHrP stimulates bone turnover creating cavities in the bone allowing tumor growth in the resulting space. PTH antagonists directed towards bone metastatic breast cancer have failed clinically due to drug turnover and failure to concentrate in bone. We synthesized two novel classes of PTHrP antagonists that consist of modifications to the native PTH peptide fused to the inert collagen binding domain (CBD) of ColG collagenase from Clostridium histolyticum. Fusion with the CBD directs PTH analog delivery to the bone matrix and blocks PTHrP action in vitro. Antagonist PTH(7-33)-CBD is an N-terminally truncated analog of PTH, and PTH([-1]-33)-CBD is an N-terminally extended analog of PTH. PTH(7-33)-CBD and PTH([-1]-33)-CBD bound to collagen type 1 in vitro. Neither compound induced cAMP accumulation in SaOS-2 osteosarcoma cells indicating no PTH agonist action. Both compounds partially antagonized PTH(1-34) agonist-induced cAMP accumulation in SaOS-2 cells. We used an established mouse model of breast cancer invasion of bone using a bone-trophic variant of estrogen receptor negative MDA-MB-231 breast cancer cells expressing luciferase (MDA-MB-231-BM/luc+) injected into the tibia marrow of nude mice (Day 1) to assess in vivo efficacy of PTH(7-33)-CBD. PTH(7-33)-CBD (1000 μg/kg administered IP on Day 7 and Day 28) reduced overall tumor burden in bone by 78% (P<0.0001) and reduced cortical bone destruction by 30% (P<0.05) compared to vehicle at 5 weeks post-tumor cell injection. Tartrate resistant acid phosphatase (TRAP) is released by osteoclasts and mediates bone resorption. PTH(7-33)-CBD reduced serum TRAP by 18% (P<0.05) compared to vehicle 12 weeks post-tumor cell injection. Importantly, PTH(7-33)-CBD treatment did not alter serum calcium levels or alter mouse body weight indicating no hypocalcemia or general toxicity on body weight. MDA-MB-231 cells and other breast cancer cells express the PTH/PTHrP receptor and PTHrP functions as an autocrine trophic factor in these cell lines. PTH(7-33)-CBD (30 nM) exhibited a direct cytotoxic effect against MDA-MB-231 cells in-vitro reducing cell number by 73% (P<0.0001) at 48 h with no effect on a series of non-tumor cells including MCF10A non-tumorigenic mammary cells, normal human fibroblasts (BJ5TA), and normal human T-cells (THP-1 monocytes). Taken together, these data demonstrate that PTH(7-33)-CBD reduces osteolysis and metastatic tumor burden in bone, and exhibits selective cytotoxicity against breast cancer cells in vitro. Support: Breast Cancer Research Program (BCRP) Breakthrough Award, BC150685 awarded to BGR and RCG. Citation Format: Brian G. Rowan, Tulasi Ponnapakkam, Murali Anbalagan, Yibin Kang, Robert C. Gensure. Bone targeted parathyroid hormone antagonist for prevention of breast cancer bone metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-259.

Silymarin Constituent 2,3-Dehydrosilybin Triggers Reserpine-Sensitive Positive Inotropic Effect in Perfused Rat Heart.

2,3-dehydrosilybin (DHS) is a minor flavonolignan component of Silybum marianum seed extract known for its hepatoprotective activity. Recently we identified DHS as a potentially cardioprotective substance during hypoxia/reoxygenation in isolated neonatal rat cardiomyocytes. This is the first report of positive inotropic effect of DHS on perfused adult rat heart. When applied to perfused adult rat heart, DHS caused a dose-dependent inotropic effect resembling that of catecholamines. The effect was apparent with DHS concentration as low as 10 nM. Suspecting direct interaction with β-adrenergic receptors, we tested whether DHS can trigger β agonist-dependent gene transcription in a model cell line. While DHS alone was unable to trigger β agonist-dependent gene transcription, it enhanced the effect of isoproterenol, a known unspecific β agonist. Further tests confirmed that DHS could not induce cAMP accumulation in isolated neonatal rat cardiomyocytes even though high concentrations (≥ 10 μM) of DHS were capable of decreasing phosphodiesterase activity. Pre-treatment of rats with reserpine, an indole alkaloid which depletes catecholamines from peripheral sympathetic nerve endings, abolished the DHS inotropic effect in perfused hearts. Our data suggest that DHS causes the inotropic effect without acting as a β agonist. Hence we identify DHS as a novel inotropic agent.

Characterization of Dahl salt-sensitive rats with genetic disruption of the A2B adenosine receptor gene: implications for A2B adenosine receptor signaling during hypertension.

The A(2B) adenosine receptor (AR) has emerged as a unique member of the AR family with contrasting roles during acute and chronic disease states. We utilized zinc-finger nuclease technology to create A(2B)AR gene (Adora2b)-disrupted rats on the Dahl salt-sensitive (SS) genetic background. This strategy yielded a rat strain (SS-Adora2b mutant rats) with a 162-base pair in-frame deletion of Adora2b that included the start codon. Disruption of A(2B)AR function in SS-Adora2b mutant rats was confirmed by loss of agonist (BAY 60-6583 or NECA)-induced cAMP accumulation and loss of interleukin-6 release from isolated fibroblasts. In addition, BAY 60-6583 produced a dose-dependent increase in glucose mobilization that was absent in SS-Adora2b mutants. Upon initial characterization, SS-Adora2b mutant rats were found to exhibit increased body weight, a transient delay in glucose clearance, and reduced proinflammatory cytokine production following challenge with lipopolysaccharide (LPS). In addition, blood pressure was elevated to a greater extent (∼15-20 mmHg) in SS-Adora2b mutants as they aged from 7 to 21 weeks. In contrast, hypertension augmented by Ang II infusion was attenuated in SS-Adora2b mutant rats. Despite differences in blood pressure, indices of renal and cardiac injury were similar in SS-Adora2b mutants during Ang II-augmented hypertension. We have successfully created and validated a new animal model that will be valuable for investigating the biology of the A(2B)AR. Our data indicate varying roles for A(2B)AR signaling in regulating blood pressure in SS rats, playing both anti- and prohypertensive roles depending on the pathogenic mechanisms that contribute to blood pressure elevation.

Beta-Endorphin 1–31 Biotransformation and cAMP Modulation in Inflammation

A large body of evidence now exists for the immune cell expression, production, and the release of beta-endorphin (BE 1–31) within inflamed tissue. The inflammatory milieu is characterised by increased acidity, temperature and metabolic activity. Within these harsh conditions BE 1–31 is even more susceptible to increased enzymatic degradation over that of plasma or other non-injured tissue. To elucidate the biotransformation pathways of BE 1–31 and provide an insight to the impact of inflamed tissue environments, BE 1–31 and three of its major N-terminal fragments (BE 1–11, BE 1–13 and BE 1–17) were incubated in inflamed tissue homogenates at pH 5.5 for 2 hrs. In addition, the potency of BE 1–31 and five main N – terminal fragments (BE 1–9, BE 1–11, BE 1–13, BE 1–17, BE 1–20) was assessed at mu-opioid receptors (MOR), delta-opioid receptors (DOR), and kappa-opioid receptors (KOR). Opioid receptor potency was investigated by examining the modulation of forskolin induced cAMP accumulation. The majority of the N-terminal fragment of BE 1–31 had similar efficacy to BE 1–31 at MOR. The shortest of the major N-terminal fragments (BE 1–9), had partial agonist activity at MOR but possessed the highest potency of all tested peptides at DOR. There was limited effect for BE 1–31 and the biotransformed peptides at KOR. Major N-terminal fragments produced within inflamed tissue have increased presence within inflamed tissue over that of the parent molecule BE 1–31 and may therefore contribute to BE 1–31 efficacy within disease states that involve inflammation.

Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO3(-)-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the Gs presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca(2+) and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.

Abstract 5388: Altered regulation of metabolic pathways by the hormone glucagon-like peptide 1 in breast cancer induces growth inhibition.

Abstract Ample data indicate an association between Type 2 diabetes mellitus (T2DM) and increased risk of breast cancer. The incretin hormone glucagon-like peptide-1 (GLP-1) is a 31-amino acids long peptide-hormone, secreted from intestinal L cells in response to food intake and, through activation of G-protein coupled receptor (GPCR) located on pancreatic β cells, serves as the major regulator of postprandial insulin secretion. In hepatocytes and adipocytes GLP-1 regulates metabolic pathways like glucose uptake and lipid biosynthesis. GLP-1 levels are reduced in T2DM, and long acting GLP-based medications are currently approved for treatment of T2DM. We have recently discovered GLP-1 as a novel player in the complex interaction between T2DM and breast cancer. GLP-1 inhibited proliferation of breast cancer cells in vitro and in vivo, induced cAMP accumulation and inhibited AKT activation. Tumor cells acquire a metabolic switch essential for their survival, allowing cells to function under stress and produce precursors needed for the synthesis of macromolecules. This phenomenon is associated with activation of the AKT pathway and inhibition of AMP activated protein kinase (AMPK). Based on our observations and on known metabolic activities of GLP-1in other cell types, we hypothesized that it may reverse the metabolic switch in breast cancer cells, thus, leading to cell death. AMPK inhibition, using compound C and siRNA, abolished GLP1-mediated MCF-7 and MDA-MB-231 cell death, implying this metabolic pathway as central to GLP-1 activity in breast cancer. Indeed, GLP1 induced AMPK phosphorylation in breast cancer cell lines. Accordingly, acetyl CoA carboxylase (ACC), an essential enzyme for fatty acid synthesis and a downstream target of AMPK, was phosphorylated by AMPK. Adenylate cyclase inhibitor 2′5′dideoxy adenosine prevented GLP-1-induced AMPK and ACC phosphorylation, suggesting cAMP as a regulator of this pathway. AMPK can be activated generally by LKB1 or CAMKK1β. As MDA-MB231 cells do not express LKB1 we suspected that GLP-1 utilizes CAMKK1β for AMPK activation in breast cancer cells. Indeed, we discovered that CAMKK1β inhibition abrogated AMPK activation by GLP-1. The mammalian target of rapamycin (mTOR) pathway is regulated by AMPK. We monitored pathway activity by measuring phosphorylation of S6-kinase and its substrate S6 in MCF-7 cells. Phosphorylation of the two p-S6Kinase isoforms (P85 and P70), and of pS6, was decreased following GLP-1 treatment. We conducted metabolomics screen using NMR and preliminary data indicated reduced lactic acid levels and increased glutamic acid levels following GLP-1 treatment. Thus, we show that regulation of AKT-mTOR and AMPK metabolic pathways by GLP-1 in breast cancer, mediate GLP-1 growth inhibitory effect. As GLP-1 levels are lower in T2DM, we suggest this activity of GLP-1 as a novel link associating T2DM and breast cancer. Citation Format: Tami Rubinek, Hagai Ligumsky, Avraham Karasik, Ido Wolf. Altered regulation of metabolic pathways by the hormone glucagon-like peptide 1 in breast cancer induces growth inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5388. doi:10.1158/1538-7445.AM2013-5388

A Gαs DREADD Mouse for Selective Modulation of cAMP Production in Striatopallidal Neurons

Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gαolf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gαs-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal Gs signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gαs signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gαs signaling in other neurobehavioral contexts.

Pharmacological profile of engineered 5-HT4 receptors and identification of 5-HT4 receptor-biased ligands

G protein-coupled receptors (GPCRs) can activate simultaneously multiple signaling pathways upon agonist binding. The combined use of engineered GPCRs, such as the receptors activated solely by synthetic ligands (RASSLs), and of biased ligands that activate only one pathway at a time might help deciphering the physiological role of each G protein signaling. In order to find serotonin type 4 receptor (5-HT4R) biased ligands, we analyzed the ability of several compounds to activate the Gs and Gq/11 pathways in COS-7 cells that transiently express wild type 5-HT4R, the 5-HT4R-D100A mutant (known also as 5-HT4-RASSL, or Rs1) or the 5-HT4R-T104A mutant, which modifies agonist-induced 5-HT4R activation. This analysis allowed completing the pharmacological profile of the two mutant 5-HT4Rs, but we did not find any biased ligand for the mutant receptors. Conversely, we identified the first biased agonists for wild type 5-HT4R. Indeed, RS 67333 and prucalopride acted as partial agonists to induce cAMP accumulation, but as antagonists on inositol phosphate production. Moreover, they showed very different antagonist potencies that could be exploited to study the activation of the Gs pathway, with or without concomitant block of Gq/11 signaling.This article is part of a Special Issue entitled Optogenetics (7th BRES)

Regulation of Cytotoxic T Lymphocyte Antigen 4 by Cyclic AMP

Recent studies indicate that cyclic AMP (cAMP) induces cytotoxic T lymphocyte antigen (CTLA) 4. CTLA4 is expressed in T cells, and is a negative regulator of T cell activation. CTLA4 expression is regulated by T cell receptor plus CD28 (adaptive immune signaling) at both the transcriptional and post-transcriptional level. Here, we examine the pathways by which cAMP regulates CTLA4 expression, focusing on transcriptional activation. Elevating intracellular cAMP levels by cell-permeable cAMP analogs, the adenylyl cyclase activator, forskolin, or phosphodiesterase inhibitors increases CTLA4 mRNA expression in EL4 murine T cells and primary CD4(+) T cells. Activation of protein kinase A (using the protein kinase A-selective agonist, N6-phenyladenosine-cAMP), but not exchange proteins activated by cAMP (using the exchange proteins activated by cAMP-selective 8-pCPT-2Me-cAMP), increases CTLA4 promoter activity. Mutation constructs of the CTLA4 promoter uncover an enhancer binding site located within the -150 to -130 bp region relative to the transcription start site. Promoter analysis and chromatin immunoprecipitation assays suggest that cAMP response element-binding is a putative transcription factor induced by cAMP. We have previously shown that CTLA4 mediates decreased pulmonary inflammation in an LPS-induced murine model of acute lung injury (ALI). We observed that LPS can induce CTLA4 transcription via the same cAMP-inducible promoter region. The immunosuppressant, rapamycin, decreases cAMP and LPS-induced CTLA4 transcription in vitro. In vivo, LPS induces cAMP accumulation in bronchoalveolar lavage fluid, bronchoalveolar lavage cells, and lung tissues in ALI. We demonstrate that rapamycin decreases cAMP accumulation and CTLA4 expression in ALI. Together, these data suggest that cAMP may negatively regulate pulmonary inflammatory responses in vivo and in vitro by altering CTLA4 expression.

Abstract 474: The essential role of Giα2 in prostate cancer progression

Abstract Basic and clinic research data have suggested that signaling of G-protein-coupled receptors (GPCRs) is amplified in malignant prostate tumors and facilitates the transition of primary cancer cells to the androgen independent stage. Oxytocin (OXT) is a neurohypophyseal peptide hormone that regulates normal prostate functions. However, the information is very limited about the effect of OXT on prostate cancer progression. The effect of OXT is mediated by a GPCR that promiscuously activates multiple G-proteins including Gqα and Giα. Recently, we have shown that OXT receptor (OXTR) is the primary target of OXT in androgen independent prostate cancer cell lines (DU145 and PC3). Moreover, activated OXTR has been shown to promote migration of PC3 cells via a pertussis toxin (PTx) sensitive Gi/o-protein dependent mechanism. In the present study, OXTR was shown to be increased in advanced prostate cancer tissues when compared with normal prostate tissues. Expression of OXTR protein is detected in DU145 and PC3 prostate cancer cell lines but not in the immortalized prostate epithelial cells (RWPE1). OXT treatment (100 nM) inhibited the foskolin (5 µM) induced cAMP accumulation in PC3 cells, as indicated by the cAMP luciferase reporter assay. This data has provided direct evidence that supports the involvement of Giα in the OXTR signaling in PC3 cells. There was no difference in expression of Giα isoforms (Giα1-3) at the mRNA level between normal (PrEC, RWPE1 and RWPE2) and prostate cancer cell lines (LNCaP, DU145, PC3, and PC3M). However, western blot analysis showed that Giα2 is the only Giα family member detectable in PC3 cells. PTx (100 ng/ml) has been shown to abolish the OXT-induced migration of PC3 cells. Overexpression of PTx resistant Giα2, but not wild type Giα2, restored the effect of OXT in PTx pretreated PC3 cells. Accordingly, genetic silencing of Giα2 expression by siRNA eliminated the OXT-induced migration of PC3 cells. Unexpectedly, knock down Giα2 also inhibited the EGF (3 ng/ml) induce migration of PC3 and DU145 cells. In conclusion, Giα2 may play an essential role in prostate cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 474. doi:1538-7445.AM2012-474

Mechanisms of Melanocortin-2 Receptor (MC2R) Internalization and Recycling in Human Embryonic Kidney (HEK) Cells: Identification of Key Ser/Thr (S/T) Amino Acids

ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPβ. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.

FGF-23/Klotho signaling is not essential for the phosphaturic and anabolic functions of PTH

Parathyroid hormone (PTH) is widely recognized as a key regulator of mineral ion homeostasis. Daily intermittent administration of PTH is the only currently available anabolic therapy for bone disorders such as osteoporosis. Recent studies have shown that PTH increases transcription and secretion of fibroblast growth factor 23 (FGF-23), another important regulator of phosphate homeostasis and skeletal metabolism. However, the full relationship between PTH and FGF-23 is largely unknown. This study evaluated the effect of FGF-23/Klotho signaling on the phosphaturic and anabolic functions of PTH. Eight-day-old wild-type (WT) Fgf23(-/-) and Kl(-/-) mice were injected with 100 µg/kg PTH(1-34) or vehicle daily for a 2-week-period and then euthanized. Intermittent injection of PTH successfully reduced the serum phosphate levels and reversed the hyperphosphatemia of Fgf23(-/-) and Kl(-/-) mice. Bone changes were analyzed in the distal femur metaphysis by peripheral quantitative computed tomography (pQCT), micro-computed tomography (µCT), and histomorphometry. PTH treatment induced substantial increases in bone mineral density (BMD) and trabecular bone volume in each mouse genotype. Expression of osteoblastic marker genes, including Runx2, Col1, Alp, Ocn, and Sost, was similarly altered. In addition, primary osteoblasts were isolated and treated with 100 nM PTH in vitro. PTH treatment similarly induced cAMP accumulation and phosphorylation of ERK1/2 and CREB in the osteoblasts from each genotype. Taken together, our results demonstrate that FGF-23/Klotho signaling is not essential for the phosphaturic and anabolic functions of PTH, suggesting that PTH can function as a therapeutic agent to improve the skeletal quality of patients even in the presence of abnormal serum FGF-23 levels.