cAMP-elevating Agents Research Articles

Cholera toxin enhances IL17 production by CD4+ T cells via cyclic AMP signaling pathway (163.5)

Abstract Cholera toxin (CT) is one of the most potent mucosal adjuvants. It has been reported to improve antigen presentation as well as suppress resting T cell activation. Recently, CT was also reported to indirectly increase the Th17 response through elevating IL-6 production from dendritic cells. This result suggests a vital role of CT in mucosal immunity. The goal of our study is to understand how cholera toxin modulates the response of CD4 T cells. We found that CT can directly enhance IL-17 production by CD4+ T cells but decrease IFN-γ, TNF-α, IL2, and IL-10 production. In addition, CD4 T cells were treated with different cAMP-elevating agents, including CT B subunit and PKA inhibitors. We found that the effect of cholera toxin on Th17 response goes through a PKA-independent but cAMP signaling dependent pathway. A more detailed study of cAMP signaling is required to fully understand how cAMP signaling controls this Th17 response. Furthermore, CT may also prevent Th17 cells from becoming Th17/Th1 cells which are more pathogenic in Th17-mediated diseases. Whether cholera toxin or cAMP signaling constrains Th17-mediated pathology will be examined.

Abstract 473: Elevated cAMP decreases migratory potential in pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, primarily resulting from inherent, aggressive dissemination. As elevated concentrations of cAMP are known to inhibit cell movement in multiple tissues, we investigated the role of cAMP in regulating migration of model pancreatic cancer cells. Increased cAMP concentrations were induced either through forskolin, an activator of adenylyl cyclase, the cAMP analog 6-bnz-cAMP, or by two phosphodiesterase cyclic nucleotide inhibitors, cilostamide or IBMX. Transwell migration assays demonstrated cAMP elevating agents significantly decreased migration of Panc1, MiaPaCa2, and HPAFII pancreatic cancer cell lines. In Panc1 cells, inhibition was dose-dependent and not significantly different between forskolin, cilostamide, or IBMX treatment. Importantly, a significant decrease in cell migration was also observed in cells treated with 6-bnz-cAMP, a specific activator of protein kinase A. The decrease in migration did not reflect alterations in cell proliferation as cell cycle analysis demonstrated little to no change in proliferation in treated compared to control cells. Similarly, the decrease in cell migration was not a function of programmed cell death as apoptosis was not elevated in cells treated with cAMP elevating agents. Consistent with its known role in F-actin rearrangement, elevated levels of cAMP increased phosphorylated RhoA and the resultant decrease in active RhoA-GTP. As expected, fluorescence microscopy indicated a significant increase in cortical actin formation along the perimeter of forskolin or cilostamide-treated cells. Taken together these data suggest that the novel use of existing anti-inflammatory drugs that target the cAMP signaling networks have the potential to block pancreatic cancer dissemination. 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 473. doi:1538-7445.AM2012-473

Abstract 1839: Phosphodiesterase 4 regulates lung tumor growth through HIF regulation, proliferation, and angiogenesis

Abstract Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Here, we elucidate the pathophysiological role of PDE4 and its therapeutic prospects in lung cancer. We exposed 10 different non-small cell lung cancer cell lines (adenocarcinoma, squamous, and large cell carcinoma) to hypoxia and assessed the expression and activity of PDE4 by quantitative real-time PCR, immunocytochemistry, western blotting, and PDE activity assays. Expression and activity of distinct PDE4 isoforms (PDE4A and PDE4D) increased in response to hypoxia in eight of the studied cell lines. Furthermore, we determined various potential hypoxia-responsive elements (HRE) in PDE4A and PDE4D. Silencing of hypoxia-inducible factor subunits (HIF1A and HIF2) by siRNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i), a cAMP-elevating agent, cAMP analogs, PKA activator/inhibitor, and EPAC activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis regulates HIF signaling as measured by HRE reporter gene assay and expression of HIF target genes (LDHA, PDK1, VEGFA). Notably, PDE4i or PDE4A-/PDE4D-selective siRNA reduced human lung tumor cell proliferation in vitro and colony formation and altered the expression of cell-cycle regulators. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in athymic nude mice by altering proliferation and angiogenesis. Collectively, our findings suggest that PDE4 is overexpressed in lung cancer, exerts crosstalk with HIF pathways, and promotes lung cancer progression; thus, PDE4 may represent a therapeutic target for lung cancer therapy. 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 1839. doi:1538-7445.AM2012-1839

Cyclic AMP induces necroptotic cell death in cardiac fibroblasts

Increased levels of the second messenger cyclic AMP (cAMP) can promote cell loss of a variety of cell types. The precise mechanism(s) for this loss is/are not clearly defined, although cAMP can promote apoptosis of certain types of cells (Insel et al, Acta Physiol in press). In the present study, we used adult rat cardiac fibroblasts (rCFs) to test the hypothesis that cAMP induces necroptosis. Our results from morphological and biochemical studies provide evidence that cAMP induces necroptosis in rCFs and is mediated by protein kinase A and not Epac, another cAMP effector. The findings include destruction of the plasma membrane of rCFs, which differs from apoptotic shrinking of the membrane, and a prominent increase in expression of HMGB1, a marker of necrosis, following treatment with cAMP‐elevating agents. In addition, such agents induce death in a caspase‐PARP independent manner. The pan‐caspase inhibitor, z‐VAD‐fmk increases cell death induced by CPT‐cAMP but the necroptosis inhibitors necrostatin‐1 and cyclosporine A reduce this effect. Taken together, these findings lead us to conclude that cAMP induces necroptosis in rCFs and that this occurs via a PKA‐mediated mechanism. cAMP/PKA‐promoted necroptosis may contribute to decrease in number of CFs in vivo and may be a mechanism of cAMP‐promoted cell death in other types of cells. (Supported by research and training grants from NIH).

Activation of Protein Kinase A and Exchange Protein Directly Activated by cAMP Promotes Adipocyte Differentiation of Human Mesenchymal Stem Cells

Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I2 (PGI2) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells.

Anti-Inflammatory Role of the cAMP Effectors Epac and PKA: Implications in Chronic Obstructive Pulmonary Disease

Cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (IL-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (ASM) cells, may contribute to the development of chronic obstructive pulmonary disease (COPD). Despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic AMP (cAMP), little is known about its exact mechanism of action. We report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by cAMP (Epac) or protein kinase A (PKA) reduced cigarette smoke extract (CSE)-induced IL-8 mRNA expression and protein release by human ASM cells. CSE-induced IκBα-degradation and p65 nuclear translocation, processes that were primarily reversed by Epac activation. Further, CSE increased extracellular signal-regulated kinase (ERK) phosphorylation, which was selectively reduced by PKA activation. CSE decreased Epac1 expression, but did not affect Epac2 and PKA expression. Importantly, Epac1 expression was also reduced in lung tissue from COPD patients. In conclusion, Epac and PKA decrease CSE-induced IL-8 release by human ASM cells via inhibition of NF-κB and ERK, respectively, pointing at these cAMP effectors as potential targets for anti-inflammatory therapy in COPD. However, cigarette smoke exposure may reduce anti-inflammatory effects of cAMP elevating agents via down-regulation of Epac1.

Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase-Dependent SOCS-3 Gene Induction Requires c-Jun, Signal Transducer and Activator of Transcription 3, and Specificity Protein 3 Transcription Factors

SOCS-3 gene induction by cAMP-elevating agents or the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), in primary HUVECs was found to require PKCη- and PKCε-dependent extracellular signal-regulated kinase (ERK) activation. The minimal, ERK-responsive element of the SOCS-3 promoter was localized to a region spanning nucleotides -107 to the transcription start site and contains conserved binding sites for AP-1 and SP1/SP3 transcription factors, as well as proximal and distal signal transducer and activator of transcription (pSTAT and dSTAT) binding elements. All three classes of transcription factor were activated in response to ERK activation. Moreover, representative protein components of each of these transcription factor binding sites, namely c-Jun, STAT3, and SP3, were found to undergo ERK-dependent phosphorylation within their respective transactivation domains. Mutational analysis demonstrated an absolute requirement for the SP1/SP3 binding element in controlling basal transcriptional activity of the minimal SOCS-3 promoter. In addition AP-1, pSTAT, and SP1/SP3 binding sites were required for ERK-dependent, PMA-stimulated SOCS-3 gene activation. The dSTAT site seems to be important for supporting activity of the AP-1 site, because combined deletion of both sites completely blocks transcriptional activation of SOCS-3 by PMA. Together these results describe novel, ERK-dependent regulation of transcriptional activity that requires codependent activation of multiple transcription factors within the same region of the SOCS-3 gene promoter.

CAMP Induces Adhesion of Microvascular Smooth Muscle Cells to Fibronectin via an Epac-Mediated but PKA-independent Mechanism

Background/Aims: Vascular smooth muscle cells (VSMCs) play important roles, both in physiologic and pathophysiologic processes. Adhesion of these cells is critical for their proper functioning, especially in resistance-sized vessels. Nonetheless, very little is known about mechanisms regulating adhesion of VSMCs. In particular, the role of cAMP and its downstream effectors, PKA and Epac, remain poorly investigated. Methods: Primary cultured cells were isolated from human dermal arterioles and adhesion to fibronectin measured. Adhesion assay was performed in the presence or absence of cAMP elevating agents and activators or inhibitors of PKA or Epac signaling. Results: cAMP increases adhesion of microvascular smooth muscle cells to fibronectin in concentration and time-dependent fashions. Pretreatment with H89, a PKA inhibitor, did not affect the adhesion, indicating a PKA-independence of the induced adhesion. Selective activation of PKA did not affect adhesion, whereas selective activation of Epac (by 8-pCPT-2’-O-Me-cAMP) increased adhesion. Moreover, expression of a dominant negative mutant of Epac abolished the induced adhesion. cAMP elevation by a physiologic agonist, isoproterenol, mimicked the results obtained with forskolin or 8-pCPT-2’-O-Me-cAMP. Blocking β1 integrins abrogated the induced adhesion. Conclusion: The results of this study demonstrate for the first time that cAMP induces adhesion of human microvascular smooth muscle cells to fibronectin via β1 integrins. This adhesion appears to be mediated by Epac, independently if PKA.

Endogenous surface expression of ΔF508‐CFTR mediates cAMP‐stimulated Cl− current in CFTRΔF508/ΔF508 pig thyroid epithelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is both an anion channel and a regulator of other transport proteins. Mutations in the CFTR gene underlie the human disease, cystic fibrosis. The most common CFTR mutation, ΔF508, produces a misfolded protein which traffics improperly. The availability of transgenic CFTR(ΔF508/ΔF508) pigs allows measurement of the impact of ΔF508 in native tissue. Thyroid epithelia respond to cAMP-elevating agents by increasing anion transport, a process reliant on functional CFTR. To assess whether endogenous levels of ΔF508-CFTR mediate thyroid transport, primary thyroid epithelial cultures (pThECs) were grown from newborn CFTR(+/+) (wild-type) and CFTR(ΔF508/ΔF508) (ΔF) pig thyroids and the stimulated, secretory components of short-circuit current (I(sc)) compared. Surface biotinylation studies assessed the surface presentation of ΔF508-CFTR. Baseline I(sc) levels of both wild-type and ΔF pThECs consisted of an amiloride-sensitive component. In ΔF pThECs, this mirrored previous measurements in CFTR(-/-) (knockout) pThECs. Surprisingly, elevation of cAMP transiently increased I(sc) to peak levels ∼65% of those achieved by wild-type. In contrast, knockout pThECs were indifferent to cAMP activation. In ΔF pThECs, total ΔF508-CFTR expression was ∼9% that of wild-type, consistent with misfolding and enhanced degradation. Surface biotinylation studies indicated that ∼4% of the total ΔF508 resided at the surface and did not increase with cAMP elevation. The present findings show that low endogenous levels of pig ΔF508-CFTR can mediate substantial anion transport by thyroid epithelia. These data suggest that both wild-type and ΔF508-CFTR regulate additional thyroid transporters, and together co-ordinate the overall I(sc) response.

Bovine Oocyte Meiotic Inhibition Before In Vitro Maturation and Its Value to In Vitro Embryo Production: Does it Improve Developmental Competence?

The efficiency of bovine in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition and supplementation. As bovine oocytes resume meiosis spontaneously when cultured, it was hypothesized that preventing meiosis in vitro before in vitro maturation (IVM) and in vitro fertilization (IVF) would allow more oocytes to acquire developmental competence. This article reviews some of the factors involved in meiotic arrest as well as the effects of meiotic inhibition before IVM on bovine oocytes developmental competence following IVF. Follicular components and cAMP-elevating agents can delay or inhibit meiosis in various proportions of oocytes; however, few studies have examined their effects on development following IVM and IVF because they are not practical (follicular components) or have a transient effect on meiosis (cAMP-elevating agents). Protein synthesis or phosphorylation inhibition prevented meiosis in high percentages of oocytes; however, these non-specific inhibitions led to lower developmental competence compared with non-arrested oocytes. Maturation promoting factor (MPF) inhibition with specific inhibitors has been examined in several studies. Despite faster maturation following removal from inhibition and some structural damage to the oocytes, MPF inhibition generally led to blastocyst rates similar to control, non-arrested oocytes. Future work will involve evaluating the effects on arrested oocytes of molecules that can improve developmental competence in non-arrested oocytes. It is also anticipated that new IVM systems that take into consideration new knowledge of the mechanisms involved in the control of meiosis will be developed. Moreover, global gene expression analysis studies will also provide clues to the culture conditions required for optimal expression of developmental competence.

Cyclic AMP dysregulates intestinal epithelial cell restitution through PKA and RhoA*

Mucosal homeostasis is dependent on the establishment and maintenance of the cell-cell contacts that comprise the physiological barrier. Breaks in the barrier are linked to multiple diseases such as inflammatory bowel disease. While increased cyclic adenosine monophosphate (cAMP) levels limit inflammation by decreasing leukocyte infiltration, the effects of elevated cAMP on intestinal epithelial repair are unknown. Restitution in animals administered rolipram was monitored by microscopic examination after laser wounding of the intestinal epithelium or in mice treated with dextran sodium sulfate (DSS). In vitro analysis was conducted using IEC6 and T84 cells to determine the role of elevated cAMP in altering Rho-dependent cellular migration signaling pathways. We show that treatment with rolipram, forskolin, and cAMP analogs decrease intestinal epithelial cell migration in vitro. In vivo cell imaging revealed that increased cAMP resulted in a decreased cellular migration rate, with cells at the edge displaying the highest activity. As expected, elevated cAMP elicited increased protein kinase A (PKA) activity, in turn resulting in the inactivation and sequestration of RhoA and decreased actin reorganization. The ablation of restitution by cAMP was not restricted to cell culture, as forskolin and rolipram treatment significantly decreased epithelial microwound closure induced by the two photon confocal injury model. Together, these data suggest that administration of cAMP-elevating agents paradoxically decrease infiltration of damage-causing leukocytes while also preventing epithelial repair and barrier maintenance. We propose that treatment with cAMP-elevating agents severely limits mucosal reepithelialization and should be contraindicated for use in chronic inflammatory bowel disorders.

Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC1

Our previous studies showed that the prototypical testicular toxic phthalate monoester, mono-(2-ethylhexyl) phthalate (MEHP), suppresses Sertoli cell TIMP2 levels and allows for the activation of MMP2 in seminiferous epithelium. Activation of MMP2 is important for triggering germ cell apoptosis and instigating germ cell detachment from Sertoli cells. These novel findings led us to examine the transcriptional regulation of the Timp2 gene that accounts for the decrease in Sertoli cell TIMP2 levels following MEHP exposure. Sequential deletion of the Timp2 5'-upstream activating sequence (1200 bp) was used to survey transcriptional activation in the Timp2 promoter region in response to MEHP. Results indicate that under control conditions in rat Sertoli cells, CCAAT enhancer-binding protein alpha (CEBPA) acts as a transactivator to initiate Timp2 gene transcription, and its action is deactivated by exposure to MEHP. By contrast, MYC protein acts as an inhibitor of Timp2 gene transcription, and its activity is increased after MEHP treatment. Addition of follicle-stimulating hormone (FSH) to cells causes translocation of CEBPA into the Sertoli cell nucleus and rescues MEHP-suppressed TIMP2 levels. Down-regulation of TIMP2 expression by MEHP exposure is blocked by forskolin (a cAMP-elevating agent), suggesting that the decrease in Sertoli cell TIMP2 expression following MEHP exposure is cAMP-dependent. Taken together, these data indicate that MEHP both disrupts the FSH-stimulated cAMP signaling pathway and activates the inhibitory signaling mediated by MYC protein, to ultimately account for the cellular mechanism underlying the decreased expression of TIMP2 in Sertoli cells.

Cyclic AMP induces apoptosis in multiple myeloma cells and inhibits tumor development in a mouse myeloma model

BackgroundMultiple myeloma is an incurable disease requiring the development of effective therapies which can be used clinically. We have elucidated the potential for manipulating the cAMP signaling pathway as a target for inhibiting the growth of multiple myeloma cells.MethodsAs a model system, we primarily used the murine multiple myeloma cell line MOPC315 which can be grown both in vivo and in vitro. Human multiple myeloma cell lines U266, INA-6 and the B-cell precursor acute lymphoblastic leukemia cell line Reh were used only for in vitro studies. Cell death was assessed by flow cytometry and western blot analysis after treatment with cAMP elevating agents (forskolin, prostaglandin E2 and rolipram) and cAMP analogs. We followed tumor growth in vivo after forskolin treatment by imaging DsRed-labelled MOPC315 cells transplanted subcutaneously in BALB/c nude mice.ResultsIn contrast to the effect on Reh cells, 50 μM forskolin more than tripled the death of MOPC315 cells after 24 h in vitro. Forskolin induced cell death to a similar extent in the human myeloma cell lines U266 and INA-6. cAMP-mediated cell death had all the typical hallmarks of apoptosis, including changes in the mitochondrial membrane potential and cleavage of caspase 3, caspase 9 and PARP. Forskolin also inhibited the growth of multiple myeloma cells in a mouse model in vivo.ConclusionsElevation of intracellular levels of cAMP kills multiple myeloma cells in vitro and inhibits development of multiple myeloma in vivo. This strongly suggests that compounds activating the cAMP signaling pathway may be useful in the field of multiple myeloma.

Cows are not mice: The role of cyclic AMP, phosphodiesterases, and adenosine monophosphate‐activated protein kinase in the maintenance of meiotic arrest in bovine oocytes

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.

Cilostazol Improves Outcome after Subarachnoid Hemorrhage: A Preliminary Report

Background: Cerebral vasospasm (VS) is the most common cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). Reversal of VS by intra-arterial infusion of cyclic adenosine monophosphate (cAMP)-elevating agents has been reported; however, the preventive role in the development of VS is not fully understood. This study is designed to evaluate the possible efficacy of using cilostazol, a selective inhibitor of phosphodiesterase type 3 and a cAMP-elevating agent, in patients with SAH. Methods: In this prospective randomized study, we enrolled 100 SAH patients who met the following criteria: neck clipping within 72 h after onset, Hunt and Hess (HH) score ≤4, modified Rankin scale (mRS) score ≤2 prior to ictus, and no serious cardiovascular complications. Patients were divided into control and cilostazol groups; we focused on the effects of cilostazol on the decrease in the incidence of symptomatic VS, cerebral infarction, and the mRS score at discharge. Result: Patients’ age, male/female ratio, mRS score prior to ictus, HH grade, Fisher group, site of the aneurysm, drugs prescribed during the observation period, and length of hospital stay were not different between the groups. Cilostazol did not significantly decrease the incidence of symptomatic VS (37.3% in the control vs. 22.4% in the cilostazol group, p = 0.183) and cerebral infarction (27.5% in control vs. 10.2% in the cilostazol, p = 0.091). However, mRS score was significantly improved at discharge (2.6 in controls vs. 1.5 in the cilostazol group, p = 0.041). Patients’ age being ≤65 years (OR = 8.47, 95% CI = 2.45–29.32, p = 0.0007), Fisher group ≤3 (OR = 4.64, 95% CI = 1.00–21.45, p = 0.049), HH grade ≤2 (OR = 4.31, 95% CI = 1.27–14.59, p = 0.019), no hydrocephalus (OR = 8.55, 95% CI = 1.72–19.23, p = 0.0046), and cilostazol use (OR = 5.52, 95% CI = 1.61–18.90, p = 0.0065) were independent predictors of good outcomes (mRS score ≤2). Conclusion: Cilostazol may improve outcomes after SAH, but further double-blind, placebo-controlled studies are required for a definitive conclusion.

Regulation of Human Vascular Protease-Activated Receptor-3 through mRNA Stabilization and the Transcription Factor Nuclear Factor of Activated T Cells (NFAT)

Thrombin promotes vascular smooth muscle cell (SMC) proliferation and inflammation via protease-activated receptor (PAR)-1. A further thrombin receptor, PAR-3, acts as a PAR-1 cofactor in some cell-types. Unlike PAR-1, PAR-3 is dynamically regulated at the mRNA level in thrombin-stimulated SMC. This study investigated the mechanisms controlling PAR-3 expression. In human vascular SMC, PAR-3 siRNA attenuated thrombin-stimulated interleukin-6 expression and extracellular signal-regulated kinases 1/2 phosphorylation, indicating PAR-3 contributes to net thrombin responses in these cells. Thrombin slowed the decay of PAR-3 but not PAR-1 mRNA in the presence of actinomycin D and induced cytosolic shuttling and PAR-3 mRNA binding of the mRNA-stabilizing protein human antigen R (HuR). HuR siRNA prevented thrombin-induced PAR-3 expression. By contrast, forskolin inhibited HuR shuttling and destabilized PAR-3 mRNA, thus reducing PAR-3 mRNA and protein expression. Other cAMP-elevating agents, including the prostacyclin-mimetic iloprost, also down-regulated PAR-3, accompanied by decreased HuR/PAR-3 mRNA binding. Iloprost-induced suppression of PAR-3 was reversed with a myristoylated inhibitor of protein kinase A and mimicked by phorbol ester, an inducer of cyclooxygenase-2. In separate studies, iloprost attenuated PAR-3 promoter activity and prevented binding of nuclear factor of activated T cells (NFAT2) to the human PAR-3 promoter in a chromatin immunoprecipitation assay. Accordingly, PAR-3 expression was suppressed by the NFAT inhibitor cyclosporine A or NFAT2 siRNA. Thus human PAR-3, unlike PAR-1, is regulated post-transcriptionally via the mRNA-stabilizing factor HuR, whereas transcriptional control involves NFAT2. Through modulation of PAR-3 expression, prostacyclin and NFAT inhibitors may limit proliferative and inflammatory responses to thrombin after vessel injury.

Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2’-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2’-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2’-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of β-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.

CAMP antagonizes ERK-dependent antiapoptotic action of insulin

Insulin has antiapoptotic activity in various cell types. However, the signaling pathways underlying the antiapoptotic activity of insulin is not yet known. This study was conducted to determine if cAMP affects the antiapoptotic activity of insulin and the activity of PI3K and ERK in CHO cells expressing human insulin receptors (CHO-IR). Insulin-stimulated ERK activity was completely suppressed by cAMP-elevating agents like as pertussis toxin (Ptx) and cholera toxin (Ctx) after 4 h treatment. Insulin-stimulated PKB/Akt activity was not affected at all. Ptx treatment together with insulin increased the number of apoptotic cells and the degree of DNA fragmentation. Ctx or 8-brcAMP treatment also increased the number of apoptotic cells and stimulated the cleavage of caspase-3 and the hydrolysis of PARP. Taken together, cAMP antagonizes the antiapoptotic activity of insulin and the main target molecule of cAMP in this process is likely ERK, not PI3K-dependent PKB/Akt.

Activation of Extracellular-Signal Regulated Kinase by Epidermal Growth Factor Is Potentiated by cAMP-Elevating Agents in Primary Cultures of Adult Rat Hepatocytes

We investigated the effects of α- and β-adrenergic agonists on epidermal growth factor (EGF)-stimulated extracellular-signal regulated kinase (ERK) isoforms in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with EGF (20 ng/ml) and/or α(1)-, α(2)- and β(2)-adrenergic agonists. Phosphorylated ERK isoforms (ERK1; p44 mitogen-activated protein kinase (MAPK) and ERK2; p42 MAPK) were detected by Western blotting analysis using anti-phospho-ERK1/2 antibody. The results show that EGF induced a 2.5-fold increase in ERK2-, but not ERK1-, phosphorylation within 3 min. This EGF-induced ERK2 activation was abolished by treatment with the EGF-receptor kinase inhibitor AG1478 (10(-7) M) or the MEK (MAPK kinase) inhibitor PD98059 (10(-6) M). The α(2)-adrenergic and β(2)-adrenergic agonists, UK14304 (10(-6) M) and metaproterenol (10(-6) M), respectively, had no effect in the absence of EGF, but metaproterenol significantly potentiated EGF-induced ERK2 phosphorylation. Moreover, the cell-permeable cAMP analog 8-bromo cAMP (10(-7) M), also potentiated EGF-induced ERK2 phosphorylation. The effects of these analogs were antagonized by the protein kinase A (PKA) inhibitor H-89 (10(-7) M). These results suggest that direct or indirect activation of PKA represents a positive regulatory mechanism for EGF stimulation of ERK2 induction.

Differences in the Response of UCP1 mRNA to Hormonal Stimulation Between Rat and Mouse Primary Cultures of Brown Adipocytes

Uncoupling protein 1 (UCP-1), the specific marker of brown adipose tissue, is transcriptionally activated in response to adrenergic stimuli and thyroid hormones are necessary for its full expression. We describe differences in the regulation of UCP-1 mRNA expression between rat and mouse brown adipocytes in culture, using norepinephrine (NE), triiodothyronine (T3), insulin and retinoic acid (RA). Results: NE and cAMP-elevating agents strongly increase UCP-1 mRNA levels in cultures of mouse adipocytes, but increases are low in those from rat. In rat adipocytes NE poorly increases UCP-1 mRNA expression and T3 markedly increases the adrenergic response of UCP-1, an effect not observed in mouse adipocytes. In the absence of insulin, T3 itself increases UCP-1 mRNA in rat adipocytes and enhances the response to NE, while in mouse adipocytes no effect of T3 is observed. RA by itself stimulates UCP-1 mRNA in mouse adipocytes, but not in those from rat. In rat cultures, RA requires the presence of NE and/or T3. Conclusions: We find important differences in the hormonal regulation of UCP-1 mRNA expression in cultured preadipocytes depending on the species used as donor; those differences are observed using identical culture conditions and should be considered when doing cultures from these species.