cAMP Stimulation Research Articles

Role of CREB on heme oxygenase-1 induction in adrenal cells: involvement of the PI3K pathway.

In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H2O2/Fe(2+) ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at -146 and -37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[-295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[-295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of the Hmox1 gene.

Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription

The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3′ reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3′ untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3′UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates.

0340 : BK (Ca) channels contribution to the control of artery tone by PDE3 and PDE4 varies depending on the mode of cAMP stimulation

In vascular smooth muscle cells (SMC), 3’-5’-cyclic adenosine monophosphate (cAMP) is a key mediator that initiates vasodilation. Downstream effectors of cAMP-induced SMC relaxation include large conductance, Ca 2+ -activated, K + channels (BK Ca ) channels. Hydrolysis of cAMP by phosphodiesterases (PDEs) controls its cellular fate.Here, we sought to establish : i) respective contributions of PDE3 and PDE4 families in regulating tone of rat coronary arteries (CA) under various cAMP stimuli; ii) the particular role of BK Ca channels under these stimuli. CA were isolated from adult male Wistar rats. Responses to vasodilators were studied on CA rings mounted on a small vessel myograph and contracted with thromboxane analogue U46619. cAMP-PDE activity was measured in CA lysate using a radioenzymatic assay. Contributions of PDE3 and PDE4 were studied using selective inhibitors, namely Ro 20-1724 (Ro, 10 μM) and cilostamide (Cil, 1 μM). Role of BK Ca channels was studied using the specific blocker iberiotoxin (IBTX, 100 nM). Contributions of PDE3 and PDE4 to the total cAMP hydrolysing activity in rat CA were 55±5% and 31±2%, respectively. Addition of Ro or Cil on contracted CA induced relaxations that were strongly blunted in the presence of IBTX. Ro and Cil potentiated relaxant responses to increasing concentrations of cAMP-elevating agents, namely β-adrenergic receptor agonist isoproterenol (ISO) or the forskolin analogue L858051 (L85), a direct activator of cAMP production. IBTX shifted to the right the relaxant response to L85 whereas it reduced maximal response to ISO. Interestingly, IBTX blocked the potentiating effect of Ro on ISO response, but not on L85 response. By contrast, BK Ca inhibition neither prevented potentiation of ISO nor L85 responses by Cil. Our data suggest that, in rat CA, the contribution of BK Ca channels to the relaxant effects of PDE3 and PDE4 inhibition varies depending on the mode of cAMP stimulation. The author hereby declares no conflict of interest

Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial ligand-gated anion channel, are associated with the lethal genetic disease cystic fibrosis. The CFTR G551D mutation impairs ATP hydrolysis and thereby makes CFTR refractory to cAMP stimulation. Both wild-type (WT) and G551D CFTR have been implicated in regulatory volume decrease (RVD), but the underlying mechanism remains incompletely understood. Here, we show that the channel activity of both WT and G551D CFTR is directly stimulated by mechanical perturbation induced by cell swelling at the single-channel, cellular, and tissue levels. Hypotonicity activated CFTR single channels in cell-attached membrane patches and WT-CFTR-mediated short-circuit current (Isc) in Calu-3 cells, and this was independent of Ca(2+)and cAMP/PKA signaling. Genetic suppression and ablation but not G551D mutation of CFTR suppressed the hypotonicity- and stretch-inducedIscin Calu-3 cells and mouse duodena. Moreover, ablation but not G551D mutation of the CFTR gene inhibited the RVD of crypts isolated from mouse intestine; more importantly, CFTR-specific blockers markedly suppressed RVD in both WT- and G551D CFTR mice, demonstrating for the first time that the channel activity of both WT and G551D CFTR is required for epithelial RVD. Our findings uncover a previously unrecognized mechanism underlying CFTR involvement in epithelial RVD and suggest that the mechanosensitivity of G551D CFTR might underlie the mild phenotypes resulting from this mutation.-Xie, C., Cao, X., Chen, X, Wang, D., Zhang, W. K., Sun, Y., Hu, W., Zhou, Z., Wang, Y., Huang, P. Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

Luminal angiotensin II stimulates rat medullary thick ascending limb chloride transport in the presence of basolateral norepinephrine

Angiotensin II (ANG II) is secreted by the proximal tubule resulting in a luminal concentration that is 100- to 1,000-fold greater than that in the blood. Luminal ANG II has been shown to stimulate sodium transport in the proximal tubule and distal nephron. Surprisingly, luminal ANG II inhibits NaCl transport in the medullary thick ascending limb (mTAL), a nephron segment responsible for a significant amount of NaCl absorption from the glomerular ultrafiltrate. We confirmed that addition of 10(-8) M ANG II to the lumen inhibited mTAL chloride transport (220 ± 19 to 165 ± 25 pmol·mm(-1)·min(-1), P < 0.01) and examined whether an interaction with basolateral norepinephrine existed to simulate the in vivo condition of an innervated tubule. We found that in the presence of a 10(-6) M norepinephrine bath, luminal ANG II stimulated mTAL chloride transport from 298 ± 18 to 364 ± 42 pmol·mm(-1)·min(-1) (P < 0.05). Stimulation of chloride transport by luminal ANG II was also observed with 10(-3) M bath dibutyryl cAMP in the bathing solution and bath isoproterenol. A bath of 10(-5) H-89 blocked the stimulation of chloride transport by norepinephrine and prevented the effect of luminal ANG II to either stimulate or inhibit chloride transport. Bath phentolamine, an α-adrenergic agonist, also prevented the decrease in mTAL chloride transport by luminal ANG II. Thus luminal ANG II increases chloride transport with basolateral norepinephrine; an effect likely mediated by stimulation of cAMP. Alpha-1 adrenergic stimulation prevents the inhibition of chloride transport by luminal ANG II.

A Dietary Medium-Chain Fatty Acid, Decanoic Acid, Inhibits Recruitment of Nur77 to the HSD3B2 Promoter In Vitro and Reverses Endocrine and Metabolic Abnormalities in a Rat Model of Polycystic Ovary Syndrome.

Hyperandrogenism is the central feature of polycystic ovary syndrome (PCOS). Due to the intricate relationship between hyperandrogenism and insulin resistance in PCOS, 50%-70% of these patients also present with hyperinsulinemia. Metformin, an insulin sensitizer, has been used to reduce insulin resistance and improve fertility in women with PCOS. In previous work, we have noted that a dietary medium-chain fatty acid, decanoic acid (DA), improves glucose tolerance and lipid profile in a mouse model of diabetes. Here, we report for the first time that DA, like metformin, inhibits androgen biosynthesis in NCI-H295R steroidogenic cells by regulating the enzyme 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase type 2 (HSD3B2). The inhibitory effect on HSD3B2 and androgen production required cAMP stimulation, suggesting a mechanistic action via the cAMP-stimulated pathway. Specifically, both DA and metformin reduced cAMP-enhanced recruitment of the orphan nuclear receptor Nur77 to the HSD3B2 promoter, coupled with decreased transcription and protein expression of HSD3B2. In a letrozole-induced PCOS rat model, treatment with DA or metformin reduced serum-free testosterone, lowered fasting insulin, and restored estrous cyclicity. In addition, DA treatment lowered serum total testosterone and decreased HSD3B2 protein expression in the adrenals and ovaries. We conclude that DA inhibits androgen biosynthesis via mechanisms resulting in the suppression of HSD3B2 expression, an effect consistently observed both in vitro and in vivo. The efficacy of DA in reversing the endocrine and metabolic abnormalities of the letrozole-induced PCOS rat model are promising, raising the possibility that diets including DA could be beneficial for the management of both hyperandrogenism and insulin resistance in PCOS.

The miR-200 Family and Its Targets Regulate Type II Cell Differentiation in Human Fetal Lung

Type II cell differentiation and expression of the major surfactant protein, SP-A, in mid-gestation human fetal lung (HFL) are induced by cAMP and inhibited by TGF-β. cAMP induction of SP-A promoter activity is mediated by increased phosphorylation and DNA binding of thyroid transcription factor-1 (TTF-1/Nkx2.1), a master regulator of lung development. To further define mechanisms for developmental induction of surfactant synthesis in HFL, herein, we investigated the potential roles of microRNAs (miRNAs, miRs). To identify and characterize differentially regulated miRNAs in mid-gestation HFL explants during type II pneumocyte differentiation in culture, we performed miRNA microarray of RNA from epithelial cells isolated from mid-gestation HFL explants before and after culture with or without Bt2cAMP. Interestingly, the miR-200 family was significantly up-regulated during type II cell differentiation; miR-200 induction was inversely correlated with expression of known targets, transcription factors ZEB1/2 and TGF-β2. miR-200 antagonists inhibited TTF-1 and surfactant proteins and up-regulated TGF-β2 and ZEB1 expression in type II cells. Overexpression of ZEB1 in type II cells decreased DNA binding of endogenous TTF-1, blocked cAMP stimulation of surfactant proteins, and inhibited miR-200 expression, whereas cAMP markedly inhibited ZEB1/2 and TGF-β. Importantly, overexpression of ZEB1 or miR-200 antagonists in HFL type II cells also inhibited LPCAT1 and ABCA3, enzymes involved in surfactant phospholipid synthesis and trafficking, and blocked lamellar body biogenesis. Our findings suggest that the miR-200 family and ZEB1, which exist in a double-negative feedback loop regulated by TGF-β, serve important roles in the developmental regulation of type II cell differentiation and function in HFL.

Abstract 1028: Prostaglandin E2 activates the mTORC1 pathway through an EP4/cAMP/PKA and EP1/calcium-mediated mechanisms in human pancreatic carcinoma cells

Abstract Obesity, a known risk factor for pancreatic cancer, is associated with inflammation and insulin resistance. The pro-inflammatory prostaglandin E2 (PGE2) that signals via EP receptors and cAMP, and insulin-like growth factor 1 (IGF-1) that is elevated in insulin resistant states and activates the Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling module, are both shown to play critical roles in pancreatic cancer progression. We aimed at exploring the potential signaling crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 pathways in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In multiple human pancreatic cancer cell lines, PGE2 exposure increased intracellular cAMP levels, indicating the activation of Gαs-coupled receptors EP2 and/or EP4. In PANC-1 cells, which showed the greatest cAMP response, PGE2 dose- and time-dependently increased the phosphorylation of mTORC1 downstream targets p70S6 kinase (p70S6K) and S6 ribosomal protein (S6rp), suggesting a crosstalk between PGE2/cAMP and mTORC1. Accordingly, the mTORC1 inhibitor rapamycin suppressed PGE2-induced p70S6K and S6rp phosphorylation (Ser235/236 and Ser240/244). Also, the effect of PGE2 on p-S6rp was mimicked by forskolin, a cAMP stimulator. Interestingly, PGE2 and forskolin had no effect on p-Akt, suggesting a link downstream of Akt. Subsequent analysis using EP2 and EP4 antagonists/ agonists revealed that the PGE2-induced responses are mainly mediated by EP4. Further, PGE2-induced mTORC1 activation paralleled an increase in the phosphorylation of cAMP response element-binding protein (CREB), a substrate of protein kinase A (PKA). Pharmacological and genetic inhibition of PKA reduced baseline and PGE2-activated p-S6rp, indicating a role of PKA in the crosstalk. Additionally, calcium signaling seemed to be also involved in PGE2-induced mTORC1 activation, as p-S6rp (Ser235/236 and Ser240/244) was significantly decreased in cells pre-incubated with BAPTA-AM, a cell permeable Ca2+ chelator. Moreover, knockdown of EP1, a receptor known to mediate PGE2-induced Ca2+ response, attenuated PGE2-stimulated mTORC1 activation. Importantly, PGE2 enhanced the effect of IGF-1 on mTORC1 activation, suggesting a positive reinforcement by the interaction between the two pathways. Together, our data reveal a previously unrecognized mechanism of PGE2-stimulated mTORC1 activation mediated by EP4/cAMP/PKA and EP1/Ca2+ signaling in pancreatic cancer cells, which may be of great importance in elucidating the promoting effects of obesity in pancreatic cancer. Ultimately, a precise understanding of these molecular links may provide novel targets for efficacious interventions devoid of adverse effects. Citation Format: Hui-Hua Chang, Steven Young, James Sinnett-Smith, Caroline Ei Ne Chou, Oscar Joe Hines, Enrique Rozengurt, Guido Eibl. Prostaglandin E2 activates the mTORC1 pathway through an EP4/cAMP/PKA and EP1/calcium-mediated mechanisms in human pancreatic carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1028. doi:10.1158/1538-7445.AM2015-1028

Abstract IA18: Obesity, inflammation, and pancreatic cancer

Abstract There is epidemiologic evidence that obesity increases the risk of cancers including pancreatic cancer. Several underlying mechanisms, e.g. inflammation and insulin resistance, are proposed. However, the driving mechanisms in pancreatic cancer are poorly understood. We developed a model of diet-induced obesity and pancreatic cancer development in a state-of-the-art mouse model, which resembles important clinical features of human obesity, e.g. weight gain and metabolic disturbances. Offspring of Pdx-1-Cre (or p48-cre) and LSL-KrasG12D crosses were allocated to either a diet high in fats and calories (HFCD; ~4,535 kcal/kg; 40% of calories from fats) or control diet (CD; ~3,725 kcal/kg; 12% of calories from fats) for various time periods. Compared to control animals, mice fed the HFCD significantly gained more weight and developed hyperinsulinemia, hyperglycemia, hyperleptinemia, and elevated levels of IGF-1. Mice fed the HFCD had a substantial increase in visceral fat as measured by micro-CT. The white visceral adipose tissue in mice fed the HFCD showed signs of marked inflammation, which was more pronounced in the peri-pancreatic fat depot. In addition, the pancreas of HFCD-fed animals showed robust signs of inflammation with increased numbers of infiltrating inflammatory cells. Detailed flow cytometry analysis showed a marked elevation of infiltrating natural killer cells, myeloid-derived suppressor cells, T-regulatory cells, and tumor-infiltrating macrophages into the pancreas of mice fed the HFCD. Multiplex analysis demonstrated a significant change in the pancreatic cytokine profile in HFCD-fed mice compared to CD-fed animals. Importantly, the HFCD led to an acceleration of PanIN development in conditional KrasG12D mice. These results demonstrate that a diet high in fats and calories leads to obesity and metabolic disturbances similar to humans and accelerates early pancreatic neoplasia in the conditional KrasG12D mouse model. Based on the notion that obesity is associated with an increase in inflammatory mediators and insulin-resistance and our previous finding that non-steroidal anti-inflammatory drugs inhibit the progression of PanIN lesions in the conditional KrasG12D mouse model, we investigated a potential crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 signaling pathways in pancreatic cancer. In multiple cell lines PGE2 increased intracellular cAMP levels, indicating the activation of Gsα-coupled EP receptors (EP2 and/or EP4). PGE2 dose- and time-dependently increased the phosphorylation of S6 ribosomal protein downstream of mTORC1, suggesting a crosstalk between PGE2/cAMP and mTORC1. In addition, the effect of PGE2 on phospho-S6rp was mimicked by Forskolin, a pharmacological cAMP stimulator. Importantly, PGE2 enhanced the effect of IGF-1 on S6rp phosphorylation, supporting the existence of a positive reinforcement. Interestingly, PGE2 and forskolin had no effect on Akt phosphorylation, suggesting a link downstream of Akt. In the presence of the PKA inhibitor H89, baseline phosphorylation of S6rp and PGE2-activated S6rp phosphorylation were reduced, indicating a role of PKA in the crosstalk. In summary, these data provide evidence of a crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 signaling pathways. Since both pathways are over-activated in obesity-associated cancers, this interaction may be of great importance in elucidating the tumor-promoting effects of obesity in pancreatic cancer. Citation Format: Guido Eibl. Obesity, inflammation, and pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA18.

Transcriptomic analysis of cyclic AMP response in bovine cumulus cells.

Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.

Physiological characterization of the human EndoC-βH1 β-cell line

In the new human EndoC-βH1 β-cell line, a detailed analysis of the physiological characteristics was performed. This new human β-cell line expressed all target structures on the gene and protein level, which are crucial for physiological function and insulin secretion induced by glucose and other secretagogues.Glucose influx measurements revealed an excellent uptake capacity of EndoC-βH1 β-cells by the Glut1 and Glut2 glucose transporters. A high expression level of glucokinase enabled efficient glucose phosphorylation, increasing the ATP/ADP ratio along with stimulation of insulin secretion in the physiological glucose concentration range. The EC50 value of glucose for insulin secretion was 10.3 mM. Mannoheptulose, a specific glucokinase inhibitor, blocked glucose-induced insulin secretion (GSIS). The nutrient insulin secretagogues l-leucine and 2-ketoisocaproate also stimulated insulin secretion, with a potentiating effect of l-glutamine. The Kir 6.2 potassium channel blocker glibenclamide and Bay K 8644, an opener of the voltage-sensitive Ca2+ channel significantly potentiated GSIS. Potentiation of GSIS by IBMX and forskolin went along with a strong stimulation of cAMP generation.In conclusion, the new human EndoC-βH1 β-cell line fully mirrors the analogous physiological characteristics of primary mouse, rat and human β-cells. Thus, this new human EndoC-βH1 β-cell line is very well suited for physiological β-cell studies.

Bioactivity assay of porcine relaxin based on cAMP accumulation in THP-1 cells quantified by LC–MS/MS

This study describes a reliable bioactivity assay of porcine relaxin (pRLX) based on cyclic adenosine 3′,5′-monophosphate (cAMP) accumulation in the human monocyte cell-line quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). As a result, the LC–MS/MS was based on a positive selected reaction monitoring of cAMP with a stable internal standard, 8-Br-cAMP and a protein precipitation procedure by HClO4. The standard curve of cAMP was linear from 5.0ngmL−1 to 992.0ngmL−1, with lower limits of detection and quantification of 0.5ngmL−1 and 5.0ngmL−1, respectively. The satisfactory validation data including stability assay were obtained. When measured by the LC–MS/MS, the pRLX sample showed a time- and dose-dependent stimulation of cAMP with the concentration for 50% of the maximal effect (EC50) of 40.6ngmL−1. The developed method indicated higher precision and selectivity than the commercial enzyme linked immunosorbent assay kits, which showed EC50 of 66.6ngmL−1.

Extracellular and intracellular factors regulating the migration direction of a chemotactic cell in traveling-wave chemotaxis

This report presents a simple model that describes the motion of a single Dictyostelium discoideum cell exposed to a traveling wave of cyclic adenosine monophosphate (cAMP). The model incorporates two types of responses to stimulation by cAMP: the changes in the polarity and motility of the cell. The periodic change in motility is assumed to be induced by periodic cAMP stimulation on the basis of previous experimental studies. Consequently, the net migration of the cell occurs in a particular direction with respect to wave propagation, which explains the migration of D. discoideum cells in aggregation. The wave period and the difference between the two response times are important parameters that determine the direction of migration. The theoretical prediction compared with experiments presented in another study. The transition from the single-cell state of the population of D. discoideum cells to the aggregation state is understood to be a specific example of spontaneous breakage of symmetry in biology.

OxeR1 regulates angiotensin II and cAMP-stimulated steroid production in human H295R adrenocortical cells

Hormone-regulated steroidogenesis and StAR protein induction involve the action of lipoxygenated products. The products of 5-lipoxygenase act on inflammation and immunity by stimulation of a membrane receptor called OxeR1. The presence of OxeR1 in other systems has not been described up to date and little is known about its mechanism of action and other functions. In this context, the aim of this study was the identification and characterization of OxeR1 as a mediator of cAMP-dependent and independent pathways. Overexpression of OxeR1 in MA-10 Leydig cells increased cAMP-dependent progesterone production. Angiotensin II and cAMP stimulation of adrenocortical human H295R cells produced an increase in StAR protein induction and steroidogenesis in cells overexpressing OxeR1 as compared to mock-transfected cells. Additionally, activation of OxeR1 caused a time-dependent increase in ERK1/2 phosphorylation. In summary, membrane receptor OxeR1 is involved in StAR protein induction and activation of steroidogenesis triggered by cAMP or angiotensin II, acting, at least in part, through ERK1/2 activation.

Chemical conversion of human fibroblasts into neuronal cells: dawn of future clinical trials

Major causes of human death in developed countries are atherosclerosis(1) and carcinogenesis.(2) Atherosclerosis finally leads to a variety of diseases such as myocardial infarction and cerebral infarction. The essence of these pathologic processes is largely due to free radical reactions or oxidative stress due to our persistent use of oxygen in the presence of iron,(3–5) and the products can be literally rust in our body.(6) Heart may be replaced with transplantation from another person with brain death under his/her own permission, but it is currently impossible to transplant brain. Additionally, highly ethical issues, severe donor deficiency and immunological rejections are always present with transplantation.(7) If brand-new cells and organs of any type to be replaced and of their own immunophenotype were freely available, all the people on earth would benefit from this novel technology. Induced pluripotent stem cells can be established from differentiated somatic cells by the forced induction of four transcription factors: Oct3/4, Klf4, Sox2 and c-Myc.(8) However, premature termination of reprogramming might lead to cancer development through altered epigenetic regulation.(9) Recently, the concept of directly converting one type of somatic cell into another has attracted much attention.(10) However, one of the major drawbacks linked to the current strategies is that they are also based on ectopic expression of important developmental genes (transcription factors, etc.),(11) which might cause unexpected undesired effects. In the present issue of Journal of Clinical Biochemistry and Nutrition, Dai et al.(12) for the first time describe a highly efficient chemical conversion of human fibroblasts to neuronal cells with defined six chemical compounds (Fig. 1). Their protocol used five inhibitors to block distinct signaling pathways including transforming growth factor (TGF)-β (SMAD pathway), bone morphologic protein (BMP), glycogen synthase kinase (GSK)-3β, MEK-ERK and p53, together with a cAMP stimulator. Moreover, the authors emphasize that this protocol shortens the time required for differentiation into neuronal cells to 3 weeks in comparison to the reported protocols(10,13) with ~90% efficiency. Fig. 1 Chemical conversion of human fibroblasts into neuronal cells. Refer to text for details. I believe that this is the dawn of regenerative neurology. In a few years or so, human clinical trials would be performed by the use of this technique. Target diseases would be neurodegenerative diseases(14) such as Alzheimer’s and Parkinson diseases, motor neuron diseases such as amyotropic lateral sclerosis, and cerebral infarction/hemorrhage. Simultaneously, we always have to consider how we can load these new neuronal cells with long-time experience and memory of the recipient if necessary.

Role of the prostaglandin E2/E-prostanoid 2 receptor signalling pathway in TGFβ-induced mice mesangial cell damage.

The prostaglandin E2 receptor, EP2 (E-prostanoid 2), plays an important role in mice glomerular MCs (mesangial cells) damage induced by TGFβ1 (transforming growth factor-β1); however, the molecular mechanisms for this remain unknown. The present study examined the role of the EP2 signalling pathway in TGFβ1-induced MCs proliferation, ECM (extracellular matrix) accumulation and expression of PGES (prostaglandin E2 synthase). We generated primary mice MCs. Results showed MCs proliferation promoted by TGFβ1 were increased; however, the production of cAMP and PGE2 (prostaglandin E2) was decreased. EP2 deficiency in these MCs augmented FN (fibronectin), Col I (collagen type I), COX2 (cyclooxygenase-2), mPGES-1 (membrane-associated prostaglandin E1), CTGF (connective tissue growth factor) and CyclinD1 expression stimulated by TGFβ1. Silencing of EP2 also strengthened TGFβ1-induced p38MAPK (mitogen-activated protein kinase), ERK1/2 (extracellular-signal-regulated kinase 1/2) and CREB1 (cAMP responsive element-binding protein 1) phosphorylation. In contrast, Adenovirus-mediated EP2 overexpression reversed the effects of EP2-siRNA (small interfering RNA). Collectively, the investigation indicates that EP2 may block p38MAPK, ERK1/2 and CREB1 phosphorylation via activation of cAMP production and stimulation of PGE2 through EP2 receptors which prevent TGFβ1-induced MCs damage. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the damage induced by TGFβ1.

Abstract 17913: Inhibition of CETP by Dalcetrapib Results in a Modest Increase in Cholesterol Efflux Capacity Associated With an Increase in Large HDL Particles, but Does Not Impact Carotid Intima-Media Thickness in a dal-PLAQUE-2 Substudy

Inhibition of cholesteryl ester transfer protein (CETP) is an approach aiming at raising HDL-cholesterol levels and reducing cardiovascular risk. The dal-PLAQUE-2 phase III study recruited 988 subjects with stable coronary artery disease. Its primary objective was to evaluate the effect of dalcetrapib on the progression of carotid atherosclerotic disease measured by intima-media thickness after one year of therapy. As dalcetrapib showed a neutral effect on cardiovascular risk in the dal-OUTCOMES study, our objective was to evaluate its effect on cIMT and markers of HDL mass, lipoprotein subclass distribution and HDL function. All subjects from dal-PLAQUE-2 who had provided serum samples at baseline and one year were included in our substudy of 193 subjects on placebo and 186 subjects on dalcetrapib. Comparisons between groups at one year were made by ANCOVA after adjustment for baseline levels. No significant differences between groups for all variables considered were observed at baseline. Dalcetrapib reduced CETP activity as measured by a fluorescent method by 30% after 1 year (p&lt;0.001), which resulted in increases in HDL-cholesterol and apoA-I levels by 32% and 11%, respectively (both p&lt;0.001). Dalcetrapib markedly increased the concentration of large HDL particles measured by NMR profiling (+59%, p&lt;0.001), at the expense of small HDL particles (-9.5%, p&lt;0.001). Cholesterol efflux capacity of serum was measured from J774 macrophages under basal conditions and after cAMP stimulation. Dalcetrapib increased basal and stimulated efflux by 7.1% and 5.5% (both p&lt;0.001), but was without effect on the ABCA1-dependent component (p=0.26). Despite these effects, dalcetrapib had no impact on mean and maximal cIMT (p=0.98 and p=0.85). While the change in cIMT was inversely correlated with basal cholesterol efflux in the placebo group (Spearman r=-0.163, p&lt;0.05), such a relationship was not found in the dalcetrapib group. Moreover, the change in total HDL particles concentration was inversely correlated with change in cIMT (r=0.181, p&lt;0.05) in the placebo arm only. In conclusion, dalcetrapib raised HDL-C and larger HDL, but this had minimal effects on cholesterol efflux capacity of patients’ serum and had a neutral effect on carotid artery plaque burden.

Perilipin 5 regulates islet lipid metabolism and insulin secretion in a cAMP-dependent manner: implication of its role in the postprandial insulin secretion.

Elevation of circulating fatty acids (FA) during fasting supports postprandial (PP) insulin secretion that is critical for glucose homeostasis and is impaired in diabetes. We tested our hypothesis that lipid droplet (LD) protein perilipin 5 (PLIN5) in β-cells aids PP insulin secretion by regulating intracellular lipid metabolism. We demonstrated that PLIN5 serves as an LD protein in human islets. In vivo, Plin5 and triglycerides were increased by fasting in mouse islets. MIN6 cells expressing PLIN5 (adenovirus [Ad]-PLIN5) and those expressing perilipin 2 (PLIN2) (Ad-PLIN2) had higher [3H]FA incorporation into triglycerides than Ad-GFP control, which support their roles as LD proteins. However, Ad-PLIN5 cells had higher lipolysis than Ad-PLIN2 cells, which increased further by 8-Br-cAMP, indicating that PLIN5 facilitates FA mobilization upon cAMP stimulation as seen postprandially. Ad-PLIN5 in islets enhanced the augmentation of glucose-stimulated insulin secretion by FA and 8-Br-cAMP in G-protein–coupled receptor 40 (GPR40)- and cAMP-activated protein kinase–dependent manners, respectively. When PLIN5 was increased in mouse β-cells in vivo, glucose tolerance after an acute exenatide challenge was improved. Therefore, the elevation of islet PLIN5 during fasting allows partitioning of FA into LD that is released upon refeeding to support PP insulin secretion in cAMP- and GPR40-dependent manners.

Expression of the cAMP binding protein EPAC1 in thyroid tumors and growth regulation of thyroid cells and thyroid carcinoma cells by EPAC proteins.

The thyrotropin receptor-cAMP pathway is central in growth regulation of thyroid cells and thyroid tumorigenesis, and it regulates expression of thyroid specific genes. Recently, 2 new protein kinase A-independent cAMP effectors named EPAC1 and 2 were described that activate additional intracellular pathways. The aim of our study was to investigate the role of EPAC proteins in growth regulation of thyroid cells and thyroid carcinomas. EPAC1 expression was investigated immunohistochemically in tissues of various thyroid tumors. Utilizing MTT assay, the effect of EPAC stimulation on proliferation in thyroid carcinoma cells and in non-transformed rat FRTL5 cells was investigated. The activation of intracellular signaling pathways was examined by RAP pull-down assay and Western blots. EPAC1 expression was strong in non-oxyphilic follicular thyroid adenomas and carcinomas and in follicular papillary thyroid carcinomas. It was moderate in oxyphilic follicular tumors and classical and tall cell papillary carcinomas. In contrast, EPAC1 expression was low in poorly differentiated carcinomas and very low in anaplastic carcinomas. Thyroid carcinoma cell lines showed no or very weak EPAC1 expression and exhibited no growth-promoting effect after EPAC stimulation. Non-transformed rat FRTL5 cells were growth-stimulated by an EPAC-specific cAMP-analogue and showed EPAC-dependent activation of RAP, ERK, and p70S6 kinase. EPAC1 expression and cellular response to EPAC activation in rat FRTL5 cells reflect cellular responses to cAMP and TSH stimulation in non-transformed thyroid cells. In undifferentiated thyroid carcinomas, loss of EPAC1 expression may be in accordance with the loss of thyroid-specific functions and the loss of responsiveness of the TSHR-cAMP pathway.

Abstract 5291: Signaling cross-talks in obesity-associated pancreatic cancer: Interaction between prostaglandin E2 signaling and mTOR pathway

Abstract Obesity is a known risk factor for human cancers, including pancreatic cancer, and is associated with inflammation and insulin-resistance. The pro-inflammatory eicosanoid prostaglandin E2 (PGE2), which signals via EP receptors and cAMP, and insulin-like growth factor 1 (IGF-1), which is elevated in insulin resistance and activates the Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling module, have both been shown to play critical roles in pancreatic cancer development and growth. The aim of this study was to investigate an unexplored crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 signaling pathways in pancreatic cancer, which may be a key to unraveling the obesity-cancer link. In our studies, human pancreatic cancer cell lines were used, and the activation of signaling molecules was mainly assessed by Western blotting analysis. In multiple cell lines (Capan-2, HPAF-II, Mia PaCa-2, and PANC-1), PGE2 exposure increased intracellular cAMP levels measured by enzyme immunoassay, indicating the activation of Gs alpha-coupled EP receptors (EP2 and/or EP4). In PANC-1 cells, which showed the greatest responsiveness of cAMP induction, PGE2 dose- and time-dependently increased the phosphorylation of S6 ribosomal protein (S6rpSer235/236) that is downstream of mTORC1, suggesting a crosstalk between PGE2/cAMP and mTORC1. In addition, the effect of PGE2 on phospho-S6rp was mimicked by Forskolin, a pharmacological cAMP stimulator. Importantly, PGE2 enhanced the effect of IGF-1 on S6rp phosphorylation, supporting the existence of a positive reinforcement by the interaction between the two pathways. Interestingly, PGE2 and forskolin had no effect on Akt phosphorylation (Thr308 and Ser473), suggesting a link downstream of Akt. The activation of the mTORC-1 pathway upon PGE2 treatment paralleled an increase in the phosphorylation of cAMP response element-binding protein (CREB), a substrate of protein kinase A (PKA). In the presence of the PKA inhibitor H89, baseline phosphorylation of S6rp and PGE2-activated S6rp phosphorylation were reduced, indicating a role of PKA in the crosstalk. In summary, our data provide the first evidence of a crosstalk between the PGE2/EP2/cAMP and IGF-1/Akt/mTORC1 signaling pathways. Since both pathways are over-activated in obesity-associated cancers, this interaction may be of great importance in elucidating the tumor-promoting effects of obesity in pancreatic cancer. Ultimately, a detailed understanding of these molecular links may provide mechanistic targets for novel efficacious interventions devoid of adverse effects for pancreatic cancer in an increasingly obese population. Citation Format: Hui-Hua Chang, Guido Eibl. Signaling cross-talks in obesity-associated pancreatic cancer: Interaction between prostaglandin E2 signaling and mTOR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5291. doi:10.1158/1538-7445.AM2014-5291