Inducible cAMP Research Articles

Salt-inducible kinase 1 (SIK1) is induced by gastrin and inhibits migration of gastric adenocarcinoma cells.

Salt-inducible kinase 1 (SIK1/Snf1lk) belongs to the AMP-activated protein kinase (AMPK) family of kinases, all of which play major roles in regulating metabolism and cell growth. Recent studies have shown that reduced levels of SIK1 are associated with poor outcome in cancers, and that this involves an invasive cellular phenotype with increased metastatic potential. However, the molecular mechanism(s) regulated by SIK1 in cancer cells is not well explored. The peptide hormone gastrin regulates cellular processes involved in oncogenesis, including proliferation, apoptosis, migration and invasion. The aim of this study was to examine the role of SIK1 in gastrin responsive adenocarcinoma cell lines AR42J, AGS-GR and MKN45. We show that gastrin, known to signal through the Gq/G11-coupled CCK2 receptor, induces SIK1 expression in adenocarcinoma cells, and that transcriptional activation of SIK1 is negatively regulated by the Inducible cAMP early repressor (ICER). We demonstrate that gastrin-mediated signalling induces phosphorylation of Liver Kinase 1B (LKB1) Ser-428 and SIK1 Thr-182. Ectopic expression of SIK1 increases gastrin-induced phosphorylation of histone deacetylase 4 (HDAC4) and enhances gastrin-induced transcription of c-fos and CRE-, SRE-, AP1- and NF-κB-driven luciferase reporter plasmids. We also show that gastrin induces phosphorylation and nuclear export of HDACs. Next we find that siRNA mediated knockdown of SIK1 increases migration of the gastric adenocarcinoma cell line AGS-GR. Evidence provided here demonstrates that SIK1 is regulated by gastrin and influences gastrin elicited signalling in gastric adenocarcinoma cells. The results from the present study are relevant for the understanding of molecular mechanisms involved in gastric adenocarcinomas.

Abstract A54: PTHR1 signaling regulates the invasion and differentiation stage of osteosarcoma

Abstract Osteosarcoma (OS) is the most common tumor of bone. Parathyroid hormone (PTH) is a major endocrine regulator of skeletal development and homeostasis. We have sought to understand the contribution of PTH and parathyroid hormone related protein (PTHrP) in the regulation of osteosarcoma behaviour. In the present study we have used shRNA to knock-down the PTH receptor (PTHR1) to understand the role of PTH/PTHrP signaling in OS. Methods: shRNA to PTHR1 were designed and screened for activity using cyclic AMP (cAMP) radioimmunoassay following stimulation with PTH(1-34) (1). The most effective shRNA was designated PTHR1.358. OS cell lines derived from Osx-Cre p53fl/flpRbfl/fl mice (2) were stably infected with PTHR1.358 or Ren.1309 (negative control) and tested for gene expression changes, cellular invasion and cell cycle parameters. Cells were tagged with luciferase and grafted subcutaneously to determine the in vivo effects of PTHR1 signaling. Results: We have made use of an engineered murine model in which p53 and Rb have been deleted in an osteoblast-specific manner using Osx-Cre. Tumors in the mouse model share the cardinal features of human OS including histology, metastatic behavior and gene expression profiles. PTHR1 and PTHrP are expressed in the primary and metastatic lesions, and OS cell cultures derived from the tumors show cAMP induction in response to PTH or PTHrP treatment. Expression analysis confirmed PTHR1 expression in human OS. PTHR1 knock-down did not affect viability, but impaired the invasive capacity of the cells and altered gene expression, notably a reduction in RANKL RNA, an increase in OPG, and increased mRNA for Phex and matrix gla protein (MGP), with the latter effects likely related to the increased mineralization in knockdown cells. Strikingly, PTHR1.358 expressing cells showed a dramatically reduced proliferation in vivo which did not appear due to profound death of the cells but rather, as revealed by microCT, the acquisition of a more differentiated phenotype in the tumors. The Pthr1.358 tumors were smaller and more highly mineralized. PTH treatment did not influence tumor growth in vivo, nor did treatment with neutralizing antibody against PTHrP (3), the likely in vivo ligand for PTHR1 in OS cells. These results together establish clearly the importance of signaling through the PTHR1 in maintaining OS proliferation, but indicate that extracellular PTHrP is not significantly contributing to this effect, making it likely that an intracrine action of PTHrP contributes to this aspect of OS biology. Conclusions: Collectively these studies demonstrate that PTHR1 signaling plays a cell autonomous role in OS, acting in an intracrine manner. Inhibition of PTHR1 signaling reduced in vitro cell invasion but most notably profoundly reduces OS proliferation in vivo and drives the acquisition of a mineralized phenotype. Therapeutic inhibition of PTHR1 may be useful at impairing OS proliferation and/or increasing differentiation. 1. R. A. Dickins et al., Nat Genet 37, 1289 (Nov, 2005). 2. C. R. Walkley et al., Genes Dev 22, 1662 (Jun 15, 2008). 3. E. Onuma et al., Anticancer Res 24, 2665 (Sep-Oct, 2004). Citation Format: Patricia WM Ho, Megan Russell, Ankita Goradia, Alistair Chalk, John Slavin, Ross Dickins, T. John Martin, Carl Walkley. PTHR1 signaling regulates the invasion and differentiation stage of osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A54.

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

β-cell induction in vivo in severely diabetic male mice by changing the circulating levels and pattern of the ratios of estradiol to androgens.

Previously we have generated transgenic (Tg) mice developing severe diabetes early in life with a profound depletion of β-cells with β-cell-directed expression of inducible cAMP early repressor-Iγ. Only male mice continue to demonstrate hyperglycemia throughout life. To investigate this sexual dimorphism, we treated severely diabetic male Tg mice with orchiectomy (ORX) or 17β-estradiol (E2) pellet implantation alone or in combination with ORX and E2-implantation to change the circulating levels and patterns of the ratio of estradiol to androgens. In the Tg-ORX group, the blood-glucose levels decreased to a certain level within several weeks but never reached the female Tg-control level. In contrast, the Tg-ORX+E2 or Tg-E2 group showed a more rapid drop in blood glucose to the basal level with a substantial increase in β-cells, thus preventing the occurrence of severe diabetes in the male mice. The β-cells, not only within islet but also in and adjacent to ducts and scattered β-cell clusters, were strongly induced by 1 week after treatment, and the islet morphology dramatically changed. Enhanced β-cell induction in the ducts occurred concomitantly with markedly increased levels of pancreatic duodenal homeobox-1 and related transcription factors. The glucose-lowering and β-cell-increasing effects were independent of the age at which the treatment is started. These data provide evidence that the circulating level of E2 and the ratio of E2 to T greatly affect the blood glucose levels, the β-cell induction, and the islet morphology in diabetic male Tg mice. This novel mechanism offers great potential for developing strategies to increase the number of β-cells in vivo.

Quercetin-3-O-glucuronide inhibits noradrenaline-promoted invasion of MDA-MB-231 human breast cancer cells by blocking β2-adrenergic signaling

Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. The adrenergic system plays a central role in stress signaling, and excessive stress was found to be associated with increased production of reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage in tissues and causes the development of diseases such as cancer. In this study, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin, which is a type of natural flavonoid, on the catecholamine-induced β2-adrenergic receptor (β2-AR)-mediated response in MDA-MB-231 human breast cancer cells expressing β2-AR. Treatment with A or NA at concentrations above 1μM generated significant levels of ROS, and NA treatment induced the gene expression of heme oxygenase-1 (HMOX1), and matrix metalloproteinase-2 (MMP-2) and -9 (MMP9). Inhibitors of p38 MAP kinase (SB203580), cAMP-dependent protein kinase (PKA) (H-89), activator protein-1 (AP-1) transcription factor (SR11302), and NF-κB and AP-1 (Tanshinone IIA) decreased MMP2 and MMP9 gene expression. NA also enhanced cAMP induction, RAS activation and phosphorylation of ERK1/2. These results suggested that the cAMP-PKA, MAPK, and ROS-NF-κB pathways are involved in β2-AR signaling. Treatment with 0.1μM Q3G suppressed ROS generation, cAMP and RAS activation, phosphorylation of ERK1/2 and the expression of HMOX1, MMP2, and MMP9 genes. Furthermore, Q3G (0.1μM) suppressed invasion of MDA-MB-231 breast cancer cells and MMP-9 induction, and inhibited the binding of [3H]-NA to β2-AR. These results suggest that Q3G may function to suppress invasion of breast cancer cells by controlling β2-adrenergic signaling, and may be a dietary chemopreventive factor for stress-related breast cancer.

Prostaglandin E2 inhibits IL-27 production by bone marrow-derived dendritic cells (CCR6P.281)

Abstract Interleukin-27 (p28/EBI3) is expressed by activated antigen presenting cells and has been shown to have immunosuppressive functions including the inhibition of Th17 cell development and the induction of type 1 regulatory T cells (Tr1). In vivo studies demonstrated the anti-inflammatory effects of IL-27 in murine models of collagen-induced arthritis (CIA), inflammatory bowel disease (IBD), and experimental autoimmune encephalomyelitis. We showed previously that PGE2 has proinflammatory effects in models of CIA and IBD through the stimulation of IL-23 production and subsequent development of Th17 cells. Here we report on a novel proinflammatory function through the inhibition of IL-27 production by LPS-stimulated murine BMDC. A similar effect was observed in the dendritic, macrophage and microglia cell lines DC2.4, RAW264.7 and BV2. We determined that the effect of PGE2 was mediated through EP2/EP4 receptors and induction of cAMP. Studies targeting various downstream signaling pathways are in progress. These studies further elucidate the mechanisms involved in the inflammatory role of PGE2, and might identify additional therapeutic targets for the treatment of inflammatory autoimmune diseases.

Deletion of transient receptor potential canonical channel 1 prevents pulmonary vascular hyper‐permeability and edema formation secondary to cAMP activation of SPHK1 (847.4)

Transient receptor potential channel (TRPC1) mediates store operated calcium (Ca2+) (SOC) entry in response to endoplasmic reticulum (ER) store depletion by IP3, and thereby signals the loss of endothelial barrier function. We show that pulmonary endothelial cells isolated from TRPC1 null mice failed to increase cytosolic Ca2+ entry, endothelial gap formation and permeability in response to thrombin and thereby rendered the mice resistant to thrombin and thapsigargin‐induced lung vascular barrier dysfunction. cAMP as well as the lipid mediator sphingosine‐1‐phosphate (S1P) stabilize adherens junctions (AJs) and prevent lung vascular permeability and inflammation. Thus, we tested the hypothesis that TRPC1 deletion enhances endothelial barrier function through induction of cAMP and S1P signaling. Intriguingly, loss of TRPC1 in lung endothelial cells augmented sphingosine kinase1 (SPHK1) activity which induces the phosphorylation of sphingosine into S1P preventing AJs disruption. We show that EPAC but not PKA induced the activation of SPHK1 in human lung endothelial cells. EPAC activation enhanced endothelial barrier function in control cells but failed to strengthen barrier function in SPHK1 knockdown cells. Our results suggest that loss of TRPC1 deletion prevents increase in endothelial permeability through EPAC up regulation of SPHK1 activity. Thus, specific inhibitors design to target TRPC1 channel by increasing cAMP and S1P signaling may provide resistance to inflammatory pulmonary diseases such as acute lung injury and acute respiratory distress syndrome.Grant Funding Source: NIH

Identification of ICER’s ubiquitination sites to distinguish its Novel Ubiquitin Ligase (LB186)

Inducible cAMP Early Repressor (ICER) is a dominant negative transcriptional repressor that expresses tumor suppressor activity. In cancer cells, ICER is abnormally expressed and becomes modified by the posttranslational modification ubiquitination. Consequentially, ICER is degraded or mislocalized from the nucleus to the cytosol where it is non‐ functional. When exogenous ICER is introduced into cancer cells, it inhibits the transformed phenotype of cancer cells. The goal of this project is to identify the ubiquitinated residues of ICER to ultimately rescue this protein from degradation and cytosolic localization. Since lysine residues are sites for ubiquitination, mutant forms of each residue were constructed using site‐directed mutagenesis. All lysine residues were substituted to arginine to create an ICER molecule without lysines. Another 10 independent mutants were generated where all but one specific lysine was substituted by arginine. All clones were subcloned in a mammalian expression vector. By using these altered forms of ICER, we have found that ICER is ubiquitinated in several lysines. This data suggested alternative modes of regulating ICER degradation and subcellular localization. The long‐term goal of this project is to identify ways of inhibiting ICER degradation in cancer cells as a new mode for cancer treatment.

Biological effects of leukotriene E4 on eosinophils

Studies demonstrate the existence of novel receptors for cysteinyl leukotrienes (CysLTs) that are responsive to leukotriene (LT) E4 and might be pathogenic in asthma. Given the eosinophilic infiltration in this disorder, we investigated eosinophil expression of P2Y12 and gpr99 and their capacity to respond to LTE4. Receptor transcript expression was investigated via quantitative PCR and surface protein expression via flow cytometry. We investigated LTE4 influences on eosinophils including Ca+2 flux, cAMP induction, modulation of adhesion molecule expression, apoptosis and degranulation. Eosinophils displayed both transcript and surface protein expression of P2Y12 and gpr99. We could not find evidence of LTE4 activation of eosinophils, however, LTE4 induced cAMP expression, and preincubation of eosinophils with LTE4 inhibited degranulation. Even though eosinophils are an important source of CysLTs in AERD, eosinophils are not themselves the pro-inflammatory biological target and, in contrast, LTE4 via cAMP primarily elicits anti-inflammatory responses.

Development of a novel antibody to calcitonin gene-related peptide for the treatment of osteoarthritis-related pain

Investigate a role for calcitonin gene-related peptide (CGRP) in osteoarthritis (OA)-related pain. Neutralizing antibodies to CGRP were generated de novo. One of these antibodies, LY2951742, was characterized invitro and tested in pre-clinical invivo models of OA pain. LY2951742 exhibited high affinity to both human and rat CGRP (KD of 31 and 246pM, respectively). The antibody neutralized CGRP-mediated induction of cAMP in SK-N-MC cells invitro and capsaicin-induced dermal blood flow in the rat. Neutralization of CGRP significantly reduced pain behavior as measured by weight bearing differential in the rat monoiodoacetate model of OA pain in a dose-dependent manner. Moreover, pain reduction with neutralization of CGRP occurred independently of prostaglandins, since LY2951742 and NSAIDs worked additively in the NSAID-responsive version of the model and CGRP neutralization remained effective in the NSAID non-responsive version of the model. Neutralization of CGRP also provided dose-dependent and prolonged (>60 days) pain reduction in the rat meniscal tear model of OA after only a single injection of LY2951742. LY2951742 is a high affinity, neutralizing antibody to CGRP. Neutralization of CGRP is efficacious in several OA pain models and works independently of NSAID mechanisms of action. LY2951742 holds promise for the treatment of pain in OA patients.

Identification of Protein-Protein Interactions Between the TatB and TatC Subunits of the Twin-Arginine Translocase System and the Redox Enzyme Maturation Protein Chaperones

The Twin-arginine translocation pathway (Tat) serves for targeting and translocation of fully folded proteins across cytoplasmic membrane in bacterial and plant chloroplast thylakoid membranes. TatA, TatB, and TatC are the core components of the Escherichia coli Tat system, where the TatB and TatC subunits are considered to form a receptor complex for Tat-bound proteins. TatB protein is composed of a transmembrane helix and extramembrane part facing the cytoplasmic side of the cell, the structure of TatC revealed six transmembrane helixes and four extramembrane loops. Redox Enzyme Maturation Proteins (REMPs) are the system specific chaperons, which play significant roles in respiratory enzyme maturation, including targeting to the Tat translocase system. We applied a bacterial two-hybrid technique to determine whether REMPs interact with the extramembrane loops of the TatBC recognition module. Individual loops were cloned and fused with one of the two catalytic domains of adenylate cyclase from Bordetella pertussis. The constructs were co-expressed in Escherichia coli where peptides interaction leads to functional complementation between the two adenylate cyclase catalytic domains resulting in increased cAMP levels and induction of beta-galactosidase expression. Our study demonstrated that the DmsD chaperone interacts with the loops 1, 2 and 4 of TatC and with the proposed unfolded domain of TatB. Other E. coli chaperones homologous to DmsD- YcdY and NarJ - also show an interaction with the TatB domain. This suggests that there may be a handoff mechanism of Tat substrates to the translocase via the REMP.

Decompensation of β-cells in diabetes: when pancreatic β-cells are on ICE(R).

Insulin production and secretion are temporally regulated. Keeping insulin secretion at rest after a rise of glucose prevents exhaustion and ultimately failure of β-cells. Among the mechanisms that reduce β-cell activity is the inducible cAMP early repressor (ICER). ICER is an immediate early gene, which is rapidly induced by the cyclic AMP (cAMP) signaling cascade. The seminal function of ICER is to negatively regulate the production and secretion of insulin by repressing the genes expression. This is part of adaptive response required for proper β-cells function in response to environmental factors. Inappropriate induction of ICER accounts for pancreatic β-cells dysfunction and ultimately death elicited by chronic hyperglycemia, fatty acids, and oxidized LDL. This review underlines the importance of balancing the negative regulation achieved by ICER for preserving β-cell function and survival in diabetes.

Regulatory T-cells and cAMP suppress effector T-cells independently of PKA–CREM/ICER: a potential role for Epac

cAMP signalling is both a major pathway as well as a key therapeutic target for inducing immune tolerance and is involved in Treg cell (regulatory T-cell) function. To achieve potent immunoregulation, cAMP can act through several downstream effectors. One proposed mechanism is that cAMP-mediated suppression, including immunosuppression by Treg cells, results from activation of PKA (protein kinase A) leading to the induction of the transcription factor ICER (inducible cAMP early repressor). In the present study, we examined CD4(+)CD25(-) Teff cell (effector T-cell) and CD4(+)CD25(+) Treg cell immune responses in Crem (cAMP-response-element modulator) gene-deficient mice which lack ICER (Crem(-/-)/ICER-deficient mice). ICER deficiency did not significantly alter the frequency or number of Treg cells and Teff cells. Treg cells or a pharmacological increase in cAMP suppressed Teff cells from Crem(+/+) and Crem(-/-)/ICER-deficient mice to an equivalent degree, demonstrating that ICER is dispensable in these functions. Additionally, activating the cAMP effector Epac (exchange protein directly activated by cAMP) suppressed Teff cells. Treg cells expressed low levels of all cyclic nucleotide Pde (phosphodiesterase) genes tested, but high levels of Epac. These data identify ICER as a redundant mediator of Treg cells and cAMP action on Teff cells and suggest that Epac may function as an alternative effector to promote cAMP-dependent Teff cell suppression.

Serotonin 2C receptor antagonists induce fast-onset antidepressant effects

Current antidepressants must be administered for several weeks to produce therapeutic effects. We show that selective serotonin 2C (5-HT2C) antagonists exert antidepressant actions with a faster-onset (5 days) than that of current antidepressants (14 days) in mice. Subchronic (5 days) treatment with 5-HT2C antagonists induced antidepressant behavioral effects in the chronic forced swim test (cFST), chronic mild stress (CMS) paradigm and olfactory bulbectomy paradigm. This treatment regimen also induced classical markers of antidepressant action: activation of cAMP response element-binding protein (CREB) and induction of brain-derived neurotrophic factor (BDNF) in the medial prefrontal cortex (mPFC). None of these effects were induced by subchronic treatment with citalopram, a prototypical selective serotonin reuptake inhibitor (SSRI). Local infusion of 5-HT2C antagonists into the ventral tegmental area was sufficient to induce BDNF in the mPFC, and dopamine D1 receptor antagonist treatment blocked the antidepressant behavioral effects of 5-HT2C antagonists. 5-HT2C antagonists also activated mammalian target of rapamycin (mTOR) and eukaryotic elongation factor 2 (eEF2) in the mPFC, effects recently linked to rapid antidepressant action. Furthermore, 5-HT2C antagonists reversed CMS-induced atrophy of mPFC pyramidal neurons. Subchronic SSRI treatment, which does not induce antidepressant behavioral effects, also activated mTOR and eEF2 and reversed CMS-induced neuronal atrophy, indicating that these effects are not sufficient for antidepressant onset. Our findings reveal that 5-HT2C antagonists are putative fast-onset antidepressants, which act through enhancement of mesocortical dopaminergic signaling.

NR4A2 Is Regulated by Gastrin and Influences Cellular Responses of Gastric Adenocarcinoma Cells

The peptide hormone gastrin is known to play a role in differentiation, growth and apoptosis of cells in the gastric mucosa. In this study we demonstrate that gastrin induces Nuclear Receptor 4A2 (NR4A2) expression in the adenocarcinoma cell lines AR42J and AGS-GR, which both possess the gastrin/CCK2 receptor. In vivo, NR4A2 is strongly expressed in the gastrin responsive neuroendocrine ECL cells in normal mucosa, whereas gastric adenocarcinoma tissue reveals a more diffuse and variable expression in tumor cells. We show that NR4A2 is a primary early transient gastrin induced gene in adenocarcinoma cell lines, and that NR4A2 expression is negatively regulated by inducible cAMP early repressor (ICER) and zinc finger protein 36, C3H1 type-like 1 (Zfp36l1), suggesting that these gastrin regulated proteins exert a negative feedback control of NR4A2 activated responses. FRAP analyses indicate that gastrin also modifies the nucleus-cytosol shuttling of NR4A2, with more NR4A2 localized to cytoplasm upon gastrin treatment. Knock-down experiments with siRNA targeting NR4A2 increase migration of gastrin treated adenocarcinoma AGS-GR cells, while ectopically expressed NR4A2 increases apoptosis and hampers gastrin induced invasion, indicating a tumor suppressor function of NR4A2. Collectively, our results uncover a role of NR4A2 in gastric adenocarcinoma cells, and suggest that both the level and the localization of NR4A2 protein are of importance regarding the cellular responses of these cells.

A novel intronic promoter of theCremgene induces small ICER (smICER) isoforms

The transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) regulate gene transcription in response to elevated cAMP levels. The Crem isoform inducible cAMP early repressor (Icer) is transcribed by the internal promoter P2 as a critical regulator of multiple cellular processes. Here, we describe a novel inducible Crem isoform, small Icer (smIcer), regulated by a newly identified promoter (P6). ChIP revealed binding of CREB to P6 in human and mouse myocardium. P6 activity was induced by constitutively active CREB or stimulation of adenylyl cyclase. In mice, smIcer mRNA was ubiquitously expressed and transiently induced by β-adrenoceptor stimulation e.g., in heart and lung. SmICER repressed both basal and cAMP-induced activities of P6 and P2 promoters. Stimulation of adenylyl cyclase induced P2 and P6 in cell type-specific manner. Alternative translational start sites resulted in three different smICER proteins, linked to increased apoptosis sensitivity. In conclusion, the Crem gene provides two distinct and mutually controlled mechanisms of a cAMP-dependent induction of transcriptional repressors. Our results suggest not only that smICER is a novel regulator of cAMP-mediated gene regulation, but also emphasize that biological effects that have been ascribed solely to ICER, should be revised with regard to smICER.

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection (1). Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Cyclic nucleotide phosphodiesterase 3A1 protects the heart against ischemia-reperfusion injury

Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5±7.8% vs. 83.9±4.7%) as well as compensatory expansion of left ventricular diameter (4.19±0.19mm vs. 3.10±0.18mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (β-AR) response in TG mice. Importantly, the myocardial infarct size (4.0±1.8% vs. 24.6±3.8%) and apoptotic cell number (1.3±1.0% vs. 5.6±1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates β-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.

MC1R and cAMP signaling inhibit cdc25B activity and delay cell cycle progression in melanoma cells

The melanocortin 1 receptor (MC1R) mediates the tanning response through induction of cAMP and downstream pigmentary enzymes. Diminished function alleles of MC1R are associated with decreased tanning and increased melanoma risk, which has been attributed to increased rates of mutation. We have found that MC1R or cAMP signaling also directly decreases proliferation in melanoma cell lines. MC1R overexpression, treatment with the MC1R ligand, or treatment with small-molecule activators of cAMP signaling causes delayed progression from G2 into mitosis. This delay is caused by phosphorylation and inhibition of cdc25B, a cyclin dependent kinase 1-activating phosphatase, and is rescued by expression of a cdc25B mutant that cannot be phosphorylated at the serine 323 residue. These results show that MC1R and cAMP signaling can directly inhibit melanoma growth through regulation of the G2/M checkpoint.