5′‐N‐ethylcarboxamide induces IL‐6 expression via MAPKs and NF‐κB activation through Akt, Ca2+/PKC, cAMP signaling pathways in mouse embryonic stem cells

Many studies suggest that adenosine modulates cell responses in a wide array of tissues through potent and selective regulation of cytokine production. This study examined the effects of adenosine on interleukin (IL)‐6 expression and its related signal pathways in mouse embryonic stem (ES) cells. In this study, the adenosine analogue 5′‐N‐ethylcarboxamide (NECA) increased IL‐6 protein expression level. Mouse ES cells expressed the A1, A2A, A2B, and A3 adenosine receptors (ARs), whose expression levels were increased by NECA and NECA‐induced increase of IL‐6 mRNA expression or secretion level was inhibited by the non‐specific AR inhibitor, caffeine. NECA increased Akt and protein kinase C (PKC) phosphorylation, intracellular Ca2+ and cyclic adenosine monophosphate (cAMP) levels, which were blocked by caffeine. On the other hand, NECA‐induced IL‐6 secretion was partially inhibited by Akt inhibitor, bisindolylmaleimide I (PKC inhibitor), SQ 22536 (adenylate cyclate inhibitor) and completely blocked by the 3 inhibitor combination treatment. In addition, NECA increased mitogen activated protein kinase' (MAPK) phosphorylation, which were partially inhibited by the Akt inhibitor, bisindolylmaleimide I, and SQ 22536 and completely blocked by the 3 inhibitor combination treatment. NECA‐induced increases of IL‐6 protein expression and secretion levels were inhibited by MAPK inhibition. NECA‐induced increase of nuclear factor (NF)‐κB phosphorylation was inhibited by MAPK inhibitors. NECA also increased cAMP response element‐binding protein (CREB) phosphorylation, which was blocked by MAPK or NF‐κB inhibitors. Indeed, NECA‐induced increase of IL‐6 protein expression and secretion was blocked by NF‐κB inhibitors. In conclusion, NECA stimulated IL‐6 expression via MAPK and NF‐κB activation through Akt, Ca2+/PKC, and cAMP signaling pathways in mouse ES cells. J. Cell. Physiol. 219: 752–759, 2009. © 2009 Wiley‐Liss, Inc.

[1]  M. Feldmann,et al.  Rac mediates TNF-induced cytokine production via modulation of NF-kappaB. , 2008, Molecular immunology.

[2]  Y. Kim,et al.  High‐Glucose‐Induced Prostaglandin E2 and Peroxisome Proliferator‐Activated Receptor δ Promote Mouse Embryonic Stem Cell Proliferation , 2008 .

[3]  Min Young Lee,et al.  Sonic Hedgehog Stimulates Mouse Embryonic Stem Cell Proliferation by Cooperation of Ca2+/Protein Kinase C and Epidermal Growth Factor Receptor As Well as Gli1 Activation , 2007, Stem cells.

[4]  D. Meldrum,et al.  Stem cell delivery to the heart: clarifying methodology and mechanism. , 2007, Critical care medicine.

[5]  Meijing Wang,et al.  STEM CELL MECHANISMS AND PARACRINE EFFECTS: POTENTIAL IN CARDIAC SURGERY , 2007, Shock.

[6]  K. Lillemoe,et al.  THE CRITICAL ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN PULMONARY VASCULAR REMODELING AFTER LUNG INJURY , 2007, Shock.

[7]  J. Heo,et al.  ATP Stimulates Mouse Embryonic Stem Cell Proliferation via Protein Kinase C, Phosphatidylinositol 3‐Kinase/Akt, and Mitogen‐Activated Protein Kinase Signaling Pathways , 2006, Stem cells.

[8]  Hajime Isomoto,et al.  Interleukin 6 upregulates myeloid cell leukemia‐1 expression through a STAT3 pathway in cholangiocarcinoma cells , 2005, Hepatology.

[9]  Matthew R. Jones,et al.  Animal models for the study of adenosine receptor function , 2005, Journal of cellular physiology.

[10]  T. Patel,et al.  Translational Regulation of X-Linked Inhibitor of Apoptosis Protein by Interleukin-6 , 2004, Cancer Research.

[11]  B. Fredholm,et al.  Adenosine receptors as targets for drug development. , 2003, Drug news & perspectives.

[12]  M. Scanlon,et al.  Adenosine‐induced IL‐6 expression in pituitary folliculostellate cells is mediated via A2b adenosine receptors coupled to PKC and p38 MAPK , 2003, British journal of pharmacology.

[13]  B. Fredholm,et al.  Signaling pathway from the human adenosine A(3) receptor expressed in Chinese hamster ovary cells to the extracellular signal-regulated kinase 1/2. , 2002, Molecular pharmacology.

[14]  K. Ravid,et al.  Overexpression of A3 adenosine receptors in smooth, cardiac, and skeletal muscle is lethal to embryos. , 2002, Microvascular research.

[15]  B. Fredholm,et al.  International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. , 2001, Pharmacological reviews.

[16]  H. Lagercrantz,et al.  Neurotransmitters and neuromodulators during early human development. , 2001, Early human development.

[17]  K. Klotz,et al.  Regulation of p42/p44 mitogen-activated protein kinase by the human adenosine A3 receptor in transfected CHO cells. , 2001, European journal of pharmacology.

[18]  J. Linden.  Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection. , 2001, Annual review of pharmacology and toxicology.

[19]  A. Wittinghofer,et al.  Mechanism of Regulation of the Epac Family of cAMP-dependent RapGEFs* , 2000, The Journal of Biological Chemistry.

[20]  M. Schwaninger,et al.  Adenosine‐induced expression of interleukin‐6 in astrocytes through protein kinase A and NF‐IL‐6 , 2000, Glia.

[21]  J. Lunz,et al.  Growth control of human biliary epithelial cells by interleukin 6, hepatocyte growth factor, transforming growth factor β1, and activin a: Comparison of a cholangiocarcinoma cell line with primary cultures of non‐neoplastic biliary epithelial cells , 2000, Hepatology.

[22]  G. Haegeman,et al.  The Nuclear Factor-κB Engages CBP/p300 and Histone Acetyltransferase Activity for Transcriptional Activation of the Interleukin-6 Gene Promoter* , 1999, The Journal of Biological Chemistry.

[23]  G. Gores,et al.  Inhibition of interleukin 6–mediated mitogen‐activated protein kinase activation attenuates growth of a cholangiocarcinoma cell line , 1999, Hepatology.

[24]  R. Corradetti,et al.  Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices , 1999, British journal of pharmacology.

[25]  I. Biaggioni,et al.  Role of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase kinase in adenosine A2B receptor-mediated interleukin-8 production in human mast cells. , 1999, Molecular pharmacology.

[26]  I. Biaggioni,et al.  Role of p 38 Mitogen-Activated Protein Kinase and Extracellular Signal-Regulated Protein Kinase Kinase in Adenosine A 2 B Receptor-Mediated Interleukin-8 Production in Human Mast Cells , 1999 .

[27]  R. Perona,et al.  Multiple Signalling Pathways Lead to the Activation of the Nuclear Factor κB by the Rho Family of GTPases* , 1998, The Journal of Biological Chemistry.

[28]  B. Fredholm.  Adenosine and neuroprotection. , 1997, International review of neurobiology.

[29]  B. Fredholm.  Chapter 11 Adenosine and Neuroprotection , 1996 .

[30]  I. Biaggioni,et al.  Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma. , 1995, The Journal of clinical investigation.

[31]  A. Rashtchian,et al.  Novel methods for cloning and engineering genes using the polymerase chain reaction. , 1995, Current opinion in biotechnology.

[32]  K. Kataoka,et al.  Adenosine enhances intracellular Ca2+ mobilization in conjunction with metabotropic glutamate receptor activation by t-ACPD in cultured hippocampal astrocytes , 1994, Neuroscience Letters.

[33]  M. Walker,et al.  Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors. , 1993, PCR methods and applications.

[34]  R. North,et al.  Functional expression of adenosine A2b receptor in Xenopus oocytes. , 1993, Molecular pharmacology.

[35]  S. D. Coleman,et al.  Interstitial Transudate Purines in Normoxic and Hypoxic Immature and Mature Rabbit Hearts , 1990, Pediatric Research.

[36]  H. van Belle,et al.  Formation and release of purine catabolites during hypoperfusion, anoxia, and ischemia. , 1987, The American journal of physiology.

[37]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.