Prostaglandin E2 activates cAMP response element-binding protein in glioma cells via a signaling pathway involving PKA-dependent inhibition of ERK
Introduction
Prostaglandin E2 (PGE2), a bioactive lipid mediator, plays a pivotal role in regulation of cellular homeostasis as well as in inflammation and tumorigenesis [1]. Under basal conditions the cellular production of PGE2 is tightly regulated by the concerted action of the cyclo-oxygenase-1 (COX-1) and the cytosolic prostaglandin E synthase (cPGES) [2], [3]. In response to inflammatory stimuli, the cellular production of PGE2 dramatically increases as a result of arachidonic acid oxidation by the inducible cyclo-oxygenase-2 (COX-2) and microsomal PGES-1 (mPGES-1) activities [4], [5]. On the other hand, constitutive expression of COX-2 and/or mPGES-1 constitutes a biological hallmark of several human tumors, and it has been associated with increased tumor aggressiveness and poor clinical outcome [6], [7]. While the effects of COX-2 in influencing tumor cell behavior were found in some instances to be independent from the production of PGE2 [8], [9] there is compelling evidence to suggest a critical role for PGE2 in regulation of tumor cell proliferation, survival, and invasion [10], [11], [12].
The signaling pathways by which PGE2 contributes to the biological behavior of tumor cells are initiated by its binding to specific membrane bound receptors that belong to the G-protein coupled receptor superfamily and are classified into four subtypes: EP-1, EP-2, EP-3 and EP-4 [13]. Activation of EP receptor-dependent signaling pathways results in phosphorylation of many substrates, including transcription factors, which in turn regulate cellular functions by altering the expression of target genes.
The cAMP response element (CRE)-binding protein CREB belongs to the basic leucine zipper motif family of transcription factors and promotes gene expression by binding to the conserved TGACGTCA promoter sequence [14]. Phosphorylation of CREB at the serine residue 133 (Ser-133) increases its transcriptional activity and leads to stimulation of CREB-dependent gene expression [14]. Activation of CREB is critical to the regulation of a wide array of cellular functions, including survival and proliferation [15], [16], [17].
Agonist-induced increases in the intracellular levels of cAMP result in phosphorylation of CREB at Ser-133 via activation of protein kinase A (PKA), a serine-threonine kinase implicated in a wide array of biological processes [18]. Under basal conditions, PKA is localized in the cytosol as an inactive enzyme composed of two regulatory (R) subunits associated with two catalytic (C) subunits [19]. In mammalian cells, two isoforms of PKA (type I and type II) have been identified based on the composition of the regulatory subunits (RI and RII) [19]. These isoforms are differentially expressed in a variety of cells and exert distinct roles in regulation of cellular processes [20], [21]. In response to the binding of cAMP to each of the regulatory subunits, the catalytic subunits of PKA are released and translocate into the nucleus to stimulate gene expression by phosphorylating CREB at Ser-133. In addition to PKA, other protein kinases, including members of the mitogen activated protein kinase and the PI3-kinase/AKT pathways, have been reported to contribute to CREB activation either by promoting its phosphorylation at Ser-133 or by stimulating the recruitment of proteins that can potentiate CREB-mediated transcription [22], [14].
Malignant gliomas are among the most aggressive primary tumors of the central nervous system [23]. Despite treatment, patients with malignant gliomas die within 12–14 months from diagnosis [23]. Alterations in the metabolism of arachidonic acid, including constitutive expression of COX-2 and mPGES-1 have been detected in human gliomas [24], [25]. Moreover, levels of PGE2 were found to be increased in tissue samples and cerebral fluid of patients with malignant gliomas [26]. While these findings suggest a critical role for PGE2 in brain tumor growth and development, surprisingly the signaling pathways by which PGE2 regulates glioma cell growth have not been yet characterized. Using the U87-MG cell line as an in vitro model to study the contribution of PGE2 to glioma cell behavior, we recently demonstrated that PGE2 promotes glioma cell proliferation via a signaling pathway involving activation of PKA [25]. The present study was undertaken to further characterize the signaling events downstream to PKA activated by PGE2 in glioma cells.
Section snippets
Reagents
Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). H-89, KT-5720, PD98059, LY294002, wortmannin, 8-4-chlorophenylthioadenosine-3′,5′-cyclic-monophosphate (8-CTP-cAMP), N6-mono-t-butyryl-carbomoyl-3′,5′-cyclic-monophosphate (6-MBC-cAMP) were purchased from Calbiochem (San Diego, CA). Forskolin, 8-methylamino-adenosine-3′,5′-cyclic-monophosphate (8-MA-cAMP), 8-piperidinoadenosine-3′,5′-cyclic-monophosphate (8-PIP-cAMP) and anti-β-actin antibodies were purchased from Sigma–Aldrich
PGE2 stimulates phosphorylation of CREB at Ser-133 and CREB-dependent transcription
We previously reported that PGE2 stimulates PKA activity in U87-MG cells [25]. Given that CREB is a downstream target of PKA, we sought to determine whether stimulation of U87-MG cells with PGE2 resulted in CREB activation. Cells were stimulated with PGE2 or vehicle, and CREB activation was assessed by Western blot analyses using antibodies that recognize CREB phosphorylated at Ser-133. Challenge of U87-MG cells with varying concentrations of PGE2 (0.01–10 μM) stimulated CREB phosphorylation in
Discussion
Activation of CREB plays a critical role in regulation of proliferation in normal and tumor cells [15], [16]. Moreover, other studies have reported increased protein expression of activated CREB in tumor tissues of patients with high-grade astrocytomas [39]. However, the upstream signals responsible for activation of CREB in glioma cells are not understood. In here, we present evidence that stimulation of U87-MG cells with PGE2 results in activation of CREB, as demonstrated by increased
Acknowledgments
We are grateful to Dr. Stanley McKnight for providing the dominant negative PKA construct and Dr. David Ginty for providing the dominant negative CREB constructs. We thank Dr. Cary Mariash for the critical reading of the manuscript. We thank Dr. Dino Rotondo and Dr. Norrie Wilson for helpful discussions. This work was supported by the Methodist Research Institute Funds to M.T.R.
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