protein kinase A signaling pathway

Advances in Neuroimmunology Vol. 5, pp. 261-269, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 096&5428/95 $29.00

Pergamon

0960-5428(95)00022-4

Regulation of astrocyte cell biology by the CAMP/protein kinase A signaling pathway Brandon S. Huneycutt and Etty N. Benveniste Department

of Cell Biology,

KeywordsSecond

University

messengers,

of Alabama

at Birmingham,

Birmingham,

AL 35294-0005,

USA

glial cells, cytokines.

Introduction Eukaryotic cells function by detecting and responding to changes in the extracellular environment. The molecular mechanisms involved in performing these tasks can be generalized into one single dynamic system: the binding and dissociation of protein subunits. Nowhere else in the biological system is the importance of protein-protein interactions better exemplified than in the second messenger cascades (for reviews see Cohen et al., 1995; Cohen, 1992; Mooibroek and Wang, 1988). The CAMP/protein kinase A (PKA) pathway, one of the best characterized of the transducing pathways, is dependent on the coordinated activation of three classes of protein (for review see Cooper et al., 1995). These include: adenylate cyclase (AC), the generators of CAMP (for review see Krupinski, 1991); PKA, the effector that directly activates the machinery of the cell through phosphorylation of protein substrates (for review see Taylor, 1989); and CAMP-specific phosphodiesterases (PDE), proteins that regulate the intracellular concentration of CAMP (Beltman et al., 1993). The sequential and parallel activation of these proteins generates a temporal pattern of signals that carry specific information about the conditions of the extracellular microenvironment. With eight differ261

ent isoforms of AC, six functional categories of phosphodiesterases, and at least two isoforms of PKA holoenzymes, the cell can increase its resolving power for the detection of minute changes in the microenvironment and, consequently, improve the precision of its cellular responses. In this review, we will briefly describe the components of the cAMP/PKA signaling pathway, and then discuss how activation of this intracellular pathway regulates the functions of an important cell type in the brain, the astrocyte. In addition, we provide examples of cross-talk between the cAMP/PKA pathway and another important signal transduction pathway, that being the activation of protein kinase C (PKC). cAMP/PKA signaling pathway Many of the seven membrane spanning receptors (prototype: P-adrenergic receptor) are linked to G proteins, a family of membrane associated heterotrimers (IX, p, y subunits) which relay changes in receptor conformation upon ligand binding to the downstream effector protein AC (for review see Strader et al., 1995). There are different types of G proteins. In this review, we will consider the G stimulatory

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(Gs) family, the isoform that activates AC. AC is an integral membrane protein that catalyzes the conversion of ATP into CAMP. CAMP, the second messenger in this pathway, activates the serine/threonine kinase PKA. PKA is a tetramer containing two CAMP binding regulatory subunits and two catalytic subunits. Two CAMP molecules bind to each regulatory subunit. As a result, a conformational change induces the dissociation of the two catalytic subunits. The catalytic subunit is an enzymatically active protein kinase only upon release from the regulatory subunits. The catalytic subunit can phosphorylate a broad spectrum of protein substrates, an event leading to changes in cell function and gene expression (see Fig. 1; this is an extremely simplified diagram of this pathway). PKA activates a broad group of genes which include the immediate early protooncogenes, fos and jun, a number of inflammatory cytokines, and growth/differentiation factors (for review see Karin, 1992). By phosphorylating a number of transcription factors, PKA

PROTEIN KINASE A

is able to control gene transcription by inducing the dissociation, translocation, reassembly and activation of specific transcription factors within relevant promoters. The first transcription factor to be characterized as a primary substrate for PKA was the CAMP response element binding protein (CREB), a transcription factor that binds to the CAMP response element (CRE) consensus sequences expressed in a number of promoters (for review see Karin, 1992). PKA activates the transcriptional activity of CREB by phosphorylating CREB at serine 133 in a domain required for transcriptional function (Gonzalez and Montminy, 1989). The concentrations of intracellular CAMP are strictly controlled by CAMP-specific PDE, enzymes that catalyze the conversion of CAMP into the non-cyclic isomer AMP. PDE is activated by an increase in cytosolic Ca2+, indicating that levels of the second messenger CAMP are controlled by another second messenger, Ca2+. The cAMP/PKA pathway does not operate in isolation, rather it can be influenced by

Protein -Phosphorylation

-

CELLULAR RESPONSE I

diagram of the cAMP/PKA signaling pathway. Ligand binding to a sevenFig. 1. Schematic membrane spanning receptor (P-adrenergic receptor) results in activation of a G protein (Gs), causing bound GDP to be replaced by GTP. The GsooGTP subunit dissociates, and then binds to and activates AC. AC converts ATP to CAMP, which is the second messenger of this pathway. Binding of CAMP to the regulatory subunits of PKA causes the catalytic subunits to dissociate. The dissociated (on serine/threonine residues) a number of catalytic subunits are active, and can phosphorylate The drug forskolin directly activates protein substrates, ultimately leading to cellular responses. AC, leading to increases in intracellular CAMP, while agents such as dbcAMP or 8-bromo-CAMP are CAMP analogs.

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components of another well understood second messenger pathway, the PKC system. In contrast to the cAMP/PKA system, the PKC pathway utilizes both Ca2f and lipids for activation, which ultimately regulate the activation of at least 11 different isoforms of PKC (for review see Nishizuka, 1992). PKC, like PKA, is also a serine/threonine kinase that regulates gene expression/function by the phosphorylation of protein substrates (see Fig. 2; this is an extremely simplified diagram of this pathway). Astrocytes Astrocytes are the predominant glial cell type in the mammalian brain and are essential for neuronal development, neuronal activity, and regulation of localized inflammatory responses. Astrocytes function to maintain an optimal environment for efficient synapse function by removing excess cations, uptake of released neurotransmitters, and providing protective signals for neurons at sites of injury by the elaboration of soluble products such as nerve growth factor (NGF), ciliary neurotropic factor (CNTF), and interleukin-6 (IL-6) (for review see

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Eddleston and Mucke, 1993). Furthermore, astrocytes contribute to both the induction and cessation of immune/inflammatory responses within the CNS by their ability to express class I and class II major histocompatibility complex (MHC) antigens, adhesion molecules such as intercellular adhesion molecule-l (ICAM-1) and vascular adhesion molecule-l (VCAM-I), and cytokines such as IL-l, TNF-a and TGF-P (for review see Benveniste, 1995). Astrocytes monitor the microenvironment of the CNS by cell surface receptors that reflect the characteristics of the CNS; these receptors bind neurotransmitters, neuropeptides and neurotrophins. Astrocytes also integrate the environment of the CNS with local inflammatory responses through the expression of receptors specific for various cytokines. The neurotransmitter class of receptors expressed by astrocytes include (Y-and P-adrenergic receptors, serotonin and dopamine receptors, and histaminergic receptors (for review see Shao et al., 1993). The specificity of neuropeptide receptors is also broad. They include receptors for vasoactive intestinal peptide (VIP), calcitonin gene related peptide, somatostatin,

PLASMA MEMBRANE

intracellularcompatment

Fig. 2. Schematic diagram of the Ca z+/PKC signaling pathway. Binding of a ligand to its receptor triggers activation of a G protein that, in turn, activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP,) to inositol 1,4,5trisphosphate (IP,) and 1,2-diacylglycerol (DAG). IP, acts to release intracellular stores of Ca2+; DAG remains in the membrane, where, in conjunction with Ca2+, it helps to activate PKC. PKC in turn phosphorylates (on serine/threonine residues) protein substrates. Phorbol esters (PMA, TPA) directly activate PKC, while Ca2+ ionophores act to increase intracellular Ca2+ levels.

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K and 6 opiates, substance P, and bradykinin (for review see Wilkin and Marriott, 1993). AC in astrocytes can be activated to generate CAMP through membrane associated trimeric G proteins coupled to ligand specific receptors (see Fig. 1). The first and historically best characterized of the G protein coupled receptors expressed by astrocytes are the B-adrenergic receptors, Bl and B2 (Salm and McCarthy, 1989). Agonists specific for the B-adrenergic receptors, isoproterenol and isoprenaline, each induces the accumulation of CAMP in the presence of the phosphodiesterase inhibitor, IBMX, as does forskolin, which is a direct activator of AC (Norris et al., 1994; Schwartz and Mishler, 1990). The natural ligand for the Badrenergic receptors, norepinephrine (NE), also is a strong inducer of CAMP in astrocytes (Norris et al., 1994). NE can also stimulate a-adrenergic receptors; the oil receptor has been shown to activate phosphoinositol hydrolysis in astrocytes (Pearce et al., 1986), while the (~2 receptor acts to inhibit CAMP production (Evans et al., 1984; Northam et al., 1989). Thus, the intracellular signals generated by NE stimulation of astrocytes include the activation/inhibition of CAMP and the activation of the PKC pathway. As mentioned previously, cross-talk between the cAMP/PKA and PKC pathways can occur by a variety of mechanisms. For example, phorbol esters, activators of PKC, can regulate the accumulation of CAMP in cells by the preferential activation of type II AC, and the type I or type III AC, which are both stimulated by Ca2+ and calmodulin, leading indirectly to increases in intracellular CAMP (Choi et al., 1993). Role of second messengers in modulating astrocyte gene expression A number of astrocyte gene products can be either positively or negatively regulated by cAMP/PKA. Some of these studies will be reviewed here as examples of how activa-

tion of PKA leads to changes in astrocyte function. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin3 (NT-3) and NT-4/5 are classical neurotrophic factors which exert their actions through high-affinity receptors residing within the cell membrane of responsive cells. The trk genes, a family of related transmembrane protein kinases, serve as high affinity receptors for the neurotrophins (for review see Thoenen, 1991). Astrocytes have the capacity to both produce neurotrophins as well as respond to them via expression of trk family members. NGF mRNA and secretion in astrocytes can be induced by a number of pharmacological agents including neurotransmitters, growth factors and cytokines, all of which activate distinct intracellular signaling pathways. Primary cultures of rat astrocytes constitutively express NGF mRNA, and treatment with the B-adrenergic agonist, isoproterenol, as well as forskolin, enhances NGF mRNA levels (Schwartz and Mishler, 1990). This increase in NGF mRNA expression is dependent on increases of CAMP in the cell induced by isoproterenol or forskolin. These results indicate that the NGF gene expression in astrocytes occurs through activation of the cAMP/PKA pathway. The PKC activator TPA is also capable of stimulating NGF secretion, as is the cytokine IL-l (Carman-Krzan and Wise, 1993). The IL-l induction of NGF is not mediated by PKC since down-regulation of PKC activity does not affect the stimulation induced by IL-l, nor is it mediated by activation of CAMP (Pshenichkin et al., 1994). The authors propose that the stimulatory action of IL-l on NGF expression may involve the inactivation of a protein phosphatase, leading to sustained activation of a unique protein kinase, which ultimately causes a net increase in the phosphorylation state of proteins involved in activation of NGF gene expression in astrocytes (Pshenichkin

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and Wise, 1995). Thus, NGF expression in astrocytes can be induced by CAMP, PKC activation, and an unknown kinase activated by IL-l. Basal levels of BDNF mRNA in astrocytes are very low, but can also be enhanced by forskolin treatment (Zafra et al., 1992). Interestingly, BDNF mRNA expression was also enhanced by treatment with the calcium ionophore ionomycin, and a strong synergistic effect between forskolin and ionomycin was noted, suggesting an involvement of both CAMP and Ca2+ in regulating BDNF expression in astrocytes. NE also significantly increased BDNF mRNA levels in astrocytes. A more recent study has extended these findings by demonstrating that BDNF and NT-3 mRNA expression in astrocytes is increased following treatment with IBMX, a phosphodiesterase inhibitor (Condorelli et al., 1994). In contrast, NT-4 mRNA expression was not elevated by IBMX treatment. Expression of neurotrophin receptors was also regulated by CAMP elevating agents; trkB mRNA was increased upon treatment with dbcAMP, S-bromo-cyclic AMP or IBMX, while trkC mRNA levels were unaffected (Condorelli et al., 1994). Thus, in astrocytes, expression of the neurotrophins NGF, BDNF, and NT3, as well as the trkB receptor, is regulated by activation of the CAMP second messenger system, with influences from Ca2+ and PKC activation. Ciliary neurotrophic factor (CNTF) has a broad range of activities such as neurotrophic properties, induction of acute phase protein expression in hepatocytes, promoting the maturation and survival of oligodendrocytes, and protecting oligodendrocytes against TNF+ induced death (Ip et al., 1992; Louis et al., 1993; Mayer et al., 1994). In astrocytes, constitutive levels of CNTF mRNA are high, but can be potently inhibited by a number of CAMP elevating agents such as isoproterenol, forskolin, VIP and 8-bromo-CAMP (Nagao et al., 1995; Rudge

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et al., 1994). This inhibition is due to stimulation of both al and P-adrenergic receptors (both pl and p2), and is thought to occur by inhibition of CNTF gene transcription. In contrast, activation of the a2 adrenergic receptor, which inhibits CAMP production in astrocytes, caused an increase in CNTF mRNA as did TPA, a PKC activator (Rudge demonstrate et al., 1994). These results that activation of the CAMP pathway in astrocytes inhibits CNTF expression, while activation of the PKC pathway enhances expression, indicating that the cAMP/PKA and PKC pathways exert differential effects on CNTF expression. Astrocytes have been implicated in contributing to localized inflammatory responses within the brain due to their ability to secrete a number of immunoregulatory cytokines such as IL-l, TNF-a, TGF-P, and IL6 (for review see Benveniste, 1995). IL6 is a cytokine that has both inflammatory actions within the CNS (astrogliosis, immunoglobulin production, T-cell activation), as well as anti-inflammatory effects, which include inhibition of TNF-(r and ICAMexpression in astrocytes (Benveniste et al., 1994, 1995; Selmaj et al., 1990; of Shrikant et al., 1995). The induction IL-6 in astrocytes has been extensively studied, particularly with regard to the signal transduction pathways involved in IL-6 expression. Astrocytes secrete IL-6 in response to a large collection of ligands; these include the cytokines IL-l, TNF-IY_ et al., 1990, 1994; and TGF-P (Benveniste Frei et al., 1989; Norris et al., 1994), the neurotransmitter NE (Maimone et al., 1993; Norris and Benveniste, 1993), the neuropeptides VIP and pituitary adenylate cyclase activating polypeptide (PACAP) (Gottschall et al., 1994; Grimaldi et al., 1994; Maimone et al., 1993), and lipopolysaccharide (Benveniste et al., 1990). NE, VIP and PACAP all stimulate IL-6 production through induction of CAMP, leading to activation of PKA (Grimaldi et

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al., 1994; Norris and Benveniste, 1993). The NE effect was predominantly mediated by activation of the B2-adrenergic receptors, with a minor contribution of et al., al-adrenergic receptors (Maimone 1993; Norris and Benveniste, 1993). In keeping with this observation, a number of pharmacological agents such as forskolin, which directly activates adenylate cyclase, isoproterenol, the B-adrenergic agonist and CAMP analogs (dbcAMP, Sbromo-CAMP), also activate IL-6 gene expression in astrocytes (Grimaldi et al., 1994; Maimone et al., 1993; Norris and Benveniste, 1993; Norris activation of et al., 1994). In addition, the PKC pathway induces IL-6 expression by astrocytes. PMA, a direct activator of PKC, alone or in conjunction with Ca2+ ionophore, stimulates IL-6 production by astrocytes (Grimaldi et al., 1995; Norris et al., 1994). We have shown that the cytokines IL-1B and TNF-o induce IL-6 expression through activation of the PKC, but not PKA pathway (Norris et al., 1994). This was demonstrated by the inhibition of IL-ll3 and TNF-a induced IL-6 expression by PKC inhibitors such as calphostin C and H7, while cAMP/PKA inhibitors were without effect (Norris et al., 1994). These studies collectively indicate that IL-6 expression in astrocytes can occur through activation of two distinct second messenger pathways, PKA or PKC, depending on the stimulus used. A number of studies have demonstrated cross-talk between the PKA and PKC pathways for IL-6 expression. For example, VIP, PACAP, and NE all synergize with IL-1B for enhanced IL-6 production (Gottschall et al., 1994; Maimone et al., 1993; Norris and Benveniste, 1993), indicating that an amplification of IL-6 expression occurs when both PKA and PKC pathways are activated simultaneously. We have begun to address the molecular basis of this synergistic interaction, and have demonstrated that costimulation of astrocytes with forskolin

(activates PKA pathway) and PMA (activates PKC pathway) enhances steady-state levels of IL-6 mRNA, but does not promote stabilization of the IL-6 message compared to PMA or forskolin alone (Huneycutt and Benveniste, 1995). These results indicate that the actions of these two signaling pathways converge at the transcriptional level.

Concluding

remarks and future directions

It is clear that the cAMP/PKA pathway in astrocytes is intimately regulated by conditions of the CNS environment in the context of both neuronal activity and states of inflammation. The activation of this pathway in astrocytes leads to expression of neurotrophic factors essential for initiating neuronal differentiation and perhaps neuronal protection under noxious conditions generated as a response to local inflammatory responses, as well as the expression of IL-6, a cytokine involved in both inflammatory and anti-inflammatory events in the CNS. These effects on astrocyte functions are also influenced by PKC activation and/or Ca*+ levels, creating a complex circuitry of intracellular activities. It will now become imperative to determine the exact mechanism of cAMP/PKA communication with other second messenger pathways; aspects of these interactions that should be addressed are the regulation of protein phosphatase activities, the role of individual isoforms of AC and PKA, and the molecular mechanisms by which cAMP/PKA and PKC pathways converge. Acknowledgments The authors thank Sue Wade for excellent secretarial assistance. This work was supported in part by National Institutes of Health Grant NS-29719 (E.N.B.). B.S.H. was supported by National Institutes of Health Postdoctoral Fellowship (NS-07335).

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