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• W2025376985 abstract "We have shown previously that nerve growth factor (NGF) down-regulates adenosine A2A receptor (A2AAR) mRNA in PC12 cells. To define cellular mechanisms that modulate A2AAR expression, A2AAR mRNA and protein levels were examined in three PC12 sublines: i) PC12nnr5 cells, which lack the high affinity NGF receptor TrkA, ii) srcDN2 cells, which overexpress kinase-defective Src, and iii) 17.26 cells, which overexpress a dominant-inhibitory Ras. In the absence of functional TrkA, Src, or Ras, NGF-induced down-regulation of A2AAR mRNA and protein was significantly impaired. However, regulation of A2AAR expression was reconstituted in PC12nnr5 cells stably transfected with TrkA. Whereas NGF stimulated the mitogen-activated protein kinases p38, extracellular regulated kinase 1 and 2 (ERK1/ERK2), and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in PC12 cells, these kinases were activated only partially or not at all in srcDN2 and 17.26 cells. Inhibiting ERK1/ERK2 with PD98059 or inhibiting SAPK/JNK by transfecting cells with a dominant-negative SAPKβ/JNK3 mutant partially blocked NGF-induced down-regulation of A2AAR expression in PC12 cells. In contrast, inhibiting p38 with SB203580 had no effect on the regulation of A2AAR mRNA and protein levels. Treating SAPKβ/JNK3 mutant-transfected PC12 cells with PD98059 completely abolished the NGF-induced decrease in A2AAR mRNA and protein levels. These results reveal a role for ERK1/ERK2 and SAPK/JNK in regulating A2AAR expression. We have shown previously that nerve growth factor (NGF) down-regulates adenosine A2A receptor (A2AAR) mRNA in PC12 cells. To define cellular mechanisms that modulate A2AAR expression, A2AAR mRNA and protein levels were examined in three PC12 sublines: i) PC12nnr5 cells, which lack the high affinity NGF receptor TrkA, ii) srcDN2 cells, which overexpress kinase-defective Src, and iii) 17.26 cells, which overexpress a dominant-inhibitory Ras. In the absence of functional TrkA, Src, or Ras, NGF-induced down-regulation of A2AAR mRNA and protein was significantly impaired. However, regulation of A2AAR expression was reconstituted in PC12nnr5 cells stably transfected with TrkA. Whereas NGF stimulated the mitogen-activated protein kinases p38, extracellular regulated kinase 1 and 2 (ERK1/ERK2), and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in PC12 cells, these kinases were activated only partially or not at all in srcDN2 and 17.26 cells. Inhibiting ERK1/ERK2 with PD98059 or inhibiting SAPK/JNK by transfecting cells with a dominant-negative SAPKβ/JNK3 mutant partially blocked NGF-induced down-regulation of A2AAR expression in PC12 cells. In contrast, inhibiting p38 with SB203580 had no effect on the regulation of A2AAR mRNA and protein levels. Treating SAPKβ/JNK3 mutant-transfected PC12 cells with PD98059 completely abolished the NGF-induced decrease in A2AAR mRNA and protein levels. These results reveal a role for ERK1/ERK2 and SAPK/JNK in regulating A2AAR expression. adenosine A2A receptor nerve growth factor glyceraldehyde-3-phosphate dehydrogenase mitogen-activated protein stress-activated protein kinase/c-Jun NH2-terminal kinase 4-(2-(7-amino-2-(2-fury)(1,2,4)triazolo(2,3-α)(1,3,5)triazin-5-amino)ethyl)phenol extracellular regulated kinase day(s) polyacrylamide gel electrophoresis Adenosine receptors are G-protein coupled receptors that mediate important physiological processes in both the central and peripheral nervous system, including vasodilation, respiratory depression, wakefulness, and spontaneous locomotor activity. There are four major adenosine receptor subtypes, A1, A2A, A2B, and A3; each is encoded by a distinct gene, and each has unique affinities for adenosine analogs and methylxanthine derivatives (1Fredholm B.B. Abbracchio M.P. Burnstock G. Daly J.W. Harden T.K. Jacobson K.A. Leff P. Williams M. Pharmacol. Rev. 1994; 46: 143-156PubMed Google Scholar, 2Collis M.G. Hourani S.M. Trends Pharmacol. Sci. 1993; 14: 360-366Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 3Daval J.L. Nehlig A. Nicolas F. Life Sci. 1991; 49: 1435-1453Crossref PubMed Scopus (128) Google Scholar). In the developing rat brain, adenosine A2A receptor (A2AAR)1 mRNA is expressed transiently in various regions (4Weaver D.R. Brain Res. Mol. Brain Res. 1993; 20: 313-327Crossref PubMed Scopus (126) Google Scholar). Moreover, a severalfold increase in A2AAR protein levels occurs during early postnatal development in a number of brain regions, whereas a decrease in A2AAR mRNA is observed in other regions (5Johansson B. Georgiev V. Fredholm B.B. Neuroscience. 1997; 80: 1187-1207Crossref PubMed Scopus (83) Google Scholar). PC12 cells, derived from a rat pheochromocytoma, have been used extensively as a model for neuronal differentiation and development (6Halegoua S. Armstrong R. Kremer N. Curr. Top. Microbiol. Immunol. 1991; 1 65: 119-170Google Scholar). In response to NGF, these cells differentiate into sympathetic-like neurons and extend neurites (6Halegoua S. Armstrong R. Kremer N. Curr. Top. Microbiol. Immunol. 1991; 1 65: 119-170Google Scholar). The signal transduction pathways activated by NGF originate at both high (TrkA) and low (p75) affinity receptors, and downstream targets of each receptor have been implicated in regulating expression of genes involved in differentiation, neurotransmission, and neuronal function (6Halegoua S. Armstrong R. Kremer N. Curr. Top. Microbiol. Immunol. 1991; 1 65: 119-170Google Scholar, 7Lee N.H. Weinstock K.G. Kirkness E.F. Earle-Hughes J.A. Fuldner R.A. Marmaros S. Glodek A. Gocayne J.D. Adams M.D. Kerlavage A.R. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar, 8Smith M.A. Fanger G.R. O'Connor L.T. Bridle P. Maue R.A. J. Biol. Chem. 1997; 272: 15675-15681Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 9Rossner S. Ueberham U. Schliebs R. Perez-Polo J.R. Bigl V. J. Neurochem. 1998; 71: 757-766Crossref PubMed Scopus (47) Google Scholar, 10Sadot E. Jaaro H. Seger R. Ginzburg I. J. Neurochem. 1998; 70: 428-431Crossref PubMed Scopus (29) Google Scholar, 11D'Arcangelo G. Halegoua S. Mol. Cell. Biol. 1993; 13: 3146-3155Crossref PubMed Scopus (136) Google Scholar, 12Lee N.H. Malek R.L. J. Biol. Chem. 1998; 273: 22317-22325Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 13Liu H. Force T. Bloch K.D. J. Biol. Chem. 1997; 272: 6038-6043Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Stimulation of the receptor tyrosine kinase TrkA results in the activation of Ras, Src, phospholipase C-γ, SNT, and phosphoinositide 3-OH kinase (14Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar, 15Kaplan D. Stephens R. J. Neurobiol. 1994; 25: 1404-1417Crossref PubMed Scopus (473) Google Scholar, 16Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (908) Google Scholar, 17Qiu M.S. Green S.H. Neuron. 1991; 7: 937-946Abstract Full Text PDF PubMed Scopus (122) Google Scholar). In PC12 cells, active Ras triggers a cascade of phosphorylation events leading to activation of ERK1/ERK2 via Raf-1 (18Wood K.W. Sarneck C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (661) Google Scholar, 19Oshima M. Sithanandam G. Rapp U.R. Guroff G. J. Biol. Chem. 1991; 266: 23753-23760Abstract Full Text PDF PubMed Google Scholar, 20Thomas S. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (504) Google Scholar) or p38 MAP kinase and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) via MAP kinase kinase kinase (21Goodman M.N. Reigh C.W. Landreth G.E. J. Neurobiol. 1998; 36: 537-549Crossref PubMed Scopus (8) Google Scholar, 22Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar, 23Morooka T. Nishida E. J. Biol. Chem. 1998; 273: 24285-24288Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). p75 activation increases ceramide production (24Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (549) Google Scholar) and activates NFκB (25Carter B.D. Kaltschmidt C. Kaltschmidt B. Offenhauser N. Bohm-Matthaei R. Baeuerle P.A. Barde Y.A. Science. 1996; 272: 542-545Crossref PubMed Scopus (614) Google Scholar). Gene products regulated by NGF in PC12 cells include several G-protein coupled receptors, such as the M4 muscarinic, secretin, and adenosine A2A receptors (7Lee N.H. Weinstock K.G. Kirkness E.F. Earle-Hughes J.A. Fuldner R.A. Marmaros S. Glodek A. Gocayne J.D. Adams M.D. Kerlavage A.R. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar, 26Arslan G. Kontny E. Fredholm B.B. Neuropharmacology. 1997; 36: 1319-1326Crossref PubMed Scopus (44) Google Scholar, 12Lee N.H. Malek R.L. J. Biol. Chem. 1998; 273: 22317-22325Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Using gene expression profiling (expressed sequence tags) coupled with Northern analysis, a decrease in A2AAR mRNA could be demonstrated as early as 3 days (3 d) posttreatment with NGF and levels remained depressed for up to 12 d (7Lee N.H. Weinstock K.G. Kirkness E.F. Earle-Hughes J.A. Fuldner R.A. Marmaros S. Glodek A. Gocayne J.D. Adams M.D. Kerlavage A.R. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar). In situ hybridization with an A2AAR oligonucleotide probe detected a 50% decrease in the number of grains per cell in NGF-differentiated PC12 cells, confirming that NGF decreases A2AAR mRNA levels (26Arslan G. Kontny E. Fredholm B.B. Neuropharmacology. 1997; 36: 1319-1326Crossref PubMed Scopus (44) Google Scholar). Corresponding to the changes in mRNA levels, immunoreactive A2AAR protein declines by more than half after 7 d of NGF treatment, and the number of binding sites for the A2AAR selective antagonist, [3H]SCH 58261, decreases by 3-fold (26Arslan G. Kontny E. Fredholm B.B. Neuropharmacology. 1997; 36: 1319-1326Crossref PubMed Scopus (44) Google Scholar). When PC12 cells are treated with A2AAR agonists, a transient down-regulation of A2AAR mRNA and protein occurs (27Saitoh O. Saitoh Y. Nakata H. Neuroreport. 1994; 5: 1317-1320PubMed Google Scholar). Despite these observations, the specific cellular mechanisms regulating A2AAR mRNA levels have not been thoroughly delineated. In this study, we provide the first insights into the downstream pathways employed by NGF to control A2AAR expression in PC12 cells. Such pathways may likewise play an important role in the regulation of A2AAR expression during brain development. Rat pheochromocytoma cells (PC12) were obtained from the American Type Tissue Culture collection (Manassas, VA). PC12nnr5, clone 106, and srcDN2 cells were a generous gift from Gordon Guroff at NICHD, National Institutes of Health (Bethesda, MD). The dominant-negative Ras cell line, 17.26, was obtained from Robert Maue at Dartmouth Medical Center (Hanover, NH). PC12 cell lines were maintained on rat tail collagen, Type IV (Upstate Biotechnology, Saranac Lake, NY) as described previously (7Lee N.H. Weinstock K.G. Kirkness E.F. Earle-Hughes J.A. Fuldner R.A. Marmaros S. Glodek A. Gocayne J.D. Adams M.D. Kerlavage A.R. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar). SrcDN2, 17.26, and clone 106 were cultured in the presence of 300 μg/ml Geneticin (Life Technologies, Inc.). Cells were treated with PD98059 or SB203580 (Calbiochem, San Diego, CA) and mouse 2.5S NGF (Promega, Madison, WI) as described below. Poly(A+) RNA was isolated, fractionated through a denaturing agarose gel, and transferred to Hybond N+ membranes (NEN Life Science Products) essentially as described previously (12Lee N.H. Malek R.L. J. Biol. Chem. 1998; 273: 22317-22325Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A32P-labeled 2.3-kilobase pairSstI/XhoI fragment from a rat A2AcDNA clone and a 1.2-kilobase pair EcoRI/XhoI fragment from a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were used as probes (7Lee N.H. Weinstock K.G. Kirkness E.F. Earle-Hughes J.A. Fuldner R.A. Marmaros S. Glodek A. Gocayne J.D. Adams M.D. Kerlavage A.R. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar). Expression levels of A2AAR mRNA were normalized to GAPDH mRNA levels. Blots were analyzed on a Molecular Dynamics PhosphorImager. Data are expressed as the mean ± S.E. of n independent experiments. At the times indicated, total RNA was isolated from PC12nnr5 cells. An RNase protection assay was performed essentially as described by Lee et al. (28Lee N.H. Earle-Hughes J. Fraser C.M. J. Biol. Chem. 1994; 269: 4291-4298Abstract Full Text PDF PubMed Google Scholar). Construction of plasmid 118GAPDHpSP73 for generating an antisense riboprobe of the GAPDH mRNA was described previously (12Lee N.H. Malek R.L. J. Biol. Chem. 1998; 273: 22317-22325Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Plasmid 296A2ApSP72 for generating an antisense riboprobe template of the A2AAR mRNA was constructed by subcloning anEcoRI/PvuII restriction fragment of 296 nucleotides into pSP72 (Promega). Riboprobes transcribed fromEcoRI-linearized 296A2ApSP72 correspond to nucleotides 596–891 of the A2AAR cDNA clone in pBluescript (Stratagene, La Jolla, CA) (7Lee N.H. Weinstock K.G. Kirkness E.F. Earle-Hughes J.A. Fuldner R.A. Marmaros S. Glodek A. Gocayne J.D. Adams M.D. Kerlavage A.R. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar). Authenticity of the plasmid construct was verified by dideoxy sequencing. PC12 cells were plated onto 100-mm dishes, grown to 70–80% confluency, and transiently transfected with 5 μg of SAPKβ(K55A) in expression vector pCMV5 (29Swantek J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar) or an empty expression vector with LipofectAMINE 2000 (Life Technologies, Inc.). At 18–24 h after transfection, cells were treated with PD98059 and/or NGF, and they were harvested for total RNA at the indicated times. PC12 cells were plated onto 35-mm collagen-coated tissue culture dishes at 90% confluency and incubated 24 h. The medium was then replaced with Dulbecco's modified Eagle's medium lacking serum, and the cells were incubated for an additional 24 h. Serum starved monolayers were washed with cold phosphate-buffered saline, and cells were harvested/lysed in 95 °C SDS-PAGE sample buffer containing 50 mm Tris, pH 6.8, 10% glycerol, 0.1% bromphenol blue, 2% SDS, 0.7 mβ-mercaptoethanol, 50 mm sodium fluoride, 2 mm sodium orthovanadate, 1 mm EDTA, 2 mm phenylmethylsulfonyl fluoride, 10.8 μg/ml aprotinin, and 10 μg/ml leupeptin. Lysates were placed on ice during sonication and reheated to 95 °C for 5 min. Proteins were separated on a 7.5% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). Membranes were processed for Western analysis as described by the New England Biolabs (Beverly, MA) protocol supplied with the antibodies. Phospho-p44/42 MAP kinase (ERK1/ERK2) antibody, phospho-p38 MAP kinase antibody, p44/42 MAP kinase antibody, SAPK/JNK antibody, and p38 MAP kinase antibody were from New England Biolabs. Phospho-JNK (G-7) monoclonal antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-TrkA (Ab-1) monoclonal antibody was from Calbiochem. Goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Upstate Biotechnology) allowed detection of proteins by the ECL+Plus detection system (Amersham Pharmacia Biotech). Treated cells were washed in ice-cold phosphate-buffered saline, lysed in immune precipitation buffer (20 mm Tris-HCl, pH 7.4, 0.15 m NaCl, 1 mm EDTA, 0.5% (v/v) Nonidet P-40, 1% Triton X-100, 2 mm sodium orthovanadate, 20 μg/ml aprotinin, 5 μg/ml leupeptin, 50 mm sodium fluoride) and disrupted by aspiration through a 21 gauge needle. Cell debris was removed by centrifugation. Supernatants were incubated with anti-p38 antibody (C20) (Santa Cruz Biotechnology) for 2 h at 4 °C. Immunocomplexes were precipitated with immune precipitation buffer equilibrated protein A-agarose (Sigma) for 2 h at 4 °C, washed three times with immune precipitation buffer and twice with kinase buffer (25 mm HEPES, pH 7.5, 10 mmMgCl2, 25 mm sodium β-glycerophosphate, 2 mm sodium orthovanadate, 0.5 mm dithiothreitol) and resuspended in kinase buffer containing 100 μm ATP, 5 μCi of [γ-32P]ATP and 2 μg of glutathioneS-transferase-ATF-2 substrate. Reactions were incubated 30 min at 30 °C and terminated by the addition of 2× SDS-PAGE loading buffer. Proteins were separated on a 7.5% SDS-PAGE gel and analyzed by autoradiography. Crude membrane preparations were obtained as described recently (30Nie Z. Mei Y. Ramkumar V. Mol. Pharmacol. 1997; 52: 456-464Crossref PubMed Scopus (35) Google Scholar). 125I-ZM241358, an A2AAR-specific antagonist was used to measure specific binding to A2AARs in crude membrane preparations. Data are expressed as the mean ± S.E. of n independent determinations. To assess the contribution of individual components of the NGF signal transduction pathway leading to regulation of A2AAR mRNA and protein, PC12 sublines lacking functional TrkA or overexpressing dominant-inhibitory forms of Ras or Src were used. The role of the three MAP kinase family members p38, ERK1/ERK2, and SAPK/JNK in NGF-mediated regulation of A2AAR mRNA was also determined. Earlier experiments have concentrated on the effects of long term (7–12 days) NGF treatment on A2AAR expression in PC12 cells (7Lee N.H. Weinstock K.G. Kirkness E.F. Earle-Hughes J.A. Fuldner R.A. Marmaros S. Glodek A. Gocayne J.D. Adams M.D. Kerlavage A.R. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar, 26Arslan G. Kontny E. Fredholm B.B. Neuropharmacology. 1997; 36: 1319-1326Crossref PubMed Scopus (44) Google Scholar). In the present study, shorter periods of treatment were examined to define initial pathways responsible for NGF regulation. Steady state A2AAR mRNA declined to 62 and 43% of untreated control cells following 1 and 3 d of NGF treatment, respectively (Fig. 1 A). Correspondingly, binding of the A2AAR antagonist 125I-ZM241358 to PC12 cells decreased by 50 and 45% following 1 and 3 d of NGF treatment, respectively (Fig. 1 B). Thus, NGF-induced down-regulation of A2AAR mRNA and protein is apparent as early as 24 h following NGF treatment, with a further decrease in A2AAR mRNA occurring at 3 d. The contribution of TrkA and p75 to the regulation of A2AAR mRNA and protein were examined in PC12nnr5 cells, which are a PC12 subline that expresses p75, lacks functional TrkA receptors, and does not differentiate in response to NGF (31Green S. Rydel R. Connolly J. Greene L. J. Cell Biol. 1986; 102: 803-843Crossref PubMed Scopus (231) Google Scholar). As the basal steady-state level of A2AAR mRNA is reduced in PC12nnr5 cells, a ribonuclease protection assay was performed to quantitate A2AAR mRNA. As shown in Fig. 1, NGF failed to down-regulate both A2AAR mRNA and protein levels. For comparison, the effects of NGF on A2AAR expression were studied in clonal cell line 106. Clone 106, derived from PC12nnr5 cells stably transfected with TrkA, has levels of 125I-NGF binding similar to those in native PC12 cells and differentiates in response to NGF (32Lazarovici P. Oshima M. Shavit D. Shibutani M. Jiang H. Monshipouri M. Fink D. Movesesyan V. Guroff G. J. Biol. Chem. 1997; 272: 11026-11034Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). When cultures of clone 106 were treated with NGF for 1 or 3 d, both A2AAR mRNA and protein levels were down-regulated to the same extent as native PC12 cells (Fig. 1). Because TrkA is implicated in A2AAR mRNA and protein regulation, potential roles for TrkA-associated signaling components, Src and Ras, were examined. As oncogenic Src mimics NGF-induced neurite outgrowth and phosphorylation of a similar set of cellular substrates, a role for Src in the signal transduction pathway initiated by NGF has been implicated (33Thomas S.M. Hayes M. D'Arcangelo G. Armstrong R.C. Meyer B.E. Zilberstein A. Brugge J.S. Halegoua S. Mol. Cell. Biol. 1991; 11: 4739-4750Crossref PubMed Scopus (49) Google Scholar). Therefore, the effects of NGF on A2AAR mRNA and protein were examined in srcDN2 cells that overexpress a dominant-negative, kinase-defective Src mutant (34Rusanescu G. Qi H. Thomas S. Brugge J. Halegoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar). Upon treatment of srcDN2 cells with NGF for 1 or 3 d, down-regulation of steady-state mRNA was not observed (92 and 100% of untreated cells, respectively) (Fig. 2). Likewise, A2AAR protein levels remained near control levels following 1 and 3 d of NGF treatment (110 and 81%, respectively). Thus, Src appears to be critical for NGF-induced down-regulation of both A2AAR mRNA and protein. PC12 cells undergo a Ras-dependent transient induction of several immediate-early genes within minutes of NGF treatment that precedes neurite outgrowth (35Szeberenyi J. Cai H. Cooper G. Mol. Cell. Biol. 1990; 10: 5324-5332Crossref PubMed Scopus (275) Google Scholar). The delayed response genes are transcriptionally active hours to days following NGF treatment (6Halegoua S. Armstrong R. Kremer N. Curr. Top. Microbiol. Immunol. 1991; 1 65: 119-170Google Scholar, 7Lee N.H. Weinstock K.G. Kirkness E.F. Earle-Hughes J.A. Fuldner R.A. Marmaros S. Glodek A. Gocayne J.D. Adams M.D. Kerlavage A.R. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8303-8307Crossref PubMed Scopus (180) Google Scholar), and the induction of several genes, such as agrin, tau, transin, and SCG10, has been shown to be Ras-dependent (8Smith M.A. Fanger G.R. O'Connor L.T. Bridle P. Maue R.A. J. Biol. Chem. 1997; 272: 15675-15681Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Sadot E. Jaaro H. Seger R. Ginzburg I. J. Neurochem. 1998; 70: 428-431Crossref PubMed Scopus (29) Google Scholar, 11D'Arcangelo G. Halegoua S. Mol. Cell. Biol. 1993; 13: 3146-3155Crossref PubMed Scopus (136) Google Scholar). Furthermore, transcriptional down-regulation of the epidermal growth factor receptor requires Ras activity (36Shibutani M. Lazarovici P. Johnson A.C. Katagiri Y. Guroff G. J. Biol. Chem. 1998; 273: 6878-6884Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The data shown in Fig. 3 demonstrate that NGF can also mediate down-regulation of A2AAR mRNA in a Ras-dependent manner. In 17.26 cells expressing a dominant-negative Ras mutant (35Szeberenyi J. Cai H. Cooper G. Mol. Cell. Biol. 1990; 10: 5324-5332Crossref PubMed Scopus (275) Google Scholar), steady state mRNA levels following 1 and 3 d of NGF treatment were 88 and 81% of untreated control cells, respectively, which is a less dramatic decrease than that seen in native PC12 (compare Figs. 1 A and3 A). A similar impairment of NGF-induced down-regulation of protein was observed in 17.26 cells (compare Figs. 1 B and3 B). The loss of NGF-mediated down-regulation of A2AAR mRNA and protein in 17.26 and srcDN2 cells was not due to a loss of TrkA receptor levels. As determined by Western analysis using an anti-TrkA antibody, expression of TrkA protein in srcDN2 and 17.26 cells was comparable to that found in native PC12 cells (data not shown). In agreement with our findings, srcDN2, 17.26, and native PC12 cells exhibited similar levels of 125I-NGF binding to TrkA (32Lazarovici P. Oshima M. Shavit D. Shibutani M. Jiang H. Monshipouri M. Fink D. Movesesyan V. Guroff G. J. Biol. Chem. 1997; 272: 11026-11034Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Taken together, these results indicate that both Src and Ras are necessary TrkA-signaling components that regulate A2AAR mRNA and protein levels. Ras has multiple downstream effectors that activate divergent signaling pathways (reviewed in Ref. 37Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Crossref PubMed Scopus (875) Google Scholar), such as Raf-1 and MAP kinase kinase kinase (22Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar). The most thoroughly described Ras activated pathway is the Raf-dependent activation of ERK1/ERK2 (38Kyriakis J.M. App H. Zhang X.F. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (980) Google Scholar, 39Dent P. Haser W. Haystead T.A. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (501) Google Scholar). More recently, Raf-independent Ras-activated MAP kinase pathways have been identified. For example, the Ras effector MAP kinase kinase kinase 1 activates SEK, which in turn activates SAPK/JNK and p38 (40Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (714) Google Scholar, 41Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar, 42Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (917) Google Scholar, 43Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (660) Google Scholar). As such, the activity of p38, ERK1/ERK2, and SAPK/JNK was examined in PC12, srcDN2, and 17.26 cells. NGF activation of p38, ERK1/ERK2, and SAPK/JNK was assessed with phosphorylation state-specific antibodies (Fig. 4). In native PC12 cells, NGF activated p38 and ERK1/ERK2 at early time points (15 and 30 min), whereas SAPK/JNK was not activated until 3 d. In agreement with previous reports, expression of dominant-negative Ras in 17.26 cells inhibited NGF activation of ERK1/ERK2 (18Wood K.W. Sarneck C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (661) Google Scholar, 20Thomas S. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (504) Google Scholar) and SAPK/JNK (22Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar), confirming that growth factor activation of these two MAP kinases is Ras-dependent. Likewise, activation of p38 above basal levels in NGF-treated 17.26 cells was not observed. As demonstrated previously (34Rusanescu G. Qi H. Thomas S. Brugge J. Halegoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar), ERK1/ERK2 was activated in srcDN2 cells albeit to a slightly lesser extent than native PC12 cells. Moreover, NGF did not effectively activate p38 or SAPK/JNK in srcDN2 cells (Fig. 4). These findings demonstrate that Src is required for the full activation of the MAP kinases. As these kinases are distal components of the NGF signal transduction pathway, and their activation was impaired or inhibited by dominant-negative signaling components more proximal to TrkA, it is possible that one or more of these MAP kinase family members plays a role in mediating the effects of NGF on A2AAR mRNA. Notably, a recent report demonstrated that two MAP kinase family members, JNK and ERK1/ERK2, had opposing effects on tau promoter activity and affected promoter activity over different time frames (10Sadot E. Jaaro H. Seger R. Ginzburg I. J. Neurochem. 1998; 70: 428-431Crossref PubMed Scopus (29) Google Scholar). To examine potential roles of the individual MAP kinases in affecting down-regulation of A2AAR mRNA by NGF, the synthetic compound SB203580 was used to inhibit p38 (44Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1981) Google Scholar). Although p38 activation was impaired in srcDN2 and 17.26 cells, this kinase does not appear to be involved in A2AAR mRNA regulation as SB203580 failed to inhibit NGF-induced down-regulation of A2AAR mRNA in native PC12 cells (Fig. 5 A). Similarly, another p38 inhibitor, SB202190, also failed to inhibit A2AAR mRNA down-regulation (data not shown). Inhibition of p38 activity in PC12 cells was verified by an in vitroimmunocomplex kinase assay. Whereas p38 from lysates of NGF-stimulated cells phosphorylated glutathione S-transferase-ATF-2 (4-fold above basal levels), p38 from NGF-stimulated cells pretreated with SB203580 did not appreciably phosphorylate its substrate (data not shown). ERK1/ERK2 activity was inhibited by employing the MAP kinase kinase inhibitor PD98059 (45Pang L. Sawada T. Decker S.J. Salteil A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). Cells co-treated with NGF and PD98059 demonstrated significantly less down-regulation of A2AAR mRNA than cells treated with NGF alone, suggesting that ERK1/ERK2 plays at least a partial role in controlling A2AAR mRNA steady state levels (compare Figs. 5 B and 1 A). As reported previously (45Pang L. Sawada T. Decker S.J. Salteil A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar), PC12 cells pretreated with PD98059 did not extend neurites following 3 d NGF as did cells treated with NGF alone (data not shown). Western analysis also confirmed that PD98059 inhibited ERK1/ERK2, but not p38 and SAPK/JNK, activity in PC12 cells treated with NGF (data not shown). To inhibit SAPK/JNK, PC12 cells were transiently transfected with SAPKβ(K55A), a kinase-defective construct of SAPKβ/JNK3. Following 1 and 3 d of NGF treatment, PC12 cells expressing SAPKβ(K55A) did not extend neurites, whereas empty vector-transfected PC12 cells extended neurites to the same extent as nontransfected cells (data not shown). These findings are in agreement with studies demonstrating that differentiation of PC12 cells requires the SAPK/JNK signal transduction pathway (46Kita Y. Kimura K.D. Kobayashi M. Ihara S. Kaibuchi K. Kuroda S. Ui M. Iba H. Konishi H. Kikkawa U. Nagata S. Fukui Y. J. Cell Sci. 1998; 111: 907-915Crossref PubMed Google Scholar). As shown in Fig. 5 C, PC12 cells expressing SAPKβ(K55A) did not undergo NGF-induced down-regulation of A2AAR mRNA following 1 d of treatment (102% of untreated cells). After 3 d of NGF incubation, SAPKβ(K55A) transfectants had a slight decrease in A2AAR mRNA (79% of untreated cells) that was not as great as that of empty vector-transfected cells. When kinase-defective SAPKβ transfectants were co-incubated with PD98059 and NGF for 3 d, down-regulation of A2AAR mRNA was completely blocked (Fig. 5 D). In contrast, when cells transfected with an empty vector were treated for 3 d with NGF alone, A2AAR mRNA was down-regulated to the same extent (42%) as wild-type PC12 cells (Fig. 1 A). As inhibition of either ERK1/ERK2 or SAPK/JNK individually results in partial inhibition of A2AAR mRNA regulation, these results indicate that ERK1/ERK2 and SAPK/JNK are both required for complete down-regulation of A2AAR mRNA. The capacity of NGF to down-regulate A2AAR mRNA following inhibition of the different MAP kinases was mimicked at the protein level (Fig. 6). It will be of interest in the future to determine whether NGF utilizes other mechanisms (besides mRNA regulation) to down-regulate A2AAR protein (e.g. ubiquitin-mediated protein degradation). To summarize, the data presented here demonstrate that NGF-induced down-regulation of A2AAR mRNA and protein levels is TrkA-, Src-, and Ras-dependent. Furthermore, the MAP kinase family members ERK1/ERK2 and SAPK/JNK are distal signal transduction components activated by NGF and are implicated here as having important roles in mediating regulation of A2AAR mRNA. Recent reports have demonstrated a role for mitogen-activated protein kinase family members in regulating mRNA stability. NGF-induced stabilization of the M4 muscarinic receptor mRNA requires ERK1/ERK2 (12Lee N.H. Malek R.L. J. Biol. Chem. 1998; 273: 22317-22325Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and p38 plays a role in stabilizing cyclooxygenase-2 mRNA (47Ridley S.H. Dean J.L. Sarsfield S.J. Brook M. Clark A.R. Saklatvala J. FEBS Lett. 1998; 439: 75-80Crossref PubMed Scopus (202) Google Scholar, 48Dean J.L.E. Brook M. Clark A.R. Saklatvala J. J. Biol. Chem. 1999; 274: 264-269Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). As NGF destabilizes A2AAR mRNA transcripts, 2R. L. Malek and N. H. Lee, unpublished observations. we are currently examining the role of ERK1/ERK2 and SAPK/JNK in NGF-mediated A2AAR mRNA destabilization. We thank Dr. G. Guroff for the PC12nnr5, clone 106, and srcDN2 cells and Dr. R. Maue for the 17.26 cells. The SAPKβ(K55A) construct in pCMV5 was kindly provided by Dr. M. Cobb." @default.
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