The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets. K E Bornfeldt, … , E G Krebs, R Ross J Clin Invest. 1997;100(4):875-885. https://doi.org/10.1172/JCI119603. Research Article Activation of the classical mitogen-activated protein kinase (MAPK) pathway leads to proliferation of many cell types. Accordingly, an inhibitor of MAPK kinase, PD 098059, inhibits PDGF-induced proliferation of human arterial smooth muscle cells (SMCs) that do not secrete growth-inhibitory PGs such as PGE2. In striking contrast, in SMCs that express the inducible form of cyclooxygenase (COX-2), activation of MAPK serves as a negative regulator of proliferation. In these cells, PDGF-induced MAPK activation leads to cytosolic phospholipase A2 activation, PGE2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which acts as a strong inhibitor of SMC proliferation. Inhibition of either MAPK kinase signaling or of COX-2 in these cells releases them from the influence of the growth- inhibitory PGs and results in the subsequent cell cycle traverse and proliferation. Thus, the MAPK pathway mediates either proliferation or growth inhibition in human arterial SMCs depending on the availability of specific downstream enzyme targets. Find the latest version: https://jci.me/119603/pdf The Mitogen-activated Protein Kinase Pathway Can Mediate Growth Inhibition and Proliferation in Smooth Muscle Cells Dependence on the Availability of Downstream Targets Karin E. Bornfeldt,* Jean S. Campbell,‡ Hidenori Koyama,* Gretchen M. Argast,‡ Christina C. Leslie,§ Elaine W. Raines,* Edwin G. Krebs,‡ and Russell Ross* *Department of Pathology, and ‡Department of Pharmacology, University of Washington, Seattle, Washington 98195; and §Department of Pediatrics, The National Jewish Center, Denver, Colorado 80206 Abstract lic and nuclear targets in a number of different cell types (1). Activation of the MAPK pathway is initiated by ligand-bind- Activation of the classical mitogen-activated protein kinase ing to the cell surface receptor, activation of the receptor, and (MAPK) pathway leads to proliferation of many cell types. binding of adapter molecules (such as GRB2) to phosphoty- Accordingly, an inhibitor of MAPK kinase, PD 098059, in- rosine residues in the activated receptor or to proteins phos- hibits PDGF-induced proliferation of human arterial smooth phorylated by the receptor, followed by activation of the muscle cells (SMCs) that do not secrete growth-inhibitory small GTP-binding protein Ras by a guanine nucleotide ex- PGs such as PGE2. In striking contrast, in SMCs that ex- change factor (e.g., SOS). Sequential phosphorylation leads to press the inducible form of cyclooxygenase (COX-2), activa- activation of the protein kinases Raf, MAP kinase kinase tion of MAPK serves as a negative regulator of prolifera- (MAPKK or MEK), and MAPK (also known as extracellular- tion. In these cells, PDGF-induced MAPK activation leads signal regulated kinase, Erk). Two isoforms of MAPK, the p44 to cytosolic phospholipase A2 activation, PGE2 release, and MAPK (Erk-1), and the p42 MAPK (Erk-2), are expressed in subsequent activation of the cAMP-dependent protein ki- most cell types. The substrates of MAPK include nuclear tran- nase (PKA), which acts as a strong inhibitor of SMC prolif- scription factors such as Ets proteins (2) and nonnuclear sub- eration. Inhibition of either MAPK kinase signaling or of strates such as the protein serine/threonine kinase p90rsk, cy- COX-2 in these cells releases them from the influence of the toskeletal proteins, and cytosolic phospholipase A2 (cPLA2) growth-inhibitory PGs and results in the subsequent cell cy- (3). cPLA2 catalyzes the release of arachidonic acid from phos- cle traverse and proliferation. Thus, the MAPK pathway pholipids in membranes and is one of the rate-limiting steps in mediates either proliferation or growth inhibition in human the synthesis of PGs, thromboxanes, leukotrienes, and other arterial SMCs depending on the availability of specific down- arachidonic acid metabolites (4, 5). stream enzyme targets. (J. Clin. Invest. 1997. 100:875–885.) Early on, the MAPK cascade was suggested to be a regula- Key words: cyclooxygenase • human • phospholipase • plate- tor of eukaryotic cell cycle progression. This concept was let-derived growth factor • prostaglandin E based on the fact that virtually all growth regulatory molecules activate MAPK, and on the striking homology of MAPK with Introduction two protein kinases, KSS-1 and FUS-3, which regulate cell cy- cle progression in yeast (6). An important role for MAPK in The classical mitogen-activated protein kinase (MAPK)1 sig- cell proliferation was later confirmed when dominant negative naling pathway is a multistep phosphorylation cascade that mutations in MAPKK, or overexpression of MAPK phospha- transmits signals from various cell surface receptors to cytoso- tase-1, which inactivates MAPK, were shown to lead to reduced DNA synthesis and proliferation (7–9), whereas overexpres- sion of a constitutively active MAPKK caused transformation- Address correspondence to Dr. Karin E. Bornfeldt, Department of associated changes (7, 10). All of these studies are consistent Pathology, Box 357470, University of Washington School of Medi- cine, Seattle, WA 98195-7470. Phone: 206-543-8523; FAX: 206-685- with the concept that MAPKK and MAPK activities are di- 3018; E-mail: [email protected] rectly correlated with proliferation. However, it is now clear Received for publication 10 January 1997 and accepted in revised that in addition to regulating proliferation, the MAPK cascade form 27 May 1997. may be involved in a variety of biological effects, such as dif- ferentiation (11), cell attachment (12), smooth muscle contrac- 1. Abbreviations used in this paper: COX, cyclooxygenase; COX-2, tion (13), and protein synthesis (14), depending on stimuli and the inducible isoform of cyclooxygenase; cPLA2, cytosolic phospholi- cell type. For example, epidermal growth factor (EGF) activa- pase A2; EC50, concentration required for half-maximal stimulation; tion of the MAPK pathway leads to proliferation, whereas IC50, concentration required for half-maximal inhibition; MAPK nerve growth factor (NGF)-stimulated MAPK activation leads (Erk), mitogen-activated protein kinase; MAPKK, mitogen-activated to differentiation in PC12 cells (11). However, it is not com- protein kinase kinase; MBP, myelin basic protein; NGF, nerve pletely understood how activation of the same MAPK path- growth factor; PDGF-BB, PDGF B-chain homodimer; PKA, cAMP- way can induce distinct biological responses. dependent protein kinase; PKI, protein kinase A inhibitor; SMC, smooth muscle cell. To address this question, we took advantage of a synthetic, cell-permeable, noncompetitive inhibitor of MAPKK that had J. Clin. Invest. been identified by screening of a compound library (15). The © The American Society for Clinical Investigation, Inc. inhibitor, PD 098059 ([2-(2Ј-amino-3Ј-methoxyphenyl)-oxa- 0021-9738/97/08/0875/11 $2.00 naphthalen-4-one]), inhibits activation of MAPKK in intact Volume 100, Number 4, August 1997, 875–885 cells (16) and has been shown to inhibit proliferation and to re- http://www.jci.org verse the transformed phenotype induced by Ras in specific Inhibition of Proliferation by Mitogen-activated Protein Kinase 875 cell lines (15). This study was undertaken to elucidate the role from ram seminal vesicles, COX-2 isolated from sheep placentas, and of MAPK activation in normal human, diploid, arterial smooth mAbs were obtained from Cayman Chem. Co. Inc. (Ann Arbor, MI). muscle cells (SMCs). Proliferation of these cells is a key event The MAPKK inhibitor PD 098059 was generously given to us by in the formation and progression of atherosclerotic lesions and Dr. Alan Saltiel and Dr. David Dudley (Research Division, Parke- in restenosis after angioplasty (17). Thus, knowledge of the in- Davis, Warner Lambert, Ann Arbor, MI), or was purchased from BIOMOL Research Labs., Inc. (Plymouth Meeting, PA). Of the tracellular signals leading to SMC proliferation may be of clin- COX inhibitors used, indomethacin (Sigma Chemical Co., St Louis, ical relevance. Using PD 098059, we show that MAPK activa- MO) inhibits both COX-1 and -2, whereas L-745,337 (5-methane- tion induced by PDGF leads to proliferation of SMCs that do sulfonamido-6-(2,4-difluorothio-phenyl)-1-indanone, kindly provided not secrete growth-inhibitory PGs. In contrast, in SMCs that by Dr. A.W. Ford-Hutchinson (Merck Frosst Canada Inc., Dorval, secrete large amounts of inhibitory PGs, activation of the same Québec), NS-398, and nimesulide (Cayman Chem. Co.) are selective pathway results in an attenuated mitogenic response due to a COX-2 inhibitors. The inhibitors were all dissolved at a concentration negative feedback mechanism. The inducible isoform of cy- of 10 mM in dimethyl sulfoxide. Arachidonic acid (eicosa-5Z, 8Z, clooxygenase (COX-2), a downstream target regulating pros- 11Z, 14Z-tetraenoic acid, 20:4, n-6), PGE2, and 8-bromoadenosine- tanoid release, is identified as the highly regulated molecule 3Ј,5Ј-cyclic monophosphate (8-Br-cAMP) were obtained from BIO- whose availability dramatically shifts the biological effects of MOL Research Labs., Inc. Measurement of DNA synthesis, cell cycle distribution, and prolif- MAPK activation in SMCs. eration. For estimation of DNA synthesis, cells were grown in 24-well trays, and were changed to DME/1% human plasma-derived serum confluence. After a 30-min preincubation with or %80 ف Methods for 48 h at without the indicated concentrations of MAPKK or COX inhibitors, Cell cultures. Human newborn (2 d–3 mo) arterial SMCs were ob- PDGF-BB or vehicle (10 mM acetic acid/0.25% BSA) was added, and tained from the thoracic aortas of infants after accidental death, the cells were incubated for an additional 18 h and subsequently la- death from Sudden Infant Death Syndrome, or from congenital de- beled with 2 ␮Ci/ml [3H]thymidine (New England Nuclear, Boston, fects. Arterial SMCs from adult normal thoracic aortas were obtained MA) for 2 h.