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Activation of Mitogen-activated Protein Kinase Pathways by Cyclic GMP and Cyclic GMP-dependent Protein Kinase in Contractile Vascular Smooth Muscle Cells

1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês

10.1074/jbc.274.48.34301

ISSN

1083-351X

Autores

Padmini Komalavilas, Paras K. Shah, Hanjoong Jo, Thomas Lincoln,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro andin vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC. Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro andin vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC. vascular smooth muscle cell cGMP-dependent protein kinase cAMP-dependent protein kinase rat aortic smooth muscle cell mitogen-activated protein extracellular signal-regulated kinase MAP kinase kinase c-Jun N-terminal kinase glutathione S-transferase platelet-derived growth factor tumor necrosis factor-α polyacrylamide gel electrophoresis bromo-cAMP Dulbecco's modified Eagle's medium chlorophenylthio- Vascular smooth muscle cell (VSMC)1 proliferation and migration are associated with several vascular diseases such as atherosclerosis and restenosis following vascular injury (1Ross R. Ann. N. Y. Acad. Sci. 1995; 748: 1-4Crossref PubMed Scopus (43) Google Scholar, 2Moss P. Campbell G. Wang Z. Campbell J. Lab. Invest. 1985; 53: 556-562PubMed Google Scholar, 3Schwartz S. Heimar R. Majesky M. Physiol. 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Ther. 1980; 214: 9-15PubMed Google Scholar), which, in turn, regulates several functions of VSMC such as smooth muscle relaxation (15Lincoln T.M. Cyclic GMP: Biochemistry, Physiology, and Pathophysiology. Landes Inc., Austin, TX1994Google Scholar). The major receptor protein for cGMP in VSMC is cGMP-dependent protein kinase (PKG), a serine/threonine kinase that catalyzes the phosphorylation of important proteins that regulate intracellular Ca2+ levels and relaxation of vascular smooth muscle (16Cornwell T.L. Lincoln T.M. J. Biol. Chem. 1989; 264: 1146-1155Abstract Full Text PDF PubMed Google Scholar). Our laboratory has recently demonstrated that PKG also plays a major role in the regulation of the phenotype and morphology of VSMC (17Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (152) Google Scholar, 18Dey N.B. Boerth N.J. Murphy-Ullrich J.E. Chang P.L. Prince C.W. Lincoln T.M. Circ. Res. 1998; 82: 139-146Crossref PubMed Scopus (57) Google Scholar). The expression of PKG is highly variable in VSMC. When adult rat aortic SMC are subcultured in vitro, PKG expression is reduced to nearly undetectable levels (16Cornwell T.L. Lincoln T.M. J. Biol. Chem. 1989; 264: 1146-1155Abstract Full Text PDF PubMed Google Scholar, 19Cornwell T.L. Soff G.A. Traynor A.E. Lincoln T.M. J. Vasc. Res. 1994; 31: 330-337Crossref PubMed Scopus (90) Google Scholar). Coincident with the loss of expression of PKG, VSMC assume the more synthetic phenotype. Transfection of the PKG-deficient VSMC with cDNAs encoding either PKG Iα or the catalytic domain of type I PKG reverted the morphology from synthetic to the original contractile morphology (17Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (152) Google Scholar, 18Dey N.B. Boerth N.J. Murphy-Ullrich J.E. Chang P.L. Prince C.W. Lincoln T.M. Circ. Res. 1998; 82: 139-146Crossref PubMed Scopus (57) Google Scholar). Mitogen-activated protein kinases are important mediators of signal transduction from the cell surface to the nucleus, thereby regulating several cellular responses such as growth and differentiation (20Karin M. Curr. Opin. Cell. Biol. 1994; 6: 415-424Crossref PubMed Scopus (359) Google Scholar, 21Robinson M.J. Cobb M.H. Curr. Opin. Cell. Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 22Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-57Crossref PubMed Google Scholar). The MAP kinase pathways are activated during proliferation (23Pages G. Lenormand P. L'Allemain G. Chambard J.C. Meloche S. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (925) Google Scholar, 24Watson M.H. Venance S.L. Pang S.P. Mak A.S. Circ. Res. 1993; 73: 109-117Crossref PubMed Scopus (35) Google Scholar, 25Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar) and differentiation (26Qui M.S. Green S.H. Neuron. 1992; 9: 705-717Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 27Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar), and it has been proposed that it is the duration of the MAP kinase activation that determines whether a stimulus evokes proliferation or differentiation (28Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Crossref PubMed Scopus (806) Google Scholar). Three distinct MAP kinase pathways have been identified in mammalian cells: extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase(JNK)/stress-activated protein kinase, and p38 kinase. MAP kinases are activated upon phosphorylation on both threonine and tyrosine residues by the dual function kinase MAP kinase kinase or MEK (mitogen-activated extracellular-regulated protein kinase kinase). The various MEKs are themselves activated by serine phosphorylation. In the case of the MEK that activates ERKs, phosphorylation on serine by Raf kinase is the first step in ERK activation. Using constitutively active and nonactive mutants of MEK, it was demonstrated that activation of MEK was necessary and sufficient for PC12 differentiation and transformation of NIH 3T3 cells (27Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar). The two isoforms of ERK, p42 and p44 (ERK2 and ERK1, respectively), are present in adult contractile mammalian VSMC at relatively high concentrations (24Watson M.H. Venance S.L. Pang S.P. Mak A.S. Circ. Res. 1993; 73: 109-117Crossref PubMed Scopus (35) Google Scholar, 29Adam L.P. Gapinski C.J. Hathaway D.R. FEBS Lett. 1992; 302: 223-226Crossref PubMed Scopus (78) Google Scholar, 30Childs T.J. Watson M.H. Sanghera J.S. Campbell D.L. Pelech S.L. Mak A.S. J. Biol. Chem. 1992; 267: 22853-22859Abstract Full Text PDF PubMed Google Scholar, 31Ishida Y. Kawahara Y. Tsuda T. Koide M. Yokoyama M.C.S. FEBS Lett. 1992; 310: 41-45Crossref PubMed Scopus (33) Google Scholar). Additionally, it has been demonstrated that ERK1 and -2 are partially activated in fully differentiated contractile smooth muscle by contractile agonists (29Adam L.P. Gapinski C.J. Hathaway D.R. FEBS Lett. 1992; 302: 223-226Crossref PubMed Scopus (78) Google Scholar, 32Khalil R.A. Morgan K.G. Am. J. Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar, 33Katoch S.S. Moreland R.S. Am. J. Physiol. 1995; 269: H222-H229PubMed Google Scholar, 34Gorenne I. Su X. Moreland R.S. Am. J. Physiol. 1998; 275: H131-H138PubMed Google Scholar). Several reports suggest that ERK activation may be involved in regulating contraction of differentiated smooth muscle (35Adam L.P. Barany M. Biochemistry of Smooth Muscle Contraction. Academic Press, Inc., San Diego1996: 167-177Crossref Google Scholar), but others have found only small effects of ERK activation on contraction (34Gorenne I. Su X. Moreland R.S. Am. J. Physiol. 1998; 275: H131-H138PubMed Google Scholar). Nevertheless, these findings suggest that the MAP kinase pathway may be involved in other functions besides the regulation of proliferation in fully contractile smooth muscle cells. Hence, it is possible that MAP kinase activation is regulatory in phenotypic modulation. Because PKG itself appears to be important for the expression of the VSMC contractile phenotype, it was of interest to examine the effects of PKG expression on MAP kinase pathways in VSMC compared with VSMC that do not express PKG. In addition, accumulating evidence indicates that PKG activation increases the expression of a number of protooncogene transcription factors in a variety of cell types (36Dhaunsi G.S. Hassid A. Cardiovasc Res. 1996; 31: 37-47Crossref PubMed Google Scholar, 37Gudi T. Huvar I. Meinecke M. Lohmann S.M. Boss G.R. Pilz R.B. J. Biol. Chem. 1996; 271: 4597-4600Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 38Peunova N. Enikolopov G. Nature. 1993; 364: 450-453Crossref PubMed Scopus (278) Google Scholar, 39Haby C. LIsovoski F. Aunis D. Zwiller J. J. Neurochem. 1994; 63: 496-501Google Scholar, 40Pilz R.B. Suhasini M. Idriss S. Meinkoth J.L. Boss G.R. FASEB J. 1995; 9: 552-558Crossref PubMed Scopus (178) Google Scholar). In particular, increased expression of c-fosand Jun-B in response to PKG activation may underlie the increases in VSMC proliferation observed by some investigators (41Hassid A. Arabshahi H. Bourcier T. Dhaunsi G.S. Matthews C. Am. J. Physiol. 1994; 267: H1040-H1048PubMed Google Scholar, 42Sciorati C. Nisitco G. Meldolesi J. Clementi E. Br. J. Pharmacol. 1997; 122: 687-697Crossref PubMed Scopus (45) Google Scholar) and endothelial cells (43Hood J. Granger H.J. J. Biol. Chem. 1998; 273: 23504-23508Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In the case of endothelial cells, Hood and Granger (43Hood J. Granger H.J. J. Biol. Chem. 1998; 273: 23504-23508Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) have shown that cGMP analogs activate Raf-1, an upstream regulator of ERK1/2. The results reported here are the first to examine the role of PKG on MAP kinase activation in contractile VSMC. Collagenase (CLS III) and elastase were from Worthington. Fetal bovine and calf serum, Dulbecco's modified Eagle's medium (DMEM) and platelet-derived growth factor BB (PDGF) were purchased from Life Technologies, Inc. Transfectam reagent was from Promega (Madison, WI). 8-(para-chlorophenylthio)-guanosine-3′,5′-monophosphate (8-CPT-cGMP) was from Biolog (La Jolla, CA). Geneticin was purchased from Sigma. Antibodies specific to the phosphorylated forms of ERK1/2, total ERK1/2, phospho-p38, total p38, phospho-MEK, and total MEK were purchased from New England Biolabs, Inc. (Beverly, MA). Anti-human JNK-1 antibodies were from PharMingen (San Diego, CA), and the anti-phospho JNK-1 antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Purified antibodies to PKG-I were produced in our own laboratory or were purchased from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). All other reagents were purchased from either Sigma or Fisher. RASMC were isolated from the thoracic and abdominal aortas of adult Harlan Sprague-Dawley rats (150–200 g, Harlan) as described previously (16Cornwell T.L. Lincoln T.M. J. Biol. Chem. 1989; 264: 1146-1155Abstract Full Text PDF PubMed Google Scholar). Rats were sacrificed by CO2 inhalation, and aortae were excised and placed in a wash medium of DMEM containing 20 mm HEPES, 1 mg/ml bovine serum albumin, 5 μg/ml amphotericin B, and 50 μg/ml gentamicin. The aortae were cleaned and placed in digestion medium (wash medium containing 1 mg/ml elastase and 130 units/ml collagenase) for 8 min. The tunicae adventitiae were removed, and the medial layers were minced and further digested for 1–2 h in digestion medium containing 200 units/ml collagenase until a single-cell suspension was obtained. Cells were washed twice with the wash medium and plated in culture dishes. Cells were maintained in DMEM containing 10% fetal bovine serum and 50 μg/ml gentamicin and grown under 10% CO2 and subcultured every 6 days. The transfection of RASMC with the bovine PKG Iα cDNA was performed as described previously (17Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (152) Google Scholar,18Dey N.B. Boerth N.J. Murphy-Ullrich J.E. Chang P.L. Prince C.W. Lincoln T.M. Circ. Res. 1998; 82: 139-146Crossref PubMed Scopus (57) Google Scholar). Cells at passage 3 were transfected with 5 μg of recombinant pcDNA1-neo/PKG Iα vector or empty (control) pcDNA1-neo vector using 10 μl of Transfectam reagent with precipitation of the DNA-liposome complex for 15 min at room temperature. The precipitate was added to the cell monolayer, and the cells were incubated for 6 h at 37 °C and 10% CO2. The transfection was terminated by adding DMEM with 20% fetal bovine serum. Stably transfected cells were selected using 500 μg/ml Geneticin (G418). After isolating the colonies, the transfected cell lines were maintained in 250 μg/ml G418. The transfected cells from passage 6–12 were used for the experiments. For the experiments reported here, at least three different clones of cells expressing physiological levels of PKG and three different clones of cells transfected with empty vector and not expressing PKG were examined, as reported previously (17Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (152) Google Scholar, 18Dey N.B. Boerth N.J. Murphy-Ullrich J.E. Chang P.L. Prince C.W. Lincoln T.M. Circ. Res. 1998; 82: 139-146Crossref PubMed Scopus (57) Google Scholar). Cells plated in 60 mm culture dishes and grown to confluence were serum-deprived in DMEM containing 1 mg/ml bovine serum albumin for 24 h, treated with various agents in serum-free medium for different time intervals, quick-frozen in liquid nitrogen, and stored at −80 °C. Cells were harvested by scraping in 0.25–0.5 ml of ice-cold extraction buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 1% Triton X-100, 10% glycerol, 1 mmsodium orthovanadate, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each leupeptin and pepstatin A, 5 μg/ml aprotinin A, and 10 nm caliculyn A), rotated for 15 min at 4 °C, and centrifuged at 20,000 × g for 10 min. The supernatants were separated, and protein contents were estimated by using the Coomassie Plus protein assay reagent (Pierce) using bovine serum albumin as standard. The activity of JNK was measured by an immunocomplex kinase assay using an antibody specific for JNK-1 (PharMingen, clone G 151–333) and c-Jun (amino acids 5–89) fused to glutathione S-transferase (GST c-Jun) as the substrate as described previously (44Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Crossref PubMed Scopus (237) Google Scholar). 200 μg of cell lysate protein was incubated with anti JNK-1 antibodies for 1 h at 4 °C, followed by an additional 1-h incubation with Protein G-agarose beads. The immunocomplexes were washed four times with the extraction buffer followed by two washes with JNK assay buffer (20 mm HEPES, pH 7.6, 20 mm MgCl2, 20 mmβ-glycerophosphate, 20 mm p-nitrophenyl phosphate, 0.1 mm sodium orthovanadate, 2 mmdithiothreitol). The immunocomplexes were further incubated in 20 μl of JNK assay buffer containing GST-c-Jun (5 μg/sample), 50 μCi of [γ-32P]ATP for 8 min at 30 °C. The reaction was terminated by the addition of 5 μl of a 5× electrophoresis stop buffer (312.5 mm Tris-Cl, pH 6.9, 10 mm EDTA, 0.5 m sucrose, 15% sodium dodecyl sulfate (SDS), 0.8m 2-mercaptoethanol, and 0.002% bromphenol blue). The samples were heated for 10 min at 100 °C, and the denatured proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a polyvinylidene difluoride membrane (Millipore) and subjected to autoradiography. The radioactivity incorporated into the phosphorylated GST-c-Jun protein bands was quantitated by scintillation counting and by densitometry of the autoradiogram. Cell lysates (10–50 μg of protein) from the treated samples were resolved by 10% SDS-PAGE, protein transferred to nitrocellulose membranes (Bio-Rad), and probed with antibodies specific to the phosphorylated forms of ERK1/2, p38, JNK-1, and MEK1/2. As a control, the amount of total ERK1/2 protein, p38 protein, JNK-1 protein, and MEK1/2 protein was determined by using antibodies that are specific to total ERK1/2, p38, JNK-1, and MEK1/2, respectively. PKG was detected using goat anti-bovine type I PKG prepared in our laboratory or purchased from StressGen. Proteins were visualized using the enhanced chemiluminescence (ECL) detection system (Pierce). The intensity of the bands was quantitated by densitometry. Experiments were performed a minimum of three times, and the data are presented as the mean ± S.E. and analyzed by Student's t test. A p value of less than 0.05 was used as the criterion of significance. It was demonstrated earlier that the level of PKG decreases with each passage when adult RASMC are cultured in vitro (16Cornwell T.L. Lincoln T.M. J. Biol. Chem. 1989; 264: 1146-1155Abstract Full Text PDF PubMed Google Scholar, 19Cornwell T.L. Soff G.A. Traynor A.E. Lincoln T.M. J. Vasc. Res. 1994; 31: 330-337Crossref PubMed Scopus (90) Google Scholar). Similar results have been reported by other laboratories for adult rat aortic and pulmonary arterial smooth muscle cells (13Chiche J.-D. Schlutsmeyer S.M. Bloch D.B. de la Monte S.M. Roberts Jr., J.D. Fillippov G. Janssens S.P. Rosenzweig A. Bloch K.D. J. Biol. Chem. 1998; 273: 24271-34263Abstract Full Text Full Text PDF Scopus (143) Google Scholar,45Wyatt T.A. Naftilan A.J. Francis S.H. Corbin J.C. Am. J. Physiol. 1998; 274: H448-H455PubMed Google Scholar). 2C. M. Brophy, unpublished observations. On the other hand, RASMC isolated from juvenile animals have been reported to maintain PKG expression under certain plating and growth conditions (46Brown C. Pan X. Hassid A. Circ. Res. 1999; 84: 655-667Crossref PubMed Scopus (72) Google Scholar). Therefore, in order to ensure that PKG expression was suppressed for the studies described below, cells were isolated from adult animals and passaged several times. The lack of PKG expression was also examined by Western blot analysis, Northern blot analysis, and enzymatic assay. Any non-transfected cell lines still expressing PKG were excluded from these studies. Transfection of RASMC with either the cDNA encoding PKG Iα or the catalytic domain of PKG-I in passages 3–5 reversed the morphology of the cells to a more contractile phenotype (17Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (152) Google Scholar, 18Dey N.B. Boerth N.J. Murphy-Ullrich J.E. Chang P.L. Prince C.W. Lincoln T.M. Circ. Res. 1998; 82: 139-146Crossref PubMed Scopus (57) Google Scholar). Fig.1 shows the morphology of RASMC stably expressing physiological levels of PKG Iα compared with control transfected not expressing PKG. The RASMC transfected with the empty vector had a synthetic morphology as demonstrated by the flattened appearance and the irregular growth pattern (panel A), whereas the RASMC transfected with pcDNA1-neo/PKG Iα had a contractile morphology as demonstrated by the spindle-shaped appearance and "hill and valley" growth pattern (panel B). Similar results were obtained by transfection of the catalytic domain (data not shown). To determine whether the phenotypic changes were associated with alterations in MAP kinase expression, RASMC deficient in PKG and those expressing PKG Iα were used to study the activation of ERK1/2, JNK, and p38. Fig. 2 demonstrates the levels of protein expression for these MAP kinases along with PKG expression in transfected RASMC. It is important to note the absence of detectable PKG expression in RASMC transfected with the empty vector. No PKG was detected by Western blot analysis when up to 50 μg of soluble protein were probed using either a polyclonal, immunopurified anti-PKG generated in our own laboratory or an antibody purchased fromStressGen. It is also important to note that the loss of PKG expression in passaged adult rat VSMC has been found by at least three other laboratories independent of our own (13Chiche J.-D. Schlutsmeyer S.M. Bloch D.B. de la Monte S.M. Roberts Jr., J.D. Fillippov G. Janssens S.P. Rosenzweig A. Bloch K.D. J. Biol. Chem. 1998; 273: 24271-34263Abstract Full Text Full Text PDF Scopus (143) Google Scholar, 45Wyatt T.A. Naftilan A.J. Francis S.H. Corbin J.C. Am. J. Physiol. 1998; 274: H448-H455PubMed Google Scholar).2 RASMC expressing PKG Iα (Iα) have somewhat higher endogenous levels of ERK1 and -2, especially with respect to the 42-kDa protein (2.9 ± 0.87-fold) compared with RASMC deficient in PKG expression. There were no significant differences in the levels of JNK and p38 kinases in these two cell types.Figure 2Expression of MAP kinases and PKG Iα in vector-transfected (C) and PKG-transfected (1α) RASMC . Shown is a representative Western blot for p38, ERK1/2, JNK-1, and PKG Iα in extracts from vector alone-transfected RASMC and PKG Iα-transfected RASMC (1α). 10–50 μg of total protein were prepared and analyzed by Western blot as described under "Experimental Procedures" using antibodies specific for these kinases. Thearrows indicate the kinases at their respective molecular sizes. The band above the 46-kDa JNK1 is a related 54–55-kDa JNK isoform.View Large Image Figure ViewerDownload (PPT) Empty vector- and PKG Iα-transfected cells were treated with an analog of cGMP, 8-CPT-cGMP, for different time intervals. Fig. 3 is the time course of activation of ERK1/2 by cGMP in vector only (lanes 1–4) and PKG Iα-transfected RASMC (lanes 5–8). Compared with empty vector-transfected cells, the untreated (time 0) RASMC transfected with PKG Iα demonstrated increased activation of ERK1/2 (phospho-ERK1/2, lane 5 compared with lane 1). Greater levels of ERK in the PKG-expressing cells did not appear to account for the increased levels of phospho-ERK, suggesting that the basal activation state of ERK is greater in PKG-expressing cells (panel B). 8-CPT-cGMP increased ERK1/2 activity at 15, 30, and 60 min with peak increases by cGMP observed at 15 min. On the other hand, no significant increases in ERK1/2 activation by 8-CPT-cGMP were observed in the vector alone-transfected cells. The statistical analysis of ERK1/2 activation in PKG-deficient and PKG-expressing cells is shown in panel D of Fig.3. The effects of PKG Iα transfection and 8-CPT-cGMP on ERK1/2 activity were reproducibly observed in three different clonally isolated cells expressing PKG compared with three different clonally isolated cells transfected with empty vector. We had previously described the controls performed to ensure that the results observed with PKG expression were not artifacts associated with the transfection and isolation procedure itself (17Boerth N.J. Dey N.B. Cornwell T.L. Lincoln T.M. J. Vasc. Res. 1997; 34: 245-259Crossref PubMed Scopus (152) Google Scholar, 18Dey N.B. Boerth N.J. Murphy-Ullrich J.E. Chang P.L. Prince C.W. Lincoln T.M. Circ. Res. 1998; 82: 139-146Crossref PubMed Scopus (57) Google Scholar). Therefore, the results observed were not due to unique characteristics of a particular cell type derived from a lone colony of RASMC, but rather reflected the consequences of PKG expression in the RASMC. Fig. 3 (panel E) is a time course of activation of JNK in PKG-deficient and PKG-expressing cells. As shown in the panel, cGMP also activates JNK kinase as represented by GST-cJun phosphorylation of the immunoprecipitated JNK (vector only cells,lanes 1–4 and PKG Iα cells, lanes 5–8). Compared with empty vector-transfected cells, the untreated control (time 0) from the PKG-Iα RASMC had an increased activity of JNK. 8-CPT-cGMP increased JNK activation at 15 min in the PKG-expressing cells but had no significant effects on JNK activation in the PKG-deficient cells. The total level of JNK protein in these two cell lines was not significantly different (panel F), and the statistical analysis of the data for JNK activation by 8-CPT-cGMP is shown in panel G. PKG transfection and 8-CPT-cGMP had no significant stimulatory effects on p38 kinase in the PKG-expressing cells compared with the PKG-deficient cells (data not shown) and was not studied further. Panel C in Fig. 3 illustrates the level of PKG in the vector alone-transfected cells (lanes 1-4) and the PKG Iα-transfected cells (lanes 5–8). Again, it is important to note the lack of detectable expression of PKG-I in vector alone-transfected adult RASMC. Sorbitol is an osmotically stressful agent to cultured cells that is known to activate MAP kinase pathways. Sorbitol significantly activated both ERK1/2 and JNK in vector alone-transfected RASMC (panels A and C and the statistical analysis in panel D in Fig.4). In PKG-expressing RASMC, sorbitol increased JNK activation more potently than did 8-CPT-cGMP. In these same cells, however, sorbitol was not more potent in increasing ERK1/2 than was 8-CPT-cGMP. The total increase in JNK activation by sorbitol in PKG-deficient and PKG-expressing cells was similar. Growth factors such as PDGF activate MAP kinases through both Ras-dependent pathways and G-protein coupled pathways in smooth muscle cells (47Berk B.C. Corson M.A. Circ. Res. 1997; 80: 607-616Crossref PubMed Scopus (283) Google Scholar). To gain fu

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