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Am. J. Respir. Cell Mol. Biol., Volume 20, Number 6, June 1999 1294-1302

Platelet-Derived Growth Factor Stimulation of Mitogen-Activated Protein Kinases and Cyclin D1 Promoter Activity in Cultured Airway Smooth-Muscle Cells
Role of Ras

Kristen Page, Jing Li, and Marc B. Hershenson

Department of Pediatrics, University of Chicago, Chicago, Illinois


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We hypothesized that in bovine tracheal myocytes, growth factor treatment induces transcription from the cyclin D1 promoter that is dependent on the activation of both Ras and extracellular signal-related kinase (ERK). We found that platelet-derived growth factor (PDGF) treatment induced substantial activation of ERK2 that was blocked by expression of a dominant-negative Ha-Ras. Further, expression of a constitutively active Ha-Ras induced substantial ERK2 activity, consistent with the notion that Ras is required and sufficient for ERK activation. PDGF treatment induced only modest activation of the Jun amino terminal kinase-1 (JNK1) and p38 mitogen-activated protein kinases (MAPKs). Active Ras induced similar responses, implying that complete activation of the JNK and p38 pathways requires additional or alternative upstream signaling intermediates besides Ras. In contrast, expression of a constitutively active Rac1, an alternative guanosine triphosphatase involved in intracellular signaling, produced a high level of JNK1 activation, suggesting that Rac1 is an important upstream activator of JNK in this system. Active Ras and MAPK/ ERK kinase-1 (MEK1) (the upstream activator of ERK) each induced cyclin D1 promoter activity, whereas active stress-activated protein kinase/ERK kinase-1 (SEK1), an upstream activator of JNK, did not. Finally, the synthetic MEK inhibitor PD98059 blocked Ras-induced cyclin D1 promoter activity. Together, these data suggest that in bovine tracheal myocytes: (1) activation of MAPK by PDGF is dependent on Ras; (2) active Ras is sufficient for ERK activation but is insufficient for maximal activation of JNK or p38; (3) activation of Rac1 is sufficient for maximal JNK activation; and (4) Ras, MEK, and ERK constitute a distinct pathway to cyclin D1 transcriptional activation.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Extracellular signal-regulated kinases (ERKs) are serine/ threonine kinases of the mitogen-activated protein kinase (MAPK) superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus. The major pathway involved in activation of ERKs 1 and 2 appears to require the sequential activation of Ras, a 21-kD membrane-bound guanosine triphosphatase (GTPase); Raf-1, a 74-kD cytoplasmic serine/threonine kinase; and MAPK/ERK kinase (MEK), a family of 42- to 45-kD dual-specificity MAPK kinases capable of phosphorylating both tyrosine and serine/threonine residues (1). Alternative pathways, however, may exist (9). We have shown in bovine tracheal myocytes that platelet-derived growth factor (PDGF)-induced activation of ERKs requires the activation of MEK1 (12). However, inhibition of Raf-1 activation by forskolin fails to attenuate ERK activity significantly (13), implying that MEK activation may occur independently of Raf-1. The requirement and sufficiency of Ras for ERK activation in cultured airway smooth-muscle cells, however, have not yet been tested.

Recently, attention has been focused on alternative families of MAPKs that are activated in response to cellular stress rather than mitogenic stimuli. The Jun amino terminal kinases (JNKs) (analogous to the rat stress-activated protein kinases, or SAPKs) are stimulated by a wide variety of cytokines and stressful stimuli (for example, tumor necrosis factor-alpha , interleukins, anisomycin, and ultraviolet light) (14). Other stressful stimuli (such as hyperosmolarity, lipopolysaccharide) activate p38, the mammalian form of yeast high-osmolarity glycerol kinase (17, 18). Data from immortalized or transformed cell lines suggest that JNKs are phosphorylated by sequential activation of Ras, MEK kinase, and either MAPK kinase (MKK)4 or MKK7, the former being the human homologue of murine SAPK/ERK kinase-1 (SEK1) (19). Recent studies suggest that stressful stimuli may also activate JNKs in a Ras-independent manner (24, 25). The p38 MAPK is activated via MKK3 and MKK6, and possibly MKK4 (20, 26, 27). The roles of Ras and other signaling intermediates in the activation of JNKs and p38 have not been studied in airway smooth muscle.

The D-type cyclins (cyclins D1, D2, and D3) are thought to be key regulators of G1 progression in mammalian cells. We have shown in bovine tracheal myocytes that mitogenic stimulation with PDGF induces cyclin D1 transcriptional activation and protein synthesis, as well as phosphorylation of Rb (28). Further, microinjection of cells with a neutralizing antibody against cyclin D1 inhibits serum- induced S-phase traversal, suggesting that cyclin D1 is required for DNA synthesis in these cells. Finally, catalytic activation of ERK regulates cyclin D1 expression (29). However, the potential contributions of Ras and JNK activation to cyclin D1 transcriptional activation in airway smooth muscle have not been examined.

In the present study, we hypothesized that in bovine tracheal myocytes, growth factor treatment induces transcription from the cyclin D1 promoter that is dependent on the activation of both Ras and ERK. We found that activation of Ras is required and sufficient for PDGF-induced ERK2 activation. We also determined that although PDGF treatment induces modest activation of JNK and p38, active Ras is insufficient for maximal activation of these stress-activated MAPKs. Finally, we found that Ras-induced activation of cyclin D1 transcriptional activation is mediated by stimulation of the ERK but not JNK pathway.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Materials

Antihuman alpha -smooth muscle actin, peroxidase-linked goat antirabbit immunoglobulin (Ig)G, protein A sepharose beads, phorbol 12,13-dibutyrate (PDBU), o-nitrophenyl-beta -D-galactoside, and myelin basic protein (MBP) were purchased from Sigma Chemical (St. Louis, MO). PDGF was obtained from Upstate Biotechnology (Lake Placid, NY). [gamma -32P]adenosine triphosphate (ATP) and an enhanced chemiluminescence kit were obtained from DuPont/NEN Research Products (Wilmington, DE). Antibodies against phosphorylated ERK and the light chain of IgG were purchased from Promega (Madison, WI) and Zymed (South San Francisco, CA), respectively. For in vitro phosphorylation assays, a monoclonal antibody against hemagglutinin (12CA5) was purchased from Babco (Berkeley, CA). A complementary DNA (cDNA) encoding a glutathione-S-transferase-JNK fusion protein was provided by Dr. James Posada (University of Vermont, Burlington, VT) (30). The p38 substrate ATF2 was purchased from New England Biolabs (Beverly, MA). Plasmid DNAs encoding a dominant-negative Ha-Ras (pEXV-N17Ras), hemagglutinin-tagged ERK2, hemagglutinin-tagged JNK1, and hemagglutinin-tagged p38 were provided by Dr. Marsha Rosner (University of Chicago, Chicago, IL). Plasmid DNAs encoding constitutively active forms of Ras (pZIP-v-Ha-Ras), MEK1 (pCMV-EE-MEK-2E) and SEK1 (pCMV-SEK-ED) were provided by Dr. Dennis Templeton (Case Western Reserve University, Cleveland, OH). Expression vectors encoding a dominant-negative Rac1 (pEXV-N17Rac1), constitutively active Rac1 (pEXV-Myc-V12Rac1), and the reporter-1745CD1LUC were provided by Dr. Richard Pestell (Albert Einstein College of Medicine, Bronx, NY). Lipofectamine was purchased from Life Technologies (Gaithersburg, MD). Lovastatin was obtained from Merck Research Laboratories (Rahway, NJ).

Cell Culture

Bovine tracheal smooth-muscle cells were isolated as described previously (1). Myocytes of passage number 5 or less were studied. Confluent cultures exhibited the typical "hill and valley" appearance and showed specific immunostaining for alpha -smooth muscle actin. At 24 h before each experiment, bovine tracheal myocytes were serum-starved in Dulbecco's modified essential medium (DMEM) without serum.

Plasmids and Transient Transfections

The constitutively active Ras, Ras-zip6, consists of v-Ha-Ras subcloned into a mammalian expression vector. The dominant-negative Ha-Ras, N17Ras, is a cDNA encoding a mutant Ras in which amino acid 17 has been changed from serine to asparagine (31). The dominant-negative Rac1, N17Rac1, encodes a mutant Rac1 in which amino acid 17 has been changed from threonine to asparagine. The constitutively active Rac1, V12Rac, encodes a mutant Rac1 in which amino acid 12 has been changed from glycine to valine (32). Hemagglutinin-tagged ERK2, JNK1, and p38 expression vectors were constructed by ligating a DNA fragment encoding the seven-amino-acid influenza hemagglutinin epitope to the 5' end of murine ERK2, JNK1, and p38, respectively (13, 19). The constitutively active MEK1, MEK-2E, is a cDNA encoding a protein in which amino acids 218 and 222 have been changed from serine to glutamine (8). Amino acids 220 and 224 of murine SEK1 were mutated from serine to glutamine and aspartate to form the constitutively active SEK-ED. To construct the reporter-1745CD1LUC, a 1,882-base pair PvuII fragment of the human cyclin D1 genomic clone was subcloned into the vector pA3 (33).

Immune Complex Assays

Cells were transiently transfected using a liposome/DNA solution. Cells were seeded into 100-mm plates at a density of 5 × 105 cells/plate and incubated in 10% fetal bovine serum (FBS) DMEM overnight. After rinsing, cells were incubated in a solution consisting of serum- and antibiotic-free medium, plasmid DNA (5 µg/plate cDNA encoding HA-tagged MAPK and 5 µg/plate cDNA encoding Ras, Rac, or empty vector) and lipofectamine (40 µl/plate). After 5 h, the solution was replaced with 10% FBS/DMEM. At 48 h after transfection, cells were serum-starved in DMEM. The next day, cells were treated with 30 ng/ml PDGF or 200 nM PDBU for 10 min (ERK) or 30 min (JNK and p38). In some instances, cells were pretreated for 48 h with 30 µm lovastatin (34). Activation of MAPK was assessed by immunoprecipitation of the epitope-tag using the antihemagglutinin antibody 12CA5, followed by an in vitro phosphorylation assay using MBP, Jun, or ATF2 as substrates (12). Treated cells were washed twice with phosphate-buffered saline (150 mM NaCl and 0.1 M phosphate, pH 7.5) and incubated in a lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 40 mM beta -glycerophosphate, 100 mM NaCl, 50 mM NaF, 2 mM ethylenediaminetetraacetic acid (EDTA), 200 µm Na3VO4, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF) (30 min at 4°C). Insoluble materials were removed by centrifugation (13,000 rpm for 10 min at 4°C). Cell lysates were then incubated for 3 h with 30 µl of protein-A sepharose beads precoupled with the 12CA5 antihemagglutinin antibody. Immunoprecipitates were washed three times with lysis buffer and twice with kinase buffer containing 20 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol, 200 µm Na3VO4, and 10 mM p-nitrophenyl phosphate. Immune complexes were resuspended in a final volume of 30 µl kinase buffer and incubated (20 min at 30°C) with 5 µCi [alpha -32P]ATP and the relevant substrate (0.25 mg/ml MBP, 0.3 mg/ml Jun, or 65 µg/ml ATF-2). Reactions were terminated by adding Laemmli buffer and boiling. Samples were resolved on a 10% sodium dodecyl sulfate (SDS) gel and the proteins transferred to a nitrocellulose membrane using a semidry transfer apparatus (Hoefer, South San Francisco, CA). After Ponceau staining, the membrane was exposed to film and substrate phosphorylation measured by optical scanning (Jandel Scientific, San Rafael, CA).

To confirm that apparent differences in MAPK activity were not related to alterations in expression of the epitope-tagged MAPK due to Ras or Rac coexpression, nitrocellulose membranes were probed with the antihemagglutinin antibody 12CA5. Signals were amplified and visualized using peroxidase-linked rat antimouse kappa  light-chain IgG and enhanced chemiluminescence.

Determination of Cyclin D1 Promoter Transcriptional Activity

Cells were transiently cotransfected with plasmids encoding the human cyclin D1 promoter subcloned into a luciferase reporter and either active Ras, MEK1, SEK1, or the appropriate empty vector. Cells were seeded into 60- mm dishes at 50 to 80% confluence and incubated in 10% FBS/DMEM overnight. After rinsing, cells were incubated with a liposome solution consisting of serum- and antibiotic-free medium, DNA (total of 1.8 µg/plate) and lipofectamine (12 µl/plate). Because cotransfection with viral promoter-driven expression vectors tends to reduce the promoter activity of cyclin D1 (29), a concentration- response curve was generated for each expression vector to determine optimal concentration, and the effect of the expression vectors on cyclin D1 promoter activity was compared with that of an equal amount of parental empty vector. In each case, concentrations of 30 to 50 ng/plate were employed. After 5 h, the liposome solution was replaced with 10% FBS/DMEM. The next day, cells were serum-starved in DMEM. Eight hours later, cells were treated with the appropriate stimulus. Finally, 16 h after treatment, cells were harvested for analysis of luciferase activity using lysis buffer provided with the Promega Luciferase Assay system. Luciferase activity was measured at room temperature using a luminometer (Turner Designs, Sunnyvale, CA). Luciferase content was assessed by measuring the light emitted during the initial 30 s of the reaction and the values were expressed in arbitrary light units. The background activity from cell extracts was typically less than 0.02 units.

Cyclin D1 promoter transcriptional activation was normalized for transfection efficiency by cotransfecting cells with a cDNA encoding beta -galactosidase (pCMV-beta -galactosidase, 30 ng/plate). beta -galactosidase activity was assessed by colorimetric assay using o-nitrophenyl-beta -D-galactoside as a substrate (35).

Anti-phosphoERK Immunoblots

In some experiments, the ERK activity of whole cell lysates was estimated by determining the level of phosphorylated ERKs. These experiments utilized a phosphospecific antibody (36) that recognizes ERKs only when phosphorylated at Thr183 and Tyr185, which are required for full enzymatic activity (37). Samples were treated with 30 ng/ml PDGF for 10 min and extracted in a lysis buffer containing 50 mM Tris (pH 7.5), 40 mM beta -glycerophosphate, 100 mM NaCl, 2 mM EDTA, 50 mM NaF, 200 µm Na3VO4, 200 µm PMSF, and 1% Triton X-100. Insoluble materials were removed by centrifugation (13,000 rpm for 10 min at 4°C). Samples were resolved on a 10% SDS gel and the proteins transferred to a nitrocellulose membrane by semidry transfer. After Ponceau staining, membranes were probed with antibody against phosphorylated ERK. Signals were amplified and visualized using peroxidase-linked goat antirabbit IgG and enhanced chemiluminescence.

Data Analysis

As noted previously, the effects of mutant protein expression on kinase or promoter activity were compared with empty vector controls. Statistical significance was assessed by one-way analysis of variance (ANOVA). Differences identified by ANOVA were pinpointed by Student-Newman-Keul's multiple-range test. Data sets that were not normally distributed (as assessed by Kolmogorov-Smirnov testing) were log transformed before ANOVA.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Ras Is Required for PDGF-Induced ERK Activation and Sufficient for Activation of ERK

To investigate the requirement of Ras for PDGF-induced ERK activation, we transiently transfected bovine tracheal myocytes with a dominant-negative Ras (N17Ras) and HA- tagged ERK2. Cells were treated with 30 ng/ml PDGF for 10 min and the ERK2 activity was assessed by in vitro phosphorylation. PDGF resulted in an increase (fivefold) in ERK activity, as measured by phosphorylation of MBP (Figure 1, upper panel). Expression of a dominant-negative Ras (N17Ras) appeared to inhibit PDGF-induced ERK activation, suggesting that Ras is required for PDGF signaling. There was no associated reduction in HA-ERK2 expression (Figure 1, lower panel), demonstrating that reductions in ERK2 activity due to N17Ras were not due to reductions in HA-ERK2 expression. Phorbol ester-induced ERK activation was not Ras-dependent, because stimulation with PDBU in the presence of N17Ras was similar to that with empty vector. In contrast to the dominant-negative Ras, expression of a dominant-negative Rac1 had no effect on PDGF-induced ERK activation (Figure 2).


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  		Figure 1.   Effect of dominant-negative Ras on PDGF-induced and phorbol ester-induced ERK activity. (a) Cells were transiently cotransfected with cDNAs encoding a hemagglutinin-tagged ERK2 and a dominant-negative Ras. After serum starvation, cells were treated with PDGF (30 ng/ml) or PDBU (200 ng/ml) for 10 min, after which cell lysates were immunoprecipitated using an antibody against hemagglutinin. Untreated cells were transfected with empty vector. Top: ERK activity was assessed by in vitro phosphorylation using MBP as a substrate. Bottom: level of expression of epitope-tagged ERK2, as determined by Western blot. (b) Group mean data (± standard error of the mean [SEM]) from three experiments (*P < 0.05, ANOVA).


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  		Figure 2.   Rac1 is not required for PDGF-induced ERK activation. Cells were transiently cotransfected with cDNAs encoding an epitope-tagged ERK2 and a dominant-negative Rac1 (N17 Rac). Cells were serum-starved and treated with PDGF as described in Figure 1. Untreated cells were transfected with empty vector. Top: ERK activity, as assessed by in vitro phosphorylation assay using MBP as a substrate. Bottom: level of expression of epitope-tagged ERK2, as determined by Western blot. Data shown are typical of three experiments.

The requirement of Ras for PDGF-induced ERK activation was confirmed by pretreatment with lovastatin, an inhibitor of cholesterol biosynthesis that blocks addition of farnesyl and geranyl groups on Ras. In this experiment, ERK phosphorylation was assessed by immunoblotting using an antibody directed against phosphorylated ERK. Lovastatin pretreatment (30 µm for 48 h) nearly abolished PDGF stimulation of ERK phosphorylation, but had no apparent effect on that induced by PDBU (Figure 3).


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  		Figure 3.   Effect of lovastatin on PDGF-stimulated phosphorylation of ERK. Cells were pretreated with lovastatin (30 µm for 48 h) before stimulation with PDGF (30 ng/ml for 10 min) or PDBU (200 ng/ml for 10 min). ERK phosphorylation was assessed by Western blotting with a phosphospecific antibody (1:10,000 dilution). This experiment was repeated twice.

To test for the sufficiency of Ras for ERK activation, bovine tracheal myocytes were cotransfected with a constitutively active Ras (Ras-zip6) and HA-tagged ERK2. Ras activation induced a substantial increase in ERK activity (Figure 4), similar to that detected with PDGF.


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  		Figure 4.   Effect of constitutively active Ras on ERK activation. (a) Cells were transiently cotransfected with an epitope-tagged ERK2 and, in one plate per experiment, Ras-zip6. After serum starvation, selected plates were treated with PDGF (30 ng/ml) for 10 min, after which cell lysates were immunoprecipitated using an antibody against hemagglutinin. Untreated cells were transfected with empty vector. Top: transfectant ERK2 activity. Bottom: level of expression of hemagglutinin-tagged ERK2. (b) Group mean data (± SEM) from three experiments (*P < 0.05, ANOVA).

PDGF Activates JNK1 and p38 in a Ras-Dependent Manner

We also studied the role of Ras in the activation of two other members of the MAPK superfamily, JNK1 and p38. Cells were cotransfected with N17Ras and either HA-tagged JNK1 or p38. JNK1 and p38 activation was assessed by in vitro phosphorylation assays using recombinant c-Jun or ATF-2, respectively. PDGF treatment induced modest levels of JNK1 and p38 activity. The levels of JNK1 and p38 activation were substantially below that induced by anisomycin, a potent activator of JNK and p38 (Figures 5 and 6). Cotransfection with N17Ras reduced the level of PDGF-induced JNK1 activation, suggesting a requirement for Ras. To test for the sufficiency of Ras for JNK and p38 activation, bovine tracheal myocytes were cotransfected with a constitutively active Ras (Ras-zip6) and either an HA-tagged JNK1 or p38. Expression of a constitutively active Ras induced only modest activation of JNK1 and p38 (Figures 5 and 6). Together, these data imply that complete activation of the JNK and p38 pathways (as likely occurs after anisomycin treatment) requires the activation of additional or alternative upstream signaling intermediates besides Ras.


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  		Figure 5.   Effects of PDGF and active Ras on JNK1 activity. (a) Cells were transiently transfected with a cDNA encoding a hemagglutinin-tagged JNK1 and, in some plates, cotransfected with either a dominant-negative Ras (N17Ras) or a constitutively active Ras (Ras-zip6). After serum starvation, selected plates were treated with PDGF (30 ng/ml for 30 min). Untreated cells were transfected with empty vector. For comparison, some cultures were treated with anisomycin (50 ng/ml for 30 min). Top: transfectant JNK activity was assessed by immunoprecipitation of the epitope-tagged JNK1, followed by an in vitro phosphorylation assay using recombinant Jun as substrate. Bottom: level of expression of hemagglutinin-tagged JNK1, as determined by Western blotting. (b) Group mean data (± SEM) for three experiments (*P < 0.05, ANOVA).


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  		Figure 6.   Effect of PDGF and active Ras on p38 activity. (a) Cells were transiently transfected with a cDNA encoding a hemagglutinin-tagged p38 and, in some plates, cotransfected with either a dominant-negative Ras (N17Ras) or a constitutively active Ras (Ras-zip6). Cells were treated as described in Figure 4. Top: transfectant p38 activity was assessed by immunoprecipitation of hemagglutinin-tagged p38, followed by an in vitro phosphorylation assay using ATF-2 as the substrate. Bottom: level of expression of epitope-tagged p38 as determined by Western blotting. (b) Group mean data (± SEM) from three experiments (*P < 0.05, ANOVA).

We tested the requirement of Rac1, a member of the Rho family of 21-kD GTPases, for JNK1 activation. Cells were cotransfected with cDNAs encoding HA-JNK1 and a dominant-negative (N17Rac1) or a constitutively active Rac1 (V12Rac1). Expression of N17Rac1 substantially inhibited anisomycin-induced JNK1 activation (Figure 7), whereas expression of a constitutively active Rac1 (V12Rac1) induced a level of JNK1 activation similar in magnitude to that generated by anisomycin treatment (Figure 7). These data suggest that Rac1 is required and sufficient for maximal activation of JNKs.


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  		Figure 7.   Effect of mutant alleles of Rac1 on JNK1 activation. Cells were transiently cotransfected with an epitope-tagged JNK1 and either the dominant-negative Rac1 (N17Rac1) or constitutively active Rac1 (V12Rac1). Selected plates were treated with anisomycin (50 ng/ml for 30 min). Untreated cells were transfected with empty vector. Top: transfectant HA-JNK1 activity. Bottom: level of expression of hemagglutinin-tagged JNK1. This experiment was repeated twice.

Ras Regulates Cyclin D1 Transcriptional Activation via ERK

We have previously demonstrated that ERK activation regulates the transcriptional activation of cyclin D1 (29), a key regulator of G1 progression in bovine tracheal myocytes (28). If Ras is required and sufficient for ERK activation, as demonstrated previously, then Ras should also regulate cyclin D1 expression. To test this, we transiently transfected cells with cDNA encoding either N17Ras or Ras-zip6 and the full-length human cyclin D1 promoter, subcloned into a luciferase reporter. Expression of the dominant-negative Ras inhibited PDGF-induced cyclin D1 promoter activation, whereas expression of the constitutively active Ras increased cyclin D1 promoter activity (Figure 8). To determine whether activation of the JNK pathway might also be responsible for Ras-induced transcriptional activation of the cyclin D1 promoter, we transfected cells with cDNAs encoding a constitutively active form of SEK1 (SEK-ED), an upstream activator of JNK. Active SEK1, the murine homologue of human MKK4, had no effect on cyclin D1 promoter activity. The activity of SEK-ED was confirmed by measurement of JNK1 activation, which was increased by expression of active SEK1 (Figure 9). In contrast to active SEK1, expression of an active MEK1 (MEK 2E), the upstream activator of ERK (12), increased cyclin D1 promoter activation. Finally, the synthetic MEK inhibitor PD98059 (30 µm) attenuated both Ras and PDGF-induced cyclin D1 promoter activity. Together, these data demonstrate that Ras, MEK, and ERK constitute a distinct pathway to cyclin D1 transcriptional activation in bovine tracheal myocytes, and that stimulation of the SEK1/JNK pathway is insufficient for cyclin D1 promoter activation.


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  		Figure 8.   Requirement and sufficiency of Ras for cyclin D1 promoter activation. Cells were transiently cotransfected with the cyclin D1 full-length promoter tagged to a luciferase reporter gene and either pEXV, EE-CMV, N17Ras, Ras-zip6, or SEK-ED. pCMV-beta -Galactosidase was also cotransfected to normalize for transfection efficiency. At 48 h after transfection and 24 h after serum starvation, cells were treated with 30 ng/ml PDGF for 16 h. One plate per experiment was also pretreated for 15 min with the synthetic MEK inhibitor PD98059 (30 µm). Untreated cells were transfected with empty vector. Cells were extracted and luciferase activity measured in a luminometer. beta -Galactosidase activity was assessed by colorimetric assay. Data are calculated as luciferase/beta -galactosidase/h, normalized to the control vector. Data shown are the means ± SEM of four experiments (*P < 0.05, ANOVA).


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  		Figure 9.   Activation of JNK1 by SEK1. Cells were transiently cotransfected with hemagglutinin-tagged JNK1 and, in one plate per experiment, a constitutively active SEK1 (SEK-ED), the murine homolog of human MKK4. Cells were treated as described in Figure 4. Top: in vitro phosphorylation assay of JNK1 activity using c-Jun as a substrate. Bottom: expression of epitope-tagged JNK1. Data shown are typical of three experiments.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In this study, we examined the role of p21 Ras in the activation of MAPKs by the growth factor PDGF. We found that: (1) Ras is required for PDGF-induced ERK activation and sufficient for activation of ERK; (2) PDGF treatment or active Ras each induces only moderate activation of JNK1 and p38, implying that complete activation of the JNK and p38 pathways requires additional or alternative upstream signaling intermediates besides Ras; (3) Rac1 is required and sufficient for maximal activation of JNKs; (4) Ras-induced activation of cyclin D1 transcriptional activation is mediated by stimulation of the ERK pathway; and (5) stimulation of the SEK1/JNK pathway is insufficient for cyclin D1 promoter activation.

The requirement and sufficiency of Ras for MAPK activation in cultured airway smooth-muscle cells have not been previously examined. Because the major pathway involved in activation of ERKs appears to require the sequential activation of Ras, Raf-1, and MEK, it is perhaps not unexpected that in bovine tracheal myocytes, inhibition of Ras by expression of either a dominant-negative mutant or lovastatin attenuates PDGF-induced ERK activation. However, our finding that ERK activation by PDBU does not require Ras suggests that agonists signaling though protein kinase C (PKC), for example endothelin-1 and bradykinin (38, 39), may activate ERK in a Ras-independent manner. Diacylglycerol and phorbol esters serve as hydrophobic anchors to recruit PKC to the cell membrane, where the enzyme is activated by interaction with phosphatidylserine residues. PKC, in turn, may phosphorylate and activate Raf-1 (40), thereby bypassing Ras. On the other hand, a recent study in COS cells showed that whereas activation of Raf-1 by PKC is not blocked by expression of N17Ras, Raf-1 mutations preventing association with Ras block activation of Raf-1 by PKC (41). Thus, it is conceivable that after PKC activation, Ras activates Raf-1 by a mechanism distinct from that initiated by receptor tyrosine kinases.

Little is known about the activation of JNKs in airway smooth muscle, or about the signaling outcomes of this pathway. Thrombin, a mitogen for rat tracheal and aortic myocytes, induces rapid activation of JNK in these cell types (30, 42). In both cell types, JNK activation is inhibited by forskolin, an activator of adenyl cyclase and inhibitor of cell proliferation. Recently, activation of JNK was demonstrated to be required for primary hepatocyte DNA synthesis (43), and expression of a dominant-negative JNK inhibits Galpha 12-induced DNA synthesis in NIH3T3 cells (44). Finally, active c-Jun has been demonstrated to induce cyclin D1 promoter activity in JEG-3 trophoblasts and COS cells (33). Together, these data imply that the JNK pathway may function as a positive regulator of cell growth. On the other hand, based on the types of signals that activate JNKs, it is possible that this pathway is involved in growth inhibition rather than mitogenesis (45). This possibility is supported by studies in guinea-pig tracheal myocytes in which treatment with sphingosine and cell-permeable ceramides was associated with JNK activation, cyclic adenosine monophosphate accumulation, and growth arrest (46). In PC12 pheochromocytoma cells, activation of SAPK and p38 and concurrent inhibition of ERK are critical for apoptosis, whereas activation of ERK inhibits apoptosis (47). Similarly, somatostatin inhibits both ERK activity and cell proliferation in human lung small-cell carcinoma cell lines, suggesting that ERKs are required for tumor growth (48), whereas ultraviolet light- stimulated JNK1 activation promotes apoptosis in these cells (49). In our study, treatment of bovine tracheal myocytes with PDGF activated both ERK2 and JNK1 through a common signaling intermediate, Ras, consistent with the notion that JNK activation is involved in the cellular response to mitogenic stimulation. However, transient transfection with a cDNA encoding a constitutively-active SEK1, an activator of JNK1, failed to increase cyclin D1 promoter activity, suggesting that JNK1 is not involved in cell-cycle progression. Interestingly, these cells do not take on the phenotypic appearance of cells undergoing apoptosis (K. Page and M. Hershenson, preliminary observations), suggesting that activation of the JNK pathway may be insufficient for either growth or apoptosis in cultured airway smooth-muscle cells. One possible explanation is that growth factor-induced JNK activation plays a permissive role, allowing apoptosis to occur only when accompanied by the stimulation of other apoptosis-related pathways.

We found that whereas PDGF and activated Ras induced activation of both JNK1 and p38, the level of activation induced by these stimuli was substantially less than that elicited by the stress-inducing protein synthesis inhibitor anisomycin. These data imply that complete activation of the JNK1 and p38 pathways requires the activation of additional or alternative upstream signaling intermediates besides Ras. To test this, we examined the requirement and sufficiency of the 21-kD Rho family GTPase Rac1 for JNK signaling. Expression of a dominant-negative form of Rac1 substantially inhibited anisomycin-induced JNK1 activation, whereas expression of active Rac1 induced a level of JNK1 activity similar to that produced by anisomycin. These data suggest that Rac1 is a major regulator of JNK activation in airway smooth-muscle cells. Rac has been demonstrated to be a potent activator of JNK in other cell systems (50, 51). However, it should be noted that there appear to be additional signaling outcomes associated with Rac activation, including cytoskeletal organization, gene expression, and progression though G1 of the cell cycle (43, 52). This diversity of Rac responses likely arises from interactions with multiple effector proteins. It is therefore conceivable that, whereas activation of JNK1 by SEK1 was insufficient to induce transcription from the cyclin D1 promoter, activation of Rac1 could induce cyclin D1 promoter activity via JNK-independent pathways.

Expression of a constitutively active Ras induced not only activation of ERK and JNK1, but also transcriptional activation of the cyclin D1 promoter. The ability of a constitutively active MEK1 also to induce transcriptional activation, as well as the attenuation of Ras-induced promoter activity by the synthetic MEK inhibitor PD98059, strongly suggests that cyclin D1 expression is stimulated via the ERK pathway. These data are consistent with previous reports demonstrating that ERK activation regulates cyclin D1 promoter transcriptional activation (29, 33, 55, 56), and infer that Ras, MEK, and ERK constitute a distinct pathway to cyclin D1 expression in airway smooth-muscle cells. On the other hand, expression of an active SEK1, an upstream activator of JNK, failed to induce cyclin D1 promoter activity in cultured bovine tracheal myocytes.

It is important to note potential limitations of our study. First, it is conceivable that lovastatin inhibits PDGF-induced ERK activation by preventing the post-translational farnesylation and geranylgeranylation of GTPases other than Ras, such as the Rho family GTPases (Rho, Rac, and Cdc42). However, expression of a constitutively active Rac1 does not induce ERK activation (K. Page and M. Hershenson, unpublished data), implying that lovastatin is unlikely to have inhibited PDGF-induced ERK activation by preventing post-translational modification of Rho family GTPases. Second, we did not corroborate the requirement of Ras for PDGF-induced MAPK and cyclin D1 promoter activation by measurement of Ras-bound guanosine triphosphate (GTP). Measurement of Ras GTP loading has been found to underestimate the role of Ras in NIH 3T3 cells (57, 58), aortic endothelial cells (59), and BALB/c 3T3 cells (T.-S. Chao and M. Rosner, unpublished data). In 3T3 cells, the requirement of Ras for PDGF-induced ERK activation and mitogenesis has been demonstrated by the use of dominant-negative N17Ras and neutralizing antibodies, yet Ras GTP/guanosine diphosphate (GDP) ratio increases less than twofold after PDGF stimulation. Apparently small changes in Ras GTP/GDP ratio are physiologically significant, perhaps because Ras effectors are highly sensitive to this proportion, or because only a small percentage of the total cellular Ras is apportioned to any particular signaling pathway. We therefore elected to defer these measurements, and instead to rely on the use of two inhibitors (N17Ras and lovastatin) to determine the requirement of Ras for MAPK and cyclin D1 promoter activation. Third, one potential problem related to the use of the dominant-negative Ha-Ras is its propensity to bind its upstream activator, Son of sevenless (Sos), thereby blocking the activity of other proteins that are activated by this nucleotide exchange factor, including K-Ras and N-Ras. It is therefore conceivable that these isoforms of Ras may also play a role in PDGF-induced MAPK activation.

We conclude that PDGF-induced activation of the ERK and JNK pathways is dependent on Ras. Further studies are needed to determine the exact signaling outcomes of these pathways in airway smooth muscle.

    Footnotes

Address correspondence to: Marc B. Hershenson, M.D., University of Chicago Children's Hospital, 5841 S. Maryland Ave., MC 4064, Chicago, IL 60637-1470. E-mail: mhershen{at}midway.uchicago.edu

(Received in original form October 27, 1998 and in revised form January 8, 1999).

Abbreviations: analysis of variance, ANOVA; complementary DNA, cDNA; Dulbecco's modified essential medium, DMEM; extracellular signal-regulated kinase, ERK; fetal bovine serum, FBS; guanosine triphosphate, GTP; guanosine triphosphatase, GTPase; hemagglutinin, HA; immunoglobulin, Ig; Jun amino terminal kinase, JNK; mitogen-activated protein kinase, MAPK; myelin basic protein, MBP; MAPK/ERK kinase, MEK; MAPK kinase, MKK; phorbol 12,13-dibutyrate, PDBU; platelet-derived growth factor, PDGF; protein kinase C, PKC; stress-activated protein kinase, SAPK; SAPK/ERK kinase-1, SEK1; standard error of the mean, SEM.

Acknowledgments: The authors thank Drs. James Posada, Dennis Templeton, Marsha Rosner, and Richard Pestell for providing materials required for this work. This work was supported by grants from the National Institutes of Health (HL54685 and HL56399) and the Blowitz-Ridgeway Foundation.
    References
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

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p38 MAP kinase negatively regulates cyclin D1 expression in airway smooth muscle cells
Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L955 - L964.
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