p38 MAP kinase negatively regulates cyclin D1 expression in airway smooth muscle cells

Kristen Page, Jing Li, and Marc B. Hershenson

Department of Pediatrics, University of Chicago, Chicago, Illinois 60637-1470


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that platelet-derived growth factor (PDGF) stimulates p38 mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes, suggesting that p38 is involved in growth regulation. We therefore examined whether p38 regulates expression of cyclin D1, a G1 cyclin required for cell cycle traversal. The chemical p38 inhibitors SB-202190 and SB-203580 each increased basal and PDGF-induced cyclin D1 promoter activity and protein abundance. Overexpression of a dominant negative allele of MAP kinase kinase-3 (MKK3), an upstream activator of p38alpha , had similar effects. Conversely, active MKK3 and MKK6, both of which increase p38alpha activity, each decreased transcription from the cyclin D1 promoter. Together, these data demonstrate that p38 negatively regulates cyclin D1 expression. We tested whether p38 regulates cyclin D1 expression via inhibition of extracellular signal-regulated kinase (ERK) activation. Chemical inhibitors of p38 induced modest ERK phosphorylation and activation. However, dominant negative MKK3 was insufficient to activate ERK, and active MKK3 and MKK6 did not attenuate platelet-derived growth factor-mediated ERK activation. These data are consistent with the notion that p38alpha negatively regulates cyclin D1 expression via an ERK-independent pathway.

bovine; extracellular signal-regulated kinase; platelet-derived growth factor; signal transduction; mitogen-activated protein kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THERE IS AMPLE EVIDENCE supporting the notion that excess airway smooth muscle mass contributes to the pathogenesis of airflow obstruction in asthma (6, 10-12, 19, 22, 49, 52, 55). Accordingly, we have examined the signaling pathways responsible for cell cycle traversal in primary bovine tracheal myocytes. Inhibition of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) reduced platelet-derived growth factor (PDGF)-induced DNA synthesis in a concentration-dependent manner, suggesting that catalytic activation of MEK-1 and ERK is required for DNA synthesis in these cells (28). In addition, we found that activation of MEK-1 is required and sufficient to activate transcription from the promoter of cyclin D1 (47), a critical regulator of G1 progression in these cells (66).

Growth factor treatment of airway smooth muscle cells also induces activation of the two stress-activated MAP kinases, p38 and c-Jun amino-terminal kinase (JNK) (41, 45, 50), consistent with the notion that these intermediates, like ERKs, play a role in the growth regulation. The precise role of p38 and JNK in airway smooth muscle cell cycle traversal has not been established, however.

The p38 MAP kinase family consists of four isoforms. p38alpha was originally identified in lipopolysaccharide-stimulated mouse macrophages and was found to have substantial homology to the yeast high-osmolarity glycerol kinase (9, 18, 36, 38). Since this original identification, three additional isoforms, beta , gamma , and delta , have been cloned (23, 24, 53, 60). p38alpha , -beta , and -delta are somewhat ubiquitously expressed, whereas p38-gamma is primarily restricted to skeletal muscle (60). p38alpha and -beta are inhibitable by pyridinylimidazole compounds such as SB-202190 and SB-203580, whereas p38gamma and -delta are not (14, 24, 31). Three MAP kinase kinases (MKKs) for p38 have been identified. MKK6 strongly activates all p38 isoforms, and MKK3 preferentially activates p38alpha and -delta (14, 21, 23, 37, 46, 57). MKK4 appears to phosphorylate and activate both JNK1 and p38alpha (9, 36).

Few studies in any cell system have addressed the potential role of p38 in the regulation of cell growth. Overexpression of p38 inhibits serum-induced NIH/3T3 cell cycle progression (39). Mutations of the MKK4 gene have been found in human pancreatic, lung, breast, testicular, and colorectal cancer cell lines (54, 56). Expression of an active MEK kinase (MEKK)-3 inhibited cell cycle progression and reversed Ras-induced transformation in NIH/3T3 cells (13). This effect was blocked by coexpression of an inactive MKK6, suggesting that cell cycle arrest was mediated by p38.

The mechanism by which signaling through the p38 pathway may negatively regulate growth has not been well studied. p38 attenuates transcription from the cyclin D1 promoter in CCL-39 hamster lung fibroblasts (33) but is necessary for optimal induction of cyclin D1 promoter activity in the human breast cancer MCF-7 cell line (34).

Recent evidence suggests that p38 exerts its effects, in part, via the negative regulation of ERK. The triggering of stress-activated kinases, concomitant with the inhibition of the ERK pathway, has been observed in a number of cell systems undergoing apoptosis (4, 65). In rat-1 fibroblasts, stimulation of alpha 1A-adrenoreceptors by phenylephrine stimulated p38 activation while inhibiting ERK phosphorylation (3). Treatment with the chemical p38 inhibitors SB-202190 or SB-203580 has been demonstrated to increase ERK activation in mouse keratinocytes (48), HepG2 cells (51), and, in a preliminary report, canine tracheal myocytes (25). These data suggest that p38 may negatively regulate the ERK pathway, although the signaling intermediates linking p38 and ERK have not been established.

We tested the hypothesis that in primary bovine tracheal myocytes, p38 negatively regulates transcription from the cyclin D1 promoter. Our results suggest that p38 negatively regulates cyclin D1 expression in an ERK-independent manner.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Anti-human alpha -smooth muscle actin, peroxidase-linked goat anti-rabbit IgG, protein A Sepharose beads, and myelin basic protein were purchased from Sigma (St. Louis, MO). SB-203580 and SB-202190 were from Calbiochem (La Jolla, CA). PDGF was obtained from Upstate Biotechnology (Lake Placid, NY). PD-98059 and recombinant activating transcription factor (ATF)-2 were obtained from New England Biolabs (Beverly, MA). [gamma -32P]ATP and an enhanced chemiluminescence kit were purchased from DuPont/NEN Research Products (Wilmington, DE). LipofectAMINE and Opti-MEM were obtained from Life Technologies (Gaithersburg, MD). An antibody against dually phosphorylated ERK and a luciferase assay kit were purchased from Promega (Madison, WI). Peroxidase-linked rat anti-mouse kappa  light chain IgG was obtained from Zymed Laboratories (South San Francisco, CA). A polyclonal antibody against cyclin D1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For in vitro phosphorylation assays, a monoclonal antibody against hemagglutinin (HA; 12CA5) was obtained from Babco (Berkeley, CA).

The full-length cyclin D1 promoter subcloned into a luciferase reporter gene, -1745CD1LUC, was a gift from R. Pestell (Albert Einstein School of Medicine, Bronx, NY) (2). cDNA encoding a dominant negative form of MKK3 (pcDNA3-HA-MKK3-AL) was a gift from J. Woodgett (Ontario Cancer Institute, Toronto, Canada) (67). Plasmids encoding constitutively active MKK3 [pcDNA3-Flag-MKK3(glu)] and constitutively active MKK6 [p6R-Flag-MKK6(glu)] were gifts from R. Davis (Univ. of Massachusetts, Worcester, MA) (46). HA-tagged p38alpha was a gift from M. Karin (Univ. of California, San Diego, CA) (64). HA-tagged ERK2 (pcDNA3-HA-ERK2) was constructed by ligating a DNA fragment encoding the seven-amino acid influenza HA epitope to the 5'-end of murine ERK2 (20).

Cell culture. Bovine tracheal smooth muscle cells were isolated as described previously (1). Myocytes from passage 5 or less were studied. Confluent cultures exhibited the typical "hill-and-valley" appearance and showed specific immunostaining for alpha -smooth muscle actin. Cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 1% nonessential amino acids, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. A7r5 and COS cells were obtained from the American Type Culture Collection (Manassas, VA).

Determination of cyclin D1 promoter transcriptional activity. Cells were seeded into 60-mm dishes at 50-80% confluence and incubated in 10% FBS-DMEM overnight. After being rinsed, the cells were incubated with a liposome solution consisting of serum- and antibiotic-free medium, plasmid DNA (total of 1.8 µg/plate), and LipofectAMINE (12 µl/plate). Cells were transiently cotransfected with plasmids encoding the human cyclin D1 promoter subcloned into a luciferase reporter and either the relevant expression vector or control vector (42). After 5 h, the liposome solution was replaced with 10% FBS-DMEM. The next day, cells were serum starved in DMEM. After 24 h of serum starvation, selected cultures were treated with chemical p38 inhibitors and/or PDGF (30 ng/ml). Finally, 16 h after PDGF treatment, cells were harvested for analysis of luciferase activity with lysis buffer provided in the Promega luciferase assay system. Luciferase activity was measured at room temperature with 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 are expressed in arbitrary light units. The background activity from cell extracts was typically >0.02 U compared with signals on the order of 102 to 103 U. Cyclin D1 promoter transcriptional activation was normalized for transfection efficiency by cotransfecting cells with a cDNA encoding beta -galactosidase (30 ng/plate). beta -Galactosidase activity was assessed by colorimetric assay with o-nitrophenyl-beta -D-galactoside as a substrate (43).

We have found that cotransfection with viral promoter-driven expression vectors tends to suppress cyclin D1 promoter activity, perhaps due to a limitation in the transcription factors available for overall gene expression. This effect seems to be pronounced in primary cells. Concentration-response curves were therefore generated for each expression vector to determine optimal concentration. In all cases, concentrations of 30-50 ng expression vector/plate were used.

Preparation of cell extracts for immunoblotting. Cells were cultured in six-well plates and serum starved for 48 h before treatment. Selected cultures were treated with 3 µM SB-202190 or SB-203580 1 h before growth factor treatment or with 30 ng/ml of PDGF (for 10 min or 16 h for ERK or cyclin D1 immunoblots, respectively). Cells were washed in PBS (150 mM NaCl and 0.1 M phosphate, pH 7.5) 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 phenylmethylsulfonyl fluoride, and 1% Triton X-100. Insoluble materials were removed by centrifugation (13,000 rpm for 10 min at 4°C), and the supernatant was transferred to fresh microcentrifuge tubes.

Western analysis of whole cell extracts. Extracts (10 µg) were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose by semidry transfer (Hoefer, San Francisco, CA). After incubation with the appropriate polyclonal antibody, signals were amplified and visualized with anti-rabbit IgG and enhanced chemiluminescence.

Measurement of ERK and p38 activation. Cells were transiently cotransfected with cDNAs encoding HA-tagged ERK2 or p38alpha and the expression vector of interest. Forty-eight hours after transfection, the cells were serum starved in DMEM. The next day, selected cultures were treated with PDGF (30 ng/ml for 10 min) or anisomycin (50 µg/ml for 30 min). Activation of ERK2 or p38alpha was then assessed by immunoprecipitation of the epitope tag followed by an in vitro phosphorylation assay with major basic protein (MBP) and ATF-2 as substrates, respectively, as described (28, 41). Treated cells were washed twice with PBS 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 EDTA, 200 µM Na3VO4, and 0.2 mM phenylmethylsulfonyl fluoride (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 anti-HA antibody. Immunoprecipitates were washed three times with lysis buffer and twice with kinase buffer containing 20 mM HEPES (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 of kinase buffer and incubated (20 min at 30°C) with 5 µCi of [alpha -32P]ATP and either 0.25 mg/ml of MBP or 0.2 mg/ml of ATF-2. Reactions were terminated by adding Laemmli buffer and boiling. Samples were resolved on a 10% SDS gel, and the proteins were transferred to a nitrocellulose membrane by semidry transfer. After Ponceau staining, the membrane was exposed to film and substrate phosphorylation assessed by optical scanning (Jandel Scientific, San Rafael, CA).

To confirm that apparent differences in ERK or p38 activity were not related to alterations in expression of the epitope-tagged ERK or p38, nitrocellulose membranes were probed with the anti-HA antibody 12CA5. Signals were amplified and visualized with peroxidase-linked rat anti-mouse kappa  light-chain IgG and enhanced chemiluminescence.

Statistical analysis. When applicable, significance was assessed by one-way analysis of variance (ANOVA). Differences identified by ANOVA were pinpointed by the Student-Newman-Keuls multiple range test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chemical inhibition of p38 negatively regulates cyclin D1 transcription and protein abundance. Inhibition of p38 has been demonstrated to enhance cyclin D1 promoter activity and protein abundance in Chinese hamster lung fibroblasts (33). To determine the effect of p38 inhibition on cyclin D1 expression in bovine tracheal myocytes, cells were transiently transfected with a cDNA encoding the full-length cyclin D1 promoter subcloned into a luciferase reporter gene (2). SB-202190 or SB-203580 (3 µM each) each increased basal and PDGF-induced transcription from the cyclin D1 promoter (Fig. 1A). Furthermore, SB-203580 increased basal and PDGF-induced cyclin D1 protein abundance (Fig. 1B).


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Fig. 1.   Effects of SB-202910 and SB-203580 on growth factor-induced cyclin D1 expression. A: cells were transiently transfected with cDNAs encoding the human full-length cyclin D1 promoter subcloned into a luciferase reporter gene. To control for transfection efficiency, selected cultures were also cotransfected with pCMV-beta -galactosidase. Forty-eight hours after transfection and 24 h after serum starvation, cells were pretreated for 1 h with 3 µM SB-202190 or SB-203580, then 30 ng/ml of platelet-derived growth factor (PDGF) were added for 16 h. Cells were lysed, and luciferase activity was measured in a luminometer. Data are means ± SE for 4-6 experiments. *Significantly greater than control value, P < 0.05 by ANOVA. **Significantly greater than PDGF, P < 0.05 by ANOVA. B: cells were pretreated with SB-203580 for 1 h before low-dose PDGF stimulation (3 ng/ml for 16 h). Whole cell extracts were probed with a polyclonal antibody against cyclin D1 (1:1,000 dilution). This experiment was repeated 3 times with similar results.

MKK3 and MKK6 regulate transcription from the cyclin D1 promoter and cyclin D1 protein abundance. MKK3 and MKK6 phosphorylate and activate p38 (14, 21, 23, 37, 46, 57). To determine whether MKK3 regulates cyclin D1 expression, we transiently transfected bovine tracheal myocytes with cDNAs encoding mutant alleles of MKK3 and the luciferase-tagged cyclin D1 promoter. Similar to the chemical p38 inhibitors, overexpression of a dominant negative form of MKK3 (MKK3-AL) increased transcription from the cyclin D1 promoter (Fig. 2A). Overexpression of a constitutively active MKK3 [MKK3(glu)] reduced basal and PDGF-induced cyclin D1 promoter activity, and overexpression of active MKK6 [MKK6(glu)] had similar effects (Fig. 2B).


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Fig. 2.   Effect of mutant alleles of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MKK)-3 and MKK6 on cyclin D1 transcription. A: cells were transiently cotransfected with cDNAs encoding the human full-length cyclin D1 promoter subcloned into a luciferase reporter gene (-1745CD1LUC) and the dominant negative allele of MKK3 (MKK3-AL). Selected cultures were treated with PDGF (30 ng/ml for 16 h). Data are means ± SE for 4 experiments. *Significantly greater than control, P < 0.05 by ANOVA. **Significantly greater than PDGF, P < 0.05 by ANOVA. B: cells were transiently cotransfected with cDNAs encoding the cyclin D1 luciferase reporter gene and constitutively active forms of MKK3 [MKK3(glu)] or MKK6 [MKK6(glu)]. Data are means ± SE for 4 experiments. *Significantly less than PDGF, P < 0.05 by ANOVA.

Because of the relatively low transfection efficiency of primary bovine tracheal myocytes, we could not determine whether inhibition of p38 by dominant negative MKK3 alters cyclin D1 protein abundance in whole cell lysates. We therefore examined this question in the A7r5 rat aortic smooth muscle cell line and in COS cells. Overexpression of MKK3-AL increased cyclin D1 protein abundance (Fig. 3).


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Fig. 3.   Effect of a dominant negative allele of MKK3 on cyclin D1 protein abundance. A7r5 vascular smooth muscle cells and COS cells were transfected with MKK3-AL. Selected cultures were treated with PDGF (30 ng/ml for 16 h) or epidermal growth factor (EGF; 30 ng/ml for 16 h). Whole cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-cyclin D1 antibody (1:1,000 dilution). This experiment was repeated twice with similar results.

Chemical p38 inhibitors inhibit p38 and activate ERK in primary bovine tracheal myocytes. In certain cell systems, chemical inhibition of p38 (with SB-202190 or SB-203580) increases ERK activity (3, 25, 51). As in other cell types (2, 33, 62), it has been demonstrated in bovine tracheal myocytes that ERK activation is an upstream activator of transcription from the cyclin D1 promoter (47). Thus inhibition of p38 could increase cyclin D1 accumulation via the activation of ERK. We therefore investigated the potential interactions between the p38 and ERK signaling pathways in bovine tracheal smooth muscle cells.

To test the effects of chemical inhibitors on p38 activity, cells were transiently transfected with a HA-tagged p38alpha (64), and p38alpha activity was assessed by immunoprecipitation of the epitope tag followed by in vitro phosphorylation with ATF-2 as a substrate. As expected, pretreatment with either SB-202190 or SB-203580 (each 3 µM) attenuated anisomycin-induced p38alpha activation (Fig. 4). To test whether p38 inhibitors increase endogenous ERK activity, we first assayed for ERK phosphorylation with an antibody against dually phosphorylated ERK. Both SB-202190 and SB-203580 modestly increased ERK phosphorylation (Fig. 5A). Next, we transiently transfected cells with a HA-tagged ERK2 (20) and assessed ERK activity by immunoprecipitation of the epitope tag followed by in vitro phosphorylation assay with MBP as a substrate. Both SB-202190 and SB-203580 were sufficient to induce modest activation of ERK2 (Fig. 5, B and C). ERK2 activation by SB-202190 was reduced by pretreatment with PD-98059, a specific inhibitor of MEK (Fig. 5D). Taken together, these data suggest that chemical p38 inhibitors activate ERK in bovine tracheal smooth muscle cells and that they do so by stimulating an upstream activator of MEK.


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Fig. 4.   Effects of SB-202190 and SB-203580 on anisomycin-induced p38alpha phosphorylation and activity. A: cells were transiently transfected with a cDNA encoding a hemagglutinin (HA)-tagged p38alpha . After serum starvation, cells were pretreated with 3 µM SB-202190 or SB-203580 for 1 h before activation with anisomycin (50 µg/ml for 30 min). Cell lysates were immunoprecipitated with an antibody against HA. Top: p38alpha activity was assessed by in vitro phosphorylation assay with activating transcription factor (ATF)-2 as a substrate. Bottom: level of expression of epitope-tagged p38alpha as determined by immunoblotting. B: in vitro phosphorylation assays. Data are means ± SE; n = 4 experiments. *Significantly less than anisomycin, P < 0.05 by ANOVA.



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Fig. 5.   Activation of extracellular signal-regulated kinase (ERK) by chemical p38 inhibitors. A: cells were pretreated with 3 µM SB-202190 or SB-203580 for 1 h. Selected cultures were stimulated with PDGF (30 ng/ml for 10 min). Phosphorylation of ERK was assessed by immunoblotting with a phospho-specific antibody against ERK (phosphoERK; 1:4,000 dilution). This experiment was repeated twice. B: cells were transiently transfected with a cDNA encoding a HA-tagged ERK2 (top). After serum starvation, selected cultures were treated for 1 h with 3 µM SB-202190 or SB-203580. Selected cultures were treated with PDGF (30 ng/ml for 10 min). Cell lysates were immunoprecipitated with an antibody against HA. ERK activity was assessed by in vitro phosphorylation with major basic protein (MBP) as a substrate. The level of HA-ERK2 expression was determined by immunoblotting (bottom). C: in vitro phosphorylation assays. Data are means ± SE; n = 4 experiments. *Significantly greater than control, P < 0.05 by ANOVA. D: after transfection (as described above), selected cultures were pretreated with PD-98059 (30 µM for 1 h) and stimulated with SB-202190 (3 µM for 1 h). A positive control is also shown (PDGF, 30 ng/ml for 10 min). ERK activity was assessed by in vitro phosphorylation with MBP as a substrate (top). The level of expression of epitope-tagged ERK2 was determined by immunoblotting (bottom). This experiment was repeated twice, with similar results.

MKK3 and MKK6 regulate p38 but not ERK in primary bovine tracheal myocytes. To examine the effects of mutant forms of MKK3 and MKK6 on p38 activity, bovine tracheal myocytes were transiently cotransfected with cDNAs encoding a HA-tagged p38alpha (64) and mutant alleles of either MKK3 [MKK3-AL and MKK3(glu)] or MKK6 [MKK6(glu)]. p38alpha activity was determined by immunoprecipitation with an anti-HA antibody followed by an in vitro phosphorylation assay with ATF-2 as a substrate. Overexpression of MKK3-AL reduced p38alpha activation (Fig. 6, A and B), whereas overexpression of MKK3(glu) and MKK6(glu) each induced significant activation of p38alpha (Fig. 6, C and D).


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Fig. 6.   Effect of mutant alleles of MKK3 and MKK6 on p38alpha activation. A: cells were transiently cotransfected with MKK3-AL and a HA-tagged p38alpha . p38alpha activity was assessed by in vitro phosphorylation with ATF-2 as a substrate (top). Selected cultures were treated with anisomycin (50 µg/ml for 30 min) before harvest. The level of expression of epitope-tagged p38alpha was determined by immunoblotting (bottom). B: in vitro phosphorylation assays. Data are means ± SE; n = 4 experiments. *Significantly less than anisomycin, P < 0.05 by ANOVA. C: cells were transiently cotransfected with either MKK3(glu), MKK6(glu), or the vector controls pcDNA3 or P6R and a HA-tagged p38alpha . p38alpha activity was assessed by in vitro phosphorylation with ATF-2 as a substrate (top). Level of expression of epitope-tagged p38 was determined by immunoblotting (bottom). D: in vitro phosphorylation assays. Data are means ± SE for; n = 4 experiments. *Significantly greater than control, P < 0.05 by ANOVA.

To test whether MKK3 or MKK6 regulates ERK activation, we cotransfected cells with mutant alleles of MKK3 or MKK6 and a HA-tagged ERK2. Unlike the chemical p38 inhibitors, inhibition of p38 by overexpression of MKK3-AL did not increase basal or PDGF-stimulated ERK activity (Fig. 7, A and B). Similarly, neither MKK3(glu) nor MKK6(glu) significantly decreased basal or PDGF-stimulated ERK activity (Fig. 7, C and D). Taken together, these data suggest that p38 negatively regulates cyclin D1 promoter activity in an ERK-independent manner.


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Fig. 7.   Effect of mutant alleles of MKK3 and MKK6 on ERK activation. A: cells were transiently cotransfected with MKK3-AL and a HA-tagged ERK2. ERK2 was assessed by in vitro phosphorylation with MBP as a substrate (top). Selected cultures were treated with PDGF (30 ng/ml for 10 min) before harvest. Level of expression of epitope-tagged ERK2 was determined by immunoblotting (bottom). B: in vitro phosphorylation assays. Data are means ± SE for; n = 4 experiments. C: cells were transiently cotransfected with MKK3(glu) or MKK6(glu) and a HA-tagged ERK2. ERK2 activity was assessed by in vitro phosphorylation with MBP as a substrate (top). Selected cultures were treated with PDGF (30 ng/ml for 10 min) before harvest. Level of expression of epitope-tagged ERK2 was determined by immunoblotting (bottom). D: in vitro phosphorylation assays. Data are means ± SE for; n = 4 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ERK activation has been shown to be required for DNA synthesis in a wide variety of cell systems including airway smooth muscle (28, 35, 40, 59, 63). On the other hand, activation of p38 MAP kinase has been associated with growth inhibition (33) and apoptosis (4, 5, 7, 15, 26, 30, 32, 58, 61, 65). We have demonstrated that PDGF induces activation of both ERK and p38alpha in bovine tracheal myocytes (41) and that ERK activation is required for PDGF-induced cyclin D1 expression and DNA synthesis in these cells (28, 41, 47). If p38 negatively regulated cyclin D1 expression, then PDGF-induced activation of p38 might limit ERK-mediated cell growth. Because excess airway smooth muscle proliferation has been noted in patients with fatal asthma, the concurrent regulation of growth-stimulatory and growth-inhibitory pathways by growth factors might constitute an important physiological mechanism limiting airflow obstruction in these patients.

In this report, we show that inhibition of p38, either by chemical inhibitors or by overexpression of a dominant negative allele of MKK3, increases cyclin D1 promoter transcription and protein abundance in cultured primary airway smooth muscle cells. These data imply a basal level of p38 activation in serum-starved cells (Figs. 4 and 6). Furthermore, selective activation of p38 by overexpression of either MKK3 or MKK6 attenuated both basal and PDGF-induced transcription from the cyclin D1 promoter. These data suggest that p38 negatively regulates cyclin D1 expression in airway smooth muscle cells. Our data confirm previous experiments in CCL-39 fibroblasts in which overexpression of MKK3 decreased basal and serum-induced cyclin D1 expression, whereas treatment with a chemical p38 inhibitor or a dominant negative form of MKK3 increased transcription from the cyclin D1 promoter (33). Furthermore, our data are consistent with recent studies suggesting that p38 negatively regulates cyclin G1 expression. In PC12 cells, hypoxia increased p38 activation while decreasing cyclin D1 levels (8). In mouse embryonic kidney cells, chemical inhibition of p38 allowed Rac1 to induce anchorage-dependent cyclin A transcription (44).

In PC12 (65) and HeLa cells (4), p38 activation occurred concomitantly with inhibition of the ERK pathway, suggesting that p38 negatively regulates ERK. Also, treatment with the pyridinyl imidazole compounds SB-203580 and SB-202190 has been noted to increase ERK activation in mouse keratinocytes (48), canine tracheal myocytes (25), and HepG2 cells (51), suggesting that inhibition of p38 activates the ERK pathway (51). We therefore examined the regulation of ERK activity by p38 in bovine tracheal myocytes. We found that treatment of bovine myocytes with the chemical p38 inhibitors SB-202190 and SB-203580 increased ERK activation. However, overexpression of a dominant negative form of MKK3, which inhibited anisomycin-induced p38alpha activation, did not increase basal or PDGF-stimulated ERK activity. Furthermore, when we overexpressed active forms of MKK3 and MKK6, both of which increase p38alpha activation, there was no reduction of either basal or PDGF-induced activation of ERK. Taken together, these data suggest that the selective inhibition or activation of p38alpha by upstream activators does not regulate ERK activation. Furthermore, they suggest that the pyridinyl imidazole compounds SB-202190 and SB-203580 may activate ERK via a MEK-dependent, p38alpha -independent pathway.

It has recently been demonstrated that SB-203580 inhibits Raf-1 in vitro (17). However, in vivo, inhibition of Raf-1 by SB-203580 is counterbalanced by a novel feedback loop, leading to Raf-1 activation (16, 27). Our study, in which ERK activation was induced by both SB-202190 and SB-203580 but not a dominant negative inhibitor of MKK3, supports the notion that pyridinyl imidazole inhibitors may have p38-independent effects. Interestingly, neither chemical inhibitor increased ERK activation in COS cells (Page and Hershenson, unpublished results), indicating that the level of compensatory Raf-1 activation may vary with cell type. Our laboratory has previously shown (29) that in bovine tracheal myocytes, an alternative MEK activator besides Raf-1 is responsible for MEK phosphorylation. Thus the finding that pyridinyl imidazole compounds activate ERK in our system suggests that these reagents may inhibit other MAP kinase kinase kinases besides Raf.

Other possible explanations for the differential effects of chemical p38 inhibitors and the dominant negative MKK3 need to be considered. The pyridinyl imidazole compounds selectively inhibit p38alpha and -beta , and therefore it is conceivable that the activation of ERK by these compounds relates to inhibition of p38beta (31). Because p38beta is activated by MKK6 but not by MKK3, experiments that employed a dominant negative mutant of MKK6 would address this possibility. However, the two dominant negative MKK6 mutants we tested, although they attenuated anisomycin-induced activation of p38, also significantly increased basal p38 activity. Nevertheless, our finding that a constitutively active MKK6 mutant that activates all isoforms of p38 (14, 23, 24, 60) had no effect on ERK activation suggests that p38beta does not regulate ERK in our system.

It is important to note potential limitations of our approach, which employed the overexpression of constitutively active and dominant negative signaling intermediates. First, it is possible that overexpression of a constitutively active protein could induce a supraphysiological outcome. For example, it is conceivable that although selective activation of p38 by MKK3/6 inhibits cyclin D1 expression in our system, p38 activation alone may not be sufficient for this effect under normal conditions and may require the activation of other negative regulatory pathways. Second, dominant negative proteins may bind downstream signaling intermediates, thereby blocking the activity of other upstream activators that bind at the same site. However, due to the difficulty of selectively activating p38, we saw no alternative but to use these reagents. For example, chemical activators of p38 such as anisomycin, hyperosmolarity, and UV irradiation tend to cause multiple stress-related effects that would likely influence our results. Nevertheless, chemical inhibitors of p38 increased cyclin D1 promoter activation and protein abundance in our system, confirming the enhancing effects of the dominant negative MKK3 on transcription from the cyclin D1 promoter. Finally, we did not examine the effects of p38 on the transcription or protein abundance of other G1 cyclins, cyclin-dependent kinases, or cyclin-dependent kinase inhibitors. Thus while the negative regulation of cyclin D1 by p38 would tend to attenuate cell cycle traversal, the net effect of p38 activation on airway cell DNA synthesis and proliferation remains to be determined.

In conclusion, our data strongly suggest that in bovine tracheal myocytes, p38alpha negatively regulates transcription from the cyclin D1 promoter via an ERK-independent pathway. Furthermore, chemical inhibitors of p38 may activate ERK in a p38alpha -independent manner.


    ACKNOWLEDGEMENTS

We thank Drs. Richard Pestell, Michael Karin, Roger Davis, and James Woodgett for kindly providing the cDNAs encoding -1745CD1LUC, p38alpha , MKK3, and MKK6, respectively. We also thank Dr. Marsha Rosner for thoughtful critique of the manuscript.


    FOOTNOTES

These studies were supported by National Heart, Lung, and Blood Institute Grants HL-54685, HL-56399, and HL-63314.

Address for reprint requests and other correspondence: M. B. Hershenson, Univ. of Chicago Children's Hospital, 5841 S. Maryland Ave., MC 4064, Chicago, IL 60637-1470 (E-mail: mhershen{at}midway.uchicago.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 June 2000; accepted in final form 17 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abe, MK, Chao TS, Solway J, Rosner MR, and Hershenson MB. Hydrogen peroxide stimulates mitogen-activated protein kinase in bovine tracheal myocytes: implications for human airway disease. Am J Respir Cell Mol Biol 11: 577-585, 1994[Abstract].

2.   Albanese, C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, and Pestell RG. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem 270: 23589-23597, 1995[Abstract/Free Full Text].

3.   Alexandrov, A, Keffel S, Goepel M, and Michel MC. Stimulation of alpha 1A-adrenoreceptors in rat-1 cells inhibits extracellular signal-regulated kinase by activated p38 mitogen-activated protein kinase. Mol Pharmacol 54: 755-760, 1998[Abstract/Free Full Text].

4.   Berra, E, Diaz-Meco MT, and Moscat J. The activation of p38 and apoptosis by the inhibition of ERK is antagonized by the phosphoinositide 3-kinase/Akt pathway. J Biol Chem 273: 10792-10797, 1998[Abstract/Free Full Text].

5.   Brenner, B, Koppenhoefer U, Weinstock C, Linderkamp O, Lang F, and Gulbins E. Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulated activation of Jun N-terminal kinase/p38 kinases and GADD153. J Biol Chem 272: 22173-22181, 1997[Abstract/Free Full Text].

6.   Carroll, N, Elliot J, Morton A, and James A. The structure of large and small airways in nonfatal and fatal asthma. Am Rev Respir Dis 147: 405-410, 1993[ISI][Medline].

7.   Chen, YR, Wang X, Templeton D, Davis RJ, and Tan TH. The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation. J Biol Chem 271: 31929-31936, 1996[Abstract/Free Full Text].

8.   Conrad, PW, Rust RT, Han J, Millhorn DE, and Beitner-Johnson D. Selective activation of p38 alpha and p38 gamma by hypoxia. Role in regulation of cyclin D1 by hypoxia in PC12 cells. J Biol Chem 274: 23570-23576, 1999[Abstract/Free Full Text].

9.   Derijard, B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, and Davis RJ. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267: 682-685, 1995[ISI][Medline].

10.   Dunnill, MS, Massarella GR, and Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis and in emphysema. Thorax 24: 176-179, 1969[ISI][Medline].

11.   Ebina, M, Takahashi T, Chiba T, and Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 148: 720-726, 1993[ISI][Medline].

12.   Ebina, M, Yaegashi H, Chiba R, Takahashi T, Motomiya M, and Tanemura M. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. A morphometric study. Am Rev Respir Dis 141: 1327-1332, 1990[ISI][Medline].

13.   Ellinger-Ziegelbauer, H, Kelly K, and Siebenlist U. Cell cycle arrest and reversion of Ras-induced transformation by a conditionally activated form of mitogen-activated protein kinase kinase kinase 3. Mol Cell Biol 19: 3857-3868, 1999[Abstract/Free Full Text].

14.   Enslen, H, Raingeaud J, and Davis RJ. Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J Biol Chem 273: 1741-1748, 1998[Abstract/Free Full Text].

15.   Frasch, SC, Nick JA, Fadok VA, Bratton DL, Worthen GS, and Henson PM. p38 mitogen-activated protein kinase-dependent and -independent intracellular signal transduction pathways leading to apoptosis in human neutrophils. J Biol Chem 273: 8389-8397, 1998[Abstract/Free Full Text].

16.   Hall-Jackson, CA, Eyers PA, Cohen P, Goedert M, Boyle FT, Hewitt N, Plant H, and Hedge P. Paradoxical activation of Raf by a novel Raf inhibitor. Chem Biol 6: 559-568, 1999[ISI][Medline].

17.   Hall-Jackson, CA, Goedert M, Hedge P, and Cohen P. Effect of SB-203580 on the activity of c-Raf in vitro and in vivo. Oncogene 18: 2047-2054, 1999[ISI][Medline].

18.   Han, J, Lee JD, Tobias PS, and Ulevitch RJ. Endotoxin induces rapid protein tyrosine phosphorylation in 70Z/3 cells expressing CD14. J Biol Chem 268: 25009-25014, 1993[Abstract/Free Full Text].

19.   Heard, BE, and Hossain S. Hyperplasia of bronchial smooth muscle in asthma. J Pathol 10: 319-332, 1973.

20.   Hershenson, MB, Chao TS, Abe MK, Gomes I, Kelleher MD, Solway J, and Rosner MR. Histamine antagonizes serotonin and growth factor-induced mitogen-activated protein kinase activation in bovine tracheal smooth muscle cells. J Biol Chem 270: 19908-19913, 1995[Abstract/Free Full Text].

21.   Holland, PM, Suzanne M, Campbell JS, Noselli S, and Cooper JA. MKK7 is a stress-activated mitogen-activated protein kinase kinase functionally related to hemipterous. J Biol Chem 272: 24994-24998, 1997[Abstract/Free Full Text].

22.   James, AL, Pare PD, and Hogg JC. The mechanics of airway narrowing in asthma. Am Rev Respir Dis 139: 242-246, 1989[ISI][Medline].

23.   Jiang, Y, Chen C, Li Z, Guo W, Gegner JA, Lin S, and Han J. Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta ). J Biol Chem 271: 17920-17926, 1996[Abstract/Free Full Text].

24.   Jiang, Y, Gram H, Zhao M, New L, Gu J, Feng L, Di Padova F, Ulevitch RJ, and Han J. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta. J Biol Chem 272: 30122-30128, 1997[Abstract/Free Full Text].

25.   Jones, CA, and Brown JK. p38 inhibition increases ERK1/ERK2 kinase activity and DNA synthesis in airway smooth muscle cells: evidence for cross-talk between mitogen-activated protein (MAP) kinase pathways (Abstract). Am J Respir Crit Care Med 159: A722, 1999[ISI].

26.   Juo, P, Kuo CJ, Reynolds SE, Konz RF, Raingeaud J, Davis RJ, Biemann HP, and Blenis J. Fas activation of the p38 mitogen-activated protein kinase signaling pathway requires ICE/CED-3 family proteases. Mol Cell Biol 17: 24-35, 1997[Abstract].

27.   Kalmes, A, Deou J, Clowes AW, and Daum G. Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB-203580. FEBS Lett 444: 71-74, 1999[ISI][Medline].

28.   Karpova, AY, Abe MK, Li J, Liu P, Rhee JM, Kuo WL, and Hershenson MB. MEK1 is required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes. Am J Physiol Lung Cell Mol Physiol 272: L558-L565, 1997[Abstract/Free Full Text].

29.   Kartha, S, Naureckas ET, Li J, and Hershenson MB. Partial characterization of a novel mitogen-activated protein kinase/extracellular signal-regulated kinase activator in airway smooth-muscle cells. Am J Respir Cell Mol Biol 20: 1041-1048, 1999[Abstract/Free Full Text].

30.   Kawasaki, H, Morooka T, Shimohama S, Kimura J, Hirano T, Gotoh Y, and Nishida E. Activation and involvement of p38 mitogen-activated protein kinase in glutamate-induced apoptosis in rat cerebellar granule cells. J Biol Chem 272: 18518-18521, 1997[Abstract/Free Full Text].

31.   Kumar, S, McDonnell PC, Gum RJ, Hand AT, Lee JC, and Young PR. Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem Biophys Res Commun 235: 533-538, 1997[ISI][Medline].

32.   Kummer, JL, Rao PK, and Heidenreich KA. Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase. J Biol Chem 272: 20490-20494, 1997[Abstract/Free Full Text].

33.   Lavoie, J, L'Allemain G, Brunet A, Muller R, and Pouyssegur J. Cyclin D1 expression is regulated positively by p42/p44MAPK and negatively by p38/HOGMAPK pathway. J Biol Chem 271: 20608-20616, 1996[Abstract/Free Full Text].

34.   Lee, RJ, Albanese C, Stenger RJ, Watanabe G, Inghirami G, Haines GK, Webster M, Muller WJ, Brugge JS, Davis RJ, and Pestell RG. pp60(v-src) induction of cyclin D1 requires collaborative interactions between the extracellular signal-regulated kinase, p38 and Jun kinase pathways. A role for cAMP response element-binding protein and activating transcription factor-2 in pp60(v-src) signaling in breast cancer cells. J Biol Chem 274: 7341-7350, 1999[Abstract/Free Full Text].

35.   Lew, DB, Dempsey BK, Zhao Y, Muthalif M, Fatima S, and Malik KU. Beta-hexosaminidase-induced activation of p44/42 mitogen-activated protein kinase is dependent on p21Ras and protein kinase C and mediates bovine airway smooth-muscle proliferation. Am J Respir Cell Mol Biol 21: 111-118, 1999[Abstract/Free Full Text].

36.   Lin, A, Minden A, Martinetto H, Claret FX, Lange-Carter C, Mercurio F, Johnson GL, and Karin M. Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2. Science 268: 286-290, 1995[ISI][Medline].

37.   Lu, X, Nemoto S, and Lin A. Identification of c-Jun NH2-terminal protein kinase (JNK)-activating kinase 2 as an activator of JNK but not p38. J Biol Chem 272: 24751-42475, 1997[Abstract/Free Full Text].

38.   Minden, A, Lin A, McMahon M, Lange-Carter C, Derijard B, Davis RJ, Johnson GL, and Karin M. Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science 266: 1719-1723, 1994[ISI][Medline].

39.   Molnar, A, Theodoras AM, Zon LI, and Kyriakis JM. Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK. J Biol Chem 272: 13229-13235, 1997[Abstract/Free Full Text].

40.   Orsini, MJ, Krymskaya VP, Eszterhas AJ, Benovic JL, Panettieri RA, and Penn RB. MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation. Am J Physiol Lung Cell Mol Physiol 277: L479-L488, 1999[Abstract/Free Full Text].

41.   Page, K, Li J, and Hershenson MB. Platelet-derived growth factor stimulation of mitogen-activated protein kinases and cyclin D1 promoter activity in cultured airway smooth muscle cells: role of Ras. Am J Respir Cell Mol Biol 20: 1294-1302, 1999[Abstract/Free Full Text].

42.   Page, K, Li J, Hodge JA, Liu PT, Vanden Hoek TL, Becker LB, Pestell RG, Rosner MR, and Hershenson MB. Characterization of a Rac1 signaling pathway to cyclin D1 expression in airway smooth muscle cells. J Biol Chem 274: 22065-22071, 1999[Abstract/Free Full Text].

43.   Parmacek, MS, Vora AJ, Shen T, Barr E, Jung F, and Leiden JM. Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow cardiac tropinin C gene. Mol Cell Biol 12: 1967-1976, 1992[Abstract].

44.   Philips, A, Roux P, Coulon V, Bellanger JM, Vie A, Vignais ML, and Blanchard JM. Differential effect of rac and cdc42 on p38 kinase activity and cell cycle progression of nonadherent primary mouse fibroblasts. J Biol Chem 275: 5911-5917, 2000[Abstract/Free Full Text].

45.   Pyne, NJ, and Pyne S. Platelet-derived growth factor activates a mammalian Ste20 coupled mitogen-activated protein kinase in airway smooth muscle. Cell Signal 9: 311-317, 1997[ISI][Medline].

46.   Raingeaud, J, Whitmarsh AJ, Barrett T, Derijard B, and Davis RJ. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol 16: 1247-1255, 1996[Abstract].

47.   Ramakrishnan, M, Musa NL, Li J, Liu P, Pestell RG, and Hershenson MB. Catalytic activation of extracellular signal-regulated kinases induces cyclin D1 expression in airway smooth muscle. Am J Respir Cell Mol Biol 18: 736-740, 1998[Abstract/Free Full Text].

48.   Rosenberger, SF, Gupta A, and Bowden GT. Inhibition of p38 MAP kinase increases okadaic acid mediated AP-1 expression and DNA binding but has no effect on TRE dependent transcription. Oncogene 18: 3626-3632, 1999[ISI][Medline].

49.   Saetta, M, Di Stefano A, Rosina C, Thiene G, and Fabbri LM. Quantitative structural analysis of peripheral airways and arteries in sudden fatal asthma. Am Rev Respir Dis 143: 138-143, 1991[ISI][Medline].

50.   Shapiro, PS, Evans JN, Davis RJ, and Posada JA. The seven-transmembrane-spanning receptors for endothelin and thrombin cause proliferation of airway smooth muscle cells and activation of the extracellular regulated kinase and c-Jun NH2-terminal kinase groups of mitogen-activated protein kinases. J Biol Chem 271: 5750-5754, 1996[Abstract/Free Full Text].

51.   Singh, RP, Dhawan P, Golden C, Kapoor GS, and Mehta KD. One way cross-talk between p38MAPK and p42/44MAPK: inhibition of p38MAPK induces low density lipoprotein receptor expression through activation of the p42/44MAPK cascade. J Biol Chem 274: 19593-19600, 1999[Abstract/Free Full Text].

52.   Sobonya, RE. Quantitative structural alterations in long-standing allergic asthma. Am Rev Respir Dis 130: 289-292, 1984[ISI][Medline].

53.   Stein, B, Yang MX, Young DB, Janknecht R, Hunter T, Murray BW, and Barbosa MS. p38-2, a novel mitogen-activated protein kinase with distinct properties. J Biol Chem 272: 19509-19517, 1997[Abstract/Free Full Text].

54.   Su, GH, Hilgers W, Shekher MC, Tang DJ, Yeo CJ, Hruban RH, and Kern SE. Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene. Cancer Res 58: 2339-2342, 1998[Abstract].

55.   Takizawa, T, and Thurlbeck WM. Muscle and mucous gland size in the major bronchi of patients with chronic bronchitis, asthma and asthmatic bronchitis. Am Rev Respir Dis 104: 331-336, 1971[ISI][Medline].

56.   Teng, DH, Perry WL, Hogan JK, Baumgard M, Bell R, Berry S, Davis T, Frank D, Frye C, Hattier T, Hu R, Jammulapati S, Janecki T, Leavit A, Mitchell JT, Pero R, Sexton D, Schroeder M, Su PH, Swedlund B, Kyriakis JM, Avruch J, Bartel P, Wong AK, and Tavtigian SV. Human mitogen-activated protein kinase kinase 4 as a candidate tumor suppressor. Cancer Res 57: 4177-4182, 1997[Abstract].

57.   Tournier, C, Whitmarsh AJ, Cavanagh J, Barrett T, and Davis RJ. Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc Natl Acad Sci USA 94: 7337-7342, 1997[Abstract/Free Full Text].

58.   Toyoshima, F, Moriguchi T, and Nishida E. Fas induces cytoplasmic apoptotic responses and activation of the MKK7-JNK/SAPK and MKK6-p38 pathways independent of CPP32-like proteases. J Cell Biol 139: 1005-1015, 1997[Abstract/Free Full Text].

59.   Walker, TR, Moore SM, Lawson MF, Panettieri RAJ, and Chilvers ER. Platelet-derived growth factor-BB and thrombin activate phosphoinositide 3-kinase and protein kinase B: role in mediating airway smooth muscle proliferation. Mol Pharmacol 54: 1007-1015, 1998[Abstract/Free Full Text].

60.   Wang, XS, Diener K, Manthey CL, Wang S, Rosenzweig B, Bray J, Delaney J, Cole CN, Chan-Hui PY, Mantlo N, Lichenstein HS, Zukowski M, and Yao Z. Molecular cloning and characterization of a novel p38 mitogen-activated protein kinase. J Biol Chem 272: 23668-23674, 1997[Abstract/Free Full Text].

61.   Wang, Y, Huang S, Sah VP, Ross J, Jr, Brown JH, Han J, and Chien KR. Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem 273: 2161-2168, 1998[Abstract/Free Full Text].

62.   Watanabe, G, Howe A, Lee R, Albanese C, Shu I, Karnezis A, Zon L, Kyriakis J, Rundell K, and Pestell R. Induction of cyclin D1 by simian virus 40 small T tumor antigen. Proc Natl Acad Sci USA 93: 12861-12866, 1996[Abstract/Free Full Text].

63.   Whelchel, A, Evans J, and Posada J. Inhibition of ERK activation attenuates endothelin-stimulated airway smooth muscle proliferation. Am J Respir Cell Mol Biol 16: 589-596, 1997[Abstract].

64.   Xia, Y, Wu Z, Su B, Murray B, and Karin M. JNKK1 organizes a MAP kinase module through specific and sequential interactions with upstream and downstream components mediated by its amino-terminal extension. Genes Dev 12: 3369-3381, 1998[Abstract/Free Full Text].

65.   Xia, Z, Dickens M, Raingeaud J, Davis RJ, and Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326-1331, 1995[Abstract].

66.   Xiong, W, Pestell RG, Watanabe G, Li J, Rosner MR, and Hershenson MB. Cyclin D1 is required for S phase traversal in bovine tracheal myocytes. Am J Physiol Lung Cell Mol Physiol 272: L1205-L1210, 1997[Abstract/Free Full Text].

67.   Zanke, BW, Rubie EA, Winnett E, Chan J, Randall S, Parsons M, Boudreau K, McInnis M, Yan M, Templeton DJ, and Woodgett JR. Mammalian mitogen-activated protein kinase pathways are regulated through formation of specific kinase-activator complexes. J Biol Chem 271: 29876-29881, 1996[Abstract/Free Full Text].


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