Department of Pediatrics, University of Chicago, Chicago, Illinois 60637-1470
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We previously demonstrated that Rac1 increased cyclin D1 promoter activity in an extracellular signal-regulated kinase (ERK)-independent, antioxidant-sensitive manner. Here, we examined the regulation of cyclin D1 expression by Cdc42 and RhoA. Overexpression of active Cdc42, but not of RhoA, induced transcription from the cyclin D1 promoter. Furthermore, dominant negative Cdc42, but not RhoA, attenuated platelet-derived growth factor-mediated activation of the cyclin D1 promoter. Overexpression of active Cdc42 increased cyclin D1 protein abundance in COS cells. Cdc42-induced cyclin D1 promoter activation was independent of ERK as evidenced by insensitivity to PD-98059, an inhibitor of mitogen-activated protein kinase/ERK kinase (MEK). Furthermore, Cdc42 was neither sufficient nor required for activation of ERK. Similar to Rac1-induced cyclin D1 expression, pretreatment with the antioxidants catalase and ebselen inhibited Cdc42-mediated transcription from the cyclin D1 promoter. Finally, like Rac1, active Cdc42 induced transactivation of the cyclin D1 promoter cAMP response element binding protein/activating transcription factor-2 binding site. Together, these data suggest that in airway smooth muscle cells, Cdc42 and Rac1 share a common signaling pathway to cyclin D1 promoter activation.
activating transcription factor-2; antioxidant; adenosine 5'-cyclic monophosphate response element binding protein; extracellular signal-regulated kinase; guanosine 5'-triphosphatase; platelet-derived growth factor; Rac1
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INCREASED AIRWAY SMOOTH MUSCLE proliferation is thought to contribute to airflow obstruction in patients with asthma (8). The signaling mechanisms underlying airway smooth muscle proliferation are not completely understood. Our laboratory and others have shown that extracellular signal-regulated protein kinases (ERKs) increase cyclin D1 expression (30) and DNA synthesis (16, 24) in bovine tracheal myocytes. However, in NIH/3T3 cells, activation of the ERK pathway does not induce all the key events required for transition from the G1 to the S phase of the cell cycle (23), suggesting that an additional signaling pathway is required for cell proliferation. Our laboratory (26) recently demonstrated that Rac1 activates transcription from the cyclin D1 promoter in an ERK-independent, antioxidant-sensitive manner. We now investigated the roles of two additional Rho GTPases, Cdc42 and RhoA, in the regulation of cyclin D1 expression.
The Rho family of GTPases (RhoA, -B, and -C, Rac1 and -2, and Cdc42) control the assembly and organization of the actin cytoskeleton (13). Rho activation leads to stress fiber formation, and Rac induces the assembly of a meshwork of actin filaments at the cell periphery to produce lamellipodia and membrane ruffles, whereas Cdc42 induces surface protrusions called filopodia. Rac1 and Cdc42 have also been reported to activate the stress-activated mitogen-activated protein (MAP) kinases, c-Jun amino-terminal kinase (JNK), and p38 (5-7, 9, 10, 20, 33, 38). In addition, there may be significant cross talk between the members of the Rho GTPase family. For example, Cdc42 can activate Rac1, whereas Rac1 can activate RhoA (13, 22, 31).
Rho GTPases have also been demonstrated to play an essential role in cell cycle progression through G1 in NIH/3T3 cells (4, 18, 23). Rac1 has been shown to activate transcription from the cyclin D1 promoter in NIH/3T3 cells (36) and, as noted above, primary bovine tracheal smooth muscle cells (26). Rac1 constitutes part of the NADPH oxidase complex that generates reactive oxygen species (1, 2). In airway smooth muscle, NADPH oxidase activity is required for platelet-derived growth factor (PDGF)- and Rac1-mediated transcription from the cyclin D1 promoter, and growth factor-induced promoter activity is antioxidant sensitive, suggesting that Rac1 activates transcription from the cyclin D1 promoter via the generation of reactive oxygen species (26).
It has also been demonstrated that active Cdc42 (12) and RhoA (36, 39) are each sufficient to induce cyclin D1 protein accumulation in NIH/3T3 cells. However, the precise mechanism that underlies this regulation has not been studied. In this report, we investigated the role of Cdc42 and RhoA in the regulation of cyclin D1 expression in airway smooth muscle cells. We found that Cdc42, but not RhoA, activates transcription from the cyclin D1 promoter in an ERK-independent, antioxidant-sensitive manner similar to that of Rac1. Furthermore, both Rac1 and Cdc42 induce transactivation of the cyclin D1 promoter cAMP response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site. These data suggest that in airway smooth muscle cells, Cdc42 and Rac1 share a common signaling pathway to cyclin D1 promoter activation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Peroxidase-linked goat anti-rabbit IgG, protein A Sepharose beads,
myelin basic protein (MBP), catalase, ebselen, and
o-nitrophenyl--D-galactoside were
purchased from Sigma (St. Louis, MO). PDGF and an anti-Myc tag antibody
(clone 9E10) were obtained from Upstate Biotechnology (Lake Placid,
NY). PD-98059 was obtained from New England Biolabs (Beverly, MA).
[
-32P]ATP and an enhanced chemiluminescence kit were
purchased from DuPont/NEN Research Products (Wilmington, DE). A
polyclonal antibody against cyclin D1 was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). A peroxidase-linked rat anti-mouse
light chain IgG was obtained from Zymed Laboratories (South San
Francisco, CA). For in vitro phosphorylation assays, a monoclonal
antibody against hemagglutinin (HA; 12CA5) was obtained from Babco
(Berkeley, CA). The MACSelect Kk-transfected cell selection
kit was purchased from Miltenyi Biotec (Auburn, CA).
Cell culture.
Bovine tracheal smooth muscle cells were isolated as described
previously (17). Myocytes of passage 5 or less
were studied. Confluent cultures exhibited the typical "hill and
valley" appearance and showed specific immunostaining for -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. In some studies, COS cells (American Type
Culture Collection, Manassas, VA) were used and cultured as described above.
Determination of cyclin D1 promoter transcriptional activity. Cells were seeded in 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; Life Technologies, Gaithersburg, MD). The transfection efficiency in primary cultures of bovine tracheal myocytes was on the order of 10-20%. Cotransfection with viral promoter-driven expression vectors tends to suppress cyclin D1 promoter activity. Therefore, concentration-response curves were generated for each expression vector to determine optimal concentration. cDNA concentrations of 30-50 ng/plate were used. Cells were transiently cotransfected with plasmids that encode the human cyclin D1 promoter subcloned into a luciferase reporter and either the relevant expression vector or the control vector. After 5 h, the liposome solution was replaced with 10% FBS-DMEM. The next day, cells were serum starved in DMEM. After 8 h of serum starvation, selected cultures were pretreated with chemical inhibitors, if necessary, for 1 h before stimulation with PDGF (30 ng/ml). Finally, 16 h after PDGF treatment, cells were harvested for analysis of luciferase activity with the lysis buffer provided with the Promega (Madison, WI) 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 the 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 that encodedSerum response factor reporter assay. Cells were transiently transfected with the serum response factor (SRF)-luciferase reporter plasmid from Stratagene and either the constitutively active or dominant negative RhoA mutants or the vector control as described in Determination of cyclin D1 promotor transcriptional activity. Selected cells were treated with 10% serum for 16 h, extracted, and assayed for luciferase activity.
Magnetic cell separation. To verify the expression of relevant plasmids in bovine tracheal myocytes, we used a magnetic cell selection system to increase the proportion of transfected cells in whole cell extracts, thereby facilitating immunostaining for the Myc epitope tag (11). Cells were transiently cotransfected as described in Determination of cyclin D1 promoter transcriptional activity with pMACS Kk (2.5 µg/plate) and either constitutively active forms of Cdc42, RhoA or Rac1, dominant negative forms of Cdc42 or RhoA, or their respective control vectors (7.5 µg/plate). After 48 h, cells were rinsed with PBS, trypsinized, pooled, and centrifuged (1,000 rpm for 3 min at room temperature). The resulting pellet was resuspended in 320 µl of PBS supplemented with 0.5% BSA and 5 mM EDTA (PBE buffer) and incubated with 80 µl of magnetic beads for 15 min at room temperature. After incubation, the volume of the respective samples was adjusted to 2 ml with PBE buffer and loaded onto the magnetized separation column in 500-µl aliquots. After collection of the flow-through (nonselected cells), the column was washed four times with 2 ml of PBE. After being washed, the column was detached from the magnet, 1 ml of PBE was added, and the selected cells were flushed out with a plunger. The collected cells were centrifuged (1,000 rpm for 3 min at room temperature), the supernatant was discarded, and the pellet was resuspended in lysis buffer.
Preparation of cell extracts for immunoblotting.
Cells were cultured in six-well plates and serum starved for 24 h
before PDGF treatment (30 ng/ml for 16 h). The 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 -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.
Immunoblotting. Cell extracts (10 µg for cyclin D1, 50 µg for Myc) were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose by semidry transfer (Hoefer, San Francisco, CA). After incubation with the relevant antibody, signals were amplified and visualized with anti-rabbit or anti-mouse IgG and enhanced chemiluminescence.
Measurement of ERK and JNK activation.
Cells were transiently cotransfected with cDNAs that encoded HA-tagged
ERK2 or HA-JNK1 and the expression vector of interest. Cells were
seeded in 100-mm plates at a density of 5 × 105
cells/plate and incubated in 10% FBS-DMEM overnight. After being rinsed, the cells were incubated in a solution consisting of serum- and
antibiotic-free medium, plasmid DNA (10 µg/plate), and LipofectAMINE (40 µl/plate). After 5 h, the solution was replaced with 10%
FBS-DMEM. 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). Activation of ERK and JNK was then assessed by
immunoprecipitation of the epitope tag followed by an in vitro phosphorylation assay with MBP or recombinant c-Jun as substrates, respectively (16, 26). 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
-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 for 20 min
at 30°C with 5 µCi of [
-32P]ATP and 0.25 mg/ml of
MBP. Reactions were terminated by adding Laemmli buffer and boiling.
Samples were resolved on a 10% sodium dodecyl sulfate gel, and the
proteins were transferred to a nitrocellulose membrane by semidry
transfer. After being studied with Ponceau stain, the membrane was
exposed to film, and substrate phosphorylation was assessed by optical
scanning (Jandel Scientific, San Rafael, CA).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Verification of plasmid expression and function in bovine tracheal
myocytes.
To verify the expression of Myc-tagged Cdc42, RhoA, and Rac1 in bovine
tracheal myocytes, we enriched the population of transfected cells
using immunomagnetic separation and then performed immunoblots to
detect protein expression. Anti-Myc immunoblots of transfected cells
demonstrated expression of the Myc-tagged Cdc42, RhoA, and Rac1
proteins (Fig. 1A).
|
Cdc42, but not RhoA, induces transcription from the cyclin D1
promoter.
Bovine tracheal smooth muscle cells were cotransfected with cDNAs
encoding the human full-length cyclin D1 promoter subcloned into a
luciferase reporter and mutant forms of Cdc42 or RhoA. Overexpression
of active Cdc42 (V12Cdc42) increased transcription from the cyclin D1
promoter, whereas overexpression of dominant negative Cdc42 (N17Cdc42)
attenuated PDGF-mediated activation of the cyclin D1 promoter (Fig.
2A). Overexpression of active Cdc42 in the presence of PDGF stimulation increased cyclin D1 transcription in an additive fashion. In contrast, overexpression of
active RhoA (V14RhoA) did not increase basal or PDGF-mediated cyclin D1
transcription. Furthermore, PDGF-mediated activation of the cyclin D1
promoter was not attenuated by overexpression of dominant negative RhoA
(N19RhoA) (Fig. 2B). These data suggest that Cdc42, but not
RhoA, regulates cyclin D1 expression.
|
Regulation of cyclin D1 by Cdc42 is ERK independent.
We asked whether Cdc42 induces transcription from the cyclin D1
promoter via activation of ERK. Cells were transiently transfected with
cDNAs encoding HA-tagged ERK2 and either constitutively active or
dominant negative Cdc42. ERK activity was assessed by
immunoprecipitation of the HA tag followed by an in vitro
phosphorylation assay with MBP as a substrate. Overexpression of
dominant negative Cdc42 did not attenuate PDGF-mediated ERK activation,
nor did overexpression of active Cdc42 increase ERK2 activity (Fig.
3A). To further examine the
requirement of ERK for Cdc42 regulation of cyclin D1 transcription, we
used the MAP kinase/ERK kinase (MEK) chemical inhibitor PD-98059. Pretreatment of cells with PD-98059 had no effect on Cdc42-mediated activation of the cyclin D1 promoter (Fig. 3B). Together,
these data suggest that Cdc42-induced activation of cyclin D1 is
independent of ERK.
|
Regulation of cyclin D1 by Cdc42 is antioxidant sensitive.
Previously, our laboratory (26) has shown that
Rac1-mediated transcription from the cyclin D1 promoter is dependent on
the generation of reactive oxygen intermediates by NADPH oxidase. We
therefore investigated the effect of antioxidants on Cdc42- induced
activation of cyclin D1 promoter activity. Treatment with catalase
attenuated Cdc42-mediated transcription from the cyclin D1 promoter in
a concentration-dependent manner (Fig.
4A). In addition, treatment
with ebselen, a glutathione peroxidase mimetic, also attenuated
Cdc42-mediated activation (Fig. 4B). These data suggest that
activation of the cyclin D1 promoter by Cdc42 is sensitive to
antioxidants.
|
Rac1 and Cdc42 each activate the cyclin D1 promoter at the
CREB/ATF-2 binding site.
We have identified the cyclin D1 promoter CREB/ATF-2 response element
located 58 to
52 bp 5' from the transcription start site to be a
Rac1 response element (28). To determine whether Cdc42 and
Rac1 activate the cyclin D1 promoter by a similar mechanism, cells were
transiently cotransfected with a luciferase reporter plasmid that
encoded the cyclin D1 promoter sequences from
66 to
40 bp under the
control of a minimal thymidine kinase promoter (CRE-TK81LUC)
(34), and active alleles of Rac1, Cdc42, or RhoA. Rac1 and
Cdc42, but not RhoA, were sufficient to activate this reporter plasmid
(Fig. 5).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Rho family of GTPases has recently been implicated in the regulation of cell cycle progression. In both NIH/3T3 cells (36) and primary bovine myocytes (26), Rac1 regulated transcription from the cyclin D1 promoter. However, the other GTPase family members have not been thoroughly studied. Reports of cross talk between the small GTPases led us to investigate the role of Cdc42 and RhoA in transcriptional regulation of cyclin D1.
In this study, we found that in primary bovine tracheal myocytes 1) overexpression of active Cdc42 increased transcription from the cyclin D1 promoter, whereas overexpression of dominant negative Cdc42 attenuated PDGF-induced activation; 2) overexpression of constitutively active or dominant negative RhoA had no effect on cyclin D1 promoter activity; 3) Cdc42 activated the cyclin D1 promoter in an ERK-independent, antioxidant-sensitive manner, similar to that of Rac1 (26); and 4) both Rac1 and Cdc42 induced transactivation of the cyclin D1 promoter at a common CREB/ATF-2 binding site. Taken together, these data suggest that in airway smooth muscle cells, Cdc42 and Rac1 share a common signaling pathway to cyclin D1 promoter activation.
Overexpression of active Cdc42 increased cyclin promoter activity in bovine tracheal myocytes two- to threefold. Although such increases may not be as great as those generated in cell lines (3), they are nearly so. Stimulation by PDGF gave similar results. Finally, observed changes in promoter activity correlated with changes in protein abundance. Taken together, these data suggest that the observed increases are meaningful.
It has previously been demonstrated (12) that Cdc42 is sufficient to induce cyclin D1 protein accumulation in NIH/3T3 cells. In addition, overexpression of Dbl family members, which serve as guanine nucleotide exchange factors for Cdc42 and RhoA, has been shown to induce transcription from the cyclin D1 promoter in NIH/3T3 cells (37), consistent with the notion that Cdc42 regulates cyclin D1 expression. In the present study, we confirm that Cdc42 is a positive regulator of cyclin D1 expression and show that this effect is mediated at the transcriptional level.
Previous reports examining the contribution of RhoA to cyclin D1 expression have been less conclusive. In NIH/3T3 cells, overexpression of active RhoA was sufficient to induce transcription from the cyclin D1 promoter (36, 39). In IIC9 cells, a subclone of Chinese hamster embryo fibroblasts, overexpression of a dominant negative RhoA did not decrease PDGF-mediated cyclin D1 protein abundance (35), suggesting that RhoA is not required for transcription from the cyclin D1 promoter. In the present study, we found that although RhoA was sufficient to induce cytoskeletal reorganization, it was neither required nor sufficient for cyclin D1 transcription. Discrepancies between these studies may be due to the use of different cyclin D1 reporter constructs (we used the full-length promoter) or, perhaps, cell type-specific differences in RhoA signaling.
Significant cross talk between Rho GTPase family members exists. In Swiss 3T3 cells, Cdc42 activates Rac1, whereas Rac1 can activate RhoA (13, 22, 31). Because Cdc42 and Rac1 regulate cyclin D1 transcription in a similar manner, we speculate that Cdc42 acts upstream of Rac1 in this signaling pathway. However, due to the suppressive effects of multiple expression vectors on cyclin D1 promoter activity, we were unable to test this hypothesis directly (e.g., by coexpressing active Cdc42 and dominant negative Rac). On the other hand, because RhoA and Rac1/Cdc42 have different effects on bovine tracheal smooth muscle cyclin D1 promoter activity, it appears that in the context of cyclin D1 expression, Cdc42 and Rac1 were not upstream activators of RhoA in our system. This model is consistent with a study (15) in NIH/3T3 cells that showed that activated forms of Cdc42 or Rac1 signal to the serum response element in a Rho-independent manner, suggesting that Rac1 and/or Cdc42 and RhoA do not necessarily function in a linked cascade.
Our laboratory (26) previously showed that Rac1-induced transcription from the cyclin D1 promoter is dependent on the generation of reactive oxygen species by NADPH oxidase. However, Rac1 and Cdc42 interact with multiple target proteins, including the JNK and p38 MAP kinases (5-7, 9, 10, 20, 33, 38). We have confirmed that Rac and Cdc42 activate both stress-activated MAP kinases in bovine tracheal myocytes (Page and Hershenson, unpublished data). Because p38 negatively regulates transcription from the cyclin D1 promoter in NIH/3T3 cells (19) and bovine tracheal myocytes (27), it is conceivable that Rac1 and Cdc42 activate both stimulatory and inhibitory pathways to cyclin D1 expression. The relative activities of these opposing pathways, in conjunction with other signaling pathways, may determine the final outcome of Rho GTPase signaling. This model may explain the divergent results described above, including differences in Rho GTPase signaling between cell types.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: K. Page, Univ. of Chicago Children's Hospital, 5841 S. Maryland Ave., MC 4064, Chicago, IL 60637-1470 (E-mail: kpage{at}peds.bsd.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 2 August 2000; accepted in final form 27 November 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abo, A,
Boyhan A,
West I,
Thrasher AJ,
and
Segal AW.
Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245.
J Biol Chem
267:
16767-16770,
1992
2.
Abo, A,
Pick E,
Hall A,
Totty N,
Teahan CG,
and
Segal AW.
Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1.
Nature
353:
668-670,
1991[ISI][Medline].
3.
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
4.
Auer, KL,
Contessa J,
Brenz-Verca S,
Pirola L,
Rusconi S,
Cooper G,
Abo A,
Wymann MP,
Davis RJ,
Birrer M,
and
Dent P.
The Ras/Rac1/Cdc42/SEK/JNK/c-Jun cascade is a key pathway by which agonists stimulate DNA synthesis in primary cultures of rat hepatocytes.
Mol Biol Cell
9:
561-573,
1998
5.
Bagrodia, S,
Derijard B,
Davis RJ,
and
Cerione RA.
Cdc42- and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation.
J Biol Chem
270:
27995-27998,
1995
6.
Collins, LR,
Minden A,
Karin M,
and
Brown JH.
G12 stimulates c-Jun NH2-terminal kinase through the small G proteins Ras and Rac.
J Biol Chem
271:
17349-17353,
1996
7.
Coso, OA,
Chiariello M,
Yu JC,
Teramoto H,
Crespo P,
Xu N,
Miki T,
and
Gutkind JS.
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway.
Cell
81:
1137-1146,
1995[ISI][Medline].
8.
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].
9.
Frost, JA,
Steen H,
Shapiro P,
Lewis T,
Ahn N,
Shaw PE,
and
Cobb MH.
Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins.
EMBO J
16:
6426-6438,
1997
10.
Frost, JA,
Xu S,
Hutchison MR,
Marcus S,
and
Cobb MH.
Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.
Mol Cell Biol
16:
3707-3713,
1996[Abstract].
11.
Gaines, P,
and
Wojchowski DM.
pIRES-CD4t, a dicistronic expression vector for MACS- or FACS-based selection of transfected cells.
Biotechniques
26:
683-688,
1999[ISI][Medline].
12.
Gjoerup, O,
Lukas J,
Bartek J,
and
Willumsen BM.
Rac and Cdc42 are potent stimulators of E2F-dependent transcription capable of promoting retinoblastoma susceptibility gene product hyperphosphorylation.
J Biol Chem
273:
18812-18818,
1998
13.
Hall, A.
Rho GTPases and the actin cytoskeleton.
Science
279:
509-514,
1998
14.
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
15.
Hill, CS,
Wynne J,
and
Treisman R.
The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF.
Cell
81:
1159-1170,
1995[ISI][Medline].
16.
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
17.
Kelleher, MD,
Abe MK,
Chao TS,
Jain M,
Green JM,
Solway J,
Rosner MR,
and
Hershenson MB.
Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis.
Am J Physiol Lung Cell Mol Physiol
268:
L894-L901,
1995
18.
Lamarche, N,
Tapon N,
Stowers L,
Burbelo PD,
Aspenstrom P,
Bridges T,
Chant J,
and
Hall A.
Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade.
Cell
87:
519-529,
1996[ISI][Medline].
19.
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
20.
Minden, A,
Lin A,
Claret FX,
Abo A,
and
Karin M.
Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs.
Cell
81:
1147-1157,
1995[ISI][Medline].
21.
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].
22.
Nobes, CD,
and
Hall A.
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81:
53-62,
1995[ISI][Medline].
23.
Olson, MF,
Ashworth A,
and
Hall A.
An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1.
Science
269:
1270-1272,
1995[ISI][Medline].
24.
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
25.
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
26.
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
27.
Page, K,
Li J,
and
Hershenson MB.
p38 MAP kinase negatively regulates cyclin D1 expression in airway smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
280:
L955-L964,
2001
28.
Page, K,
Li J,
Wang Y,
Kartha S,
Pestell RG,
and
Hershenson MB.
Regulation of cyclin D1 expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells.
Am J Respir Cell Mol Biol
23:
436-443,
2000
29.
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].
30.
Ramakrishnan, MN,
Musa L,
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
31.
Ridley, AJ,
Paterson HF,
Johnston CL,
Diekmann D,
and
Hall A.
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
Cell
70:
401-410,
1992[ISI][Medline].
32.
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
33.
Teramoto, H,
Crespo P,
Coso OA,
Igishi T,
Xu N,
and
Gutkind JS.
The small GTP-binding protein rho activates c-Jun N-terminal kinases/stress-activated protein kinases in human kidney 293T cells. Evidence for a Pak-independent signaling pathway.
J Biol Chem
271:
25731-25734,
1996
34.
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
35.
Weber, JD,
Hu W,
Jefcoat SC, Jr,
Raben DM,
and
Baldassare JJ.
Ras-stimulated extracellular signal-related kinase 1 and RhoA activities coordinate platelet-derived growth factor-induced G1 progression through the independent regulation of cyclin D1 and p27.
J Biol Chem
272:
32966-32971,
1997
36.
Westwick, JK,
Lambert QT,
Clark GJ,
Symons M,
Van Aelst L,
Pestell RG,
and
Der CJ.
Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways.
Mol Cell Biol
17:
1324-1335,
1997[Abstract].
37.
Whitehead, IP,
Lambert QT,
Glaven JA,
Abe K,
Rossman KL,
Mahon GM,
Trzaskos JM,
Kay R,
Campbell SL,
and
Der CJ.
Dependence of Dbl and Dbs transformation on MEK and NF-kB activation.
Mol Cell Biol
19:
7759-7770,
1999
38.
Zhang, S,
Han J,
Sells MA,
Chernoff JA,
Knaus UG,
Ulevitch RJ,
and
Bokoch GM.
Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1.
J Biol Chem
270:
23934-23936,
1995
39.
Zohn, IE,
Symons M,
Chrzanowska-Wodnicka M,
Westwick JK,
and
Der CJ.
Mas oncogene signaling and transformation require the small GTP-binding protein Rac.
Mol Cell Biol
18:
1225-1235,
1998