help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Page, K.
Right arrow Articles by Hershenson, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Page, K.
Right arrow Articles by Hershenson, M. B.
Am. J. Respir. Cell Mol. Biol., Volume 23, Number 4, October 2000 436-443

Regulation of Cyclin D1 Expression and DNA Synthesis by Phosphatidylinositol 3-Kinase in Airway Smooth Muscle Cells

Kristen Page, Jing Li, Yan Wang, Sreedharan Kartha, Richard G. Pestell, and Marc B. Hershenson

Department of Pediatrics, University of Chicago, Chicago, Illinois; and Albert Einstein Cancer Center, Department of Medicine and Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We have shown in bovine tracheal myocytes that extracellular signal-regulated kinase (ERK) and Rac1 function as upstream activators of transcription from the cyclin D1 promoter. We now examine the role of phosphatidylinositol (PI) 3-kinase in this process. PI 3-kinase activity was increased by platelet-derived growth factor (PDGF) and attenuated by the PI 3-kinase inhibitors wortmannin and LY294002. These inhibitors also decreased cyclin D1 promoter activity, protein abundance, and DNA synthesis. Overexpression of the active catalytic subunit of PI 3-kinase (p110PI 3-KCAAX) was sufficient to activate the cyclin D1 promoter. Wortmannin and LY294002 failed to attenuate PDGF-induced ERK activation, and overexpression of p110PI 3-KCAAX was insufficient to activate ERK. p110PI 3-KCAAX-induced cyclin D1 promoter activity was not blocked by PD98059, an inhibitor of mitogen-activated protein kinase/ERK kinase. We next examined whether PI 3-kinase and the 21-kD guanidine triphosphatase Rac1 regulate cyclin D1 promoter activity by similar mechanisms. p110PI 3-KCAAX-induced cyclin D1 promoter activity was decreased by two inhibitors of Rac1-mediated signaling, catalase and diphenylene iodonium. Further, PDGF, PI 3-kinase, and Rac1 each activated the cyclin D1 promoter at the cyclic adenosine monophosphate response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site, as evidenced by expression of a CREB/ATF-2 reporter plasmid. Finally, PI 3-kinase and Rac1-induced CREB/ATF-2 transactivation were each inhibited by catalase. Together, these data suggest that in airway smooth muscle (ASM) cells, PI 3-kinase regulates transcription from the cyclin D1 promoter and DNA synthesis in an ERK-independent manner. Further, PI 3-kinase and Rac1 regulate ASM cell cycle traversal via a common cis-regulatory element in the cyclin D1 promoter.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Excess airway smooth muscle (ASM) cell proliferation is thought to contribute to airflow obstruction in patients with asthma (1). The signaling mechanisms underlying ASM proliferation are not completely understood. We have previously investigated the role of extracellular signal-regulated kinases (ERKs), cytosolic serine/threonine kinases of the mitogen-activated protein kinase (MAPK) superfamily, in bovine tracheal myocyte DNA synthesis. ERK activation is required for platelet-derived growth factor (PDGF)-induced DNA synthesis (2), and also regulates the transcriptional activation of cyclin D1 (3), a critical regulator of G1 progression in these cells (4).

Recently, we examined the importance of the Rho family guanidine triphosphatase (GTPase) Rac1 for cyclin D1 promoter transcriptional activation in bovine tracheal myocytes (5). Overexpression of active Rac1 induced transcription from the cyclin D1 promoter, whereas PDGF-induced transcription was inhibited by a dominant-negative allele of Rac1, suggesting that Rac1 functions as an upstream activator of cyclin D1 in this system. Rac1 forms part of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex that generates reactive oxygen species (ROS) such as H2O2 (6, 7). PDGF treatment of bovine tracheal myocytes stimulated a substantial increase in intracellular ROS, and pretreatment with antioxidants and the flavoprotein inhibitor diphenylene iodonium (DPI) each attenuated Rac1-mediated, but not ERK-mediated, cyclin D1 promoter activation. Overexpression of an N-terminal fragment of p67phox, a component of NADPH oxidase that interacts with Rac1, attenuated PDGF-induced cyclin D1 promoter activity, whereas overexpression of the wild-type p67phox did not. Finally, Rac1 was neither required nor sufficient for ERK activation. Together, these data suggest a model by which two distinct signaling pathways, the ERK and Rac1 pathways, positively regulate cyclin D1 and smooth-muscle growth.

Phosphatidylinositol (PI) 3-kinase is a lipid kinase comprised of an 85-kD regulatory subunit and a 110-kD catalytic subunit that phosphorylates PI at the D-3 hydroxyl of the inositol ring, forming the phosphatidylinositides PI 3-phosphate, PI  3,4-diphosphate, and PI  3,4,5-triphosphate. At least three different isoforms of the 110-kD subunit of PI 3-kinase have been identified in humans (8). PI 3-kinase, by virtue of its SH2-containing regulatory subunit, may interact with a number of mitogenic signaling intermediates including the PDGF receptor (9), Ras (10), and Src family kinases (11). Further, D-3 phosphorylated phosphoinositide products of PI 3-kinase may induce the translocation of additional intermediates to the cell membrane via their pleckstrin homology domains, including Akt (protein kinase B) and guanine nucleotide exchange factors, the upstream activators of GTPases (15). Another intermediate, phosphoinositide-dependent kinase-1, is recruited by PI 3-kinase for activation of PKB and the 70-kD ribosomal S6 kinase (16, 17). Activation of S6 kinase also appears to require FK506 binding protein-rapamycin-associated protein, the mammalian homolog of the yeast target of rapamycin proteins (18). S6 kinase, through the phosphorylation of the 40S ribosomal protein, upregulates the translation of messenger RNAs (mRNAs) containing an oligopyrimidine tract at their transcriptional start site, including ribosomal proteins and elongation factors (19).

Studies have demonstrated a requirement for PI 3-kinase activation in ASM cell cycle traversal. Chemical PI 3-kinase inhibitors decrease bovine (20, 21) and human (22) ASM DNA synthesis. Rapamycin also attenuates ASM DNA synthesis (20, 22), suggesting that PI 3-kinase regulates cell cycle traversal via the translational control of protein synthesis. However, the precise mechanism by which PI 3-kinase exerts its effects on ASM cell cycle control has not been studied.

It has recently been shown in NIH 3T3 cells that overexpression or activation of PI 3-kinase is sufficient to induce mRNA and protein expression of cyclin D1 (23, 24), a critical regulator of G1 progression in ASM and other mammalian cells (4, 25, 26). It is therefore conceivable that PI 3-kinase regulates ASM cell cycle traversal by inducing transcription from the cyclin D1 promoter.

We therefore examined whether activation of PI 3-kinase is required or sufficient for transcription from the cyclin D1 promoter in bovine tracheal myocytes, and whether PI 3-kinase and Rac1 might activate cyclin D1 promoter activity by similar mechanisms. We found that PI 3-kinase is required for PDGF-induced cyclin D1 promoter activity, and that overexpression of the active catalytic subunit of PI 3-kinase (p110PI 3-KCAAX) is sufficient to activate transcription from the cyclin D1 promoter. Further, PI 3-kinase was neither required nor sufficient for ERK activation, and inhibition of MAPK/ERK kinase (MEK)-1 failed to attenuate p110PI 3-KCAAX-induced cyclin D1 promoter activity. Finally, both active PI 3-kinase and Rac1 induced transactivation of the cyclin D1 promoter cyclic adenosine monophosphate response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site, and transactivation by each signaling intermediate was attenuated by antioxidants. Together, these data suggest that in ASM cells, PI 3-kinase regulates transcription from the cyclin D1 promoter and DNA synthesis in an ERK-independent manner. Further, PI 3-kinase and Rac1 appear to regulate ASM cell cycle traversal via a common cis-regulatory element in the cyclin D1 promoter.

    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, PI, PI monophosphate, phosphatidylserine, myelin basic protein, LY294002, catalase, DPI, o-nitrophenyl-beta -D-galactoside, and bromodeoxyuridine (BrdU) were purchased from Sigma Chemical (St. Louis, MO). Protein G sepharose beads were purchased from Pharmacia (Piscataway, NJ). PDGF was obtained from Upstate Biotechnology (Lake Placid, NY). PD98059 was obtained from New England Biolabs (Beverly, MA). Anti-[gamma -32P]adenosine triphosphate (ATP) and an enhanced chemiluminescence kit were purchased from DuPont/NEN Research Products (Wilmington, DE). Antibodies were purchased to detect phosphotyrosine (Upstate Biotechnology), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), dually phosphorylated ERK (Promega, Madison, WI), and BrdU (Becton-Dickinson, San Jose, CA). A peroxidase-linked rat antimouse kappa  light-chain IgG was obtained from Zymed Laboratories (South San Francisco, CA). For in vitro phosphorylation assays, a monoclonal antibody against hemagglutinin (12CA5) was obtained from Babco (Berkeley, CA).

Plasmids encoding a constitutively active Rac1 (pEXV-Myc-V12Rac1) (27) and a constitutively active p110 subunit of PI 3-kinase (pSG5-Myc-p110PI 3-KCAAX) (28) were gifts from Audrey Minden (Columbia University, New York, NY) and Julian Downward (Imperial Cancer Research Fund, London, UK), respectively. A hemagglutinin-tagged ERK-2 (pcDNA3-HA-ERK2) was constructed by ligating a DNA fragment encoding the seven amino-acid influenza hemagglutinin epitope to the 5' end of murine ERK-2 (29). The construction of luciferase reporter plasmids encoding the full-length human cyclin D1 promoter (-1745CD1LUC) and the promoter sequences -66 to -40 base pairs (bp) 5' from the transcriptional start site, under the control of a minimal thymidine kinase promoter (CRE-TK81LUC), were reported previously (30, 31). CRE-TK81LUC includes the cyclin D1 promoter CREB/ATF-2 binding site (-58 to -52 bp 5' from the start site).

Cell Culture

Bovine tracheal smooth muscle cells were isolated as previously described (32). 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. Cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 10% fetal bovine serum (FBS), 1% nonessential amino acids, penicillin (100 U/ml), and streptomycin (100 µg/ml).

Determination of PI 3-Kinase Activity

Cells were seeded into 100-mm dishes and incubated in 10% FBS/ DMEM. After growth to confluence, serum-starved cells were treated with PDGF (30 ng/ml; 15 min). Selected cultures were pretreated with wortmannin (100 nM for 30 min) or LY294002 (15 µM for 15 min). Cells were extracted in lysis buffer (50 mM Tris [pH 7.5], 40 mM beta -glycerophosphate, 100 mM NaCl, 2 mM ethylenediaminetetraacetic acid [EDTA], 50 mM NaF, 200 µM Na3VO4, 200 µM phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100), and lysates were incubated with protein G beads precoupled with antiphosphotyrosine antibody (2 h at 4°C). Immunoprecipitates were washed once with lysis buffer and three times with kinase buffer (10 mM Tris, pH 7.5, 0.2 mM ethyleneglycol-bis- (beta -aminoethyl ether)-N,N'-tetraacetic acid, 100 mM NaCl, and 2 mM MgCl2). Immune complexes were resuspended in 50 µl kinase buffer and freshly sonicated PI (250 µg), PI monophosphate (250 µg), and phosphatidylserine (50 µg), and then incubated with 10 µCi [gamma -32P]ATP (30 min at 30°C). Reactions were terminated by adding 100 µl of chloroform/methanol/HCl (100:200:2, vol/vol). After adding an additional 100 µl chloroform and 100 µl of water, the lipid phase was extracted twice with chloroform/methanol/HCl (2:1:2, vol/vol) and dried. The lipids were dissolved in chloroform/ methanol (95:5), and PI 3-phosphate was resolved by silica gel thin layer chromatography in 1-propanol/acetic acid (2 M) (65:35, vol/ vol). The dried plates were exposed to film.

Determination of Cyclin D1 Promoter Transcriptional Activity

Cells were seeded into 60-mm dishes at 50 to 80% confluence and incubated in 10% FBS/DMEM overnight. Cells were transiently transfected using a liposome solution, as described previously (3). Cells were cotransfected with 1745CD1LUC or CRE-TK81LUC and either the expression vector of interest or relevant control vector. Selected cultures were treated with PDGF (30 ng/ml), wortmannin (100 nM), or catalase (100 to 1,000 U/ml). At 16 h after PDGF treatment, cells were harvested using lysis buffer provided with the Promega Luciferase Assay system, and 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, compared with signals on the order of 102 to 103 units.

Cyclin D1 promoter transcriptional activation was normalized for transfection efficiency by cotransfecting selected cultures with a complementary DNA (cDNA) encoding beta -galactosidase (30 ng/ plate). beta -galactosidase activity was assessed by colorimetric assay using o-nitrophenyl-beta -D-galactoside as a substrate (33).

Immunoblotting

Cells were cultured in six-well plates and serum-starved for 24 h before PDGF treatment (30 ng/ml for 16 h). Cells were washed in phosphate-buffered saline (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 PMSF, and 1% Triton X-100. Lysates were centrifuged (13,000 rpm for 10 min at 4°C) and the supernatant was transferred to fresh microcentrifuge tubes. Extracts (10 µg) were resolved on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to nitrocellulose by semidry transfer (Hoefer, San Francisco, CA). After incubation with the appropriate antibody (anti-cyclin D1 or anti-phosphoERK), signals were amplified and visualized using antirabbit IgG and enhanced chemiluminescence.

Fractional Labeling with BrdU

Subconfluent bovine tracheal myocytes were serum-starved in DMEM for 24 h. At 8 h after PDGF treatment, cells were incubated with BrdU (10 µM) and fluorodeoxyuridine (1 µM). In some wells, wortmannin (100 nM) was added 15 min before growth factor treatment. At 16 h later, myocytes were fixed in periodate lysine paraformaldehyde buffer and the DNA was precipitated with 2 M HCl. After acid neutralization with 0.1 M borate buffer (pH 9.0), cells were permeabilized with 0.2% Triton X-100. Cells were then immunostained with fluorescein isothiocyanate (FITC)- labeled anti-BrdU and counterstained with propidium iodide.

Statistical Analysis

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

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

PDGF Activates PI 3-Kinase in Bovine ASM Cells

To confirm the observation that PDGF activates PI 3-kinase in bovine tracheal myocytes (20), PI 3-kinase activity was assessed by in vitro phosphorylation assay and thin layer chromatography. PDGF treatment increased the level of PI 3-phosphate (Figure 1). Pretreatment with the PI 3-kinase inhibitors wortmannin (100 nM) and LY294002 (30 µM) each attenuated the signal.


View larger version (36K):
[in this window]
[in a new window]
  		Figure 1.   PDGF activates PI 3-kinase in bovine ASM cells. Selected cultures were pretreated with wortmannin (100 nM for 15 min) or LY294002 (30 µM for 15 min), and stimulated with PDGF (30 ng/ml for 15 min). PI 3-kinase activity was assessed by immunoprecipitating cell extracts with an antiphosphotyrosine antibody and measuring the in vitro phosphorylation of PI. Samples were resolved by thin layer chromatography. This result was typical of two separate experiments.

PI 3-Kinase Is Required for PDGF-Induced Cyclin D1 Promoter Activity and Protein Accumulation

To test for the requirement of PI 3-kinase for cyclin D1 promoter activity, cells were transiently transfected with a cDNA encoding the full-length cyclin D1 promoter subcloned into a luciferase reporter gene. Wortmannin inhibited PDGF-induced activation of the cyclin D1 promoter (Figure 2A). Wortmannin (100 nM) and LY294002 (15 and 25 µM) each inhibited PDGF-induced cyclin D1 protein abundance (Figure 2B).


View larger version (14K):
[in this window]
[in a new window]
 

View larger version (11K):
[in this window]
[in a new window]
  		Figure 2.   Effects of PI 3-kinase inhibitors on PDGF- and Rac1-induced cyclin D1 promoter activity and protein abundance. (A) Wortmannin attenuates PDGF- but not Rac1-induced transcription from the cyclin D1 promoter. Cells were transiently transfected with cDNAs encoding the full-length human cyclin D1 promoter subcloned into a luciferase reporter gene (-1745CD1LUC). Selected cultures were pretreated with wortmannin (100 nM; 15 min) and stimulated with PDGF (30 ng/ml for 16 h). Additional cultures were cotransfected with active Rac1 (pEXV-myc-V12Rac1) or vector control (pEXV). To control for transfection efficiency, selected cultures were also cotransfected with pCMV-beta -galactosidase. Data are calculated as luciferase/beta -galactosidase/h normalized to the control vector (mean ± standard error for five to nine experiments). Wortmannin pretreatment significantly decreased PDGF- but not Rac1-induced transcription from the cyclin D1 promoter (P < 0.05, ANOVA/SNK multiple range test). (B) PI 3-kinase inhibitors attenuate cyclin D1 protein abundance. Cells were pretreated either with wortmannin (100 nM, left panel) or LY294002 (15-25 µM, right panel) for 15 min before stimulation with PDGF (30 ng/ml for 16 h). Cellular proteins were resolved by SDS-polyacrylamide gel electrophoresis transferred to nitrocellulose, and probed using a polyclonal antibody against cyclin D1 (1:1,000 dilution). These results were typical of two separate experiments.

Overexpression of Active PI 3-Kinase Is Sufficient for Transcription from the Cyclin D1 Promoter

To test whether PI 3-kinase was sufficient for cyclin D1 promoter activation, cells were transiently cotransfected with cDNAs encoding the luciferase-tagged cyclin D1 promoter and a constitutively active catalytic subunit of PI 3-kinase (p110PI 3-KCAAX). Activation of PI 3-kinase induced cyclin D1 promoter activity (Figure 3).


View larger version (11K):
[in this window]
[in a new window]
  		Figure 3.   Overexpression of PI 3-kinase is sufficient for transcription from the cyclin D1 promoter and is not attenuated by pretreatment with the chemical MEK inhibitor PD 98059. Cells were transiently cotransfected with cDNAs encoding the full-length cyclin D1 promoter subcloned into a luciferase reporter gene (-1745CD1LUC) and either empty vector (pSG5) or constitutively active p110 (pSG5-p110PI 3-KCAAX). Selected cultures were pretreated with PD98059 (30 µM, 30 min) and stimulated with PDGF (30 ng/ml for 16 h). Data are calculated as luciferase/ beta -galactosidase/h normalized to the control vector (mean ± standard error for five experiments). PD98059 pretreatment significantly decreased PDGF-induced but not PI 3-kinase-induced transcription from the cyclin D1 promoter (P < 0.05, ANOVA/ SNK multiple range test).

PI 3-Kinase Activation of Cyclin D1 Promoter is ERK-Independent

We have previously demonstrated in bovine tracheal myocytes that ERK is required for PDGF-induced transcription from the cyclin D1 promoter, and that overexpression of a constitutively active MEK-1, the upstream activator of ERKs (2), is sufficient for cyclin D1 promoter activity (3). We therefore examined potential crosstalk between PI 3-kinase and the ERK pathway. First, pretreatment of PDGF-stimulated cells with the PI 3-kinase inhibitors wortmannin (100 nM) and LY294002 (25 µM) each failed to inhibit ERK phosphorylation (Figure 4A). Second, overexpression of p110PI 3-KCAAX, although sufficient for activation of the cyclin D1 promoter (Figure 3), was insufficient to induce phosphorylation of ERK2, as determined by immunoprecipitation and anti-phosphoERK immunoblot (Figure 4B). Next, we examined the effect of the specific MEK inhibitor PD98059 on cells transiently transfected with the active catalytic subunit of PI 3-kinase. Although previous studies have demonstrated that PD98059 pretreatment attenuates PDGF-induced ERK activity (2) and cyclin D1 promoter activation (3, 5), it had no effect on promoter activity due to active PI 3-kinase (Figure 3). Together, these data demonstrate that PI 3-kinase is neither required nor sufficient for ERK activation, and that PI 3-kinase activates transcription from the cyclin D1 promoter in a MEK-1/ERK-independent manner.


View larger version (27K):
[in this window]
[in a new window]
 

View larger version (28K):
[in this window]
[in a new window]
  		Figure 4.   PI 3-kinase is not required or sufficient for ERK activation. (A) PI 3-kinase inhibitors do not block PDGF-induced ERK phosphorylation. Cells were pretreated with LY294002 (25 µM) or wortmannin (100 nM) for 15 minutes before stimulation with PDGF (30 ng/ml for 10 min). Cell extracts were assessed for ERK phosphorylation by immunoblotting with an ERK phosphospecific antibody (1:10,000 dilution). Immunoblots shown are representative of two separate experiments. (B) Overexpression of active PI 3-kinase does not increase ERK-2 activity. Cells were transiently cotransfected with hemagglutinin-tagged ERK-2 (pcDNA3-HA-ERK) and the constitutively active PI 3-kinase (pSG5-p110PI 3-KCAAX) or empty vector (pSG5). Selected cultures were treated with PDGF (30 ng/ml for 10 min). ERK phosphorylation was assessed by immunoprecipitation using an antihemagglutinin antibody (12CA5) and immunoblotting with anti-phosphoERK (upper panel). The level of hemagglutinin-tagged ERK2 expression was determined by immunoblotting with 12CA5 (bottom panel). Immunoblots shown are representative of two separate experiments.

Transcriptional Activation of Cyclin D1 by PI 3-Kinase Is Antioxidant-Sensitive

We recently found that the GTPase Rac1 functions as an upstream activator of cyclin D1 in bovine tracheal myocytes (5). Further, the effect of Rac1 was mediated by NADPH oxidase, as evidenced by an increase in intracellular ROS and by attenuation of cyclin D1 promoter activity by antioxidants and the flavoprotein inhibitor DPI. We therefore examined whether PI 3-kinase might also induce transcription from the cyclin D1 promoter that is sensitive to antioxidants. First, we tested the effects of two inhibitors of the Rac1 pathway, catalase and DPI, on PI 3-kinase (p110PI 3-KCAAX)-induced cyclin D1 promoter activity. Both catalase and DPI attenuated promoter activity (Figure 5).


View larger version (13K):
[in this window]
[in a new window]
  		Figure 5.   PI 3-kinase-induced transcription from the full-length cyclin D1 promoter is antioxidant-sensitive. Cells were transiently cotransfected with cDNAs encoding the full-length cyclin D1 promoter subcloned into a luciferase reporter gene (-1745CD1LUC) and either empty vector (pSG5) or constitutively active PI 3-kinase (pSG5-p110PI 3-KCAAX). Selected cultures were treated with catalase (300 or 1,000 U/ml for 16 h) or DPI (25 µM for 16 h). Data are calculated as luciferase/beta -galactosidase/h normalized to the control vector (mean ± standard error for five to 10 experiments). Treatment with either catalase (1,000 U/ml) or DPI significantly reduced CAAX-induced transcription from the cyclin D1 promoter (P < 0.05, ANOVA/SNK multiple range test).

PI 3-Kinase and Rac1 Each Activate the Cyclin D1 Promoter CREB/ATF-2 Binding Site

Previously, we localized the Rac1 responsive element in the cyclin D1 promoter to a region 66 bp 5' to the transcriptional start site myocytes (5). A comprehensive analysis of this region has revealed the Rac1 responsive element to be a CREB/ATF-2 binding site located -58 to -52 bp 5' from the transcription start site, and electromobility shift assays indicate binding of the nuclear transcription factor CREB-1 (Page and Hershenson, manuscript in preparation). To further examine whether, in the context of the cyclin D1 promoter, PI 3-kinase and Rac1 function on the same pathway, we transfected cells with a luciferase reporter plasmid encoding the cyclin D1 promoter sequences from -66 to -40, under the control of a minimal thymidine kinase promoter (CRE-TK81LUC) (31). These sequences include the cyclin D1 promoter CREB/ATF-2 binding site, but not the -39 to -30 nuclear factor-kappa B site (34). PDGF, V12Rac1, and p110PI 3-KCAAX were each sufficient to activate this promoter (Figure 6). Pretreatment with wortmannin inhibited PDGF-induced CREB/ATF-2 transactivation (Figure 6A). Finally, V12Rac1- and p110PI 3-KCAAX-induced activation of CRE-TK81LUC were each attenuated by catalase pretreatment (Figures 6B and 6C). Together, these data suggest that PI 3-kinase and Rac1 share a common downstream target regulating transcription from the cyclin D1 promoter.


View larger version (10K):
[in this window]
[in a new window]
 

View larger version (14K):
[in this window]
[in a new window]
 

View larger version (13K):
[in this window]
[in a new window]
  		Figure 6.   PI 3-kinase and Rac1 each activate the cyclin D1 promoter CREB/ATF-2 binding site. Cells were transfected with a luciferase reporter plasmid encoding the cyclin D1 promoter sequences from -66 to -40, under the control of a minimal thymidine kinase promoter (CRE-TK81LUC). These sequences include the -58 to -52 CREB/ATF-2 binding site. (A) Effect of PDGF on transactivation of the cyclin D1 promoter CREB/ATF-2 binding site. Selected cultures were pretreated with wortmannin (100 nM for 15 min) before PDGF treatment (30 ng/ml for 16 h). Wortmannin significantly decreased PDGF-induced transactivation of CRE-TK81LUC (P < 0.05, ANOVA/SNK multiple range test). (B) Effect of catalase pretreatment on V12Rac1-induced transactivation of the cyclin D1 promoter CREB/ATF-2 binding site. Treatment with catalase (either 300 or 1,000 U/ml for 16 h) significantly reduced Rac1-induced transactivation of CRE-TK81LUC (P < 0.05, ANOVA/SNK multiple range test). (C) Effect of catalase pretreatment on p110PI 3-KCAAX-induced transactivation of the cyclin D1 promoter CREB/ATF-2 binding site. Treatment with catalase (either 300 or 1,000 U/ml for 16 h) significantly reduced CAAX-induced transactivation of CRE-TK81LUC (P < 0.05, ANOVA/SNK multiple range test). Data are calculated as luciferase/beta -galactosidase/h normalized to the control vector (means ± standard error for three or four experiments).

PI 3-Kinase Regulates DNA Synthesis in Bovine ASM Cells

Because PI 3-kinase is required for PDGF-induced cyclin D1 expression, and cyclin D1 is required for DNA synthesis in bovine tracheal myocytes (4), we asked whether PI 3-kinase is required for PDGF-induced DNA synthesis. DNA synthesis was assayed by fractional labeling with BrdU. PDGF activated DNA synthesis in over 20% of the cells, whereas pretreatment with wortmannin abolished this increase (Figure 7).


View larger version (11K):
[in this window]
[in a new window]
  		Figure 7.   Wortmannin abolishes PDGF-induced S-phase traversal. Selected cultures were pretreated with wortmannin (100 nM) 15 min before stimulation with PDGF (30 ng/ ml for 24 h). At 8 h after the initiation of PDGF treatment, cells were incubated with BrdU (10 µM) and fluorodeoxyuridine (1 µM). After fixation, cells were stained with a FITC-labeled monoclonal antibody against BrdU and counterstained with propidium iodide. Wortmannin significantly decreased PDGF-induced BrdU labeling (P < 0.05, ANOVA/SNK multiple range test). Data shown are the means ± standard error of three experiments.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

PI 3-kinase appears to play an important role in the proliferation of airway myocytes and other mesenchymal cells. Epidermal growth factor, a potent mitogen for human tracheal myocytes, stimulates PI 3-kinase activation in these cells (35). Chemical PI 3-kinase inhibitors have been demonstrated to attenuate PDGF-induced DNA synthesis in bovine (20, 21) and human ASM cells (22). Activation of PI 3-kinase has also been demonstrated to be required for DNA synthesis in fibroblasts, adipocytes, and vascular smooth-muscle cells (36). Although the sufficiency of PI 3-kinase activation for ASM cell cycle progression has not been tested, expression of active PI 3-kinase was sufficient to promote entry into S phase in CHO cells (40) and fibroblasts (41). Together, these studies suggest that PI 3-kinase is an important regulator of progression through G1 of the cell cycle.

The precise mechanism by which PI 3-kinase exerts its effects on cell cycle control is not well understood. It has recently been demonstrated in growth factor-deprived NIH3T3 cells that overexpression of wild-type p110 is sufficient to induce mRNA and protein expression of cyclin D1 (23), a critical regulator of G1 progression in ASM and other mammalian cells (4, 25, 26). Further, growth factor- induced cyclin D1 expression and DNA synthesis were inhibited by LY294002 but not rapamycin, suggesting that S6 kinase does not serve as the principal mediator of cyclin D1 expression. Finally, PI 3-kinase activation of cyclin D1 expression was not inhibited either by the synthetic MEK inhibitor PD98059 or by expression of a dominant-negative ERK (23). In the present study, we found that inhibition of PI 3-kinase by the chemical inhibitor wortmannin attenuates PDGF-induced transcription from the cyclin D1 promoter in cultured ASM cells. In addition, overexpression of a constitutively active form of p110 PI 3-kinase was sufficient to induce cyclin D1 promoter activity. Finally, PI 3-kinase was neither required nor sufficient for activation of ERK, and pretreatment with PD98059 did not attenuate PI 3-kinase-induced cyclin D1 transcription. Together, these data confirm that PI 3-kinase functions as an upstream activator of cyclin D1 in primary ASM cells, and that this activation may occur independently of the ERK pathway.

Studies in fibroblast cell lines (42) and rat hepatocytes (45) have demonstrated a requirement for Rac1 in G1 progression. In NIH3T3 cells, Rac1 activates transcription from the cyclin D1 promoter (46, 47), suggesting a mechanism by which Rac1 signaling may regulate G1 progression. We recently examined the importance of the Rho family GTPase Rac1 for cyclin D1 promoter transcriptional activation in bovine tracheal myocytes (5). Overexpression of active Rac1 induced transcription from the cyclin D1 promoter, whereas PDGF-induced transcription was inhibited by a dominant-negative allele of Rac1, suggesting that Rac1 functions as an upstream activator of cyclin D1 in this system. Overexpression of a dominant-negative p67phox, a component of NADPH oxidase complex, and pretreatment with antioxidants and the flavoprotein inhibitor DPI each attenuated Rac1-mediated cyclin D1 promoter activation, consistent with the notion that the generation of ROS by NADPH oxidase is required for promoter activity. Because PI 3-kinase may function as an upstream activator of Rac1 (48), we examined whether PI 3-kinase and Rac1 induce transcription from the cyclin D1 promoter via similar mechanisms. Pretreatment of bovine tracheal myocytes with either catalase or DPI each attenuated PI 3-kinase- induced activation of the cyclin D1 promoter. Further, PDGF, active PI 3-kinase, and active Rac1 each activated the cyclin D1 promoter at the CREB/ATF-2 binding site, as evidenced by expression of a luciferase reporter plasmid linked to a cyclin D1 promoter sequence including the -58 to -52 CREB/ATF-2 site. Finally, PI 3-kinase and Rac1-induced CREB/ATF-2 transactivation were each inhibited by catalase, and PDGF-induced transactivation was attenuated by wortmannin. Together, these data suggest that PI 3-kinase and Rac1 regulate ASM cell cycle traversal via a common cis-regulatory element in the cyclin D1 promoter.

Although PI 3-kinase and Rac1 each induce transcription from the cyclin D1 promoter by similar mechanisms, we are unable at this time to determine whether PI 3-kinase induces promoter activity via activation of Rac1. Given the low transfection efficiency (< 50%) in our primary cell system, we were unable to measure the effect of p110PI 3-KCAAX on Rac1 guanidine triphosphate loading. However, even in cell lines, this measurement may be a relatively insensitive measure of Rac1 activation (50). Also, we were unable to test the effect of a dominant-negative Rac1 on p110PI 3-KCAAX-induced cyclin D1 promoter activity, owing to the nonspecific inhibition of endogenous promoter activity by multiple expression vectors. Finally, it is conceivable that the abilities of PI 3-kinase and Rac1 to activate CRE-TK81LUC depend on the binding of two separate transcription factor targets, and therefore represent two separate signaling pathways. However, the sensitivity of both PI 3-kinase and Rac1-induced transactivation to antioxidants makes this possibility less likely. Also, recent studies indicate CREB to be a downstream transcription factor target of PI 3-kinase (52).

Recent studies suggest that cyclin D1 protein abundance may be regulated not only by gene transcription but also by changes in mRNA translation and protein degradation. In the MCF breast cancer cell line, growth factor-induced D-type cyclin protein abundance most closely correlated with changes in protein synthesis, not mRNA expression (53). Cyclin protein accumulation was blocked by wortmannin and rescued by expression of an activated form of Akt. On the other hand, pretreatment of cells with the synthetic MEK inhibitor PD98059 had no effect on cyclin levels. Cyclin D1 levels may also be regulated by ubiquitin-dependent proteasomal degradation (54). In NIH3T3 cells, phosphorylation of cyclin D1 by glycogen synthase kinase-3 triggered rapid cyclin D1 turnover, which was blocked by wortmannin and stabilized by overexpression of active Akt but not by a constitutively active mutant of MEK-1 (55). Together with the present study, these data suggest that PI 3-kinase may regulate cyclin D1 transcription, translation, and proteolysis, and that this regulation occurs in an ERK-independent manner.

Much of the aforementioned work depends in part on the transient overexpression of mutant alleles of signaling intermediates, and therefore results should be interpreted with caution. For example, overexpression of constitutively active proteins is likely to induce supraphysiologic outcomes. Indeed, given our observation that PI 3-kinase- induced transcription from the cyclin D1 promoter is ERK-independent, it is implausible that PI 3-kinase and MEK-1 are each required and sufficient for promoter activity. Nevertheless, when evaluated in the context of additional experiments using inhibitors of PI 3-kinase, we believe that our results strongly suggest that PI 3-kinase is an upstream activator of cyclin D1 promoter activity in ASM cells. Whether activation of this signaling pathway is sufficient to do so under physiologic conditions, or requires the additional activation of other pathways (such as the ERK pathway), will require further investigation.

The present study confirms previous work by other investigators (20, 35) emphasizing the importance of the PI 3-kinase signaling pathway for ASM proliferation. Together with additional data demonstrating the requirement of ERK activation for ASM growth (2, 56), these data suggest a model by which two signaling pathways, the ERK pathway and the PI 3-kinase/Rac1 pathway, regulate ASM growth.

    Footnotes

Address correspondence to: Marc B. Hershenson, M.D., University of Chicago Children's Hospital, 5841 S. Maryland Ave., MC4064, Chicago, IL 60637-1470. E-mail: mhershen{at}midway.uchicago.edu

(Received in original form September 23, 1999 and in revised form May 18, 2000).

Abbreviations: analysis of variance, ANOVA; airway smooth muscle, ASM; activating transcription factor, ATF; base pairs, bp; bromodeoxyuridine, BrdU; complementary DNA, cDNA; cyclic adenosine monophosphate response element binding protein, CREB; Dulbecco's minimum essential medium, DMEM; diphenylene iodonium, DPI; extracellular signal-regulated kinase, ERK; guanidine triphosphatase, GTPase; mitogenactivated protein kinase/ERK kinase, MEK; messenger RNA, mRNA; nicotinamide adenine dinucleotide phosphate, NADPH; the active catalytic subunit of PI 3-kinase, p110PI 3-KCAAX; platelet-derived growth factor, PDGF; phosphatidylinositol, PI; reactive oxygen species, ROS; Student-Newman-Keul, SNK.

Acknowledgments: The authors thank Dr. Audrey Minden (for active Rac1) and Dr. Julian Downward (for active PI 3-kinase). M.B.H. is especially grateful to Dr. Marsha Rosner for her ongoing advice and support. These studies were supported by National Institutes of Health Grants HL07605 to K.P.; HL54685, HL56399, and HL63314 to M.B.H.; and CA70897, CA75503, and CA13330 to R.G.P.; as well as grants from the Blowitz-Ridgeway Foundation to M.B.H. and the Susan G. Komen Breast Cancer Foundation to R.G.P.
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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

2. Karpova, A. Y., M. K. Abe, J. Li, P. Liu, J. M. Rhee, W. L. Kuo, and M. B. Hershenson. 1997. MEK1 is required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes. Am. J. Physiol. 272: L558-L565 [Abstract/Free Full Text].

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

4. Xiong, W., R. G. Pestell, G. Watanabe, J. Li, M. R. Rosner, and M. B. Hershenson. 1997. Cyclin D1 is required for S-phase traversal in bovine tracheal myocytes. Am. J. Physiol. 272: L1205-L1210 [Abstract/Free Full Text].

5. Page, K., J. Li, J. A. Hodge, P. T. Liu, T. L. Vanden, Hoek, L. B. Becker, R. G. Pestell, M. R. Rosner, and M. B. Hershenson. 1999. Characterization of a Rac1 signaling pathway to cyclin D1 expression in airway smooth muscle cells. J. Biol. Chem. 274: 22065-22071 [Abstract/Free Full Text].

6. Abo, A., E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal. 1991. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 353: 668-670 [Medline].

7. Abo, A., A. Boyhan, I. West, A. J. Thrasher, and A. W. Segal. 1992. 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 [Abstract/Free Full Text].

8. Keith, C. T., and S. L. Schreiber. 1995. PIK-related kinases: DNA repair, recombination and cell cycle checkpoints. Science 270: 50-51 .

9. Claesson-Welsh, L.. 1994. Platelet-derived growth factor receptor signaling. J. Biol. Chem. 269: 32023-32026 [Free Full Text].

10. Marshall, M. S.. 1995. Ras target proteins in eukaryotic cells. FASEB J. 9: 1311-1318 [Abstract].

11. Liu, X., L. E. Marengere, C. A. Koch, and T. Pawson. 1993. The v-Src SH3 domain binds phosphatidylinositol 3'-kinase. Mol. Cell. Biol. 13: 5225-5232 [Abstract/Free Full Text].

12. Corey, S., A. Eguinoa, K. Puyana-Theall, J. B. Bolen, L. Cantley, F. Mollinedo, T. R. Jackson, P. T. Hawkins, and L. R. Stephens. 1993. Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells. EMBO J. 12: 2681-2690 [Medline].

13. Yamanashi, Y., Y. Fukui, B. Wongsasant, Y. Kinoshita, Y. Ichimori, K. Toyoshima, and T. Yamamoto. 1992. Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling. Proc. Natl. Acad. Sci. USA 89: 1118-1122 [Abstract/Free Full Text].

14. Pleiman, C. M., W. M. Hertz, and J. C. Cambier. 1994. Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit. Science 263: 1609-1612 [Abstract/Free Full Text].

15. Lemmon, M. A., K. M. Ferguson, R. O'Brien, P. B. Sigler, and J. Schlessinger. 1995. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain. Proc. Natl. Acad. Sci. USA 92: 10472-10476 [Abstract/Free Full Text].

16. Pullen, N., P. B. Dennis, M. Andjelkovic, A. Dufner, S. C. Kozma, B. A. Hemmings, and G. Thomas. 1998. Phosphorylation and activation of p70s6k by PDK1. Science 279: 707-710 [Abstract/Free Full Text].

17. Alessi, D. R., M. T. Kozlowski, Q. P. Weng, N. Morrice, and J. Avruch. 1998. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr. Biol. 8: 69-81 [Medline].

18. Brown, E. J., M. W. Albers, T. B. Shin, K. Ichikawa, C. T. Keith, W. S. Lane, and S. L. Schreiber. 1994. A mammalian protein targeted by G1- arresting rapamycin-receptor complex. Nature 369: 756-758 [Medline].

19. Jefferies, H. B., S. Fumagalli, P. B. Dennis, C. Reinhard, R. B. Pearson, and G. Thomas. 1997. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO J. 16: 3693-3704 [Medline].

20. Scott, P. H., C. M. Belham CM, J. al-Hafidh, E. R. Chilvers, A. J. Peacock, G. W. Gould, and R. Plevin. 1996. A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells. Biochem. J. 318:965-971.

21. Walker, T. R., S. M. Moore, M. F. Lawson, R. A. J. Panettieri, and E. R. Chilvers. 1998. 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 [Abstract/Free Full Text].

22. Krymskaya, V. P., R. B. Penn, M. J. Orsini, P. H. Scott, R. J. Plevin, T. R. Walker, A. J. Eszterhas, Y. Amrani, E. R. Chilvers, and R. A. J. Panettieri. 1999. Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation. Am. J. Physiol. 277: L65-L78 [Abstract/Free Full Text].

23. Takuwa, N., Y. Fukui, and Y. Takuwa. 1999. Cyclin D1 expression mediated by phosphatidylinositol 3-kinase through mTOR-p70(S6K)-independent signaling in growth factor-stimulated NIH 3T3 fibroblasts. Mol. Cell. Biol. 19: 1346-1358 [Abstract/Free Full Text].

24. Gille, H., and J. Downward. 1999. Multiple ras effector pathways contribute to G1 cell cycle progression. J. Biol. Chem. 274: 22033-22040 [Abstract/Free Full Text].

25. Lukas, J., J. Bartkova, M. Rohde, M. Strauss, and J. Bartek. 1995. Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity. Mol. Cell. Biol. 15: 2600-2611 [Abstract].

26. Quelle, D. E., R. A. Ashmun, S. A. Shurtleff, J. Y. Kato, D. Bar-Sagi, M. F. Roussel, and C. J. Sherr. 1993. Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev. 7: 1559-1571 [Abstract/Free Full Text].

27. Ridley, A. J., H. F. Paterson, C. L. Johnston, D. Diekmann, and A. Hall. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70: 401-410 [Medline].

28. Khwaja, A., and J. Downward. 1997. Lack of correlation between activation of Jun-NH2-terminal kinase and induction of apoptosis after detachment of epithelial cells. J. Cell Biol. 139: 1017-1023 [Abstract/Free Full Text].

29. Hershenson, M. B., T. S. Chao, M. K. Abe, I. Gomes, M. D. Kelleher, J. Solway, and M. R. Rosner. 1995. Histamine antagonizes serotonin and growth factor-induced mitogen-activated protein kinase activation in bovine tracheal smooth muscle cells. J. Biol. Chem. 270: 19908-19913 [Abstract/Free Full Text].

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

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

32. Abe, M. K., T. S. Chao, J. Solway, M. R. Rosner, and M. B. Hershenson. 1994. 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 [Abstract].

33. Parmacek, M. S., A. J. Vora, T. Shen, E. Barr, F. Jung, and J. M. Leiden. 1992. Identification and characterization of a cardiac-specific transcriptional regulatory element in the slow cardiac tropinin C gene. Mol. Cell Biol. 12: 1967-1976 [Abstract/Free Full Text].

34. Joyce, D., B. Bouzahzah, M. Fu, C. Albanese, M. D'Amico, J. Steer, J. U. Klein, R. J. Lee, J. E. Segall, J. K. Westwick, C. J. Der, and R. G. Pestell. 1999. Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway. J. Biol. Chem. 274: 25245-25249 [Abstract/Free Full Text].

35. Krymskaya, V. P., R. Hoffman, A. Eszterhas, V. Ciocca, and P. A. Panettieri. 1997. TGF-1 modulates EGF-stimulated phosphatidylinositol 3-kinase activity in human airway smooth muscle cells. Am. J. Physiol. 273: L1220-L1227 [Abstract/Free Full Text].

36. Roche, S., M. Koegl, and S. A. Courtneidge. 1994. The phosphatidylinositol 3-kinase alpha is required for DNA synthesis induced by some, but not all, growth factors. Proc. Natl. Acad. Sci. USA 91: 9185-9189 [Abstract/Free Full Text].

37. Jhun, B. H., D. W. Rose, B. L. Seely, L. Rameh, L. Cantley, A. R. Saltiel, and J. M. Olefsky. 1994. Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression. Mol. Cell. Biol. 14: 7466-7475 [Abstract/Free Full Text].

38. Cheatham, B., C. J. Vlahos, L. Cheatham, L. Wang, J. Blenis, and C. R. Kahn. 1994. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol. Cell Biol. 14: 4902-4911 [Abstract/Free Full Text].

39. Bacqueville, D., F. Casagrande, B. Perret, H. Chap, J. M. Darbon, and M. Breton-Douillon. 1998. Phosphatidylinositol 3-kinase inhibitors block aortic smooth muscle cell proliferation in mid-late G1 phase: effect on cyclin-dependent kinase 2 and the inhibitory protein p27KIP1. Biochem. Biophys. Res. Commun. 244: 630-636 [Medline].

40. McIlroy, J., D. Chen, C. Wjasow, J. Michaeli, and J. M. Backer. 1997. Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways. Mol. Cell. Biol. 17: 248-255 [Abstract].

41. Klippel, A., M. A. Escobedo, M. S. Wachowicz, G. Apell, T. W. Brown, M. A. Giedlin, W. M. Kavanaugh, and L. T. Williams. 1998. Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation. Mol. Cell. Biol. 18: 5699-5711 [Abstract/Free Full Text].

42. Olson, M. F., A. Ashworth, and A. Hall. 1995. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science 269: 1270-1272 [Abstract/Free Full Text].

43. Lamarche, N., N. Tapon, L. Stowers, P. D. Burbelo, P. Aspenstrom, T. Bridges, J. Chant, and A. Hall. 1996. 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 [Medline].

44. Joneson, T., and D. Bar-Segi. 1998. A Rac1 effector site controlling mitogenesis through superoxide production. J. Biol. Chem. 273: 17991-17994 [Abstract/Free Full Text].

45. Auer, K. L., J. Contessa, S. Brenz-Verca, L. Pirola, S. Rusconi, G. Cooper, A. Abo, M. P. Wymann, R. J. Davis, M. Birrer, and P. Dent. 1998. 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 [Abstract/Free Full Text].

46. Westwick, J. K., Q. T. Lambert, G. J. Clark, M. Symons, L. Van Aelst, R. G. Pestell, and C. J. Der. 1997. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways. Mol. Cell. Biol. 17: 1324-1335 [Abstract].

47. Zohn, I. E., M. Symons, M. Chrzanowska-Wodnicka, J. K. Westwick, and C. J. Der. 1998. Mas oncogene signaling and transformation require the small GTP-binding protein Rac. Mol. Cell. Biol. 18: 1225-1235 [Abstract/Free Full Text].

48. Hall, A.. 1998. Rho GTPases and the actin cytoskeleton. Science 279: 509-514 [Abstract/Free Full Text].

49. Wennstrom, S., P. Hawkins, F. Cooke, K. Hara, K. Yonesawa, M. Kasuga, T. Jackson, L. Claesson-Welsh, and L. Stephens. 1994. Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr. Biol. 4: 385-393 [Medline].

50. Hawkins, P. T., A. Eguinoa, R.-G. Qiu, D. Stokoe, F. T. Cooke, R. Walters, S. Wennstrom, L. Claesson-Welsh, T. Evans, M. Symons, and L. Stephens. 1995. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr. Biol. 5: 393-403 [Medline].

51. Reif, K., C. D. Nobes, G. Thomas, A. Hall, and D. A. Cantrell. 1996. Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho- dependent effector pathways. Curr. Biol. 6: 1445-1455 [Medline].

52. Wang, J. M., J. R. Chao, W. Chen, M. L. Kuo, J. J. Yen, and H. F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19: 6195-6206 [Abstract/Free Full Text].

53. Muise-Helmericks, R. C., H. L. Grimes, A. Bellacosa, S. E. Malstrom, P. N. Tsichlis, and N. Rosen. 1998. Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J. Biol. Chem. 273: 29864-29872 [Abstract/Free Full Text].

54. Diehl, J. A., F. Zindy, and C. J. Sherr. 1997. Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. Genes Dev. 11: 957-972 [Abstract/Free Full Text].

55. Diehl, J. A., M. Cheng, M. F. Roussel, and C. J. Sherr. 1998. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes. Dev. 12: 3499-3511 [Abstract/Free Full Text].

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




This article has been cited by other articles:


Home page
 Proc Am Thorac SocHome page
J. K. Bentley and M. B. Hershenson
Airway Smooth Muscle Growth in Asthma: Proliferation, Hypertrophy, and Migration
Proceedings of the ATS, January 1, 2008; 5(1): 89 - 96.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S.-D. Ha, D. Ng, S. L. Pelech, and S. O. Kim
Critical Role of the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase-3 Signaling Pathway in Recovery from Anthrax Lethal Toxin-induced Cell Cycle Arrest and MEK Cleavage in Macrophages
J. Biol. Chem., December 14, 2007; 282(50): 36230 - 36239.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. C. Shepherd, S. M. Duffy, T. Harris, G. Cruse, M. Schuliga, C. E. Brightling, C. B. Neylon, P. Bradding, and A. G. Stewart
KCa3.1 Ca2+Activated K+ Channels Regulate Human Airway Smooth Muscle Proliferation
Am. J. Respir. Cell Mol. Biol., November 1, 2007; 37(5): 525 - 531.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. Xie, M. B. Sukkar, R. Issa, N. M. Khorasani, and K. F. Chung
Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-beta
Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L245 - L253.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Kiyatkin, E. Aksamitiene, N. I. Markevich, N. M. Borisov, J. B. Hoek, and B. N. Kholodenko
Scaffolding Protein Grb2-associated Binder 1 Sustains Epidermal Growth Factor-induced Mitogenic and Survival Signaling by Multiple Positive Feedback Loops
J. Biol. Chem., July 21, 2006; 281(29): 19925 - 19938.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
Y. Liu and B. L. Fanburg
Serotonin-Induced Growth of Pulmonary Artery Smooth Muscle Requires Activation of Phosphatidylinositol 3-Kinase/Serine-Threonine Protein Kinase B/Mammalian Target of Rapamycin/p70 Ribosomal S6 Kinase 1
Am. J. Respir. Cell Mol. Biol., February 1, 2006; 34(2): 182 - 191.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. K. Brown, M. D. Hollenberg, and C. A. Jones
Tryptase activates phosphatidylinositol 3-kinases proteolytically independently from proteinase-activated receptor-2 in cultured dog airway smooth muscle cells
Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L259 - L269.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
P. R. A. Johnson, J. K. Burgess, Q. Ge, M. Poniris, S. Boustany, S. M. Twigg, and J. L. Black
Connective Tissue Growth Factor Induces Extracellular Matrix in Asthmatic Airway Smooth Muscle
Am. J. Respir. Crit. Care Med., January 1, 2006; 173(1): 32 - 41.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
A. J. Halayko, S. Kartha, G. L. Stelmack, J. McConville, J. Tam, B. Camoretti-Mercado, S. M. Forsythe, M. B. Hershenson, and J. Solway
Phophatidylinositol-3 Kinase/Mammalian Target of Rapamycin/p70S6K Regulates Contractile Protein Accumulation in Airway Myocyte Differentiation
Am. J. Respir. Cell Mol. Biol., September 1, 2004; 31(3): 266 - 275.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. A. Pulver, P. Rose-Curtis, M. W. Roe, G. C. Wellman, and K. M. Lounsbury
Store-Operated Ca2+ Entry Activates the CREB Transcription Factor in Vascular Smooth Muscle
Circ. Res., May 28, 2004; 94(10): 1351 - 1358.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.