1 Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611; and 2 Centro de Investigación del Cáncer, Universidad de Salamanca, 37007 Salamanca, Spain
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ABSTRACT |
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Recently it has
been described that dopamine (DA), via dopaminergic type 2 receptors
(D2R), activates the mitogen-activated protein kinase
extracellular signal-regulated kinase (MAPK/ERK) proteins in alveolar
epithelial cells (AEC), which results in the upregulation of
Na+-K+-ATPase. In the present report, we used
AEC to investigate the signaling pathway that links DA with ERK
activation. Incubation of AEC with DA resulted in rapid and transient
stimulation of ERK activity, which was mediated by Ras proteins and the
serine/threonine kinase Raf-1. Pretreatment of AEC with Src
homology 3 binding peptide, which blocks the interaction between Grb2
and Sos, did not prevent DA activation of ERK. Diacylglycerol
(DAG)-dependent protein kinase C (PKC) isoenzymes, involved in the
DA-mediated activation of ERK proteins as pretreatment with
either bisindolylmaleimide or Ro-31-8220, prevented the
phosphorylation of Elk-1, and quinpirole, a D2R activator,
stimulates the translocation of PKC. Together, the data
suggest that DA activated MAPK/ERK via Ras, Raf-1 kinase, and
DAG-dependent PKC isoenzymes, but, importantly and contrary to the
classical model, this pathway did not involve the Grb2-Sos complex formation.
alveolar epithelial cell; mitogen-activated protein kinase/extracellular signal-regulated kinase; protein kinase C
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INTRODUCTION |
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DOPAMINE (DA) regulates ion channels to modulate the excitability of neurons and also activates second messenger systems that in turn activate protein kinase cascades and transcription factors (46). DA, via dopaminergic type 1 receptor (D1R), has been reported to increase lung liquid clearance in normal and injured lungs (2, 3, 47). Recently we also observed that stimulation of dopaminergic type 2 receptors (D2R) in alveolar epithelial cells (AEC) resulted in activation of Na+-K+-ATPase via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) proteins by yet undescribed signaling pathways (19).
The MAPK/ERK cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. Activation of ERK proteins classically involves ligand binding to a receptor tyrosine kinase (22), although ERK proteins can also be activated by G protein-coupled receptors (GPCR), such as the dopaminergic receptors (21, 62). Activated receptors may interact with adaptor proteins, such as Grb2, which in turn, by Src homology 3 (SH3) domain-mediated interaction, bind and recruit the guanine nucleotide exchange factor (GEF) Sos to the plasma membrane in proximity to Ras to exchange the GDP for GTP on Ras proteins (12, 28). Once in the activated GTP-bound state, Ras associates with the serine/threonine kinase Raf-1, which is then activated by phosphorylation (14, 31, 53). Raf-1 phosphorylates and activates the downstream kinase MAPK kinase (MAPKK or MEK), the direct activator of MAPK/ERK proteins. ERK proteins (ERK1/2) have many substrates, including further downstream kinases such as p90rsk (5) and mitogen-activated protein kinase-activated protein kinases (54), as well as transcription factors such as Elk-1 (32, 44). The protein kinase C (PKC) family of proteins also participates in signaling cascades triggered by hormones and growth factors and mediates multiple cellular functions. (35, 38, 39). It has been shown that PKC proteins can activate ERK (23, 58), probably by phosphorylating Raf-1 kinase (31, 53), and mediate processes such as differentiation and proliferation (63, 64).
The PKC family comprises at least three subfamilies based on their
homology and sensitivity to activators (37, 42). Members of the classical subfamily (c-PKC: ,
I,
II, and
) require calcium, phosphoserine, and diacylglycerol (DAG) for activation. Members of the novel subfamily (n-PKC:
,
,
, and
) do not require calcium for activation. Finally, members of the atypical or
a-PKC subfamily,
and
/
, are insensitive to DAG, phorbol esters, and calcium.
DA has an inhibitory role on the ERK pathway in most cell types (40, 60), although recent reports suggest that DA activates ERK proteins in Chinese hamster ovary (CHO) cells (62), neurons (21), and AEC (19). However, the pathways of ERK activation by DA in lung epithelial cells have not been elucidated.
Thus we investigated the pathways involved in DA activation of the ERK proteins in AEC. Our results demonstrate that DA regulates ERK activity via a pathway that is mediated by Ras and Raf-1, but not the classical Grb2-Sos complex, and involves DAG-dependent PKC isoenzymes.
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MATERIALS AND METHODS |
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Materials. Dopamine, epidermal growth factor (EGF), forskolin, phorbol 12-myristate 13-acetate (PMA), and Geneticin (G-418) were purchased from Sigma (St. Louis, MO). Quinpirole was from Research Biochemical International-Sigma (Natick, MA). PD-98059 was purchased from New England Biolabs (Beverly, MA). Adenosine-3',5'-cyclic monophosphothioate, human (h) Sos (1,149-1,158) N10 DH binding domain [Src homology 3 binding peptide (SH3bP)], farnesyl transferase inhibitor (FTI)-I, FTI-II, bisindolylmaleimide, and Ro-31-8220 were from Calbiochem (La Jolla, CA). U-0126 was purchased from Promega (Madison, WI). Ras activation assay kit was purchased from Upstate Biotechnology (Lake Placid, NY).
Cell isolation and culture. Alveolar type II (ATII) cells were isolated from pathogen-free male Sprague-Dawley rats as previously described (41, 45). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase (30 U/ml). The ATII cells were purified by differential adherence to IgG-pretreated dishes and suspended in DMEM containing 10% fetal bovine serum with 2 mM L-glutamine, 400 µg/ml gentamicin, 100 U/ml penicillin, and 100 µg/ml streptomycin. The day of isolation and plating is designated culture day 0. All experimental conditions were tested in day 2 cells.
ERK assay. ATII cells were serum starved for 18-24 h and treated with regulators for the desired time. The ERK activity was determined by two different approaches: phosphorylating the ERK substrate Elk-1, using a p44/p42 MAPK assay kit (New England Biolabs), and measuring the amount of phosphorylated ERK with an antiphospho-ERK antibody (Promega).
Generation of stable cell lines overexpressing ras genes. A549 cells, derived from human AEC (ATCC CCL 185), were transfected with 2-5 µg of plasmid DNA by using SuperFect reagent (Qiagen, Valencia, CA) and selected in the presence of 600 µg/ml of G-418. DNAs used in the transfections included the neo-selectable mammalian expression vector pMEXneo and the pMEXneo-derived construct, pMEXneoH-rasAsn17, coding for the dominant-negative H-ras N17 mutant (4).
Ras activity assay. ATII cells were grown to confluence in 100-mm dishes and serum starved for 20-24 h followed by incubation with DA or EGF in the presence or absence of FTI-I. After treatment, cells were washed twice with cold PBS and lysed in 500 µl of Mg2+ lysis/wash buffer (MLB) containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 1 mM Na3VO4, 25 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Fresh cell lysates were diluted to ~1 µg/µl total cell protein with MLB, and the lysates were precleared with glutathione agarose. After that, 500 µg-1 mg of cell lysate was incubated with 15 µl of Raf-1 Ras-binding domain (RBD) agarose conjugate per assay, and the mixture was gently rocked at 4°C for 30 min. Agarose beads were collected by pulsing, and the supernatant was drained off. The beads were washed three times with MLB, resuspended in appropriate amount of Laemmli sample buffer, and boiled for 5 min. Supernatants were collected and loaded on a 12% SDS-PAGE. The gel was transferred to nitrocellulose membrane and probed with 1 µg/ml of anti-Ras, clone RAS10 (Upstate Biotechnology), overnight at 4°C. A dilution of horseradish peroxidase-conjugated antimouse antibody was used as the secondary antibody, and the enhanced chemiluminescence reagents were used for the final protein detection.
Measurement of Raf-1 kinase activity. ATII cells were serum starved for 20-24 h before stimulation with DA (10 µM) for 10 min or with 200 ng/ml of EGF for 20 min. Raf-1 kinase activity was determined using the method described elsewhere (50), based on the phosphorylation of syntide-2 (Santa Cruz Biotechnologies, Santa Cruz, CA), a Raf-1 substrate (25).
Subcellular fractionation.
Serum-starved cells were treated with different agonists and
antagonists at the indicated times. Cells were washed twice with ice-cold PBS and scrapped into homogenization buffer containing 20 mM
Tris · HCl, pH 7.4, 2 mM EGTA, 2 mM EDTA, 1 mM PMSF, 10 mM
-mercaptoethanol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. After a 10-min incubation, cells were homogenized with 30 strokes of a
Dounce homogenizer using a tight-fitting pestle. Nuclei were pelleted
by centrifugation at 500 g for 5 min, and the low-speed supernatant was centrifuged at 100,000 g for 30 min. The high-speed supernatant constituted the cytosolic
fraction. The pellet was washed three times and extracted in ice-cold
homogenization buffer containing 1% Triton X-100 for 1 h. The
Triton-soluble component (membrane fraction) was separated from the
Triton-insoluble material (cytoskeletal fraction) by centrifugation at
100,000 g for 15 min. The cytoskeletal fraction was washed
three times with homogenization buffer, resuspended in the same buffer,
and dispersed by sonication.
PKC translocation. Serum-starved cells were treated with the corresponding agonist at the indicated times and analyzed by Western blotting, after subcellular fractionation was performed to study the distribution of the different PKC isoenzymes in the cytosolic and membrane fractions.
Statistical analysis. Data are represented as means ± SE. When multiple comparisons were made, a one-way analysis of variance was used, followed by a multiple comparison test (Tukey) when the F statistic indicated significance. Results were considered significant when P < 0.05.
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RESULTS |
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DA activates MAPK/ERK in ATII cells.
Figure 1 shows that incubation of
subconfluent, serum-starved ATII cells with 10 µM DA induced a two-
to fourfold increase in ERK activity, with peak activity at 5 min,
returning to basal values by 10 min. This activation was abolished when
cells were pretreated with the specific MEK inhibitors PD-98059 (Fig.
1, A and C) and U-0126 (Fig. 1, B and
C) 1 h before DA stimulation. On the basis of these
results, we stimulated cells with 10 µM DA for 5 min in the
subsequent experiments.
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Ras proteins participate in the stimulation of the ERK activity by
DA in AEC.
To investigate whether Ras proteins mediate the activation
of ERK by DA in AEC, three different approaches were used:
1) permanent transfectants of A549 cells expressing
pMEXneoH-rasAsn17, a neomycin-selectable
dominant-negative ras mutant, were used to study the
activation of ERK by DA. DA-mediated activation of ERK in A549 cells
has been demonstrated to be comparable with ERK activation in ATII
cells (5). Figure
2A shows that RasN17 blocked
the DA-mediated ERK activation. 2) To further demonstrate the involvement of Ras proteins in the DA-ERK pathway, serum-starved ATII cells were pretreated with two FTI, FTI-I and FTI-II, before DA
stimulation. Figure 2B shows that both inhibitors blocked
the stimulatory effect of DA on ERK. 3) Finally we performed
a Ras activation assay based on the method by Taylor and Shalloway
(55), which uses the known specificity of the interaction
between Ras-GTP and the RBD of Raf-1. Results in Fig. 2C
show that treatment of serum-starved ATII cells with 10 µM DA for 5 min increased by 2.5-fold the affinity precipitation of Ras-GTP by the
agarose-conjugated RBD. We used EGF as a positive control of Ras
activation and FTI as negative regulators of Ras activation.
These experiments support the conclusion that Ras proteins participate
in the DA-mediated ERK activation in AEC.
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The complex Grb2-Sos does not participate in the DA-Ras-ERK
pathway.
To determine whether DA-mediated ERK activation involved the Grb2-Sos
complex formation, we used SH3bP, a peptide corresponding to the SH3
binding sequence of the Ras GEF hSos
([1,149-1,158], N10 DH binding domain), which has a
strong affinity for the NH2-terminal SH3 domain of the
adapter protein Grb2, blocking the interaction Grb2-Sos. Figure
3 shows that preincubation of
serum-starved ATII cells with SH3bP did not block DA-mediated ERK
activation. Under the same conditions, ERK activation by EGF was
completely blocked by SH3bP. These results suggest that the activation
of ERK proteins by DA in ATII cells was mediated by Ras proteins that
are activated by a mechanism that does not involve the classical
Grb2-SOS complex.
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Raf-1 kinase is activated by DA and participates in the DA-ERK
pathway.
Forskolin has been described to be an activator of the enzyme
adenylcyclase, which, via cAMP, activates the cAMP-dependent protein
kinase A (PKA). In turn, PKA phosphorylates and inactivates Raf-1
kinase, probably by reducing the apparent affinity by which Raf-1 binds
to Ras (11). To determine whether Raf-1 participates in
the DA-ERK pathway, we treated serum-starved ATII cells with forskolin
(50 µM) or PKA inhibitor adenosine-3',5'-cyclic monophosphothioate (PKA-I; 10 µM) before DA stimulation. The concentration of forskolin used was based on its IC50 value (25 µM for MAPK
inhibition); the concentration of PKA-I chosen (11 µM) was based on
its Ki value. As shown in Fig.
4A, forskolin inhibited the
DA-dependent ERK activity. Also, inhibition of PKA further increased
the stimulatory effect of DA on ERK activation. These results suggest
that Raf-1 proteins participate in the DA-ERK pathway.
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Stimulation of ERK by DA requires PKC proteins.
To investigate whether PKC is a component of the DA-ERK signal
transduction pathway, we treated serum-starved ATII cells with 1 µM
bisindolylmaleimide, a PKC inhibitor, 15 min before DA stimulation. We
observed that this inhibitor completely blocked ERK activation induced
by DA as well as by the PKC activator PMA, used as a positive control
of PKC-mediated ERK activation (56). These results suggest that the DA-mediated ERK activation is dependent on PKC activity (Fig.
5). To determine which specific PKC
isoforms are involved in this pathway, we treated starved ATII cells
with Ro-31-8220, which specifically inhibits DAG-dependent PKC isoforms
(c-PKC, n-PKC). This inhibitor blocked the DA-dependent ERK activation in a way similar to that observed with bisindolylmaleimide, suggesting the involvement of classical and/or novel PKC isoforms (Fig.
6). Recently we showed that DA activates
ERK via D2R (19). To determine whether
atypical PKC isoforms participate in the DA-ERK pathway, ATII cells
were serum starved and depleted of classical and novel PKC isoforms by
incubation with PMA for 24 h. After this treatment, cells were
treated with quinpirole, a D2 agonist, or PMA. As shown in
Fig. 7, quinpirole did not activate ERK
proteins in depleted cells, which indicates that atypical PKC
isoenzymes are not involved in the DA-mediated ERK activation in ATII
cells.
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DISCUSSION |
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The major findings of the present study are that DA regulates ERK activity in AEC via Ras, Raf-1, and novel PKC proteins, but not the classical Grb2-SOS complex.
This study expands on our report that demonstrated the role of the DA-ERK pathway in the regulation of Na+-K+-ATPase in the alveolar epithelium (19). DA regulates Na+-K+-ATPase in the short term in AEC via D1R, whereas D2R appear to regulate the Na pump long term. Contrary to D2R, which activate ERK in AEC, D1R do not increase (as they appear to inhibit) ERK activity in AEC. Thus it is possible that there may be even an antagonistic effect of both pathways on ERK activation.
DA and other catecholamines have been shown to regulate ERK activity in different cells (13, 21, 40, 60, 62). Most of the studies regarding regulation of ERK by DA have been in cells of neuronal origin, where DA activates ERK proteins via a pathway that involves D2R, pertussis toxin-sensitive G proteins (30, 62), and Ras proteins (1, 30), but this is the first report that describes the pathways of ERK stimulation by DA in AEC. Results in Fig. 1 suggest that the activation of ERK by DA in AEC is transient, similar to most systems (22, 26), which may suggest the participation of protein phosphatases (PP) in the regulation of ERK activity. There is evidence that PP may play a role in our system. First, DA, via D1R, activates the serine/threonine phosphatase PP2A in AEC (27). Second, the dual-specificity phosphatases, a subclass of the protein tyrosine phosphatase family that dephosphorylates the phosphothreonine and phosphotyrosine residues within MAPK, are an important regulatory mechanism of the ERK function (10). Further work is warranted to determine which specific phosphatases participate in the DA-ERK pathway in AEC.
Sos proteins are ubiquitously expressed Ras-GEF that are recruited to the plasma membrane by the docking protein Grb2 and mediate Ras activation, via tyrosine kinase receptors (12) and GPCR (13). Activation of Ras has been shown to result in the activation of ERK proteins (8). As shown in Fig. 2, our data suggest that Ras participates in the DA-ERK pathway. First, a ras dominant-negative mutant (ras N17) inhibited the effect of DA on ERK activation in a permanent, transfected AEC line. Second, two different inhibitors of the farnesyl-transferase enzyme (FTI-I and FTI-II), necessary for the attachment to the membrane and subsequent activation of Ras proteins (49), blocked the stimulatory effect of DA on ERK activation. Third, results shown in Fig. 2C provide a direct demonstration that DA activates Ras by increasing the levels of the GTP-bound form, strongly supporting the involvement of Ras in the DA-ERK pathway.
The inhibitory effect of the ras N17 on the stimulation of ERK by EGF, a known activator of the Grb2-Sos-Ras-ERK cascade (33), is of a lesser magnitude than that on the stimulatory effect of DA (see Fig. 2). A possible explanation is that the activation of ERK by DA is weaker compared with the activation by EGF and can be abolished by the dominant-negative ras mutant, which only partially inactivated the stronger signal induced by EGF. Also, it has been shown that, in some cell types, EGF can bypass Ras and Raf-1 and directly activate MEK (7, 18). Nevertheless, the validity of EGF as a positive control of ERK activation via GRb2-Sos-Ras is justified by the results in Figs. 2, B and C, and 3, which show that 1) FTI blocked the activation of ERK by EGF; 2) EGF increased Ras-GTP-bound form; and 3) EGF-dependent ERK activation was inhibited by SH3bP, although this peptide did not inhibit the stimulatory effect of DA on ERK activity.
Sos proteins are ubiquitous Ras-GEFs that activate Ras by exchanging the GDP, bound to inactive Ras, for GTP that binds to and activates Ras (12, 28). Although Sos proteins are the most common Ras-GEFs, Ras proteins can also be activated by other GEFs, such as Ras-guanine nucleotide-release factor (GRF) (17) and the recently described Ras-guanyl nucleotide-releasing protein (GRP), regulated by DAG and calcium (16). Both Ras-GRF and Ras-GRP have been described to be Ras activators specific to the brain (51), although a recent report showed that Ras-GRF is also abundantly expressed in lung tissue (20). Additionally, it has been shown that Ras-GRF, which does not bind to the adaptor Grb2, has the ability to mediate Ras activation by signals mediated through GPCR (52).
DA activates ERK via Raf-1 kinase. cAMP exerts opposite effects on cell proliferation depending on the cell type. For example, cAMP inhibits MAPK activation in Rat-1 cells, smooth muscle cells, CHO cells, and adipocytes and stimulates the MAPK pathway in COS, PC12, and Swiss 3T3 cells (18). cAMP activates PKA, which inhibits Raf-1 kinase by phosphorylation on Ser43 and Ser259. Phosphorylation of Raf-1 in these residues may interfere with binding of Ras to its amino terminus and hence with kinase activation (50). In all cell types, cAMP inhibits Raf-1 kinase, even in the cells where cAMP activates ERK. In these cases, cAMP is able to bypass Ras and Raf-1 and directly activate MEK (18). Only in one report, thyroid-stimulating hormone (TSH), the physiological regulator of thyroid epithelial cells, stimulates proliferation in Wistar rat thyroid cells through a cAMP pathway that involves Ras activation (57), although another study in primary dog thyrocytes demonstrates that TSH and cAMP do not signal through Ras activation (59). Thus we used forskolin, an activator of the cAMP-PKA pathway to study the involvement of Raf-1 kinase in the DA-mediated ERK activation in AEC, on the basis of the data by Schramm and collaborators (50).
The data depicted in Fig. 4 suggest that Raf-1 kinase participates in the DA-ERK pathway. First, DA-dependent ERK activity was inhibited by forskolin. Second, inhibition of PKA further increased the DA-mediated ERK activation, a result that is in agreement with the inhibitory effect of the cAMP-PKA pathway on ERK activation. Third, DA stimulated Raf-1 kinase activity, measured as phosphorylation of syntide-2. Additionally, results in Fig. 4B further confirm that the activation of Raf-1 kinase and subsequently ERK by DA is independent on the Grb2-Sos complex.
PKC proteins control many ATII cell functions, including regulation of surfactant phospholipid secretion (29, 48), modulation of arachidonic acid metabolism (43), or stimulation of ion transporters (61). Nevertheless, there are no studies linking PKC proteins to ERK activation in ATII cells, although a recent report has described the participation of PKC proteins in ERK activation in A549 cells (34).
It is generally accepted that stimulation of GPCR activates MAPK/ERK via the PKC pathway (5, 6, 36). On the other hand, it has been shown that PKC proteins are able to phosphorylate and activate Raf-1 kinase in cooperation with Ras (31, 53), and a more recent report demonstrates the role of DAG-regulated PKCs in the activation of Raf-1 (9).
Results shown in Fig. 5 suggest that PKC proteins are essential in the
DA-ERK pathway in ATII cells, as inhibition of PKC by
bisindolylmaleimide, which competitively inhibits the ATP-binding site,
completely blocked ERK activation by DA. Moreover, Ro-31-8220, an
inhibitor of the DAG-dependent PKC isoforms (15, 24), also blocked DA-mediated ERK activation (Fig. 6), which suggests that classical and/or novel PKCs, but not atypical PKC isoenzymes, are the
mediators of this effect. Data depicted in Fig. 7 further support this
reasoning, as inhibition of DAG-dependent PKC isoforms by prolonged
incubation with PMA inhibits the activation of ERK by quinpirole, a
D2R agonist. Quinpirole has the same effect as DA in
activating ERK in ATII cells; we and others have shown that DA
activates ERK proteins via D2R (19, 62).
Finally, data depicted in Fig. 8 suggest that PKC- is activated by
D2R in the time frame of ERK activation and thus may
participate in the DA-mediated ERK activation.
In summary, these data suggest that in AEC, DA regulates ERK proteins
via D2R, Ras, Raf-1, and novel PKC isoenzymes, but not the
classical Grb2-Sos complex (see Fig. 9).
Future studies are warranted to determine the Ras-GEF that is
responsible for DA-mediated Ras activation, as well as the components
that signal from DA to PKC in the activation of ERK1/2 proteins in the
alveolar epithelium.
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ACKNOWLEDGEMENTS |
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We thank Dr. Aparajita Ghosh for technical support and Dr. Eugenio Santos for providing us with the pMEXneoH-rasN17 construct and for valuable discussions.
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FOOTNOTES |
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This research was supported in part by National Heart, Lung, and Blood Institute Grant HL-65161 and by the American Lung Association of Metropolitan Chicago.
Address for reprint requests and other correspondence: J. I. Sznajder, Pulmonary & Critical Care Medicine, Northwestern Univ., 300 E. Superior St., Tarry Bldg. 14-707, Chicago, IL 60611 (E-mail: j-sznajder{at}northwestern.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.
10.1152/ajplung.00178.2001
Received 17 May 2001; accepted in final form 17 December 2001.
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