1 Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104; and 2 Department of Radiation Oncology, Stritch School of Medicine, Loyola University Medical Center, Maywood, Illinois 60153
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ABSTRACT |
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Elucidating the factors that inhibit
the increase in airway smooth muscle (ASM) mass may be of therapeutic
benefit in asthma. Here, we investigated whether interferon-
(IFN-
), a potent inducer of growth arrest in various cell types,
regulates mitogen-induced ASM cell proliferation. IFN-
(1-100
U/ml) was found to markedly decrease both DNA synthesis and ASM cell
number induced by the mitogens epidermal growth factor (EGF) and
thrombin. Interestingly, IFN-
had no effect on mitogen-induced
activation of three major mitogenic signaling pathways,
phosphatidylinositol 3-kinase, p70S6k, or mitogen-activated
protein kinases. Mitogen-induced expression of cell cycle regulator
cyclin D1 was increased by IFN-
, whereas no effect was observed on
degradation of p27Kip1. Expression array analysis of 23 cell cycle-related genes showed that IFN-
inhibited EGF-induced
increases in E2F-1 expression, whereas induction of c-myc,
cyclin D2, Egr-1, and mdm2 were unaffected. Induction of E2F-1 protein
and Rb hyperphosphorylation after mitogen stimulation was also
suppressed by IFN-
. In addition, IFN-
decreased activation of
cdk2 and expression of cyclin E, upstream signaling molecules
responsible for Rb hyperphosphorylation in the late G1 phase. IFN-
also increased levels of IFI 16 protein, whose mouse homolog p202 has
been associated with growth inhibition. Together, our data indicate
that IFN-
is an effective inhibitor of ASM cell proliferation by
blocking transition from G1-to-S phase by acting at two different
levels: modulation of cdk2/cyclin E activation and inhibition of E2F-1
gene expression.
cytokines; airway remodeling; signal transduction; airway smooth muscle hyperplasia
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INTRODUCTION |
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AIRWAY REMODELING, characterized by increases in airway smooth muscle (ASM) mass, due in part to ASM hyperplasia, was one of the earliest findings described in the pathology of patients with chronic severe asthma (31). The mechanisms that regulate cell mitogenesis in ASM cells remain unknown, but we showed that activation of the mitogen-activated protein kinase (MAPK) family, phosphatidylinositol 3-kinase (PI3K), and p70S6k is necessary for transducing growth signals in response to mitogens such as epidermal growth factor (EGF) and thrombin (31). Although considerable research effort has focused on the cellular and molecular mechanisms that stimulate smooth muscle cell proliferation, few studies have examined the factors that suppress ASM cell proliferation. This is an important question since no therapy is currently available for the treatment of ASM hyperplasia in asthma.
Previous studies in human ASM cells showed that interferon-
(IFN-
), a potent lymphokine secreted by activated T cells, regulates many cellular responses. This includes the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1
(22), secretion of chemokines interleukin (IL)-8 and
RANTES (14, 15), and induction of cyclooxygenase
2 (35) or CysLT1 receptor (4), showing that
IFN-
may participate in the pathogenesis of asthma by exhibiting
immunomodulatory properties (42). Because of its ability
to inhibit cell growth in various cell types, IFN-
may also serve as
a potential antiproliferative agent in ASM cells, although no studies
have yet investigated such an interesting hypothesis. Both in
transformed and in nontransformed cells, the antiproliferative effects
of IFN-
include a blockade in G1 phase of the cell cycle, although
the precise underlying mechanisms are still controversial. In primary
cells, i.e., mammary epithelial cells or in macrophages, IFN-
induced G1 arrest by modulating the expression of cell cycle inhibitors
p21Cip1 and p27Kip1 (12, 45). In
transformed tumor cell lines, the mechanisms of cell cycle arrest by
IFN-
seem to involve cell type-specific pathways that are
p21Cip1 dependent, p27Kip1 dependent
(17), and p27Kip1 independent
(44). Finally, a previous study in bronchial
epithelial cells reported that IFN-
-induced G1 arrest was associated
with a decreased expression of the transcription factor E2F-1 proteins (38). In noncycling cells, E2F-1 proteins repress E2F
target genes because of the presence of retinoblastoma protein (Rb), which acts as a transcriptional repressor. After mitogen stimulation, release of Rb by phosphorylation leads to the derepression of gene
transcription by E2F-1, which in turn regulates many growth-promoting and growth-responsive genes such as thymidine kinase and DNA
polymerase-
(20, 26). Together, these studies show that
the effect of IFN-
on cell growth is complex and cell specific.
In this study, we show that IFN- inhibits both EGF- and
thrombin-induced human ASM cell proliferation without affecting
critical signaling events thought to be necessary to mediate ASM
mitogenesis, such as the signaling pathways PI3K or p70S6k
or the cell cycle regulators cyclin D1 and p27Kip1. Cell
cycle expression array showed that IFN-
specifically inhibited upregulation of E2F-1 and proliferator cell nuclear antigen (PCNA), important G1 regulatory proteins. IFN-
also abrogated
mitogen-induced expression of E2F-1 proteins and Rb
hyperphosphorylation and decreased the activation of cdk2 and
expression of cyclin E, upstream regulatory proteins regulating Rb
hyperphosphorylation. In addition, IFN-
increased IFI 16, a member
of the p200 family that inhibits cell proliferation via the interaction
with cell cycle regulators (6). Our study identifies
IFN-
as an effective inhibitor of ASM cell proliferation, an effect
that specifically involves at least two different pathways, i.e., the
modulation of cell cycle regulator E2F-1 expression and a reduction of
Rb hyperphosphorylation, possibly via the modulation of cyclin E/cdk2 activation.
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MATERIALS AND METHODS |
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Cell culture. Human tracheal tissue culture was obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. The culture of human ASM cells was performed as described elsewhere (33).
Thymidine incorporation assays. DNA synthesis was measured with a thymidine incorporation assay, as described previously (33). Cells were growth arrested by incubating the cultures in serum-free medium consisting of Ham's F-12 medium with 5 ng/ml insulin and 5 ng/ml transferrin. After 48 h in serum-free medium, the cells were stimulated with either 10 ng/ml EGF, 10% FBS, or 1 U/ml thrombin. After 16-18 h of stimulation, human ASM cells were labeled with 3 µCi/ml [methyl-3H]thymidine (40-60 Ci/mmol; Amersham Pharmacia, Arlington Heights, IL) for 24 h. The cells were then scraped and lysed, and the protein or DNA was precipitated with 10% trichloracetic acid. The precipitant was aspirated on glass filters and extensively washed, dried, and counted.
Cell counting experiments. Cell count assays were performed as described previously (34). Briefly, ASM cells were lifted with 0.5% trypsin-EDTA solution and counted in triplicate using the Coulter Z1 (Beckman Coulter, Miami, FL). The data represented as the percent change from basal (i.e., unstimulated cells) are means ± SE from a minimum of three flasks from triplicate experiments.
p70S6K and PI3K activities. An in vitro activity assay of p70S6K and PI3K was performed as described previously (18, 19).
SDS-PAGE and Western blot analysis.
Immunoblot analyses of cyclin D1, p27Kip1, IFI 16, cyclin
E, and cdk2 (both phosphorylated and nonphosphorylated forms) using whole cell lysates were performed as described previously (1, 2,
21). E2F-1 and Rb immunoblot analysis was performed using total
nuclear protein extracts and HeLa cell lysates as positive controls
(Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, ASM cells were
lysed in buffer containing 10 mM Tris · HCl (pH 7.6), 60 mM KCl, 1 mM EDTA, 1 mM NaF, 0.1 mM sodium orthovanadate, 1 mM
PMSF, 10 mM -glycerophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1% Triton X-100. Nuclei were separated from the cytoplasm by centrifugation at 2,500 rpm for 5 min, washed briefly, resuspended in nuclear extract buffer (20 mM
Tris · HCl, pH 8, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol), lysed by adding NaCl to a final
concentration of 400 mM, incubated on ice for 20 min, vortexed, and
centrifuged for 10 min at 14,000 rpm. The supernatant contains total
nuclear protein extract.
Gene expression analysis.
Cell cycle gene expression profiling was performed using an GEAarray
kit that contains a set of two reusable nylon membranes (3 times) with
cDNA fragments from 23 critical genes associated with cell cycle
regulation (SuperArray, Bethesda, MD). Briefly, 5 µg total RNA
extracted from human ASM cells using an RNeasy mini kit (Qiagen) were
used as the template for biotin-labeled cDNA probe synthesis using the
following reagents: Biotin-16-dUTP (200 µM; Boehringer Mannheim),
Moloney murine leukemia virus RT (5 U/µl; Promega), and RNase
inhibitor (4 U/µl; Promega). All subsequent array hybridation and
chemiluminescent detection procedures were performed according to the
manufacturer's protocol using reagents provided in the GEarray kit
except for the 20× SSC (AccuGENE; Biowhittaker). Densitometric
measurements of scanned membrane were performed using the Kodak 1D
image analyzer software (Rochester, NY). The densitometric values were
then calculated by normalizing the signal of each transcript (in
duplicate) against the signals of the housekeeping genes GAPDH (average
signal of 6 per membrane) and -actin (average of 2 per membrane).
Normalized values for each gene were then used to make comparisons
between membranes.
RT-PCR analysis. Total RNA was extracted from human ASM cells using an RNeasy mini kit (Qiagen) according to the manufacturer's instructions. RT-PCR reactions were performed using E2F-1 and GAPDH primers for semiquantitative analysis, as previously described (3). The E2F-1 primers for PCR analysis were 5'-TGACCTGCTGCTCTTCG-3' (sense) and 5'-GTTCAGGTCGACGACAC-3' (antisense; see Ref. 25). Each of 30 cycles of the PCR was programmed to carry out denaturation at 94°C for 30 s, primer annealing at 55°C for 45 s, extension at 72°C for 45 s, and a final extension at 72°C for 10 min. The semiquantitative PCR approach of E2F-1 mRNA was performed in parallel by investigating human GAPDH mRNA levels with the following primers: 5'-ATGGATGATGATATCGCCGC-3' (sense) and 5'-TTAATGTCACGCACGATTTC-3' (antisense), as described previously (4).
Data analysis. Data points from individual assays represent the mean values of triplicate measurements. Significant differences among groups were assessed with ANOVA (Bonferroni-Dunn test) or by t-test analysis, with values of P < 0.05 sufficient to reject the null hypothesis for all analyses. Each set of experiments was performed with a minimum of three different human ASM cell lines.
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RESULTS |
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IFN- inhibits EGF- and thrombin-induced DNA synthesis.
To determine the effects of IFN-
on mitogen-induced DNA synthesis in
human ASM cells, cell monolayers were stimulated with 10 ng/ml EGF or 1 U/ml thrombin in the presence of a specified dose of IFN-
or diluent
alone. The doses of EGF and thrombin used were those that maximally
induce DNA synthesis, as previously described (32).
Although IFN-
alone had no effect on thymidine incorporation,
IFN-
inhibited thrombin- and EGF-induced DNA synthesis in a
dose-dependent manner (Fig. 1, A and
B). Maximal inhibition of EGF-
and thrombin-induced DNA synthesis occurred with 10 U/ml IFN-
; this
dose inhibited thrombin- and EGF-induced DNA synthesis by 60 ± 8 and 85 ± 5%, respectively. To confirm that IFN-
inhibited cell proliferation, cell-counting assays were performed with cells treated with EGF or thrombin in the presence or absence of IFN-
. As
shown in Fig. 2, EGF or thrombin
increased cell number by 20 ± 2.77 and 13 ± 1.8%,
respectively. These growth responses were almost completely inhibited
in the presence of IFN-
(10 U/ml). IFN-
had no effect of
apoptosis of ASM, as determined by caspase staining (data not
shown). Collectively, these data suggest that IFN-
inhibits human
ASM cell proliferation stimulated by the activation of both tyrosine
kinase receptor (EGF) and G protein-coupled receptors (thrombin).
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IFN- does not inhibit EGF- or thrombin-induced PI3K or p70S6K
activation.
Current evidence suggests that PI3K and p70S6 kinase, a PI3K downstream
effector, play critical roles in mediating mitogen-induced human ASM
cell growth (24, 25). We therefore studied the effect of
IFN-
on either mitogen-induced PI3K or p70S6 kinase activation. As
shown in Fig. 3A, EGF and
thrombin activated PI3K, as previously reported (19).
IFN-
had little effect on basal PI3K activity and on EGF- or
thrombin-induced PI3K activation. Figure 3B shows that p70S6
kinase activation at basal levels is low and that IFN-
appears to
have little effect on basal p70S6k activation. EGF or
thrombin markedly activates p70S6 kinase. Neither EGF- nor
thrombin-induced p70S6 kinase activity was affected by pretreating the
cells with 100 U/ml IFN-
. These data suggest that the inhibitory
effect of IFN-
on cell growth occurs independently of the activation
of these critical signaling molecules.
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Mitogen-induced expression of cyclin D1 and degradation of
p27Kip1 are not affected by IFN-.
Mitogen stimulation of ASM cell growth increases expression of cyclin
D1 and degradation of the cell cycle inhibitor p27Kip1
(1, 21). To address whether the effects of IFN-
on
human ASM are mediated by inhibition of cyclin D1 expression or
p27Kip1 degradation, cells were treated with EGF or
thrombin in the presence or absence of indicated doses of IFN-
. As
shown in Fig. 4, cyclin D1 levels in the
basal state were low, and, after stimulation with EGF or thrombin,
there was a marked increase in cyclin D1 expression. Interestingly,
IFN-
(100 U/ml) induced a slight but significant increase in cyclin
D1 expression and even enhanced by 28% the response induced by EGF
(Fig. 4, A and B) and by thrombin (data not
shown). We also studied the effect of IFN-
on the expression of
p27Kip1. Basal levels of p27Kip1 were high,
and, after stimulation with EGF, there was a marked degradation in
p27Kip1 that was not prevented by treatment with IFN-
(Fig. 4, C and D). These data suggest that
IFN-
exerts unexpected effects on cell cycle modulators, i.e.,
enhancing cyclin D1 expression and not preventing degradation of
p27Kip1, suggesting that inhibition of ASM mitogenesis by
IFN-
is not regulated at the level of cyclin D1 expression or
p27Kip1 degradation.
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IFN- modulates EGF-induced gene expression of various cell cycle
regulators.
To further examine the molecular mechanisms by which IFN-
regulates
cell mitogenesis, we used commercially available expression arrays to
determine if mRNA levels obtained from cells treated with EGF (10 ng/ml) were modulated in the presence of IFN-
(100 U/ml). Cell cycle
gene expression arrays contain cDNA fragments from 23 important cell
cycle regulatory genes that include cyclins (A, B, C, D2, D3, E), cdks
(1, 2, 4, and 6), transcription factor E2F-1 and cell cdk inhibitors
(p19, p21, p57), and other genes such as Rb and PCNA. Analysis of gene
expression array revealed three classes of genes based on their
expression as per ASM cell stimulation with EGF and/or IFN-
. Genes
whose expression was not affected by EGF and IFN-
included those
encoding for cdks (cdk1, cdk4, and cdk6), cyclins (A, B1, C), Gadd45 (a
maker of DNA damage) and cdk inhibitors (p19, p57), and p53. As
expected, EGF significantly increased other genes critical for
promoting cell progression in other cell types, such as
c-myc protooncogene (3.8-fold), cyclin D2 (7.7-fold), cyclin
E (1.6-fold), PCNA (negligibly expressed in basal conditions), the
transcription factor egr-1 (20-fold), E2F-1 (6.22-fold), and the E3
ligase mdm2 (5-fold), an inhibitor of the tumor suppressor p53.
Interestingly, EGF also induced the expression of p21Cip1
(4.78-fold), which is a potent cell cycle inhibitor. Among these, IFN-
suppressed EGF-induced expression of E2F-1 by 66% and PCNA by
79%. Because expression of E2F transcription factors are critically important for regulating genes that regulate G1-to-S transition (26), these data suggest that IFN-
abrogated
mitogen-induced cell cycle progression potentially by decreasing E2F-1
gene expression.
Mitogen-induced phosphorylation of Rb and expression of E2F-1 is
suppressed by IFN-.
In noncycling cells, hypophosphorylated Rb functions as a cell cycle
repressor by interacting with E2F proteins that inhibit E2F-mediated
gene transcription. Mitogen-induced hyperphosphorylation of Rb releases
E2F-1 protein, which in turn initiates cell cycle progression by
activating many growth-responsive and -promoting genes
(26). We found that EGF induced Rb hyperphosphorylation in
a time-dependent manner, an effect that was almost completely inhibited
by IFN-
at 48 h (Fig. 5). Similar
results were also observed in cells stimulated with thrombin (data not
shown). We also investigated whether IFN-
modulates mitogen-induced
E2F-1 expression. EGF significantly increases levels of E2F-1 total mRNA (Fig. 6A) and E2F-1
protein (Fig. 6B). Those responses were significantly
decreased by IFN-
(Fig. 6, A and B). Together, these data suggest that suppression of E2F-1 expression, probably because of a blockade of Rb hyperphosphorylation, represents one potential mechanism by which IFN-
suppresses mitogen-induced ASM
proliferation.
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IFN- decreases EGF-induced phosphorylation of cdk2 and
expression of cyclin E.
Maintaining Rb in a hyperphosphorylated state is ensured by the
activation of cdk2 by cyclin E, synthesized in the late G1 phase.
Activation of cdk2 occurs in two steps, first upon binding of cyclin E
and second upon phosphorylation of Thr160 by a
cdk-activating kinase required for complete activation of cdk2
(36). Interestingly, we found here that IFN-
significantly reduced EGF-induced cdk2 phosphorylation at
Thr160 by 60%, whereas little effect was observed on
levels of total cdk2 protein (Fig. 7, A and
B). In addition, IFN-
reduced cyclin E levels induced by EGF by 72% (Fig. 7, C
and D). These data strongly suggest that IFN-
may
decrease EGF-induced Rb hyperphosphorylation by possibly modulating the
cdk2/cyclin E complex.
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IFN- increases IFI 16 expression.
Because IFN-
inhibited cell growth, we tested whether IFN-
increased levels of IFN-inducible protein IFI 16, a human homolog of
the mouse p202 family that exhibits strong antimitogenic effects via
the modulation of cell cycle regulators (6). IFI 16 contains three different-sized isoforms called A, B, and C (molecular
mass from 85 to 95 kDa) that result from alternative RNA splicing
(16). SDS-PAGE analysis of cell lysates from HeLa cells
that constitutively express all three isoforms showed that IFI 16 A is
the slowest-migrating isoform and IFI 16C is the fastest one. As shown
in Fig. 8, we found that ASM cells
predominately express IFI 16 B in basal conditions, whereas induction
of all IFI 16 isoforms was observed after 24 h of stimulation with
IFN-
. Similar observations were observed in two different cell
lines. This observation is consistent with the notion that increased
levels of IFI 16 may contribute to IFN-
-mediated inhibition of ASM
cell growth. This is the first demonstration in nontransformed cells
that IFN-
increases IFI 16 expression.
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DISCUSSION |
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Significant efforts have been aimed at defining the cellular and
molecular mechanisms that regulate ASM cell growth (31). In the current study, we show that IFN- is a novel and effective inhibitor of ASM mitogenesis induced by stimulation of either growth
factors (EGF) or contractile agonists (thrombin). The antiproliferative effect of IFN-
does not involve the modulation of three major mitogenic signaling pathways (PI3K, MAPK, and p70S6 kinase) thought to
be important in stimulating ASM cell proliferation. Interestingly, IFN-
modulates mitogen-induced Rb hyperphosphorylation and E2F-1 expression, key events that initiate cell cycle progression.
The ability of IFN- to modulate mitogen-induced ASM cell
proliferation is an important observation, since only a few studies have examined extracellular stimuli that inhibit ASM proliferation. Previous evidence showed that other cytokines such as tumor necrosis factor-
and IL-1
modulate the growth of ASM cells (5, 41, 46), although the underlying mechanisms were not investigated. Here, we found that IFN-
had no effect on mitogen-induced PI3K or
p70S6 kinase, two major pathways that are critically important in
integrating growth signals in ASM cells (18, 19). In some cell types, PI3K, however, seems to play an essential role in mediating
IFN-
-induced cellular responses. For example, PI3K mediates STAT-1
activation and gene expression induced by IFN-
in T98G glioblastoma
cells (27) or participates in IFN-
-induced cellular
protection from viral infections in mouse fibroblasts (37). In addition, activation of PI3K by IFN-
may
regulate integrin-dependent migration in B cells (10). Our
present study shows that IFN-
ablates mitogen-induced ASM cell
proliferation possibly by modulating targets downstream or parallel to
PI3K or p70S6 kinase. In addition, IFN-
was unable to prevent
mitogen-induced ERK1/2 activation (Y. Amrani, personal observation),
a pathway that was also regarded to be important in
mitogen-induced ASM mitogenesis (28-30).
In several cell types, IFN- is antiproliferative and, most commonly,
induces G1 cell cycle arrest. Cell cycle progression is usually
promoted by a family of cyclins and their specific partners,
cyclin-dependent kinases (CDK). Critical events during the G1-to-S
transition are induction of D-type cyclins (D1-3) and a decrease
in CDK inhibitors, such as p21Cip1, p27Kip1,
and p57Kip2 (20, 26). Reports showed that
IFN-
increases the CDK inhibitor p21Cip1
(45) or reduced cell proliferation by impairing the
downregulation of p27Kip1 (12, 23). The
observation that IFN-
greatly reduces mitogen-induced DNA synthesis
strongly suggests that IFN-
may interfere with the progression of
ASM cells through the cell cycle by preventing transition from G1 to
the S phase. In agreement with previous reports (1, 9,
21), we found that cyclin D1 upregulation and
p27Kip1 degradation were key events associated with
mitogenesis in ASM cells. Such responses, however, were not modulated
by IFN-
. Instead, we found that IFN-
alone increased cyclin D1
levels, enhanced mitogen-induced increased expression of cyclin D1, and
do not prevent mitogen-induced degradation of p27Kip1
protein levels. The latter observation interestingly contrasts with
studies that showed that upregulation of p27Kip1 by IFN
inhibits cell growth (12, 23). However, the differential effect of IFNs on p27Kip1 expression seems to be cell
specific, since previous studies in human mesothelioma cell lines
(44) or in H82, a lung cell carcinoma (24),
showed that IFNs decreased p27Kip1 content, although the
underlying mechanisms of such differential modulation are not known.
Similarly, evidence also shows that induction of D-type cyclins may
occur in the absence of cell proliferation (43) and that
IFN-
was shown to block colony-stimulating factor-1-induced proliferation of a macrophage cell line while inducing expression of
both cyclin D1 and D2 (8). Collectively, this evidence
shows that the antimitogenic mechanisms of IFN-
are complex and
cell-type specific, that expression of D-type cyclins in ASM cells may
not be predictive of mitogenesis, and that other regulators of cell growth also need to be explored.
Using expression array, we investigated the expression profile of
various genes that promote cell cycle progression from G1 to S phase in
response to EGF. We found that EGF increased levels of various
regulators that are important for cell cycle activation. Those included
activators of cdks, such as cyclin D2 and E, and PCNA, which is
required for DNA synthesis during the S phase (20). EGF
also upregulated some critical transcription factors, such as erg-1, an
immediate early gene expressed in response to mitogens (39), and E2F-1, which is essential for regulating cell
cycle progression, particularly the G1-to-S transition. E2F proteins are composed of one subunit of both the E2F (E2F-1 to E2F-5) and DP
(DP-1 to DP-3) families and act by controlling the transcription of
several important genes that are directly involved in DNA replication and cell proliferation. Those gene products include key enzymes such as
DNA polymerase and thymidine kinase, as well as cell cycle regulators
such as E2F-1 proteins, cyclins, and PCNA, which is essential for DNA
replication and processing (20). Among all the genes
increased by EGF in human ASM cells, IFN- specifically reduced E2F-1
and PCNA by 66 and 79%, respectively. Previous reports in some but not
all tumor cell lines showed that both IFN-
and IFN-
inhibited the
expression and/or function of E2F-1 (13, 38). Here, using
RT-PCR and Western blot analyses, we confirm that IFN-
suppresses
the expression of both E2F-1 mRNA and protein induced by EGF,
suggesting that a similar growth-inhibitory mechanism occurs in
nontransformed cells. The inhibitory mechanisms remain unknown, but
IFN-
may be directly repressing E2F-1 promoter activity, as
previously reported with IFN-
(11), although additional studies are needed to support this interesting hypothesis.
In quiescent cells, E2F-1/3 proteins are inactive in many E2F target
genes because of both E2F-4,5 and Rb, which act as transcriptional repressors. During G1 phase, phosphorylation of Rb initiated by cyclin
D/ckd4-6 releases Rb that triggers the subsequent degradation of
E2F-4 and -5, thereby allowing the derepression of gene transcription by E2F-1/3. Later in the G1 phase, maximal Rb phosphorylation is
ensured by another complex, cyclin E/cdk2 (26). In ASM
cells, previous reports showed indirect evidence of E2F-1 activation by
the ability of thrombin to induce hyperphosphorylation of Rb (9,
40). Here we found that IFN- inhibited EGF-induced
hyperphosphorylation of Rb, suggesting that IFN-
may promote G1
arrest by blocking the release of the transcription repressor Rb, thus
preventing the expression of S phase regulatory E2F-1-dependent genes.
The underlying mechanisms are unknown, but we found that IFN-
suppressed mitogen-induced expression of cyclin E, another important
E2F-1-dependent protein. The accumulation of cyclin E before the
G1-to-S transition is an important event for cdk2 activation to sustain
hyperphosphorylation of Rb that is required for transcription of S
phase genes (26, 36). The decrease in cyclin E levels by
IFN-
may therefore impair the function of the cyclin E/cdk2 complex.
Consistent with this hypothesis is the suppression by IFN-
of
EGF-induced cdk2 phosphorylation, an essential step that is required
for cyclin E/cdk2 complete activation (36). The modulation
of cyclin E/cdk2 activation by IFN-
may explain, at least in part,
the inhibitory effect of IFN-
on Rb phosphorylation. IFI 16, an
IFN-
-inducible nuclear protein belonging to the HIN-200 family,
consists of three sizes of isoforms (A, B, and C) that are derived from
alternative mRNA splicing (7, 16). We show here that
IFN-
increased all three IFI 16 proteins. Although the role of IFI
16 in regulating cell growth remains unknown, the mouse homolog p202 is
a potent cell growth inhibitor because of its capacity to interfere
with many cell cycle regulators, including E2F-1/DP-1 and E2F-4/DP-1 (6), thus raising the possibility that IFI 16 may play a
role in IFN-
-induced ASM cell growth inhibition. Additional studies are needed to better characterize the growth-inhibitory effects of
IFN-
in human ASM cells.
Here, we demonstrated that IFN- inhibits ASM mitogenesis, an effect
that does not involve inhibition of three major promitogenic signaling
pathways. The growth-inhibitory effect of IFN-
seems to involve a
modulation of the E2F-1/Rb pathways, either by interfering with
upstream cyclin E/cdk2 complexes or by decreasing E2F1 expression or
activity. Additional studies are clearly needed to fully investigate the growth-inhibitory mechanisms of IFN-
in ASM cells, which may
lead to the design of a new treatment for ASM hyperplasia, a
pathological feature in chronic asthma.
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ACKNOWLEDGEMENTS |
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We thank Dr. Marcelo G. Kazanietz for critical reading of the manuscript and Mary McNichol for assistance in the preparation of the manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants 2R01-HL-55301 (R. A. Panettieri, Jr.), 1P50-HL-67663 (R. A. Panettieri, Jr.), 1R01-HL-64042 (A. L. Lazaar), and CA-69031 (D. Choubey) and by American Lung Association Grant RG-062-N (Y. Amrani). Y. Amrani is a Parker B. Francis Fellow in Pulmonary Research.
Address for reprint requests and other correspondence: Y. Amrani, Pulmonary, Allergy, and Critical Care Division, Univ. of Pennsylvania Medical Center, 421 Curie Blvd., 848 BRB II/III, Philadelphia, PA 19104-6160 (E-mail: amrani{at}mail.med.upenn.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.
First published February 14, 2003;10.1152/ajplung.00363.2002
Received 29 October 2002; accepted in final form 28 January 2003.
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