Departement de Pneumologie Pediatrique, Institut National de la Santé et de la Recherche Médicale Unité 515, Hôpital Trousseau Assistance Publique-Hôpitaux de Paris, Université Paris VI, 75012 Paris, France
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ABSTRACT |
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Retinoids, including retinol and retinoic acid (RA) derivatives, have been shown to be involved in the processes of lung development as well as of lung repair after injury. Recently, we have provided evidence that RA could stimulate proliferation of lung alveolar type 2 epithelial cells (E. Nabeyrat, V. Besnard, S. Corroyer, V. Cazals, and A. Clement. Am. J. Physiol. Lung Cell. Mol. Physiol. 275: L71-L79, 1998). To gain some insight into the mechanisms involved in the mitogenic action of RA, we focused in the present study on the effects of RA on the expression of G1 phase cyclins and their cell cycle-dependent kinases (Cdks). Experiments were performed with serum-deprived cells cultured in the absence and presence of RA. The results showed no effects of RA on the expression of either cyclins or Cdks. In contrast, RA treatment was found to prevent the decrease in cyclin E-Cdk2 activity observed when cells were growth arrested by serum deprivation. The observation that changes in cyclin E-Cdk2 activity were not associated with modifications in the amount of complexes formed led to the suggestion that the Cdk inhibitory protein (CKI) was involved. Study of the CKI p21CIP1 revealed marked differences in its expression in the absence and presence of RA, with a dramatic downregulation observed in RA-treated cells. Interestingly, immunoprecipitation experiments provided evidence that the decreased levels of p21CIP1 were associated with a reduced interaction of this CKI with cyclin E-Cdk2 complexes. These data together with previous results obtained in various situations of type 2 cell growth arrest emphasize the role of p21CIP1 in the control of lung alveolar epithelial cell proliferation.
cell cycle; cyclin E; cyclin-dependent kinase-2; cyclin-dependent kinase inhibitor; mitogenesis
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INTRODUCTION |
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THE INVOLVEMENT OF VITAMIN A in fetal development as well as in the regulation of cell proliferation and differentiation during postnatal life is well established (16). In the respiratory system, there is now evidence that vitamin A can affect many processes associated with lung development and maturation as well as with lung repair after injury. Consequently, vitamin A and its biological derivative retinoid acid (RA) are considered important molecules in lung physiology and physiopathology (8, 34).
Among the various structures that comprise the lung, the airway epithelium represents a specific target for the actions of vitamin A. In several reports (6, 30) with animal models or cell systems, it has been shown that vitamin A deficiency was associated with the development of extensive cellular pathological changes of the epithelial structure that could be reversed by RA treatment. A study (7) of the mechanisms involved demonstrated a role for vitamin A in the proliferation and differentiation of lung epithelial cells.
In a previous work, Nabeyrat et al. (25) have examined the effects of RA on proliferation of the stem cells of the alveolar epithelium (type 2 cells) and provided evidence that RA treatment could stimulate cell growth in a dose-dependent manner. These results were obtained in cells cultured under basal conditions in serum-free medium, supporting the view that RA was able to directly affect the expression of factors involved in cell cycle progression. The demonstration that RA could promote type 2 cell proliferation is consistent with data reported by Massaro and Massaro (23) showing that postnatal treatment with RA increased the number of pulmonary alveoli in rats and, in addition, was able to prevent the inhibitory effect of dexamethasone on alveolar formation.
To gain some insight into the mechanisms involved in the proliferative action of RA on type 2 epithelial cells, we focused in the present work on the key regulators of the cell cycle machinery, the cyclin-dependent kinase (Cdk) system. The aim of the study was to determine the molecules that could act as specific targets of RA. Progression through the cycle is controlled by checkpoints that ensure the order of cell cycle events. The influence of extracellular growth regulatory signals is observed in the G1 phase, and the decision to advance toward mitosis occurs as cells pass the restriction point late in the G1 phase (29). After this point, the cells become refractory to extracellular signals. Passage through the restriction point and entry into the S phase is controlled by several Cdks and their associated cyclins (20). The G1 phase cyclins include mainly cyclins D and cyclin E. The D-type cyclins form complexes mostly with Cdk4 and Cdk6, and cyclin E forms a complex with Cdk2. Activity of the Cdks can be regulated by several mechanisms that include the levels of expression of cyclins, Cdks, or the Cdk-activating kinase (CAK) as well as of proteins called Cdk inhibitors (CKIs). CKIs can inhibit Cdk activity by physical association with their target cyclins, Cdks, or cyclin-Cdk complexes (27). Several CKIs have been identified. The first family includes p15Ink4B, p16Ink4A, p18Ink4C, and p19Ink4D (27). These Ink4 inhibitors are specific for Cdk4 and Cdk6 and interfere with cyclin D binding to these kinases. The second family includes p21CIP1 (also known as WAF1), p27KIP1, and p57KIP2 and acts on a wide range of cyclin-Cdks (17).
To determine the molecular targets of RA action, we evaluated in the present work the effects of RA treatment on G1 phase cyclin, Cdk, and CKI expression as well as on activity of cyclin-Cdk complexes. Experiments were performed with a rat type 2 cell line that has been shown in previous studies (9, 10) to regulate some aspects of proliferation in a fashion similar to that of primary type 2 epithelial cells. The results revealed that the levels of cyclins and Cdks were not affected by RA. In contrast, a dramatic downregulation of p21CIP1 expression was found with RA treatment. The experiments reported herein provided evidence that the decreased levels of p21CIP1 was associated with a reduced interaction of p21CIP1 with cyclin E-Cdk2 complexes.
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MATERIALS AND METHODS |
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Cells and Cell Culture Conditions
The type 2 cell line used in this study was derived from rat primary neonatal type 2 cells and had been extensively characterized (9, 10). The cells were grown in Earle's MEM (GIBCO BRL, Life Technologies, Grand Island, NY) supplemented with 4 mM glutamine, 50 U penicillin/ml, 50 µg streptomycin/ml, and 10% fetal bovine serum in a 5% CO2-95% air atmosphere at 37°C.For the study of the effects of RA, exponentially growing cells were
washed and cultured in serum-free medium containing 1 µM RA (Sigma,
St. Louis, MO) for the indicated dura- tions, the medium being changed
every 24 h. Stock solutions of RA were prepared at a concentration of
10 mM in 100% ethanol and stored at 80°C.
RNA Isolation and Analysis
Total cellular RNA was isolated with the guanidium isothiocyanate procedure described by Chirgwin et al. (6). The precipitated RNA was resuspended in sterile H2O and quantified by absorbance at 260 nm. Twenty micrograms of RNA were fractionated by electrophoresis through 1% agarose-2.2 M formaldehyde gels and blotted onto nylon membranes (Stratagene, La Jolla, CA). The integrity of RNA was assessed by visual inspection of the ethidium bromide-stained 28S and 18S rRNA bands. The blots were prehybridized and hybridized to 32P-labeled probes, washed, and exposed to film as previously described (9). The relative intensity of the bands was quantified by scanning densitometry by comparison to the 18S rRNA band intensity.The probes were generated by labeling plasmid inserts with
[-32P]dCTP by random oligonucleotide priming
(Amersham). Plasmids containing inserts for rat p21CIP1 and
transforming growth factor (TGF)-
1 were obtained as previously described (4, 11). The plasmids containing the rat type I and type II
TGF-
receptors were kindly provided by Dr. J. S. Brody (Boston
University, Boston, MA).
Protein Studies
Cellular extract preparations. Total cellular extracts were prepared as previously described (24). The cells were washed three times with cold PBS (20 mM Tris · HCl, pH 7.6, and 137 mM NaCl) and lysed by addition of a volume, adjusted for the cell number, of lysis buffer [250 mM NaCl, 50 mM HEPES, 5 mM EDTA, 1 mM dithiothreithol (DTT), and 0.1% Nonidet P-40 (NP-40)] with freshly added protease inhibitors (1 µg/ml of leupeptin, 5 µg/ml of aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation at 10,000 g for 10 min at 4°C and stored atFor the preparation of cytosolic and nuclear extracts, the cells were
harvested with trypsin-EDTA (0.5 g/l of trypsin and 0.5 mM EDTA; GIBCO
BRL) after three washings in cold PBS. The cells were then counted with
a hemacytometer and pelleted by centrifugation at 2,000 g for 5 min. The cellular pellet was resuspended in a volume, adjusted for the
cell number, of buffer A (10 mM HEPES, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, and 1% NP-40) with freshly added protease inhibitors for 5 min at 4°C with agitation. Membrane lysis was accomplished by the presence in buffer A of 1%
NP-40. The nuclei were then collected by centrifugation at 2,000 g for 5 min, and the supernatant (cytosolic extract) was stored
at 80°C until analysis by immunoblotting. The nuclear pellet
was resuspended in a volume, adjusted for the cell number, of
buffer B (20 mM HEPES, 1.5 mM MgCl2, 300 mM KCl,
0.5 mM DTT, 0.2 mM EDTA, and 25% glycerol) with freshly added protease
inhibitors as described above and agitated vigorously at 4°C for 30 min. Nuclear debris were collected by centrifugation at 15,000 g for 30 min, and the supernatant (nuclear extract) was stored
at
80°C until analysis by immunoblotting and kinase assays.
Immunoprecipitation. Cellular extracts (3 × 106 cells) were incubated at 4°C overnight with either
anti-cyclin E antibody or anti-Cdk2 antibody. Cyclin-Cdk complexes were
isolated by incubation at 4°C for 1 h with 50 µl of protein A
Sepharose 6MB beads (Pharmacia, Piscataway, NJ). The beads were then
washed and resuspended in 40 µl of reaction buffer (50 mM
Tris · HCl, pH 7.4, 10 mM MgCl2, and 1 mM
DTT) and 40 µl of 2× SDS sample buffer (62.5 mM
Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 0.025%
bromphenol blue, and 5% -mercaptoethanol). The samples were then
boiled for 5 min, separated by SDS-PAGE (11% acrylamide), and analyzed
by immunoblotting.
Protein electrophoresis and immunoblotting. Equal volumes of samples were loaded for each experimental condition, and the proteins were separated by SDS-PAGE (11% acrylamide). Western blots were prepared by transferring the proteins onto 0.45-µm nitrocellulose membranes (Bio-Rad, Richmond, CA) for 1 h 30 min at 130 V. Immunoblotting was performed by first saturating the nitrocellulose sheet for 2 h at room temperature in PBS containing 0.2% Tween (PBS-T) and 10% powdered milk. This was followed by incubation with diluted antiserum in 5% milk-PBS for 20 h at 4°C. The antisera used were rabbit anti-Cdc2 antibody from Dr. Christiane Guguen-Guillouzo (INSERM U522, Hôpital Pontchaillou, Rennes, France); rabbit anti-cyclin D3 antibody from Dr. Charles J. Sherr (Howard Hughes Medical Institute, Memphis, TN); rabbit anti-cyclin E, anti-p21CIP1, anti-Cdk4, anti-p27KIP1, and mouse anti-cyclin D1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then washed three times in PBS-T buffer and incubated for 1 h at 37°C with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G or anti-mouse immunoglobulin G (Amersham) diluted 1:6,000 in milk-PBS. The membranes were washed three times in PBS-T, after which they were incubated for 1 min at room temperature in chemiluminescence reaction detection reagents (ECL Western Blotting, Amersham). The membranes were then exposed to autoradiography film (Hyperfilm-ECL, Amersham).
Kinase Assays
For kinase assays, nuclear extracts (8 × 105 cells) were incubated at 4°C overnight with either anti-cyclin E antibody, anti-Cdk2 antibody, anti-Cdk4 antibody, or anti-cyclin D1 antibody. Cyclin-Cdk complexes were then isolated by incubation at 4°C for 1 h with 50 µl of protein A Sepharose 6MB beads (Pharmacia). The beads were then washed and incubated for 30 min at 30°C in 25 µl of reaction buffer (50 mM Tris · HCl, pH 7.4, 10 mM MgCl2, and 1 mM DTT) in the presence of either 5 µg of histone H1 (Boehringer Mannheim) or 1 µg of glutathione S-transferase (GST)-retinoblastoma protein (pRB) substrate (a gift from Dr. Mark E. Ewen, Dana Farber Institute, Boston, MA) and 1 µCi of [Statistical Analysis
The results are means ± SE. Data were analyzed with ANOVA followed, when adapted, by the Mann-Whitney U-test for multiple comparisons against control conditions. Significance was assigned for P < 0.05. ![]() |
RESULTS |
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Effects of RA on Type 2 Cell Proliferation
On the basis of previously reported data (25), a concentration of 1 µM RA was used in the experiments performed in the present work. Exponentially proliferating cells were incubated in serum-free medium containing either 1 µM RA or 0.01% ethanol for the indicated durations. In previous studies, Clement et al. (10) and Mouhieddine et al. (24) have shown that when type 2 cells were serum deprived, the fraction of cells that could initiate DNA synthesis decreased progressively, with a fall in labeling index. This decrease in thymidine incorporation was associated with no increase in cell number and, after 48 h of culture, with cell loss. When serum-deprived type 2 cells were treated with RA, a stimulatory effect on proliferation was observed, with a significant increase in cell number and the persistence of a high percentage of labeled nuclei (25). These results were confirmed in the present work. A stimulatory effect of RA on the proliferative response of cells evaluated by an increase in cell number is shown in Fig. 1.
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Effects of RA on G1 Phase Cyclin Expression
To determine the consequences of RA treatment on expression of the Cdk system, we first investigated whether RA-induced proliferation of type 2 cells was associated with changes in cyclin D1, D3, and E expression at both the cytoplasmic and nuclear levels using Western blotting analysis. In these experiments, cells were cultured in serum-free medium without and with RA for the indicated durations, and cytosolic and nuclear extracts were isolated. Laser densitometric analysis of six independent experiments indicated that no significant changes in the levels of cyclin D1, D3, or E proteins could be observed in the extracts of cells cultured in serum-free medium for 24-48 h in the absence and presence of RA (Fig. 2). In our cell system, no expression of cyclin D2 could be detected (11, 12).
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Effects of RA on Cdk4, Cdk2, and Cdc2 Expression
The effects of RA on the levels of Cdk2 and Cdk4 were also analyzed with cytosolic and nuclear extracts from six independent experiments. Compared with the levels obtained in proliferating cells, a decrease in the expression of nuclear Cdk2 and Cdk4 was found in serum-deprived cells without regard to the absence or presence of RA. Analysis of proteins in the cytosolic compartment showed a reduction in the levels of Cdk2 in all conditions compared with the levels found in proliferative cells (Fig. 3). Interestingly, study of Cdc2 expression, a kinase mainly involved in the entry into mitosis, showed no significant changes.
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Effects of RA on Cyclin-Cdk Activities
To determine whether RA-induced proliferation of type 2 cells was associated with changes in the activities of G1 phase cyclin-Cdk complexes, we performed in vitro kinase assays (Fig. 4). Cyclin D1-associated kinase activity and Cdk4 complex activity were assayed for their kinase activity toward pRB. When GST-pRB was used as a substrate in immunoprecipitation experiments performed with antibodies to Cdk4 or cyclin D1, nuclear extracts from the cells treated with RA for 24 or 48 h showed levels of kinase activities that were not different from the levels observed in the cells cultured in serum-free medium without RA. In the next set of experiments, Cdk2 complex activity and cyclin E-associated kinase activity were analyzed with histone H1 as the substrate. Cdk2 complex activity data indicated a significant decrease in the extracts from the cells cultured in serum-free medium for 48 h compared with the activity observed in proliferating cells (decrease of 54 ± 8%). This decrease was no longer observed when the cells were cultured in serum-free medium in the presence of RA. Similar findings were observed for cyclin E-associated kinase activity.
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Effects of RA on Cyclin-Cdk Complex Formation
The observations that the activities of the complexes formed with cyclin E and Cdk2 were modified with RA treatment without significant changes in the protein levels raised the possibility of modifications in the amount of cyclin E-Cdk2 complexes. To address this question, we performed immunoprecipitations with anti-Cdk2 antibody followed by immunoblotting with anti-cyclin E antibody. The results shown in Fig. 5 indicated that despite changes in cyclin E-Cdk2 activity, no variation in complex formation could be found with RA treatment.
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Effects of RA on CKI Expression
In previous studies, Corroyer and colleagues (11, 12) have reported an increase in the expression of CKI in growth-arrested cells. The stimulatory effect of RA on the activity of cyclin E-Cdk2 complexes prompted us to ask whether changes in the expression of CKIs could be observed in RA-treated cells. We first examined the consequences of RA treatment on p21CIP1 expression at the protein level in the cytosolic and nuclear compartments by Western blotting. As shown in Fig. 6, a dramatic induction of p21CIP1 protein level in cytosolic and nuclear extracts was observed in cells growth arrested by serum deprivation. In contrast, the induction of p21CIP1 protein level was reduced when serum-deprived cells were cultured in the presence of RA in nuclear extracts. We also examined the consequences on expression of p27KIP1. However, the level of expression of this protein was barely detectable under the present experimental conditions (data not shown).
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To determine whether the decreased expression of p21CIP1 in
RA-treated cells was associated with changes at the level of mRNA, RNA
from cells cultured in serum-free medium without and with RA was
extracted and studied by Northern blotting. As shown in Fig.
7, induction of p21CIP1 mRNA
was documented in cells growth arrested by serum deprivation. This
induction was no longer observed when serum-deprived cells were
cultured in the presence of RA.
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Effects of RA on the Association of p21CIP1 With Cyclin E-Cdk2 Complexes
In these experiments, the proteins in cellular extracts were first immunoprecipitated with either anti-Cdk2 antibody or anti-cyclin E antibody, and the immunoprecipitates were then analyzed by immunoblotting with anti-p21CIP1 antibody. Dramatic differences in the amount of p21CIP1 associated with cyclin E and Cdk2 were found depending on the absence or presence of RA. When serum-deprived cells were treated with RA, the presence of p21CIP1 in cyclin E and Cdk2 complexes was barely detectable (Fig. 8).
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Taken together, these data provide evidence that p21CIP1 is
an important modulator of cell cycle progression in lung alveolar epithelial cells. Based on the observations reported by several authors
(12, 27) that TGF- was a potent stimulator of p21CIP1
expression, we asked whether the TGF-
pathway could be involved in
the changes in p21CIP1 induced by RA treatment. In contrast
to the results obtained in a previous study (4) that clearly documented
a dramatic increase in TGF-
and type I and type II TGF-
receptors
in lung alveolar epithelial cells growth arrested by oxidants, we were not able to detect in the present work any expression of TGF-
as
well as any changes in the levels of type I and type II TGF-
receptor expression in serum-deprived cells in the absence or presence
of RA (data not shown).
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DISCUSSION |
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RA is an important regulator of epithelial cell biology as demonstrated by its involvement in the processes of both growth and differentiation in several epithelial cell systems. In the present work, we focused on the mechanisms used by RA to modulate proliferation of lung epithelial cells. The data reported herein indicate that RA can interfere with the activity of late G1 phase cyclin-dependent complexes and that this action is associated with a downregulation of the CKI p21CIP1.
From many reports in the literature (5, 18, 28, 32), it
was established that RA can activate or suppress progression through
the cell cycle. The mechanisms underlying these opposite actions on
cell growth are complex. The inhibition of cell proliferation has been
reported in a number of cancer cells, and the anticarcinogenic properties of RA represent interesting possibilities to suppress the
development of tumors. The growth inhibitory action of RA was also
demonstrated in other cell systems independently of the process of
malignancy. In normal human mammary epithelial cells, Seewaldt et al.
(28) showed that RA treatment inhibited cell proliferation with a block
of progression to the S phase of the cell cycle but without induction
of apoptosis. A study of the factors involved showed that
G1 phase arrest was temporarily associated with a decrease
in the levels of hyperphosphorylated pRB. Regulation of pRB
phosphorylation may allow RA to prevent malignant transformation of
normal mammary epithelial cells. If RA can suppress mitogenesis, a
number of studies also documented its stimulatory effects on proliferation in various cell systems. In colon cell lines, a dose-dependent stimulation of growth by RA was observed, with a maximal
effect at 108 M, a physiological concentration for
RA (18). Similar results were obtained in primary cultures of rat
hepatocytes with a potent stimulatory action of 9-cis-RA on DNA
synthesis (26). Treatment of rat aortic smooth muscle cells with RA
also induced growth stimulation (5). Results of these studies share
similarities with findings by Nabeyrat et al. (25) of a mitogenic
effect of RA on lung alveolar epithelial cells. Interestingly,
Takahashi et al. (30) provided data documenting the role of vitamin A in the maintenance of epithelial cells of the respiratory tract. They
showed that vitamin A treatment was associated with an increase in the
labeling index of these cells, with the most prominent effect being
observed at the alveolar level.
RA affects cell cycle progression by acting mainly on regulatory proteins involved in the G1 phase and in the G1/S phase transition. This is supported by the results of growth inhibitory experiments documenting a G1 phase arrest with RA treatment (33). In addition, in the present work, we have shown that RA was able to reverse the block of proliferation induced by culture in serum-free medium. In many cell systems including lung alveolar epithelial cells, serum deprivation has been reported to prevent entry into the S phase (10, 24). This leads us to suggest that RA could induce growth-arrested cells to resume proliferation by interfering with inhibitory events occurring in the G1 phase. In the literature, studies (2, 19, 31, 33) of the effects of RA on the expression of the components of the cyclin-Cdk system involved in the G1 phase showed various profiles of response. As an example, in rat aortic smooth muscle cells, Chen and Gardner (5) observed that RA could either activate or suppress mitogenesis. When they studied cyclin D1 expression, they observed that cyclin D1 was induced in situations of growth stimulation, whereas no effect could be observed when cells were growth inhibited. Our present data showing that RA treatment of lung epithelial cells was not associated with significant changes in cyclin or Cdk expression are therefore not surprising. In addition to the various reported effects of RA on either cyclin or Cdk levels, the cyclin-Cdk complexes in which activities were affected with RA treatment appeared to vary depending on the cell types. A decrease in Cdk4 activity was reported in many cells (35, 36). In MCF-7 cells, RA-mediated growth inhibition was associated with a decrease in Cdk2 activity, whereas Cdk4 activity remained similar in control and RA-treated cells (31). Interestingly, Crowe and Shuler (13) reported in human squamous cell lines an increase in Cdk2 activity. Their observation shares similarities with the results obtained in the present work, indicating an effect of RA on cyclin E-associated kinase activity and Cdk2 complex activity. In contrast, no changes in Cdk4 complex activity or cyclin D1-associated kinase activity could be found when growth-arrested lung epithelial cells were stimulated by RA.
After the observation that the changes in cyclin E-associated kinase activity and Cdk2 complex activity in RA-treated lung epithelial cells could not be explained by modifications in the levels of cyclin E or Cdk2 as well as in the amounts of cyclin-Cdk complexes, the possibility emerged that modulation of kinase activity may be mediated by a decrease in the CKIs that bind to cyclin-Cdk complexes and function to inhibit their enzymatic activity. Interestingly, the results reported herein showed a downregulation of p21CIP1 when serum-deprived cells were allowed to resume proliferation in the presence of RA. Most important, they also showed a decrease in the association of p21CIP1 with cyclin E-Cdk2 complexes. These data fit in well with the results of previous studies (11, 12) that have demonstrated in several situations of growth arrest of alveolar epithelial cells, including oxidant exposure and glucocorticoid treatment, a decrease in cyclin E-Cdk2 activity in relation to an increase in p21CIP1. A role for p21CIP1 in mediating the inhibitory effect of RA on cell growth has been reported in several cell systems such as smooth muscle cells and lung carcinoma cells (1, 5). In a recent work, Crowe and Shuler (13) provided data documenting increased Cdk2 kinase activity with RA treatment of squamous cell carcinoma cell lines in relation to a decreased expression of p21CIP1. p21CIP1 has been demonstrated to play a crucial role in cell cycle checkpoint in the G1 phase and functions by inhibiting Cdk activity in the G1/S phase transition. It is involved in G1 phase arrest in response to signals of cellular stress, particularly in response to agents causing DNA damage, and its induction is mainly mediated by p53. Its action is also important during mammalian development as well as during differentiation of a variety of cell lines in culture through p53-independent pathways (17). Thus the current understanding of p21CIP1 action supports a role for this CKI in the modulation of cell proliferation of alveolar epithelial cells in various situations including exposure to RA.
Several studies have addressed the question of the mechanisms by which
RA could regulate p21CIP1 expression. Regulation of
p21CIP1 can occur at different levels. Posttranscriptional
control has been documented in several cell systems and involved mainly
changes in protein stability (21). Recently, it has been reported that p21CIP1 abundance could be controlled by
proteasome-mediated proteolysis (3). However, if there is increasing
evidence of possible posttranscriptional regulation, the mechanism of
control of p21CIP1 expression occurs mainly at the level of
transcription. Studies reported in the literature on the changes in the
expression of p21CIP1 with RA treatment support involvement
of transcriptional mechanisms (1). Indeed, p21CIP1 is a
RA-responsive gene mediated by RA receptor/retinoid X receptor (RXR)
binding to its cognate promoter element (22). In squamous cell
carcinoma cells, it has been shown that introduction of RXR- regulated expression of RA-responsive genes and led to a significant reduction in the levels of p21CIP1, resulting in increased
Cdk2 activity (13). From these results, it is proposed that additional
transcription factors could interact with RA receptors and RXRs to
repress transcription from RA-responsive promoters. This may apply to
the data reported in the present work because RA treatment was
associated with the inhibition of p21CIP1 expression at
both the protein and mRNA levels. In addition to retinoid response
element activation, recent studies (5, 32) also suggested involvement
of other transactivation mechanisms in RA-induced growth stimulation.
Wan et al. (32) reported enhancement of human lung carcinoma cell
growth in serum-free medium by RA in relation to increased activator
protein-1 activation. Studies are currently being pursued to
characterize the factors and the sequence of events leading to
modulation of p21CIP1 expression by RA in lung alveolar
epithelial cell. From the present results, it is likely that the
TGF-
pathway is not involved.
To conclude, the results obtained in various situations of modulation of cell growth emphasize the role of p21CIP1 in the control of lung alveolar epithelial cell proliferation. The factors involved in p21CIP1 expression differ depending on the environmental signals. Characterization of the pathway leading to downregulation of p21CIP1 in RA-treated cells is critical for the understanding of the mechanisms used by vitamin A to favor lung development and repair.
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ACKNOWLEDGEMENTS |
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We thank Dr. Katarina Chadelat for valuable discussions. We also thank Marie-Claude Miesch and Jacqueline Chandelier for technical assistance.
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FOOTNOTES |
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This work was supported by the Association Claude Bernard, Ligue Nationale contre le Cancer (Comite de Paris), Association pour la Recherche contre le Cancer, Fondation Lancardis, and Chancellerie des Universites (Legs Poix).
E. Nabeyrat was supported by a fellowship from the Association pour la Recherche contre le Cancer.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Clement, Departement de Pneumologie Pediatrique, Hôpital Trousseau, 26, Ave. Dr. Netter, 75012 Paris, France (E-mail: annick.clement{at}trs.ap-hop-paris.fr).
Received 15 March 1999; accepted in final form 2 September 1999.
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