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Physiology Department, Trousseau Hospital, St. Antoine Medical School, University of Paris, 75012 Paris, France
Address all correspondence and requests for reprints to: Annick Clement, M.D., Ph.D., Physiology Department, Trousseau Hospital, 26 avenue Dr. Netter, 75012 Paris, France.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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From the current understanding of the processes involved in the parenchymal growth of the lung, it is proposed that alteration of alveolar epithelial cell proliferation plays a central role in the abnormal lung development observed after glucocorticoid treatment (10). Studies of the mechanisms involved in the modulation of cell proliferation by glucocorticoids indicate that events associated with the G1 phase of the cell cycle are a major target for the actions of these hormones. Indeed, in a recent work, we observed that lung alveolar epithelial cells underwent reversible late G1 arrest in the presence of glucocorticoids (11). Focusing on the mechanisms involved in this inhibition of cell replication, we have been able to show that some components of the insulin-like growth factor (IGF) system, which is known to act in cell cycle progression just before entry into S phase, were associated with the negative control of alveolar epithelial cell proliferation (11). These components included IGF-II, the type 2 IGF receptor, and the binding protein, IGFBP-2. These findings raised the question of the mechanisms by which these factors may be involved in the inhibition of DNA synthesis in lung epithelial cells treated with glucocorticoids and, therefore, may be linked to the key regulators of the cell cycle machinery: the cell cycle-dependent kinases (CDKs) (12, 13, 14).
CDKs consist of a regulatory subunit termed cyclin and a catalytic subunit (a cyclin-dependent kinase). The G1 cyclin-dependent kinases phosphorylate substrates that, in turn, regulate the initiation of DNA replication. One of these substrates is the product of the retinoblastome (Rb) tumor suppressor gene. The G1 cyclins include cyclins D (mainly D1, D2, and D3) and cyclin E. The D-type cyclins form complexes mostly with CDK4 and CDK6, and cyclin E activates CDK2 (14). The G1 CDKs can be regulated by changes in cyclin or CDK levels, by the activating kinase CAK, and also by proteins that inhibit CDK activity by physical association with their target cyclins, CDKs, or cyclin-CDK complexes (15, 16). Two families of CDK inhibitors (CKIs) have been identified. The first family consists of p15Ink4B, p16Ink4A, p18Ink4C, and p19Ink4D (17, 18). 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 (19, 20, 21).
To gain some insights into the mechanisms involved in the growth arrest of lung alveolar epithelial cells by glucocorticoids, we focused in the present study on the effects of these hormones on the expression of the G1 cyclins and their CDKs (22, 23). We observed that when cells were blocked in their proliferation by dexamethasone treatment, no changes in the expression of the various G1 cyclins could be documented. Also, the levels of CDK2 and CDK4 in glucocorticoid-treated cells did not exhibit significant modifications compared with the levels in proliferating cells. By contrast, differences in the activity of cyclin E-CDK2 complexes were found, with a profound decrease in the extracts of cells growth arrested by dexamethasone. Studies of the factors potentially implicated in the inactivation of these complexes strongly suggested a role for p21CIP1, as a dramatic accumulation of this protein was observed in cells treated with dexamethasone. The mechanisms involved in this process are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the growth study, exponentially growing cells plated at a density of 9 x 103 cells/35-mm culture dish were used. Cell proliferation was evaluated by measurement of cell number as previously described: cells were harvested with trypsin-EDTA and counted in triplicate using a hemocytometer (24).
RNA isolation and analysis
Total cellular RNA was isolated using the guanidium
isothiocyanate procedure described by Chirgwin et al. (25).
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 ribosomal RNA (rRNA) bands.
The blots were prehybridized and hybridized to 32P-labeled
probes, washed, and exposed to film as previously described (26). The
relative intensity of the bands was quantified by scanning densitometry
using comparison with 18S rRNA band intensity.
The probes were generated by labeling plasmid inserts with
[
-32P]deoxy (d)-CTP using random oligonucleotide
priming (Amersham, Aylesbury, UK). Plasmids containing inserts for rat
cyclin E, CDK2, CDK4, and p21CIP1 were obtained using the
following construction. Coding region segments of these genes were
amplified using the PCR and rat complementary DNAs (cDNAs) as template
(27). These cDNAs were synthesized from type 2 cell messenger RNA
(mRNA) with an oligo(deoxythymidine) primer. Amplification of the
various genes was performed using specific primers selected from
sequences deposited in GenBank (accession numbers: cyclin E, X75888;
CDK2, M68520; CDK4, L11007; and p21CIP1, U03106). The
primers used were for cyclin E (5'-CAG TTC TTC TGG ACT GGC TG-3' and
5'-TCA GCC AGG ACA CAA TGG TC-3'), CDK2 (5'-GGG GAA TTC GGT CCT CCA CCG
AGA CCT TA-3' and 5'-GGGCAA TTC TGG CTT GGT CAC ATC CTG GA-3'), CDK4
(5'-TGG TGT CGG TGC CTA TGG GA-3' and 5'-GGT AGC TGT AGA TTC TGG
CT-3'), and p21CIP1 (5'-AGG CCG CTC AGA CAC CAG-3' and
5'-GGT CCC GTG GAC AGT GA-3'). Each primer had an EcoRI site
on its 5'-end. The PCR reaction contained 100 ng cDNA; 2
µM of each primer; 200 µM each of dATP,
dCTP, dGTP, and dTTP; 10 x PCR reaction buffer (100
mM Tris, pH 8.3, 500 mM KCl; and 1 U
Taq polymerase (Perkin-Elmer/Cetus, Foster City, CA) in a
total volume of 100 µl. Amplification was carried out in a PCR
thermocycler (Perkin-Elmer/Cetus) and included 30 cycles (denaturation
at 92 C, for 1 min, reannealing at 55 C for 1 min, and primer extension
at 72 C for 2 min). An aliquot of each PCR reaction was analyzed by
agarose gel electrophoresis. The size of the corresponding bands was
299 bp for cyclin E, 511 bp for CDK2, 477 bp for CDK4, and 307 bp for
p21CIP1. The PCR products were purified from the agarose
band and cloned into the EcoRI site of the plasmid pCRII
(Invitrogen, San Diego, CA). Individual clones were subjected to DNA
sequence analysis (Sequenase, U.S. Biochemical Corp., Cleveland,
OH).
The plasmids containing rat cyclin D1 and D3 cDNA were kindly provided by Dr. Guguen-Guillouzo (INSERM U-49, Rennes, France). The plasmid containing mouse p27KIP1 cDNA was a gift from Dr. Masagué (Howard Hughes Medical Institute, New York, NY).
Protein electrophoresis and immunoblotting
Cellular proteins were analyzed as previously described (28).
Duplicate dishes were used for each experimental condition. One dish
was used for cell number determination. The cells from the other dish
were washed with cold PBS and scraped in 2 x Laemmli buffer; the
volume of buffer used was adjusted to cell number. Equal volumes of
samples were loaded for each experimental condition, and 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 and 30 min at 130 V.
Immunoblotting was performed by first saturating the nitrocellulose
sheet for 2 h at room temperature in TBS (20 mM
Tris-HCl, pH 7.6, and 137 mM NaCl) containing 0.2% Tween
(TBS-T) and 10% powdered milk. This was followed by incubation with
diluted antiserum in 5% milk-TBS for 20 h at 4 C. The antisera
used were the following: rabbit antimouse cyclin D1, cyclin D3
antibodies, and antimouse CDK4 antibody from Dr. Scherr (Howard Hughes
Medical Institute, Memphis, TN); rabbit anti-CDK2 and rabbit anti-CDC2
antibodies from Dr. Guguen-Guillouzo; and rabbit anticyclin E,
anti-p21CIP1, and anti-p27KIP1 antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then
washed three times in TBS-T buffer and incubated for 1 h at 37 C
with horseradish peroxidase-conjugated goat antirabbit IgG (Amersham),
diluted 1:1000 in milk-TBS. The membranes were then washed three times
in TBS-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).
Immunoprecipitation and kinase assays
Cells (800 x 103) were washed three times with
cold PBS and lysed by addition of 40 µl lysis buffer [250
mM NaCl, 50 mM HEPES (pH 7.0), 5 mM
EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 10 µg/ml
leupeptin, 10 µg/ml apoprotinin, 50 µg/ml
phenylmethylsulfonylfluoride, 2 mM sodium pyrophosphate,
and 1 mM sodium orthovanadate]. The lysates were clarified
by centrifugation at 10,000 x g for 10 min at 4 C and
incubated at 4 C overnight with anticyclin E antibody, anti-CDK2
antibody, anti-CDK4 antibody, or anti-CDC2 antibody. Cyclin-CDK
complexes were then isolated by incubation at 4 C for 1 h with 50
µl of either protein A-Sepharose beads 6MB (Pharmacia, Piscataway,
NJ) or glutathione (GST)-agarose beads (Pharmacia). The beads were then
washed and incubated for 30 min at 30 C in 25 µl reaction buffer [50
mM Tris-HCl (pH 7.4), 10 mM MgCl2,
and 1 mM dithiothreitol] in the presence of either 5 µg
histone H1 (Boehringer Mannheim, Mannheim, Germany) or 1 µg GST-pRb
substrate (a gift from Dr. Ewen, Dana Farber Institute, Boston, MA), 1
µCi [
-32P]ATP (4500 mCi/mmol), and 50
µM ATP (29, 30, 31). Reactions were stopped by adding 40 µl
2 x SDS sample buffer [62.5 mM Tris-HCl (pH 6.8),
2% SDS, 10% glycerol, 0.025% bromophenol blue, and 5%
ß-mercaptoethanol]. The samples were then boiled for 5 min and
analyzed by 10% SDS-PAGE. 32P-Labeled proteins were
detected by autoradiography and quantified with a densitometry
scanner.
Statistical analysis
Results were reported as the mean ± SEM. Data
were analyzed using ANOVA, followed, when adapted, by Mann-Whitney U
test for multiple comparisons against control conditions. Significance
was assigned for P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, treatment of proliferating cells with
dexamethasone led to a rapid inhibition of the increase in cell
number.
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, the level of cyclin D1 mRNA increased in cells
treated with dexamethasone in a dose-dependent manner. For cyclins D3
and E, the absence of modifications in mRNA levels was observed in
dexamethasone-treated cells. A study of the effects of dexamethasone on
G1 cyclins at the protein level by Western blotting is shown in Fig. 2B: no changes in the levels of cyclins D1, D3, or E could be observed
in cells growth arrested by dexamethasone treatment. In our cell
system, no expression of cyclin D2 could be detected.
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, the levels of either CDK4 or CDK2 mRNA were not
modified in the experimental conditions of glucocorticoid treatment.
Results of Western analysis of the corresponding proteins are shown in
Fig. 3B. No changes in CDK2 and CDK4 protein levels were observed after
dexamethasone treatment. For comparison, in these experiments, we
included a study of CDC2 expression, the prototypic member of the CDK
family that controls entry into mitosis. As shown in Fig. 3, A and B,
glucocorticoid treatment was associated with a decrease in CDC2
expression at the levels of mRNA and protein.
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). In the next set of experiments, cyclin E was
immunoprecipitated from cells cultured in the absence or presence of
glucocorticoids, and kinase activity in the immunoprecipitates was
measured using histone H1 as a substrate. A decrease in activity was
apparent when cells were cultured with dexamethasone, with a maximal
inhibitory effect in the immunoprecipitates from cells growth arrested
by glucocorticoid treatment for 48 h (Fig. 4B). Similar results of
kinase activity using histone H1 were obtained in the
immunoprecipitates using antibodies to CDK2 (Fig. 4C). Finally, and as
expected, a decrease in CDC2 complex activity was found in cells in the
presence of dexamethasone (Fig. 4D).
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). By
contrast, significant modifications were observed at the level of
proteins (Fig. 5B), with a dramatic induction of p21CIP1 in
the extracts of cells cultured in the presence of dexamethasone.
Opposite effects of glucocorticoids were found for
p27KIP1.
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. In cells cultured under
control conditions, the amount of p21CIP1 mRNA was reduced
after culture with actinomycin D. In contrast, in cells treated with
dexamethasone for 48 h, the levels of p21CIP1 mRNA did
not change in the presence of actinomycin D, suggesting a marked change
in p21CIP1 mRNA stability.
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shows the results of the kinase
activities measured in the immmunoprecipitates using histone H1 as a
substrate. No differences were observed in activation of cyclin
E-associated kinase in exponentially growing cells and in cells
cultured for 48 h in the absence of serum (Fig. 7A). The results
of CDK2 kinase activities are shown in Fig. 7B, with a dramatic
decrease in the extracts of cells serum deprived for 48 h.
Analysis of kinase activity associated with CDC2 indicated similar
results, with a profound reduction in cells cultured without serum
(Fig. 7C).
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, glucocorticoid
treatment led to a dramatic increase in p21CIP1 protein
levels. It is likely that this effect is not influenced by the presence
or absence of serum in the culture medium. Indeed, the levels of
p21CIP1 protein, evaluated by immunoblotting experiments,
were not modified in the extracts of serum-deprived cells compared with
those in proliferating cells (Fig. 8). The same results
were found for p27KIP1; no changes between extracts of
serum-deprived cells and proliferating cells were observed in this
immunoblotting experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of glucocorticoids on DNA replication and consequently on cell proliferation have been shown to vary depending on the cells on which they act (1). If some cells appear to be growth stimulated by these steroids, in the majority of the reports an inhibition of DNA synthesis has been observed (32, 33, 34, 35). Several of these studies have provided evidence that the growth arrest process induced by glucocorticoids occurs in the G1 phase of the cell cycle rather than in the G0 state (36, 37, 38, 39). These conclusions have been supported by various experimental approaches, including kinetic studies, flow cytometry data, as well as characterization of a number of cell cycle markers in cells treated with dexamethasone. However, if G1 arrest appears to be a common response of cells to glucocorticoids, the mechanisms involved in this response seem to vary depending on the cell type. In rat hepatoma cells, glucocorticoids have been shown to induce a specific block in cell cycle progression during the first 2 h of the G1 phase (36). Similar conclusions were obtained in studies using P178 lymphoma cells (40). By contrast, in mouse L cells from the L929 cell line, glucocorticoids induced growth arrest at a very late point in G1, as indicated by the study of thymidine kinase (39). Thymidine kinase is one of several proteins whose expression is regulated during cell cycle progression, and accumulation of thymidine kinase mRNA occurs before the onset of S phase. In L929 cells treated with dexamethasone, the transcription of thymidine kinase gene was sustained despite inhibition of [3H]thymidine incorporation. Similarly, in previous studies, we provided data indicating that lung alveolar epithelial cells underwent late G1 arrest in the presence of glucocorticoids (11).
Block of type 2 cell proliferation late in G1 in the presence of glucocorticoids is further supported by the studies of cyclins and their CDKs. The results presented herein showed that dexamethasone-induced growth arrest of alveolar epithelial cells was associated with a decrease in the activity of cyclin E-CDK2 complexes. Moreover, the magnitudes of the decreases in cyclin E-associated kinase and CDK2 complex activities appeared similar, suggesting that CDK2 was mostly complexed with cyclin E when type 2 cells were arrested in their cell cycle by dexamethasone. By contrast, activation of cyclin D-CDK4 complexes was not modified in glucocorticoid-treated cells. Recently, much has been learned about the role and the sequence of involvement of the G1 cyclins, and it is now established that cyclins D1 and E displayed distinct roles (41). Cyclin E appears to be a regulator of the G1/S transition, acting after cyclin D1 (42). A loss of cyclin D1-CDK activity before the restriction point, defined as the point in late G1 after which cells become refractory to extracellular mitogenic signals, prevents cells from entering the S phase (43, 44). By contrast, loss of D1-CDK activity after the restriction point is without effect on cell cycle progression (45). In the present work, glucocorticoids appeared to be selectively associated with inactivation of cyclin E-CDK2 complexes, strongly suggesting that they blocked type 2 epithelial cell progression in the cell cycle late in G1, after passage through the restriction point. As discussed above, it is likely that the pathways used by glucocorticoids to inhibit cell growth may vary depending on the cell type. In P1798 murine T lymphoma cells, glucocorticoids have been reported to act earlier in G1 and to inhibit expression of cyclin D3 and CDK4 (37).
Another important point of discussion of the results presented herein relates to the observation that the effects of dexamethasone appeared to differ from the effects induced by serum deprivation. When cells were placed in medium without serum and growth arrested as documented by the inhibition of CDC2 activity, they displayed a dramatic reduction in histone H1 activity of CDK2 complexes. In contrast, in the extracts of these cells, no changes in histone H1 activity of cyclin E complexes could be observed. These data strongly suggest that in the situation of serum deprivation, cyclins other than cyclin E may be involved in CDK2 complex activity. The molecule that has been reported to activate CDK2 around the start of S phase is cyclin A. Recent reports indicated that cyclin A would activate CDK2 shortly after cyclin E, and this appeared to be concomitant with the onset of DNA synthesis (29, 46, 47). If cyclin E and cyclin A are essential for the G1 to S phase transition, an order of action seems to exist with first cyclin E and then cyclin A. Ohtsubo et al. provided evidence that the appearance of cyclin E-associated kinase activity occurs earlier in the cell cycle than nuclear expression of cyclin A and accumulation of cyclin A-associated kinase activity (48, 49). Taken together these reports give support to the hypothesis that in lung epithelial cells, glucocorticoids and serum deprivation may block entry into S phase through distinct regulators of the G1 to S transition.
Studies of the mechanisms responsible for the decrease in cyclin E-CDK2 activity in glucocorticoid-treated type 2 cells strongly suggest involvement of the CKI p21CIP1, as a dramatic increase in p21CIP1 protein was observed in cells growth arrested by dexamethasone. By contrast, participation of p27KIP1 seems unlikely, as a decrease in the amount of this protein was found upon glucocorticoid treatment. p21CIP1 and p27KIP1 belong to the p21 family of inhibitors that can inhibit all the G1 cyclin-CDK complexes (50). In addition, p21CIP1 associates with and inhibits the DNA replication and repair factor-proliferating cell nuclear antigen (PCNA) (51). The inhibitors of cyclin-CDK have been reported to act in different ways. They can prevent CDK activation by cyclin activating kinase, and they can also bind to cyclin activating kinase-phosphorylated cyclin-CDK complexes (15, 16). Recent studies have begun to provide some insights into the mechanisms by which these CKIs inhibit the cyclin-CDKs. p21CIP1 has two cyclin-binding sites, termed Cy1 and Cy2, and one site for CDK, named the K site. p21CIP1 uses the Cy1 or Cy2 site for association with cyclin E and the K site for association with CDK2. Chen et al. showed that the Cy sites stabilize the interaction of the K site and inhibit kinase activity directly (52). This simultaneous interaction with cyclin and CDK seems to be essential for optimal kinase inhibition. By contrast, isolated cyclin D or CDK4 does not associate with p21CIP1, and the cyclin D-CDK4 complex exclusively uses the Cy1 site for interaction with p21CIP1. In this situation, only one site is used, which will result in partially inhibited kinase being complexed with p21CIP1. Interestingly, the Cy2 site overlaps the PCNA-binding site of p21CIP1, establishing a link between the activity of p21CIP1 on cyclin E-CDK2 and PCNA. These new data provide important insights into the mechanisms by which cells may respond to growth inhibitory agents through the induction of specific factors capable of arresting cell cycle progression late in G1 and also of blocking DNA replication. In lung epithelial cells growth arrested by glucocorticoids, the finding that inactivation of cyclin E-CDK2 was associated with a dramatic induction of p21CIP1 fits well with the concept that p21CIP1 may function as an important G1 to S phase regulator in this situation. Interestingly, it is likely that in situations where entry into S phase is blocked, which involved inhibition of CDK2 complexes formed with molecules other than cyclin E, such as in the experimental conditions of serum deprivation, lung epithelial cells may use distinct inhibitors, as no changes in the levels of p21CIP1 could be documented. This observation shares similarities with the findings of Ohtsubo et al., who reported similar amounts of p21CIP1 in proliferating and serum-starved fibroblasts (49).
Another comment that could be drawn from the results presented herein is that induction of p21CIP1 in lung epithelial cells upon dexamethasone treatment is regulated mostly at the posttranscriptional level. Indeed, type 2 cells treated with glucocorticoids accumulated increased amounts of p21CIP1 protein compared with nontreated cells. In contrast, glucocorticoids caused no changes in p21CIP1 mRNA. From recent studies, much has been learned about the molecular mechanisms of action of glucocorticoids. Their effects are mediated by the cytosolic glucocorticoid receptor, which, upon binding of ligand, dissociates from heat shock proteins, translocates into the nucleus, and participates in the regulation of gene expression (53). The glucocorticoid receptor has been reported to have the ability to function in several ways, mostly transcriptional, but also in some situations using posttranscriptional mode of actions, including stabilization of certain mRNAs. As an example, Frost et al. reported in mouse L929 cells a control of thymidine kinase by these hormones at the posttranscriptional level and suggested a direct action of hormone-receptor complex on cytoplasmic stabilization of mRNA (39). Various posttranscriptional mechanisms should be considered in the discussion of the effects of dexamethasone on p21CIP1 in lung epithelial cells, including translational control and turnover of protein. In the present study, experiments performed with actinomycin D suggested that modifications in p21CIP1 mRNA stability in dexamethasone-treated cells may participate in the increase in p21CIP1 protein levels. Recently, posttranscriptional control of CIP1/WAF1 gene has been reported in other cell systems. Li et al. showed that a novel retinoic acid, CD437, enhanced the stabilization of the message in human breast carcinoma cells (54). Similar conclusions were obtained by Schwaller et al. in differentiating human leukemic cells (55). Interestingly, it is likely that this mode of control may participate in the regulation of other CKIs. A posttranscriptional regulation of p27KIP1 was recently reported by Hengst et al. (56). These researchers showed that control at the level of translation more than at the level of protein turnover was responsible for the regulation of p27KIP1 in HeLa cells under various conditions. They suggested that this mechanism may be essential for the rapid increase in p27KIP1 that is necessary for negative regulation of G1 progression in response to antiproliferative signals.
The results presented in this study indicate that growth arrest of lung alveolar epithelial cells by glucocorticoids is associated with an inhibition of cyclin E-CDK2 complex activities. Mechanisms regulating this process strongly suggest involvement of p21CIP1. These findings share similarities with recent data on the effects of oxidants on type 2 cell proliferation, documenting an arrest of the cells in late G1 (57). However, in the situation of oxidative stress, the induction of p21CIP1 with, as a consequence, inactivation of cyclin E-CDK2 appeared to be controlled mostly at the level of transcription. Discussion of the sequence of events leading to block of proliferation in cells exposed to oxidants suggested an initial role of transforming growth factor-ß (TGFß) whose induction would trigger an increase in the expression of p21CIP1, as documented in several reports (58). Based on the observation that in some cells, such as T cells, dexamethasone could induce the production of TGF-ß, a role of TGF-ß in lung epithelial cells growth arrested by glucocorticoids was suggested. However, no induction of TGF-ß and its receptors could be documented under these conditions in the present study (data not shown). This indicates that distinct signaling pathways are involved in the induction of p21CIP1 in lung alveolar epithelial cells blocked in their proliferation by oxidants or glucocorticoids. Interestingly, these pathways converge to an increase in p21CIP1 by transcriptional or posttranscriptional mechanisms, strongly suggesting a central role for p21CIP1 in the control of lung alveolar epithelial cell proliferation. Further understanding of the signaling pathways involved in the regulation of p21CIP1 after treatment with glucocorticoids appears essential in the perspective of development of therapeutic strategies that could reverse the detrimental effects of these steroids on lung development and repair.
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Acknowledgments |
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Footnotes |
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Received February 27, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Mol Endocrinol 7:1121–132[Abstract]
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