Department of Pediatric Pulmonology, Institut National de la Santé et de la Recherche Médicale U142, Trousseau Hospital, St. Antoine Medical School, University of Paris, 75012 Paris, France
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ABSTRACT |
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Retinoids, including retinol and retinoic acid
(RA) derivatives, are important molecules for lung growth and
homeostasis. The presence of RA receptors and of RA-binding proteins in
the alveolar epithelium led to suggest a role for RA on alveolar
epithelial cell replication. In the present study, we examined the
effects of RA on proliferation of the stem cells of the alveolar
epithelium, the type 2 cells. We showed that treatment of
serum-deprived type 2 cells with RA led to a stimulation of cell
proliferation, with an increase in cell number in a dose-dependent
manner. To gain some insights into the mechanisms involved, we studied
the effects of RA on the expression of several components of the
insulin-like growth factor (IGF) system that have been shown to be
associated with the growth arrest of type 2 cells, mainly the
IGF-binding protein-2 (IGFBP-2), IGF-II, and the type 2 IGF receptor.
We documented a marked decrease in the expression of these components
upon RA treatment. Using conditioned media from RA-treated cells, we
provided evidence that the proliferative response of type 2 cells to RA was mediated through production of growth factor(s) distinct from IGF-I. We also showed that RA was able to reduce the decrease in cell
number observed when type 2 cells were treated with transforming growth
factor (TGF)-1. These results together with the known stimulatory
effect of TGF-
1 on IGFBP-2 expression led to suggest that RA may be
associated with type 2 cell proliferation through mechanisms
interfering with the TGF-
1 pathway.
alveolar epithelial cell; retinoic acid; insulin-like growth
factor; transforming growth factor-
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INTRODUCTION |
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LUNG DEVELOPMENT AND LUNG repair after injury require a complex series of controlled interactions involving genetic influences, hormonal stimulation, and cell-cell communication. These interactions are under the influence of many factors that control growth and cell differentiation. Among the molecules that participate in lung development and homeostasis are retinoids.
Retinoids, including retinol and retinoic acid (RA) derivatives, have been shown to be involved in the process of formation of the tracheal and bronchopulmonary tree (6, 37). Their participation in embryogenesis and organogenesis of the lung is sustained by studies analyzing the morphological malformations in the offspring when retinol deficiency was induced in pregnant rats, as well as by organ cultures. In fetal lung culture, addition of RA was associated with changes in the pattern of lung development characterized by more growth of proximal airways (2). A number of studies have contributed to progress in the understanding of how these molecules exert their actions with identification of specific binding proteins and receptors (15). Several types of cellular binding proteins that include cellular retinol-binding protein and cellular retinoid acid-binding protein are now characterized. Their role is to facilitate the intracellular transport of retinol and RA (26). The receptors that bind the retinoids are the RA receptors (RARs) and the retinoid X receptors (RXR) (31, 33). These intracellular receptors form either heterodimers (RAR-RXR) or homodimers (RXR) and act as DNA-binding proteins. Consistent with a role of retinoids in lung morphogenesis is the documented expression of the cellular binding proteins and the RAR in early embryonic development and fetal lung (6, 16).
The involvement of retinoids in the postnatal growth of the lung is supported by experimental and clinical studies. Recently, Massaro and Massaro (23) reported that treatment with RA prevented the low number of alveoli caused by dexamethasone in newborn rats. This result suggests that dexamethasone and RA can act antagonistically in the lung. In infants, some clinical trials have reported a decrease in the incidence and severity of bronchopulmonary dysplasia (BPD) with supplemental vitamin A (32). Interestingly, some of the morphological descriptions of the lung of children with BPD who died reproduced the modifications observed in animals deprived of vitamin A with the development of squamous metaplasia in the tracheobronchial epithelium and the loss of ciliated cells (5). These studies led to suggest that retinoids play a role in postnatal lung growth and also in the processes of lung repair after injury to maintain lung integrity. The presence of RARs in the adult lung is consistent with this hypothesis (5).
The mechanisms by which RA can be involved in lung growth and repair remain poorly understood, but it is likely that the pulmonary epithelium represents a major target. Tschantz et al. (35) reported that administration of glucocorticoids in newborn animals resulted in an acceleration of alveolar wall thinning, with a decrease in the alveolar surface area and an impairment of type 2 alveolar epithelial cell replication. The observation that RA treatment of rats that received glucocorticoids simultaneously was able to prevent the action of dexamethasone strongly suggests that RA may play a role in alveolar epithelial cell proliferation (23). Consistent with this hypothesis are the presence of RARs in alveolar epithelial cells and the modifications of type 2 cell activity upon RA treatment (38).
In the present work, we focused on the effects of RA on the
proliferative response of the stem cells of the alveolar epithelium, the type 2 cells. We showed that RA treatment of serum-deprived type 2 cells led to a stimulation of cell proliferation with an increase in
cell number. To gain some insights into the mechanisms involved, we
studied the effects of RA on the expression of several components of
the insulin-like growth factor (IGF) system that have been shown to be
associated with the growth arrest of type 2 cells, mainly IGF-binding
protein-2 (IGFBP-2), IGF-II, and the type 2 IGF receptor. We reported a
marked decrease in the expression of these components upon RA
treatment. Moreover, studies of the processes involved in the
proliferative response of type 2 cells to RA treatment strongly suggest
a link with the transforming growth factor (TGF)-1 pathway.
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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 has been extensively characterized (9). Cells were grown in Earle's MEM (GIBCO-BRL, Grand Island, NY) supplemented with 4 mM glutamine, 50 units of penicillin/ml, 50 µg of streptomycin/ml, and 10% fetal bovine serum (FBS) in 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 various concentrations of RA (Sigma, St. Louis, MO) for the indicated durations, 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.
In some experimental conditions, cells were treated with TGF-1
(Sigma). TGF-
1 was prepared in a 4 mM filtered HCl solution containing 1 mg/ml BSA (Biomérieux) and used at a concentration of 5 ng/ml.
For each protocol, three or four independent experiments were performed.
Proliferation Assay
For the growth study, exponentially growing cells were plated at a density of 4 × 103 cells/cm2. Three days later, cells were washed and cultured as described above.Cell number assay. 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 (9).
DNA synthesis assay. For autoradiography of labeled nuclei, cells were incubated for 24 h in medium containing 2 µCi/ml [methyl-3H]thymidine (60-70 Ci/mmol), as previously described (8). The plates were then washed three times with cold phosphate-buffered saline (PBS; 20 mM Tris · HCl, pH 7.6, and 137 mM NaCl), fixed with methanol, air-dried, and coated with NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). Twenty-four hours later, they were developed with Kodak rapid fix. After being stained with Giemsa, an average of 300 cells in random fields were examined at a magnification of ×400, and the percentage of labeled nuclei was calculated.
RNA Isolation and Analysis
Total cellular RNA was isolated using the guanidium isothiocyanate procedure described by Chirgwin et al. (4). 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 (7). The relative intensity of bands was quantified by scanning densitometry using comparison with 18S rRNA band intensity.The probes were generated by labeling plasmid inserts with
[-32P]dCTP using
random oligonucleotide priming (Amersham). The plasmids containing
inserts for IGF-I, IGF-II, type 1 IGF receptor, type 2 IGF receptor,
and IGFBP-2 were obtained as previously described (3, 25).
Protein Studies
Immunoblotting. For the study of secreted IGFBP-2, cells were washed in basal medium and incubated in serum-free medium for the indicated durations. The conditioned medium was then centrifuged (1,000 g for 10 min) to remove debris and unattached cells, then was desalted on Sephadex G-25 columns (Pharmacia-LKB) and lyophilized. The pellet was dissolved in a volume of 2× Laemmli buffer adjusted to the cell number. Equal volumes of samples were loaded for each experimental condition and were analyzed on SDS-PAGE (11% acrylamide). Western blots were prepared by transferring the proteins onto 0.45-µm nitrocellulose membranes (Bio-Rad, Richmond, CA) for 90 min at 130 volts. Immunoblotting was performed by first saturating the nitrocellulose (NC) 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 rabbit antibovine IGFBP-2 antiserum was used at a 1:1,000 dilution (UBI, Lake Placid, NY). 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 (Amersham) diluted 1:6,000 in 5% 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). The membranes were then exposed to autoradiography film (Hyperfilm-ECL; Amersham).For the study of type 2 IGF receptor, cellular proteins were analyzed as previously described (25). 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× Laemmli buffer, the volume of buffer used adjusted to cell number. Equal volumes of samples were loaded for each experimental condition, and proteins were separated by SDS-PAGE (7% acrylamide). Immunoblotting studies for type 2 IGF receptor were performed as above using rabbit anti-rat type 2 IGF receptor antiserum at a 1:2,000 dilution (a gift from Dr. Caroline D. Scott, Camperdown, Australia; see Refs. 3, 25).
Western ligand blotting. Conditioned media were collected after incubation of cells in basal medium and prepared as indicated above. The ligand blotting experiments were performed as previously described (25). Briefly, the lyophilized samples containing the secreted proteins were dissolved in a volume of 1× Laemmli buffer adjusted to cell number and analyzed on SDS-PAGE (11% polyacrylamide) under nonreducing conditions. The proteins were electrotransferred onto an NC filter, and the membranes were washed for 1 h at 4°C in TBS (5 mM Tris · HCl, pH 7.4 and 150 mM NaCl) containing 0.2% Tween, then incubated for 48 h at 4°C with a mixture of 125I-IGF-I and 125I-IGF-II (200,000 counts/min each) in TBS and 1 mg/ml BSA. After being washed, the binding proteins were visualized by autoradiography. Relative molecular weights were estimated by running a prestained molecular-weight standard.
IGF Assays
These assays were performed using the methods described by Babajko and Binoux (1). Lyophilized samples corresponding to 2.5-12.5 ml culture medium were gel filtered in 1 M acetic acid on columns of Ultrogel AcA 54 (Biosepra, Marlborough, MA) to separate IGFs from IGFBPs. The eluates were lyophilized and, before being assayed, desalted on Sephadex G-25 disposable columns (Pharmacia-LKB) in assay buffer. IGF-I was assayed by RIA using an anti-IGF-I antibody (gift from Dr. F. Frankenne and Dr. G. Henen, Liege, Belgium). IGF-II was measured by competitive protein binding assay using IGFBPs extracted from cerebrospinal fluid, which have a selective affinity for IGF-II. The IGF preparations used for radiolabeling and as standards were recombinant human IGF-I and IGF-II, kindly provided by Ciba Geigy (Basel, Switzerland).Statistical Analysis
Results were reported as means ± SE. 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. ![]() |
RESULTS |
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Effects of RA on Type 2 Cell Proliferation
To determine whether RA treatment was associated with changes in the proliferative response of type 2 cells, exponentially proliferating cells were incubated in serum-free medium containing either RA (at a concentration of 0.1 or 1 µM) or 0.01% ethanol (control conditions) for the indicated durations. As described previously, when type 2 cells were serum deprived, the fraction of cells that could initiate DNA synthesis decreased progressively, with a fall in labeling index (9, 25). This was associated with no increase in cell number and after 48 h of culture with cell loss (Fig. 1, A and C).
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When serum-deprived type 2 cells were treated with either 0.1 or 1 µM RA, a stimulatory effect on proliferation was observed that appeared to be maximal at 72 h, with a fourfold increase in cell number compared with the initial plating (Fig. 1A). This was associated with persistence of a high percentage of labeled nuclei (Fig. 1C). No further increase could be observed when cells were incubated with 0.1 or 1 µM RA for 96 h. To document the dose effect of RA on type 2 cell proliferation, exponentially growing cells were incubated in serum-free medium with various concentrations of RA (1 nM to 10 µM) for 72 h. As shown in Fig. 1B, a stimulatory effect of RA on the proliferative response of type 2 cells evaluated by an increase in cell number was observed for concentrations as low as 1 nM RA (P < 0.05 vs. control conditions). A maximal proliferative response was obtained when cells were treated with 0.1 and 1 µM RA. The stimulatory effect of RA on type 2 cell proliferation was not observed under the experimental conditions when cells were treated with RA in the presence of 10% FBS (data not shown).
Effects of RA on IGFBP-2 Expression
In previous studies, we have reported that type 2 cell growth arrest induced by either serum deprivation, oxidant exposure, or glucocorticoid treatment was constantly associated with an increase in the expression of several components of the IGF system, mainly IGFBP-2 (3, 24, 25). The stimulatory effect of RA on type 2 cell proliferation prompted us to ask whether changes in the expression of IGFBP-2 could be observed in RA-treated cells. We first examined the consequences of RA treatment on IGFBP-2 expression at the level of mRNA. For these experiments, RNA from cells cultured in serum-free medium without RA (control conditions) or with RA (0.1 and 1 µM) was extracted and studied by Northern blotting. As shown in Fig. 2 and as previously reported, an induction of IGFBP-2 expression was observed in growth-arrested cells by serum deprivation (25). Interestingly, the induction of IGFBP-2 mRNA was reduced when serum-deprived cells were cultured in the presence of RA. From the observations that the effects of RA on type 2 cell proliferation were similar in the presence of 0.1 or 1 µM RA but that changes in IGFBP-2 mRNA were more pronounced in the experimental conditions of 1 µM RA, we chose to use the concentration of 1 µM RA for the subsequent experiments.
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To determine whether the decreased expression of IGFBP-2 mRNA in RA-treated cells was associated with changes at the level of protein, we studied the accumulation of IGFBP-2 in the culture medium of cells using Western immunoblot analysis. For these experiments, cells were cultured in serum-free medium with or without RA for the indicated durations, and conditioned media were collected. IGFBP-2 antiserum recognized a single 32-kDa band with no additional fragments. The abundance of IGFBP-2 in the conditioned medium increased when cells were incubated in serum-free medium in the absence of RA (Fig. 3), as previously described (25). By contrast, when cells were cultured in the presence of RA, the production of IGFBP-2 and its accumulation in the conditoned media were significantly reduced. Results obtained using ligand blotting analysis with labeled IGF-I and IGF-II confirmed that a band of 32 kDa was the major IGFBP accumulated in the conditoned media of type 2 cells as previously described (25). The use of blots of the ligand experiments for immunoblotting experiments confirmed that the 32-kDa IGFBP band was IGFBP-2 (data not shown).
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Effects of RA on IGF-II and Type 2 IGF Receptor Expression
In previous studies, we have reported that type 2 cell growth arrest involved not only increased expression of IGFBP-2 but also induction of IGF-II and type 2 IGF receptor (25). To determine whether RA-induced proliferation was associated with changes in the expression of these components of the IGF system, we first examined the consequences of RA treatment on IGF-II expression at the level of mRNA. For these experiments, RNA from cells cultured in serum-free medium without or with RA was extracted and studied by Northern blotting. As shown in Fig. 4 and as previously reported, IGF-II mRNA was induced in cells growth arrested by serum deprivation (25). The induction of IGF-II was not observed when serum-deprived cells were cultured in the presence of RA. Experiments using IGF-II assays indicated that the levels of IGF-II in the conditioned medium were below detection (<1 ng/ml).
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A similar pattern of changes in type 2 IGF receptor expression upon RA treatment was observed. Results of immunoblotting experiments documented an induction of type 2 IGF receptor in growth-arrested serum-deprived cells (Fig. 5). This induction was not observed when cells were treated with RA.
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As previously described, the level of expression of type 1 IGF receptor and IGF-I was barely detectable in proliferating type 2 cells (25). With the use of Northern blotting experiments, no signals could be obtained in the experimental conditions of serum-deprived cells in the absence or presence of RA (data not shown). Furthermore, with the use of IGF-I assays, no IGF-I could be detected in the various conditioned media.
Effects of Conditioned Medium From RA-Treated Cells
To gain some insights into the mechanisms involved in the proliferative response of type 2 cells upon RA treatment, we asked whether factor(s) present in the conditioned medium of RA-treated cells could be involved. To approach this question, we evaluated the effects of conditioned medium from RA-treated cells on type 2 cell proliferation. For these experiments, exponentially growing cells were washed three times with basal medium and cultured for 24 and 48 h with conditioned medium from RA-treated cells. The experimental protocol was as follows: cells cultured for 48 h without or with RA (0.1 or 1 µM) were washed three times and were incubated overnight in basal medium without RA. The conditioned media were then collected and added to exponentially growing cells for the indicated durations. As shown in Fig. 6, a stimulation of type 2 cell proliferation evaluated by cell number was observed in cells incubated with conditioned medium from RA-treated cells compared with cells incubated with conditioned medium from control cells. A proliferative response was observed after 24 h of culture and was significant for both concentrations of RA (0.1 and 1 µM; Fig. 6). No statistical difference could be documented when RA concentrations of 0.1 and 1 µM were compared. These results led to suggest that the effects of RA on the proliferative response of type 2 cells may involve production of growth factor(s). These factors remain to be identified, but from the results presented above it is likely that they do not include IGF-I.
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Effects of RA on TGF-1-Induced Growth Arrest of
Type 2 Cells
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DISCUSSION |
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Several lines of evidence support a role for vitamin A in the
development, maturation, and maintenance of the lung. In the present
study, we focused on the effects of RA on proliferation of the
epithelial cells of the pulmonary alveolus. Experiments reported herein
indicate that RA treament led to a stimulation of type 2 cell
replication. Furthermore, it appeared that this effect was associated
with a decrease in the expression of three components of the IGF
system: IGFBP-2, IGF-II, and type 2 IGF receptor, molecules that have
been previously shown to be upregulated in various situations of growth
arrest of type 2 cells. We also provided data that showed that RA could
reduce the inhibition of proliferation induced by TGF-.
The effects of RA on cell proliferation have been shown to vary depending on the cells on which they act. The ability of RA to inhibit growth has been documented in a number of cancer cells, and much has been learned about its involvement in the transcriptional regulation of several genes that directly or indirectly result in block of proliferation. In nontransformed cells, retinoid-induced growth inhibition has also been reported (12, 36). By contrast, in several other reports, RA has been shown to exert a growth stimulatory effect. LaMonica and Thomas (21), using colon adenocarcinoma cell lines, showed that some colon cancer cell lines responded to RA by enhancing the amount of DNA. Increased proliferation upon RA treatment was also reported by Ohmura et al. (30) in hepatocytes. Their data were recently extended by reports showing an induction of DNA synthesis in the pancreas and kidneys of rats by 9-cis-RA (29). In an in vivo model, Takahashi et al. (34) provided evidence that vitamin A was able to increase the labeling index of epithelial cells of the bronchioles and cells of the alveoli in rats, the most prominent effect being observed at the alveolar level.
These results fit in well with the data reported herein. To document the effect of RA on type 2 cell replication, we focused in the present work on the model of serum deprivation, which represents a widely used system to study cell proliferation. In previous studies, we have shown that culture of type 2 cells in medium without serum led to a progressive decrease in labeling index and block of proliferation (9, 25). We have also observed that, when cells were refed with serum after culture in serum-free medium for up to 96 h, they rapidly resumed proliferation in the first 24 h of restimulation. Our present data indicated that treatment of serum-deprived type 2 cells with RA was associated with the maintenance of a high percentage of labeled nuclei. Furthermore, we observed that this effect was associated with a significant increase in cell number compared with the cell counts of the initial plating before culture in serum-deprived medium.
To progress in the understanding of the mechanisms that are involved in the control of type 2 cell proliferation, we have developed in the past few years several models of reversible cellular growth arrest. These models included serum deprivation, exposure to oxidants, and treatment with glucocorticoids (3, 8, 9, 24, 25). In these various situations, we have observed that block of entry into S phase was associated with induction of several components of the IGF system, mainly IGFBP-2, IGF-II, and the type 2 IGF receptor. Moreover, when cells were allowed to resume proliferation, a decrease in the expression of these components was always observed. Based on several studies in the literature which indicated that RA was able to modulate the expression of several components of the IGF system, the present results on stimulation of type 2 cell proliferation upon RA treatment prompted us to ask whether this effect could be associated with modifications of IGFBP-2, IGF-II, and the type 2 IGF receptor expression. Data reported herein showed a decrease in the expression of these components in RA-treated cells. These results are of interest because they provide additional evidence that IGFBP-2, IGF-II, and the type 2 IGF receptor are involved in the negative control of type 2 cell proliferation.
The effects of RA on the IGF system have been mostly investigated in cellular models where RA treatment was associated with growth inhibition. In breast cancer cells, Gucev et al. (17) provided evidence that IGFBP-3 mediated the actions of RA. They showed that antisense IGFBP-3 oligodeoxynucleotide inhibited IGFBP-3 expression and attenuated the inhibitory effect on growth in RA-treated cells. An induction of IGFBP-3 by RA was also documented in dermal papilla cells (19). Increased expression of other IGFBPs, including IGFBP-5 and IGFBP-6, was reported in osteoblast cells, as well as in skeletal cells and neuroblastoma cells (1, 13, 39). In these various situations of growth inhibition, it has been proposed that RA may mediate its effects by increasing the production and secretion of IGFBPs and consequently by decreasing the bioavailability and activity of the IGFs in the cellular microenvironment. In addition to its ability to modulate expression of IGFBPs, RA may also regulate the synthesis of IGFs (14). It has been reported in human neuroblastoma cells that RA treatment led to an increase in IGF-II expression (1). This was associated with a proteolysis of IGFBP-2 followed by an increased production of IGFBP-6. The consequence on growth was a stimulation of proliferation in the first hours of RA treatment followed by a decrease in cell number. This model illustrates the possibility of RA to modulate growth through distinct regulation of various IGFBPs.
In lung alveolar epithelial cells, the effect of RA was only documented on IGFBP-2 (3, 24, 25). Indeed, in previous studies, we have shown, using ligand blotting experiments, that IGFBP-2 was the IGFBP predominantly produced by these cells. The presence of other IGFBPs was barely detectable in the various experimental conditions tested. In addition to IGFBP-2, a modulation of IGF-II expression was also observed in RA-treated epithelial type 2 cells. However, in contrast to the stimulatory effects of RA on IGF-II documented in the studies discussed above, RA treatment resulted in a decrease in IGF-II mRNA. Again, these data support a role for IGF-II in the inhibition of type 2 cell proliferation.
The mechanisms by which RA can modulate cell proliferation are under
extensive investigations and are likely to include several pathways. In
the present study, we focused on a possible link with the TGF-1
pathway. TGF-
1 is a potent growth inhibitor of epithelial cells, and
in previous studies, we have provided evidence that this factor was
strongly induced in growth-arrested type 2 cells (3). This induction
was associated with an increased expression of the two types of
TGF-
1 receptors known to be essential for signal transduction: the
type I and the type II TGF-
1 receptors. Moreover, we have reported
that, in experimental conditions of growth arrest, addition of
anti-TGF-
1 antibody was able to partially reverse the inactivation
of cyclin E-cyclin-dependent kinase complexes, which are known to play
a central role in the G1-to-S transition (10). In addition,
several studies in the literature indicated that RA could modulate
TGF-
1 and TGF-
1 receptor expression mainly through modifications
of mRNA stability, translation, or protein activation (11, 18). Based
on these observations, we hypothesized that part of the stimulatory
effect of RA on type 2 cell proliferation could be linked to TGF-
1
signaling events. To test this hypothesis, we cultured type 2 cells in
serum-free medium in the presence of either TGF-
1, RA, or the
combination of TGF-
1 and RA. Our results showed that addition of RA
to TGF-
1 reduced the decrease in cell number observed when cells
were treated with TGF-
1 alone. This led to suggest that RA could
interfere with the TGF-
1 pathway and could partially reverse the
inhibitory effect of TGF-
1 on type 2 cell proliferation.
Studies are currently being pursued to characterize the processes
involved in the stimulation of proliferation induced by RA in type 2 cells and to define the molecular targets of the cell cycle machinery
involved. The experiments using conditoned media indicate that RA
treatment may be associated with the production of growth factor(s)
that do not include IGF-I as discussed above. Studies of the processes
involved in RA-induced proliferation need also to integrate TGF-1
and IGFBPs. In several cell systems including type 2 cells, a
link between TGF-
1 and IGFBPs has been reported. In situations
associated with TGF-
1-induced growth arrest, an induction of IGFBP-2
or IGFBP-3 was observed, and the use of antisense IGFBP oligonucleotide
was shown to attenuate the inhibition caused by TGF-
1 (3, 28).
Therefore, among the events leading to stimulation of type 2 cell
proliferation, RA treatment may impair the expression and/or
activation of TGF-
1, which in turn could result in the
downregulation of IGFBP-2. From recent data, it is likely that the
function of IGFBPs is not restricted to binding IGFs in the
extracellular environment, and it is now proposed that IGFBPs could
travel to the nucleus instead of remaining outside the cell (20, 22).
If this applied to IGFBP-2 in type 2 cells, then the key question to be
addressed would be the intracellular role of IGFBP-2 and how this
protein may be connected to the cell cycle machinery.
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ACKNOWLEDGEMENTS |
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We thank Katarina Chadelat for valuable discussions. We also thank Marie Claude Miesch for technical assistance.
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FOOTNOTES |
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This work was supported by Association Claude Bernard, Ligue Nationale contre le Cancer (Comite de Paris), Fondation Lancardis, Chancellerie des Universites de Paris (Legs Poix to A. Clement), Ministere de la Recherche Grant 92 C 0884, Ministere de la Sante PHRC94, Association Recherche et Partage, UPRES EA1531 University of Paris VI.
Address for reprint requests: A. Clement, Dept. of Pediatric Pulmonology, Trousseau Hospital, 26, Ave Dr. Netter, 75012 Paris, France.
Received 29 December 1997; accepted in final form 24 March 1998.
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