Perinatal expression of IGFBPs in rat lung and its hormonal regulation in fetal lung explants

J. Koenraad Van De Wetering1,3, Robert H. Elfring1,3, Marja A. Oosterlaken-Dijksterhuis2,3, Jan A. Mol2,3, Henk P. Haagsman1,3, and Joseph J. Batenburg1,3

1 Laboratory of Veterinary Biochemistry, 2 Department of Clinical Sciences of Companion Animals, and 3 Graduate School of Animal Health, Utrecht University, 3508 TD Utrecht, The Netherlands

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

To gain more insight into the regulation of the expression of insulin-like growth factor (IGF) binding proteins (IGFBPs) in the lung, the developmental patterns of the abundance of the mRNAs encoding IGFBPs were measured in the perinatal rat lung and in explant cultures of fetal rat lung. In hormone-free explant cultures, the levels of the mRNAs encoding IGFBP-2 through -5 changed with a pattern similar to that occurring in vivo (although in the case of IGFBP-3 to -5 at a faster rate), indicating that the developmental regulation of the expression of these IGFBPs in perinatal lung is mimicked in the explants. For the IGFBP-6 mRNA level, the pattern in vitro differed from that in vivo. In the explant cultures, dexamethasone decreased the production of IGFBP-3 and -4 and decreased the abundance of the mRNAs encoding IGFBP-2 to -5 but increased the abundance of IGFBP-6 mRNA. These observations indicate that glucocorticoids may be involved in the developmental regulation of the expression of these components of the IGF system and that the IGF system may be involved in the physiological effects of glucocorticoids on lung development. No appreciable effects of 3,3',5-triiodothyronine on the expression of the IGFBPs were observed.

insulin-like growth factor binding protein; glucocorticoids; thyroid hormones; lung development; cell interactions

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

STRUCTURAL AND FUNCTIONAL DEVELOPMENT of the lung depends on epithelial-mesenchymal interactions on a suitable extracellular matrix, but at this moment, the identity of the growth factors involved is not fully known (16, 20). Several lines of evidence indicate that the insulin-like growth factor (IGF) system, consisting of the IGF-I and IGF-II and their receptors and IGF binding proteins (IGFBPs), plays a role in lung development. IGF-I and IGF-II are mitogenic polypeptides homologous to proinsulin. They are important regulators of growth and differentiation in mammalian tissues (9). In many tissues, they are believed to function in an autocrine or paracrine fashion (9). Both IGF-I and IGF-II bring their effects about via interaction with the type 1 IGF receptor (9). A second receptor, the type 2 IGF receptor, which is identical to the mannose 6-phosphate receptor, may function primarily in the degradation of IGF-II (9). The actions of the IGFs are modulated through binding to a family of IGFBPs, six members of which have now been characterized (9, 26). The first indication for a role of the IGF system in the regulation of pulmonary development is its presence in the lung: IGF-I and IGF-II and their mRNAs have been demonstrated in lung tissue (23), whereas fetal lung tissue or cells maintained in vitro produce IGF-I (28). Lung tissue also contains type 2 IGF receptors (15), mRNA encoding type 1 and type 2 IGF receptors (23), and mRNA encoding IGFBP-2 to -6 (17, 23). Both fetal lung fibroblasts and epithelial cells produce IGFBP-2 to -4 (21). With regard to the actions of the IGF system, it has been observed that mitogenesis in adult and fetal pulmonary cells in vitro is stimulated by IGF-I (28, 29). Moreover, IGF-I stimulates collagen formation in cultured human lung fibroblasts (6) and tropoelastin synthesis in neonatal rat pulmonary fibroblasts (19). Mice carrying a null mutation of the gene encoding the type 1 IGF receptor are born alive but die at birth due to respiratory failure (10). A recent study (15) with congenic mice that are genetically identical except for a region of chromosome 17 containing the gene for the type 2 IGF receptor showed an inverse relationship between the amount of this receptor in the lung and the rate of embryonic lung maturation.

A large body of evidence indicates that development of the fetal lung is also influenced by circulating glucocorticoids and thyroid hormones, of which the concentrations in the fetal blood increase during the prenatal period (20). In vivo these hormones accelerate the developmental increase in the production of surfactant phospholipids in the fetal lung (20). The developmental increase in the activity of antioxidant enzymes in the fetal rat lung is enhanced by glucocorticoid but is depressed by thyroid hormone (27). Treatment with glucocorticoid or thyroid hormone in vivo was found to decrease fetal lung weight (27) and to alter lung structure during the perinatal period (13, 14). The effects of glucocorticoids and thyroid hormones on the maturation of the surfactant system appear to be due to modulation of epithelial-mesenchymal interaction (20).

In view of the indications that the IGF system plays a role in lung development and of the observations that glucocorticoids and thyroid hormones influence lung maturation at least partly via modulation of epithelial-mesenchymal interaction, it seemed of interest to study the developmental patterns of the expression of IGFBPs in the perinatal rat lung in vivo, to compare the patterns in vivo with those observed in hormone-free cultures of fetal rat lung explants, and to investigate the effects of dexamethasone and 3,3',5-triiodothyronine (T3) on the expression of IGFBPs in fetal rat lung tissue in explant culture.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tissue collection. Timed-pregnant Wistar rats (term is at day 22) were obtained from the Central Animal Laboratory, Utrecht University (The Netherlands). At the indicated developmental stages, they were killed by CO2 asphyxiation, and the fetuses were dissected from the uterus. Neonates were born spontaneously on day 22 of gestation and remained with their mother until they were killed. On the day of term, both fetuses and neonates were studied. No effort was made to determine exactly how much time had elapsed since the moment of birth of the neonates. Both fetuses and neonates were killed by decapitation. The lungs were immediately removed and freed of large airways. Lung tissue from adult rats was obtained after CO2 asphyxiation. For isolation of RNA, the lung tissue was quickly frozen in liquid nitrogen and stored at -70°C. Adult lung tissue was cut into pieces with scissors before freezing. Tissue from each litter or adult animal was stored together in one vial. Random samples (~0.1 g of tissue) were taken from these vials for RNA isolation.

Fetal lung explant culture. Fetal rat lung explants were cultured as described by Gross et al. (7). Briefly, the lungs obtained under sterile conditions from rat fetuses at day 18 of gestation were chopped into 1-mm3 cubes. Approximately 80 mg wet weight of the cubes were placed in four 60-mm tissue culture dishes with 2 ml/dish of serum-free Waymouth MB 752/1 medium (GIBCO, Paisley, UK) containing penicillin (100 U/ml) and streptomycin (100 µg/ml) and were incubated on a rocking platform under a humidified atmosphere of 5% CO2-95% air at 37°C for 6, 24, or 48 h in the presence of 100 nM dexamethasone, 100 nM T3, or a combination of these two hormones or in the absence of the hormones. The media were refreshed after 24 h of culture. At the end of the desired culture periods, the culture media were collected, and the explants were scraped from the dishes with a rubber policeman, rinsed four times with ice-cold 0.9% NaCl, quickly frozen in liquid nitrogen, and stored at -70°C.

Isolation and Northern blot hybridization of RNA. Total RNA was isolated from lung tissue and explants with RNAzol-B (guanidinium thiocyanate, phenol, and 2-mercaptoethanol; Cinna/Biotecx, Friendswood, TX) according to the manufacturer's instructions. RNA was quantified by measuring the absorbance at 260 nm. Aliquots containing 30 µg of total RNA were resolved by electrophoresis through a 1.2% agarose-2.2 M formaldehyde gel (11). After separation, the RNA was transferred to nylon membrane (Nytran-N, Schleicher and Schuell, Dassel, Germany) by capillary blotting. Staining with methylene blue showed that equal amounts of RNA were present on the blot in each lane. Subsequently, the RNA was cross-linked to the blot with ultraviolet light (125 mJ) and stored at 4°C.

Plasmids containing cDNA encoding mouse IGFBP-2 to -6 (generously provided by Dr. J. W. van Neck, Erasmus University, Rotterdam, The Netherlands) (24) were used to make cDNA probes. The six IGFBPs share amino- and carboxy-terminal regions with strong homology (25). The DNA probes made correspond to the nonhomologous middle region. The following DNA fragments were made: for IGFBP-3, a Pst I-Ava II restriction fragment corresponding to bases 448-715 (24); for IGFBP-4, a Taq I-Ava I restriction fragment corresponding to bases 504-659 (24); and for IGFBP-5, a Sac II-Tth111 I restriction fragment corresponding to bases 352-543 (24). A DNA fragment specific for IGFBP-2 was generated by polymerase chain reaction with 5'-GTGAAAAGAGACGCGTGGGC-3' [identical to bases 443-462 of rat IGFBP-2 cDNA (12)] and 5'-GCAGGAGGTGGGCGCAGCT-3' [complementary to bases 686-704 of rat IGFBP-2 cDNA (12)] as the primers and mouse IGFBP-2 cDNA (24) as the template. A DNA fragment specific for IGFBP-6 was generated by polymerase chain reaction with 5'-GCCAGAGGGCCGTCGGAAG-3' [identical to bases 299-318 of rat IGFBP-6 cDNA (25)] and 5'-CAGGGGCCCATTTCACCATC-3' [complementary to bases 428-448 of rat IGFBP-6 cDNA (25)] as the primers and mouse IGFBP-6 cDNA (24) as the template. A 0.4-kb cDNA insert encoding human beta -cytoplasmic actin was excised from the clone pHFbeta A-3'UT-HF (4). The 32P-labeled probes (~4 × 108 counts · min-1 · µg DNA-1) were prepared from these cDNA fragments by random priming with [alpha -32P]dATP (3,000 Ci/mmol; Amersham, Little Chalfont, UK).

Before hybridization, the blots were prehybridized at 42°C for 3 h in 6× standard saline-phosphate-EDTA (SSPE; 1× SSPE is 0.15 M NaCl, 1 mM EDTA, and 10 mM NaH2PO4; pH 7.4), 1% sodium dodecyl sulfate (SDS), 10× Denhardt's solution [1× Denhardt's solution is 0.2 mg each of bovine serum albumin (BSA), Ficoll, and polyvinylpyrrolidone/ml water], and 0.1 mg/ml of denatured phenol-extracted salmon sperm DNA. Hybridization was carried out at 42°C for 16-18 h in 50 ml of a medium containing 6× SSPE, 1% SDS, 5× Denhardt's solution, 0.1 mg/ml of denatured phenol-extracted salmon sperm DNA, 10% dextran sulfate, 50% formamide, and 0.125 µg of 32P-labeled cDNA probe. After hybridization, the blots were washed two times at room temperature for 5 min in 6× SSPE-1% SDS, two times at 42°C for 20 min in 2× SSPE-0.3% SDS, and two times at 45-50°C for 30 min in 0.2× SSPE-0.2% SDS. Autoradiograms of the blots were made at -80°C. The intensities of the hybridization signals were quantified by phosphorimaging with a Fujix Bas 1000 bioimaging analyzer system (Fuji Photo Film). After visualization of the radioactive signal, the blots were analyzed with Tina Version 2.08 beta software (Ray Test Isotopenmessgeräte, Straubenhardt, Germany). After the radioactive signal was measured, the membranes were stripped by washing two times for 30 min in 0.05× SSPE-1% SDS at 100°C and were rehybridized to human beta -actin cDNA as a control.

Western ligand blotting of IGFBPs. IGFBPs were analyzed by Western ligand blotting as described by Hossenlopp et al. (8) with some modifications. Briefly, 15 µl of the explant medium were mixed with 15 µl of the sample buffer for SDS-polyacrylamide gel electrophoresis (PAGE; 2× concentration). The samples were boiled for 5 min, cooled, and applied to a 12% polyacrylamide gel along with a set of kaleidoscope-prestained molecular-weight markers (Bio-Rad, Richmond, CA). After one-dimensional SDS-PAGE, the proteins were transferred electrophoretically from the gels to nitrocellulose membranes (Hybond-C, Amersham). The membranes were dried, incubated overnight at 4°C with 125I-labeled IGF-II (2 × 106 counts · min-1 · 25 ml-1), and analyzed by phosphorimaging as described in Isolation and Northern blot hybridization of RNA.

Western immunoblotting of IGFBPs. For immunoblot analysis, 300 µl of explant medium were concentrated with Microcon-10 microconcentrators (Amicon, Beverly, MA), after which the proteins were separated and blotted as described in Western ligand blotting of IGFBPs. IGFBPs were detected by immunostaining with the amplified alkaline phosphatase goat anti-rabbit immunoblot assay kit (Bio-Rad) according to the manufacturer's instructions. In short, the membranes were dried and immersed in 20 mM tris(hydroxymethyl)aminomethane (Tris) · HCl-500 mM NaCl [Tris-buffered saline (TBS)], pH 7.5. After the nonspecific binding was blocked with 5% nonfat milk in TBS containing 0.05% Tween 20 (TTBS), the membranes were washed and incubated overnight with the primary antibodies (rabbit anti-bovine IGFBP-2, 1:2,000, Upstate Biotechnology, Lake Placid, NY; rabbit anti-human IGFBP-3, 1:400, and rabbit anti-human IGFBP-4, 1:500, Schützdeller, Tübingen, Germany) in TTBS with 0.1% BSA. The membranes were washed two times, incubated for 1 h with biotinylated goat anti-rabbit antibodies (1:3,000) in TTBS with 0.1% BSA, washed, and incubated for 1 h with streptavidin-biotinylated alkaline phosphatase complex (1:3,000 dilution for each in TTBS). After several washes with TTBS and finally with TBS, the immune complexes were visualized with the alkaline phosphatase color development reagents 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium chloride. No bands were seen when incubation with the primary antibody was omitted.

Protein determination. Protein was determined with the Bio-Rad Bradford protein assay with Ortho 2 bovine control serum (Ortho Diagnostic Systems, Beerse, Belgium) as the standard.

Statistical analysis. Unless indicated otherwise, statistical analysis of the data was carried out by two-way analysis of variance. In those cases in which the F-test indicated that there was a significant difference (P < 0.05) among groups, comparisons with the control condition were made with Dunnett's test.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Abundance of the mRNAs encoding IGFBPs in the developing lung. Earlier publications (17, 23) reported that IGFBP-1 mRNA is undetectable in rat lung. Therefore, we made no effort to quantify this mRNA. The levels of the mRNAs encoding IGFBP-3 (2.4 kb), IGFBP-4 (2.6 kb), IGFBP-5 (6 kb), and IGFBP-6 (1.3 kb) increased over the period studied (Fig. 1). That of IGFBP-2 (1.6 kb) decreased between fetal day 18 and term. The level of IGFBP-3 mRNA showed a peak at birth. This peak was not observed for the other IGFBP mRNAs. In contrast to the levels of the mRNAs for IGFBP-3 and -6, those for IGFBP-2, -4, and -5 increased from fetal day 22 to neonatal day 2 (Fig. 1). The level of beta -actin mRNA was relatively constant over the period studied, indicating that the observed differences in the levels of IGFBP mRNAs were not due to the experimental procedures.


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Fig. 1.   Relative levels of mRNAs encoding insulin-like growth factor binding proteins (IGFBPs) and beta -actin in rat lung during development. Results were obtained in 4 series with preparations of independent litters or adult animals. Values in each series are means ± SE; n = 4 litters or adult animals. Where no error bar is shown for fetal or neonatal data, error bar is smaller than data symbol. Vertical dashed lines, term (22 days fetal age). Data points to left of line, results with animals not yet born on day of term; data points to right of line, results with animals born spontaneously on that day. Statistical analysis was performed on raw data with repeated-measures analysis of variance (MANOVA). MANOVA applied to the whole period studied showed that there is a significant increase in levels of mRNAs encoding IGFBP-3 to -6 (P < 0.05) but not in level of beta -actin mRNA. Level of IGFBP-2 mRNA showed a significant decline between fetal day 18 and birth (P < 0.01 by MANOVA). There is a significant rise in levels of mRNAs encoding IGFBP-2, -4, and -5 between fetal day 22 and neonatal day 2 (P < 0.05 by MANOVA) but not in those of IGFBP-3 and -6 and beta -actin. Comparison of fetuses and neonates at day of term showed that IGFBP-3 mRNA increased significantly at birth (P < 0.01 by Student's t-test). Adult value differed significantly from value at fetal day 18 for IGFBP-2, -5, and -6 (P <=  0.03 by Student's t-test).

IGFBP mRNAs in rat lung explants under various hormonal conditions. The relative abundance of IGFBP mRNAs was studied by Northern blot hybridization in fetal rat lung explants after culture without the addition of hormones (control) or in the presence of 100 nM dexamethasone and/or 100 nM T3 (Fig. 2). For a more quantitative analysis, the intensities of the hybridization signals were quantified by phosphorimaging, and the mean value for each condition as a percentage of the value at time (t) = 0 h was calculated (Fig. 3). As seen in Figs. 2 and 3, the expression of IGFBP-2 in the control cultures decreased with time, whereas the expression of IGFBP-3 to -5 increased. Dexamethasone decreased the level of IGFBP-2 mRNA even further on culture for 24 or 48 h. This hormone also depressed the levels of IGFBP-3 to -5 mRNAs relative to the control values on culture for 24 or 48 h. On the other hand, dexamethasone increased the level of IGFBP-6 mRNA compared with the control culture. The effect of dexamethasone after 24 h of culture on the levels of the IGFBP mRNAs was dose dependent (Fig. 4). T3 had no effect on IGFBP mRNAs (Fig. 3).


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Fig. 2.   Northern blot hybridization analysis of relative levels of mRNAs encoding IGFBPs and beta -actin in fetal rat lung explants cultured in presence (+) or absence (-) of dexamethasone (Dex; 100 nM) or 3,3',5-triiodothyronine (T3; 100 nM). Samples of 30 µg of total RNA were resolved, blotted, hybridized, and autoradiographed as described in METHODS.


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Fig. 3.   Relative levels of mRNAs encoding IGFBPs and beta -actin in fetal rat lung explants cultured in presence or absence of Dex or T3. For each experiment, abundance of a particular mRNA measured for a particular set of conditions is expressed as a percentage of abundance at time (t) = 0 h. Data are means ± SE of 4-8 independent experiments. Open bars, control; hatched bars, Dex (100 nM); crosshatched bars, T3 (100 nM); solid bars, Dex + T3. a Significant difference between control (hormone-free) cultures and t = 0 h (P < 0.05 by Dunnett's test). b Significant difference from control at the same culture time (P < 0.05 by Dunnett's test).


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Fig. 4.   Effect of Dex concentration (conc.) on relative levels of mRNAs encoding IGFBPs and beta -actin in fetal rat lung explants. For each experiment, level of a particular mRNA was measured after 24 h of culture in presence of a particular concentration of Dex. For IGFBP-3 and -4 mRNAs, experiment was carried out once. For IGFBP-2, -5, and -6 and beta -actin mRNAs, data are means ± SE; n = 2 experiments for IGFBP-2, 3 experiments for IGFBP-5, and 4 experiments for IGFBP-6 and beta -actin.

IGFBPs produced by fetal rat lung explants. The presence and relative amounts of IGFBPs in the culture media collected from the explants after the first and second 24-h culture periods were determined by Western ligand blotting. Figure 5 shows a representative result. The media contained IGFBPs with estimated masses of 43-47, 31-33, and 24 kDa. The 43- to 47-kDa multiplet can be identified as IGFBP-3 because this protein, which is variably glycosylated, is the only IGFBP known to migrate at these apparent sizes on SDS-PAGE (9, 26). Moreover, the 43- to 47-kDa multiplet was positively stained on Western immunoblots with rabbit anti-human IGFBP-3 antibodies (data not shown). Unglycosylated IGFBP-4 generally migrates with a relative molecular weight (Mr) of 24,000 on SDS-PAGE (26). In our hands, immunoblot analysis of the Western blots with a primary antibody raised against human IGFBP-4 resulted in staining of the 24-kDa band and the 31- to 33-kDa multiplet in Fig. 5 (data not shown). The 24-kDa band in Fig. 5, therefore, probably represents IGFBP-4. The 31- to 33-kDa multiplet may contain a variety of IGFBPs. An apparent Mr of 32,000 on SDS-PAGE has been reported for IGFBP-1 (26), -2 (26), and -5 (21). In addition to IGFBP-1, -2, and -5, glycosylated IGFBP-4 also migrates at ~31-33 kDa (26). Our observation (data not shown) that there was also staining of the 31- to 33-kDa multiplet on immunoblotting with rabbit anti-human IGFBP-4 antibodies (31 kDa) and with rabbit anti-bovine IGFBP-2 antibodies (33 kDa) indicates that part of the material in that multiplet is IGFBP-2 and glycosylated IGFBP-4. Because specific antibodies for IGFBP-1 and -5 are not available to us, we cannot differentiate between these binding proteins and IGFBP-2 and -4 in the 31- to 33-kDa multiplet. Rat IGFBP-6 migrates with an Mr of 26,500 (5) and is apparently not present in the culture media at a concentration sufficient for detection by the ligand blot method.


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Fig. 5.   Ligand blot analysis of IGFBPs in media from fetal rat lung explants. After culture for 24 h, media containing no hormone (control), Dex (100 nM), T3 (100 nM), or Dex + T3 were removed from explants. Ligand blot analysis was carried out as described in METHODS.

Quantification of the IGFBP concentrations from the Western ligand blots revealed no appreciable differences between the first and second 24-h culture periods (Fig. 6). During both culture periods, dexamethasone significantly depressed the secretion of IGFBP-3 (43-47 kDa), IGFBP-4 (24 kDa), and 31- to 33-kDa IGFBP into the medium, whereas T3 had no statistically significant effect. Similar to the effect on the levels of the IGFBP mRNAs (Fig. 4), the effect of dexamethasone on the IGFBP secretion was dose dependent (Fig. 7).


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Fig. 6.   Relative levels of IGFBP-3, IGFBP-4, and 31- to 33-kDa IGFBP in culture media from fetal rat lung explants cultured in presence or absence of Dex or T3. Open bars, control; hatched bars, Dex (100 nM); crosshatched bars, T3 (100 nM); solid bars, Dex + T3. For each experiment, intensity of a particular band (IGFBP-3, IGFBP-4, or 31- to 33-kDa multiplet) is expressed as a percentage of intensity of control value. Data are means ± SE of 4-6 independent experiments. Statistical analysis was carried out with raw data. a Significant difference from control (P < 0.05 by Dunnett's test).


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Fig. 7.   Effect of Dex concentration on relative levels of IGFBPs secreted into media by fetal rat lung explants. For each experiment, intensity of a particular IGFBP band measured after 24 h of culture in presence of a particular concentration of Dex is expressed as a percentage of control value. Data are means ± SE; n = 2 experiments.

Treatment with dexamethasone did not depress the overall protein production. The mean protein content of the culture medium after dexamethasone treatment amounted to 65 ± 29 (SE) µg/ml, which is not different from the control incubation that had a protein concentration of 69 ± 25 µg/ml (n = 3). Also, no differences were noticed after silver staining of the Western blots (data not shown).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

As described in RESULTS, we observed developmental increases in the levels of IGFBP-3 to -6 mRNA in the perinatal rat lung, with a peak for IGFBP-3 mRNA at birth and an increase between fetal day 22 and neonatal day 2 for IGFBP-4 and -5 mRNAs. IGFBP-2 mRNA decreased from fetal day 18 until term but increased between fetal day 22 and neonatal day 2. While these experiments were in progress, two other articles (1, 17) concerning the developmental pattern of IGFBP expression in the rat lung appeared. However, our paper differs from those publications. Unlike the present paper, Moats-Staats et al. (17) did not report measurements in the immediate neonatal period, whereas Batchelor et al. (1) only reported on IGFBP-2 and -4. The main differences between the patterns of IGFBP expression presented here and those presented by others are that we could not confirm the decrease in IGFBP-3 to -5 mRNAs on fetal day 20 (17) or the dip in IGFBP-4 mRNA on fetal day 22 (1). In addition, we did not find a general low abundance of IGFBP-2 to -5 mRNAs in the mature lung compared with lungs on fetal day 18, in contrast to the data reported by others (17). We agree with Moats-Staats et al. (17) on the low expression of IGFBP-2 and the high expression of IGFBP-6 in the adult lung, but we did not find a significant difference in mRNA levels of IGFBP-3 and -4 between fetal day 18 and adulthood and found an increased rather than a decreased level of IGFBP-5 mRNA in adult tissue. The differences in the patterns of IGFBP expression between the present data and those of Moats-Staats et al. (17) may be related to the normalization of IGFBP mRNA levels to ubiquitin mRNA expression in the latter paper. Moats-Staats et al. presented no data to show that the level of ubiquitin mRNA in the rat lung is constant from fetal day 16 until adulthood. In contrast to their data, our data indicate that not only elevated IGFBP-6 concentrations but also increased IGFBP-5 levels may play a role in maintaining the degree of development in the adult lung. Because IGFBP-6 has a 60-fold greater affinity for IGF-II than for IGF-I and appears to mainly inhibit the effects of IGF-II (22), the high expression of IGFBP-6 in the adult lung may be required for the inhibition of IGF-II action in one or more of the cell types at that stage of development.

IGFBP-2 also has a higher affinity for IGF-II than for IGF-I (9, 22). The decline in IGFBP-2 expression at term observed here and in other studies (1, 17) may therefore be related to the decrease in IGF-II expression in the same period (3) and to enhanced IGFBP-6 expression. The decrease in IGF-II expression, together with enhanced IGFBP-6 expression, may diminish the need for buffering of IGF-II action by IGFBP-2. In the developing rat lung, IGFBP-2 mRNA was only found in epithelial cells (23), and the decline in IGFBP-2 expression between fetal day 18 and term might therefore be involved in the differentiation of epithelial cells in that period.

At birth, the rat has no alveoli but breathes with smooth-walled air channels and saccules, which correspond to the prospective alveolar ducts and alveolar sacs (2). The first phase of postnatal rat lung development, lasting from day 1 to day 4, is a period of lung expansion. During this period, the total volume of the air spaces increases while there is hardly any increase in tissue mass (2). At the same time, there is an increase in the proportion of cells that are synthesizing DNA. However, the proliferative pattern of the three main cell populations, as determined from their [3H]thymidine incorporation, is distinctly different (2). One could speculate that the increased expression of IGFBP-3 immediately after birth and that of IGFBP-2, -4, and -5 on postnatal day 2 relative to the expression at term (Fig. 1) may be required for the regulation of the thinning of the primary septa present at birth. With the consideration of the observations that IGFBP-3 and -4 generally decrease IGF-stimulated cell proliferation, whereas both inhibitory and potentiating effects have been observed for IGFBP-2 and -5 (9), the increase in each of these binding proteins in the first 2 days of life might reflect the necessity to regulate the developmental processes involved in septal thinning differently in different cell types and/or in different areas of the lung.

Although results from a number of studies indicated that, in late gestation, hormones, particularly glucocorticoids and thyroid hormones, are involved in the regulation of lung maturation as reflected in the acceleration of the synthesis of both the phospholipid and protein moieties of pulmonary surfactant, these hormones do not appear to initiate differentiation (20). Morphological findings and the determinations of surfactant phospholipid content and composition and surfactant protein A content, as well as assays of choline incorporation into phosphatidylcholine (the major constituent of pulmonary surfactant), indicated that, in fetal lung explants, maturation continues in the absence of hormones with a pattern similar to that occurring in vivo (20, 30), although, depending on the animal species and the developmental stage, not always at the same speed (20). One of the purposes of the present investigation was to study whether, in fetal lung explants cultured in the absence of hormones, the abundance of the mRNAs encoding IGFBPs also changed over time with a pattern similar to that occurring in vivo. Comparison of Fig. 1 with Fig. 3 shows that this was indeed the case for IGFBP-2: the abundance of IGFBP-2 mRNA, which decreased in vivo from day 18 until term, also decreased when lung tissue at day 18 of gestation was cultured for 2 days in hormone-free medium. The levels of the mRNAs encoding IGFBP-3 to -5 increased in the explants like they did in vivo. However, in the explants, the increase in the abundance of these IGFBP mRNAs was faster than in the lung in vivo. These observations that in the explants the levels of the mRNAs encoding IGFBP-2 to -5 changed with a pattern similar to that in vivo, albeit in some cases at a different rate, indicate that the developmental regulation of the expression of IGFBP-2 to -5 in the late fetal lung is governed by factors residing in the tissue and/or by signals in the systemic circulation that decrease with the development in vivo and from whose influence the lung tissue is released on explant culture. For the IGFBP-6 mRNA level, the pattern in vitro differed from that in vivo: whereas the level in vivo increased significantly with development, the level in vitro tended to decrease with culture time.

Figure 3 also shows that the levels of the IGFBP mRNAs in the lung explants were influenced by dexamethasone. This hormone decreased the levels of mRNAs encoding IGFBP-2 to -5 and increased that of IGFBP-6 mRNA. At the protein level, dexamethasone depressed the levels of IGFBP-3, IGFBP-4, and 31- to 33-kDa IGFBP (Fig. 6). These observations indicate that, in the late fetal period, in addition to other factors, circulating glucocorticoids may be involved in the developmental regulation of pulmonary IGFBPs. They also indicate that changes in the expression of the IGFBPs may be involved in the effects of glucocorticoids on lung development (see introduction). As discussed above, it is possible that the increased expression of IGFBP-4 and -5, among others, immediately after birth is involved in the regulation of lung expansion in that period. Although postnatal dexamethasone treatment was found to accelerate postnatal alveolar wall thinning in the rat, prenatal dexamethasone administration was observed to diminish the postnatal increase in gas-exchange surface area (14). It is conceivable that the depression by dexamethasone of the expression of IGFBP-4 and -5 observed in the explant cultures (Fig. 3) also occurred on treatment of the rats with dexamethasone and was involved in the diminishing effect of dexamethasone on the postnatal increase in gas-exchange surface area (14).

The depressing effect of dexamethasone on the abundance of IGFBP-3 had earlier been reported for fibroblasts and epithelial cells isolated from fetal rat lung (21) and had also been found in other cell types (22). Dexamethasone was also observed to inhibit the production of IGFBP-4 in fetal lung epithelial cells (21). However, in contrast to the decreasing effect of dexamethasone on IGFBP-2 expression in the fetal lung explants observed in the present study, dexamethasone was found to increase the amount of IGFBP-2 mRNA and protein in immortalized neonatal rat type II cells (18). To our knowledge, this is the first report on the effect of dexamethasone on the expression of IGFBP-5 and -6 in the fetal lung.

Unlike the levels of the other IGFBP mRNAs in the explants, that of IGFBP-6 mRNA was increased by dexamethasone. It would be interesting to study by means of an immunoassay whether the increased IGFBP-6 mRNA level is accompanied by an increase in IGFBP-6 protein level because, in theory, the expression of IGFBP-6 could be regulated by dexamethasone at a posttranscriptional step in such a way that the abundance of IGFBP-6 would decrease despite an increase in the IGFBP-6 mRNA level. A parallel increase by glucocorticoids of IGFBP-6 expression at the mRNA and protein levels was recently reported for cultured osteoblasts (5).

In contrast to dexamethasone, T3 had no significant effect on IGFBP expression (Figs. 3 and 6). This argues against a physiological role for the thyroid hormone in the developmental regulation of IGFBP expression in the fetal lung in late gestation.

In summary, the data in the present paper show that in hormone-free explant cultures of fetal rat lung the levels of the mRNAs encoding IGFBP-2 to -5 changed with a pattern similar to that occurring in vivo, albeit in some cases at a faster rate. This indicates that the developmental regulation of the expression of these IGFBPs in the late fetal lung is mimicked in the explants. A similarity in the patterns in vivo and in vitro was not observed for the IGFBP-6 mRNA level. Dexamethasone influenced the expression of IGFBP-2 to -6 in the explants. Because fetal glucocorticoid levels rise at the end of gestation (20), the observation of these effects of dexamethasone indicates that glucocorticoids may be physiologically involved in the developmental regulation of the expression of these components of the IGF system in the fetal lung and that the IGF system may be involved in the physiological effects of glucocorticoids on lung development.

    ACKNOWLEDGEMENTS

We thank Dr. J. A. J. Faber (Center for Biostatistics, Utrecht University, The Netherlands) for statistical analysis of the results, Dr. J. W. van Neck (Erasmus University, Rotterdam, The Netherlands) for providing the plasmids containing the insulin-like growth factor binding protein cDNAs, and Dr. L. M. G. van Golde for critical reading of the manuscript.

    FOOTNOTES

This investigation was supported in part by the Netherlands Foundation for Chemical Research (SON), with financial aid from the Netherlands Organization of Scientific Research (NWO).

Address for reprint requests: J. J. Batenburg, Laboratory of Veterinary Biochemistry, Utrecht Univ., PO Box 80176, 3508 TD Utrecht, The Netherlands.

Received 3 December 1996; accepted in final form 8 September 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(6):L1174-L1181
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