Direct effects of corticotropin-releasing hormone andthyrotropin-releasing hormone on fetal lung explants
R. L. Emanuela, J. S. Tordayb, N. Asokananthanc, M. E. Sundayc,*aDepartment of Medicine, Children’s Hospital & Harvard Medical School, Boston, MA 02115, USA
bDepartment of Pediatrics, University of California at Los Angeles, Los Angeles 21201, USAcDepartment of Pathology, Children’s Hospital, Brigham & Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA
Investigation of hormonal regulation of lung maturationis of particular importance because it may reveal novelforms of treatment for preventing or minimizing the severityof hyaline membrane disease (HMD) and bronchopulmo-nary dysplasia (BPD). Mammalian bombesin-like peptide(BLP) was identified as the first neuropeptide localized toPNECs [1], with highest levels in mid-gestation human fetallung [2]. BLP promotes widespread cell proliferation andtype II cell differentiation during fetal lung development[3,4].
Similar to BLP, the hypothalamic neuropeptide cortico-tropin-releasing hormone (CRH) and its high-affinity recep-tor (CRH-R) have been demonstrated to be expressed andfunctional on human lung cancer cell lines [5] suggesting apotential role for CRH in the regulation of cell proliferationand/or differentiation. Consistent with this hypothesis, CRHcan stimulate increased cAMP levels and the clonal growthof lung cancer cell lines [6]. Although the cellular source ofCRH in lung has not been precisely mapped, CRH mRNAappears to be transiently expressed from embryonic day12.5 (e12.5) to e17.5 in developing mouse lung in the
vicinity of the branching bronchioles, corresponding to thelate pseudoglandular and early canalicular phases of lungdevelopment [7]. CRH could have a direct role in lungdevelopment since CRH-null mice obtained by targeteddeletion in embryonic stem cells demonstrate profound ab-normalities in alveolarization and lung epithelial matura-tion. Examination of homozygous CRH-deficient newbornmice revealed thickened alveolar septae, a paucity of airspaces and reduced mRNA levels of surfactant apoprotein B(SP-B) when compared to wild type animals [8]. Althoughthese defects appear to be corrected by maternal adminis-tration of glucocorticoids during pregnancy, it remains pos-sible that CRH itself might have a direct role independentlyof glucocorticoids.
In the present study, we investigate the effect of CRH oncell proliferation and type II cell differentiation in explantsfrom developing baboon lung. This same model system hasrecently been utilized to analyze effects of BLP on baboonlung development [9]. As specificity controls, we have com-pared the effects of CRH to those of: 1. Dex as a positivecontrol for type II cell differentiation and decreased cellproliferation; and 2. thyrotropin-releasing hormone (TRH),another hypothalamic peptide, which was expected to be anegative control because its reported effects on lung matu-ration in vivo [2,10–12] have been hypothetically due tostimulation of prolactin production and thyroid axis activa-tion in utero [13].
Prematurely delivered baboons were provided by theBronchopulmonary Dysplasia Resource Core of the South-west Foundation for Biomedical Research. Normal fetalbaboons were delivered by cesarean section at 60–175 daysgestation. Lung tissues were harvested, with one lobe snapfrozen and stored at -70°C (for RNA analyses) and thesecond lobe inflated aseptically with Waymouth’s media(see below), shipped on wet ice and used less than 24 h aftersacrifice for organ culture.
2.2. Lung explants
Fetal lungs were chopped into 0.5 mm cubes and cul-tured in 6-well tissue culture dishes in 0.5 ml Waymouth’smedia supplemented with 0.5% fetal calf serum (FCS).CRH, TRH, bombesin (BN) (Peninsula Laboratories, Bel-mont, CA) or dexamethasone (Dex, Sigma, St. Louis, MO)were added as a 1003 solution in the same media. Controlwells were treated with 0.5 ml media alone. Cultures weremaintained for 48 h (for3H-choline and3H-thymidine in-corporation analyses), or 5 days (for RNA and immunoper-oxidase analyses) at 37°C in 5%CO2/air on a rocking plat-form at 3 oscillations/min [3]. In selected experiments, 5day cultures were also used for RNA analyses and/or im-munostaining.
On the day of harvesting the cultures, tissue was incu-bated with3H-thymidine (NEN, Boston, MA, 4mCi/ml) or3H-choline (NEN, Boston, MA, 16mCi/ml) for 4 h at37°Cin 5% CO2/air on a rocking platform. Cultured tissues wereharvested and analyzed for3H-labeled DNA, DNA content,3H-labeled saturated phosphatidylcholine (termed DSPC toavoid confusion with surfactant protein-C, SP-C) and pro-tein content.3H-thymidine incorporation into acid-precipi-table counts was carried out as described previously [3].DNA was assayed after trichloroacetic acid precipitation bythe method of Burton [14]. For determination of3H-DSPC,lipids were extracted from cell homogenates with chloro-form/methanol and3H-choline incorporation into phos-phatidylcholine was determined as described previously [3].Protein was determined by the method of Bradford usingbovine serum albumin (BSA) asstandard [15]. Experimentalvalues were normalized by defining the mean of the controlgroups as baseline and expressing values as percentage changeabove or below the baseline.
2.4. RT-PCR analyses
Total RNA was prepared from frozen fetal lung tissueusing the TRI reagent (MRC, Cincinnati, OH). RT-PCR
reactions were carried out using 1mg total RNA as describedpreviously [16,17] using 35 cycles of PCR for CRH receptortype I (CRH-RI), cytidylyltransferase (CYT), surfactantprotein C (SP-C), and TRH receptor type I (TRH-RI), and18 cycles for 18S rRNA orb-actin (to normalize the PCRsignal for CRH-RI, CYT and SP-C), each cycle includingdenaturation (3 min, 93°C), annealing (0.5 min, 55°C) andextension (3 min, 72°C). Synthetic oligodeoxynucleotidepairs were designed to span at least one intron, correspond-ing to conserved sequences of human CRH-R1 (yielding a1.1 kb product), human SP-C (yielding a 494 bp product),human CYT (yielding an 842 bp product), human TRH-RI(yielding a 415 bp product), human 18S rRNA (yielding a567 bp product) or humanb-actin (yielding a 194 bp prod-uct). PCR primers were synthesized by Oligos etc, Wilson-ville, OR. The sequences are as follows:
RT-PCR products were separated on 1.5% agarose-ethidium bromide gels according to standard methods [18].Southern blots of the PCR products were probed with thecorresponding end-labeled internal oligonucleotides. Theinternal oligonucleotide probes were end labeled with T4kinase [18] and hybridized at 42°C for 18 h in 63 saline-sodium citrate (SSC; 13 SSC is 0.15 M NaCl and 0.015 Msodium citrate), 53 Denhardt’s solution (1003 Denhardt’ssolution is 20 mg/ml Ficoll 400, 20 mg/ml polyvinylpyrro-lidone and 20 mg/ml BSA), 0.1% SDS and 250mg/mlsalmon sperm DNA. Blots were washed in 23 SSC/0.1%SDS at 42–50°C before exposure to Kodak Biomax film(Kodak, Rochester NY) at -70°C for 2–16h.
2.5. Immunoperoxidase analyses
After 5 days in culture to allow for detectable phenotypicdifferentiation, lung explants were fixed for 4 h in 4%paraformaldehyde in phosphate-buffered saline (PBS) be-fore being routinely processed into paraffin. 5mm serialsections were used for immunostaining for proSP-C and
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PCNA using the avidin-biotin complex technique with dia-minobenzidine as substrate and methyl green counterstainas described previously [19]. The rabbit polyclonal proSP-Cantiserum was generously donated by Dr. Whitsett at theChildren’s Hospital of Cincinnati.
2.6. Statistical analyses
For group comparisons, statistical analyses were doneusing the Student’st test (unpaired) and one-way ANOVA.Results are given as mean values6SEM.
3. Results
3.1. Effects of CRH on lung growth and type II cellmaturation
To determine whether CRH could be acting directly ondeveloping baboon lung we used explants prepared fromlungs of various gestational ages. This lung organ culturesystem has been previously utilized in our laboratory toanalyze the effects of other growth factors on fetal lungdevelopment [9]. Cultures were maintained for 48 h with0.1, 1, 10 or 100 nM CRH or media alone before beingcollected for measurement of choline and thymidine incor-poration into DSPC and DNA respectively. In certain ex-periments, cultures were maintained for 5 days to poten-tially maximize elicitation of a phenotypic change.
The effect of CRH on cell maturation in 48h cultures isshown in Fig. 1A: in these short-term cultures CRH inhib-ited choline incorporation at e125 (,25% inhibition) butstimulated choline incorporation at e140 with a peak effectwith 100 nM CRH (21% increase,P , 0.001). In contrast,when explants were maintained for 5 days (Fig. 1B), CRHstimulated choline incorporation at both e125 and e140,with the maximal effect occurring at e140 with 1nM CRH(100% increase,P , 0.001).
CRH also stimulated cell proliferation in lung explants,measured as the amount of3H-thymidine incorporation intonuclear DNA, as shown in Fig. 1C: at e125, 1–10 nM CRHstimulated thymidine incorporation by;30% over baseline,and at e140, both 1 nM and 100 nM CRH increased thy-midine incorporation (21% and 45%,P , 0.001). There wasa consistent absence of growth stimulation by 10 nM CRHat e140 in lung tissue from all 3 baboons (Fig. 1C).
To determine whether the effects of CRH on choline andthymidine incorporation were similar to those of Dex, lungexplants were treated with 0.1 to 100 nM Dex (Fig. 2).Similar to CRH, Dex induced increased choline incorpora-tion at all gestational ages tested (e90-e160), with maximaleffects in e140 lung cultures (n 5 4) treated with 10 nMDex (125% above baseline,P , 0.001, Fig. 2A). However,in contrast to CRH, Dex treatment resulted in a significant,dose-dependent decrease in thymidine incorporation in mostexplants (Fig. 2B). The only exception to the growth inhi-
bition induced by Dex was modest stimulation of thymidineincorporation in e140 lung cultures treated with low-doseDex (1 nM). In contrast, there was essentially no effect ofDex in cultures of the one available e60 lung, in spite ofsignificant bombesin-induced choline incorporation usingthe same lung explants [9]. The one e80 lung did demon-strate increased choline incorporation with 10 nM Dex after120h in culture (32% above baseline,P , 0.05, data notshown).
We then tested whether CRH would be additive or syn-ergistic with other peptide growth factors capable of induc-ing type II cell differentiation. We chose to combine CRHwith bombesin, which is known to have a positive effect oncholine incorporation in baboon fetal lung explants [9].Combination treatment with low doses of CRH and BN hada synergistic effect on promoting choline incorporation incultures of e140 (Fig. 3A) and e160 lung (data not shown).In e140 cultures, CRH (0.1 nM) plus BN (0.01 nM) stim-ulated choline incorporation by 32% (P , 0.001) comparedto 4% individually (Fig. 3A). CRH plus BN also stimulatedcholine incorporation in e160 lung by 11–15% (P , 0.05),whereas the same dose of either BN or CRH alone wasinhibitory (;10–15% decrease compared to baseline, datanot shown). In cultures of the same lungs, results of thymi-dine incorporation studies following combined treatmentwith CRH plus BN were not significantly different fromthose with either agent alone at e125 and e140 (Fig. 3B).
3.2. Ontogeny of CRH receptor gene expression in fetalbaboon lung
To determine whether the CRH receptor gene is ex-pressed in fetal lung, semiquantitative RT-PCR was carriedout using baboon lungs of different ages. As shown in Fig.4A, CRH receptor transcripts are present and expressed inlung tissue from several fetuses beginning at e125. One oftwo e175 animals and one of two adult animals tested alsoexpressed CRH receptor transcripts (Fig. 4A). We analyzedRNA from 9 additional e125 lung samples and found that 3out of 9 expressed detectable CRH receptor transcripts us-ing the given RT-PCR conditions (Fig. 4B).
3.3. Effects of TRH on lung growth and maturation
As another specificity control (in addition to Dex), wetested thyrotropin releasing hormone (TRH) for direct ef-fects on developing baboon lung explants in parallel withthe above experiments with CRH and Dex. TRH directlystimulated lung maturation similarly at all gestational agestested (Fig. 5A). A consistent increase in choline incorpo-ration was observed in lung tissue from four fetal baboonsat e140, with the peak effect (.50% increase over baseline)at 10 nM. This strong and statistically significant effect ofTRH was greater in magnitude than that observed with CRHin the same 2 day cultures (Fig. 1). A smaller but statisti-cally significant effect was demonstrated in lung tissue from
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seven fetal baboons at e125 (,25% increase with 0.1 nMTRH). Only a minimal effect on choline incorporation oc-curred at e160 (2 baboons available at this time point, datanot shown), which had a peak increase, 20% with 100 nMTRH. TRH induced the largest magnitude of choline incor-poration in lung cultures from two animals: e80 and e90. Ine90 lung explants, 1nM TRH induced a 110% increase incholine incorporation, and in e80 lung, 0.1 nM TRH in-duced a 50% increase in choline incorporation increased50% (P , 0.001 for both e80 and e90, data not shown).
TRH augmented cell proliferation in cultures of e140lungs, measured as the amount of3H-thymidine incorpora-
tion into nuclear DNA (Fig. 5B).3H-thymidine incorpora-tion was increased over baseline at all TRH doses with themaximal effect occurring at 1 nM (39% increase,P ,0.001). In contrast, 0.1–1 nM TRH resulted in modest(,25%) inhibition of cell proliferation at e125 (Fig. 5B) andat e90 (data not shown). Again, these effects of TRH onthymidine incorporation differed from those observed withCRH, which stimulated thymidine incorporation between0.1–10 nM in both e125 and e140 cultures (Fig. 1).
In further contrast to CRH, TRH treatment of one e125explant and one e140 lung explant for 5 days increasedmRNA levels for CYT . 10-fold (Fig. 6 and data not
Fig. 1. Effect of CRH on Choline and Thymidine Incorporation in Lung Explants. Lungs collected at various gestational ages were cultured with or withoutCRH for 48h (panels A and C) or for 5 days (panel B).3H-DSPC (panels A and B) and -DNA (panel C) were measured as described in Methods. Resultsare expressed as percentage increase over baseline controls (4 to 28 values per group) and represent the mean1 SEM. The number of animals at eachgestational age are indicated in parentheses.P values are given with respect to baseline controls. Typical average values for absolute3H-choline incorporation(cpm/mg protein) in negative control cultures (48h) were: 80d: 1396 5; 90d: 35236 306; 125d: 48846 264; 140d: 63716 911.
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shown) at 10 nM TRH (Fig. 6, lane 4) and variably at 1 nMTRH (Fig. 6, lane 3) as compared to cultures treated withmedia alone (Fig. 6, lane 1). This effect of TRH was greaterthan the induction of CYT mRNA by 10 nM Dex (;2-fold),which was our positive control (Fig. 6, lane 2). A third e140lung harvested for RNA did not demonstrate increased CYTmRNA by TRH in spite of a small (;1.5-fold) increase inmRNA levels by Dex (data not shown). There was noconsistent effect of TRH on SP-C mRNA levels, with onlyone (e125) of the three baboons demonstrating an increase(data not shown). In contrast to TRH, there was no effect ofCRH on either CYT or SP-C mRNA levels in lung explantsfrom the same three e125 baboons (data not shown). Wewere unable to demonstrate transcripts encoding TRH re-ceptor type II in the same lung samples as given in Fig. 4(data not shown), in spite of good positive control mRNAobtained from baboon fetal pituitary analyzed in parallel.
3.4. Effects on ProSP-C and PCNA immunostaining infetal lung explants
To provide additional parameters for confirming effectson type II cell differentiation and cell proliferation, wecarried out immunostaining of fetal baboon lung explantsfor pro-surfactant protein C (pro-SP-C) and proliferatingcell nuclear antigen (PCNA). Explants were treated for 5days with or without CRH (0.1 and 1 nM) or TRH (0.1, 1,and 10 nM). Results of a representative experiment aregiven in Fig. 7, which demonstrates explants from one ofseven baboons cultured (experiments were carried out usinglung tissue from 3 baboons at e140, 3 at e125, and one ate80). All photomicrographs were taken with the pleuralsurface at the top of the picture. As shown in Fig. 7A, tissuecultured with media alone (Fig, 7A) had minimal evidenceof cytoplasmic proSP-C immunostaining, most of which
Fig. 2. Effect of Dexamethasone on Choline and Thymidine Incorporation in Lung Explants. Similar to Fig. 1, lungs were cultured with or without Dex for48h.3H-DSPC (panel A) or -DNA (panel B) were measured as described in Methods. Values represent the means6 SEM. The number of animals used foreach experiment are indicated in parentheses.
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was distributed in a diffuse, faint cytoplasmic pattern. Whenthe same explants were treated with 1 nM CRH, there wasa consistent and marked induction of SP-C immunostainingin all 3 e125 lungs and all 3 e140 lungs examined (Fig. 7B).The same CRH-treated lung explants did not demonstrateimmunostaining when normal rabbit serum was substitutedfor the primary rabbit antiserum.
The one e80 lung also had increased SP-C immunostain-ing with 1 nM CRH (data not shown), which was present incuboidal epithelial cells lining the primitive alveoli (Fig.7E) distributed throughout the cytoplasm and as perinuclearand apical granules. This effect of CRH was slightly weakerthan that induced by 10 nM Dex in the same lungs (data notshown).
In the same lung explants (3 at e125, 3 at e140), PCNAimmunostaining was carried out to provide independentconfirmation of the thymidine incorporation experiments.Compared to untreated controls (Fig. 8A, demonstrating arepresentative e140 lung explant), CRH (0.1 and/or 1 nM)resulted in prominent stimulation of mesenchymal cell pro-
liferation (from ,5% to .25% PCNA-positive cells) (Fig.8B). There was no appreciable effect on epithelial cellPCNA positivity, which was equally prevalent in cultures6CRH. These results are consistent with those observed withthymidine incorporation (Fig. 1C).
In contrast, TRH induced proliferation of both epithelialcells (from ;75% to .90% PCNA-positive) and mesen-chymal cells (from,5% to.25% PCNA-positive) at e140(data not shown). However, at e125 TRH (1 nM) led tomodestly diminished epithelial PCNA labeling (from;75%to ;60% of cells) whereas there was a slight increase inmesenchymal cell labeling (from,5% to 5–10% positivecells). These data are consistent with the results of thethymidine incorporation experiments (Fig. 1B).
4. Discussion
The present study demonstrates the direct stimulation oftype II cell differentiation and cell proliferation in fetal
Fig. 3. Effect of Bombesin (BN) and CRH in Combination on Choline and Thymidine Incorporation in Fetal Lung Explants. Cultures were treated with 0.01nM BN, 0.1 nM CRH, or BN1CRH for 48h.3H-DSPC (panel A) or -DNA (panel B) were measured as described in the Methods section. The number ofanimals used for each gestational age is shown in parentheses.
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baboon lung explants treated with CRH and TRH, hypotha-lamic hormones that were previously believed to promotelung maturation only through indirect mechanisms [2,8,10–12]. Our investigation was initially based on the knownpresence of high levels of CRH mRNA in developing lungprior to type II cell differentiation [7]. Lung maturation wasassessed using3H-choline incorporation into saturatedphosphatidylcholine and immunoperoxidase analyses forproSP-C protein. Cell proliferation was evaluated using3H-thymidine incorporation into DNA and PCNA immuno-staining.
CRH stimulated choline incorporation at all gestationalages with the maximal effect occurring with 1 nM at e140.Intriguingly, the CRH effect on type II cell differentiationwas delayed, being most marked in 5 day explants, and onlymarginal in 2 day explants, including both choline incorpo-ration and SP-C immunostaining. Dex and TRH stimulatedboth 2 day and 5 day cultures similarly (data not shown).These observations suggest that the mechanism of CRH-induced type II cell differentiation might involve more thanone cell type, or simply that the type II cells have to bepartially differentiated to respond to CRH. The CRH effecton cell maturation was synergistically potentiated by BN:
when added together, BN plus CRH increased choline in-corporation at all gestational ages.
We cannot totally exclude the possible contribution ofincreased choline incorporation being due to increased num-bers of type II cells secondary to CRH-induced cell prolif-eration. However, the dose-response curves for increasedcholine incorporation versus thymidine incorporation do notconsistently run in parallel, except for choline and thymi-dine incorporation stimulated by 100 nM CRH at the 140day time point (Fig. 1 A, C). Furthermore, the observationof increased SP-C immunostaining in primitive alveoli inlung explants cultured with CRH supports the interpretationthat at least part of the choline incorporation response is dueto type II cell differentiation.
We demonstrate mRNA encoding CRH receptor type I[20] in baboon fetal lung at e125 in 30% of the animalstested using the given RT-PCR conditions. It is possible thatthe animals without detectable CRH-RI either have lowerlevels of these transcripts than in the other animals, or thatthey express other CRH receptors, such as CRH-R type II[21] or novel CRH receptors [22]. These data support thehypothesis that CRH effects require at least partial epithelialcell differentiation for CRH receptor expression before
Fig. 4. Ontogeny of CRH-R Transcripts in Developing Baboon Lung. A. Lungs were harvested from fetal baboons (e60-e175) and adult animals and totalRNA was prepared as detailed in Methods. RNA from each animal was reverse transcribed and cDNA was amplified by PCR using primers specific forCRH-R, and 18S rRNA. The CRH-R primers correspond to 150–167 and 1262–1283 bp of human CRH receptor sequence [36]. Southern blots of the CRH-RRT-PCR products were probed with an end-labeled internal oligonucleotide specific for CRH-R (see Methods). The ethidium bromide-stained Southern geldemonstrates the 18S rRNA transcripts. P, proximal; D, distal. Where there are 2 animals per time point, each animal is designated numerically. Thus,“1P”and “1D” refer to proximal and distal lung, respectively, both from baboon #1 at a given time point. B. Expression of CRH-R mRNA in 9 fetal baboon lungsat 125d gestation. Total RNA from 125d baboon lungs was prepared as described in Methods. RT-PCR products were identified with end-labeled internaloligonucleotides specific for CRH-R or human beta actin as described in Methods. Each baboon is designated by a code including a letter and year, such as“O95.”
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CRH effects on type II cell differentiation are observed. Asecond peak of CRH receptor detected late in gestationmight represent CRH receptor up-regulation induced by theincreased maternal plasma CRH near term [23,24].
Thyrotropin releasing hormone (TRH) is another hypo-thalamic neuropeptide which has been utilized both clini-cally and in animal models [2,10–12] because it readilycrosses the placenta from the maternal into the fetal circu-lation. TRH promotes lung maturation in premature ani-mals, hypothetically due to stimulation of prolactin produc-tion and thyroid axis activation in utero [13]. TRH given in
vivo is known to evoke a variety of physiological responsesin the lung, including increased pulmonary surfactant pro-duction, functional and morphologic evidence of type II celldifferentiation, and increased survival of newborn rabbitsfollowing premature delivery [11]. Using a TRH analogwhich does not induce the production of prolactin or thyroidstimulating hormone from the anterior pituitary, Devaskaret al. [11] provided evidence supporting a role for TRH infetal lung development that is either direct or mediated vianeurotransmitter stimulation of parasympathetic nervoussystem activity [25,26].
Fig. 5. Effect of TRH on Choline and Thymidine Incorporation in Fetal Baboon Lung Explants. Lungs collected at e125 and e140 were cultured with orwithout TRH for 48h. On the day of harvest, explants were incubated with either3H-choline (16mCi/ml) or 3H-thymidine (4mCi/ml) for 4h and subsequentlyanalyzed for3H- (panel A) or -DNA (panel B) (after normalization for protein or DNA content, respectively), as detailed in Methods.
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We examined TRH as a specificity control for CRH, andmade the interesting independent observation that TRH alsohas direct effects on parameters of lung development in
cultured explants, but that these effects differ from those ofCRH. TRH significantly stimulated type II cell maturationat all gestational ages in fetal baboon lung explants, includ-ing increased choline incorporation (;50% above baseline)and marked induction of cytidylyltransferase (CYT) geneexpression. In contrast to CRH, TRH significantly increasedcholine incorporation after only 2 days of culture. The peakcholine incorporation response to CRH (110% increase)was observed in the one e90 baboon available for study(data not shown). Low doses of BN combined with lowdoses of TRH acted additively in increasing choline incor-poration, similar to CRH (data not shown). Although TRHis known to stimulate surfactant production and improvedlung mechanics in newborn lambs in vivo [2,12], the presentstudy is the first to indicate direct stimulation of lung mat-uration and growth by TRH. Although we were unable todemonstrate TRH-RI [27] in lung tissue samples from ourbaboons, it is plausible that there might be lower levels ofTRH-RI present, or that additional TRH receptors are in-volved, such as TRH-RII [28,29].
Both TRH and CRH had growth stimulatory activity inbaboon fetal lung explants. CRH triggered an increase inmesenchymal cell proliferation exclusively as assessed byPCNA immunostaining, whereas TRH stimulated prolifer-ation of both epithelial and mesenchymal cells. The maxi-mal effect of 1–10 nM CRH on cell proliferation wasobserved on e125. At e140, only 100 nM CRH induced the
Fig. 6. Effect of TRH and Dex on CYT mRNA Levels. Lung tissuecollected at e140 (male baboon) was cultured with: media alone (lane 1),10 nM Dex (lane 2), 1.0 nM TRH (lane 3), or 10 nM TRH (lane 4) for 48h.Total RNA prepared from each well was reverse transcribed and amplifiedby PCR using CYT and 18S primers. Southern blots of the PCR productswere probed with CYT-specific end-labeled internal oligonucleotides, asdetailed in Methods.
Fig. 7. Immunostaining for Pro-SP-C in CRH-treated Lung Explants. Lung tissue was collected from 7 separate animals (one at e80, 3 at e125, 3 at e140),and explant cultures were maintained for 60 h with media alone (as represented by one e125 explant shown in panel A) or 1 nM CRH (the same e125 explantas in panel A is shown in panels B and C). Explant tissue was then processed into paraffin and immunostained for proSP-C (Panels A and B) or with normalrabbit serum as a negative control (Panel C). Airspaces, indicated by asterisks (*), are lined by epithelial cells which are weakly SP-C-positive in A, stronglySP-C-positive in B, and negative in C. (methyl green counterstain, magnification3100).
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same effect on thymidine incorporation suggesting that theCRH receptor might be downregulated at this gestationalage, consistent with undetectable CRH receptor mRNAlevels at this fetal age using the described RT-PCR condi-tions. BN and CRH were additive in inhibiting cell growth,consistent with receptor transmodulation.
The present study demonstrates effects of CRH on fetallung explants which markedly differ from those of Dex:Dex is growth inhibitory [30], stimulates choline uptakeafter only 2 days in culture, and induces CYT gene expres-sion. Corticotropin has also been reported to be secreted byneuroendocrine cells in the lung [31,32] and to have potenteffects on stimulating lung development and/or cell differ-entiation both in vivo [33] and in vitro [34]. However,elevated ACTH levels in mice deficient in glucocorticoidreceptor does not compensate for perinatal lethality due toinadequate alveolization [35]. Cumulatively, these observa-tions support a major role for the hypothalamic-pituitary-adrenal axis, especially CRH and Dex, in promoting normallung maturation and/or growth.
Acknowledgments
We thank the NIH for the support of the U10 Collabo-rative Program in Bronchopulmonary Dysplasia, headed byDr. Jacqueline Coalson, without which this work would nothave been possible. Key U10 support staff include VickiWinter and the Pathology staff at University of TexasHealth Sciences Center at San Antonio, and the animalproduction and NICU staff at the Southwest Foundation forBiomedical Research. We are grateful to Drs. Mary EllenAvery and Joseph Majzoub for encouragement and helpfuldiscussions. Supported by NIH grants #U10-HL52638(M.E.S.) and HL52636 (Resource Grant).
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