![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, December 2003, p. 8542-8552, Vol. 23, No. 23
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.23.8542-8552.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Medical Nutrition, Karolinska Institute, NOVUM, Huddinge University Hospital, SE14186 Huddinge, Sweden,1 Center MPL, Department of Pharmacological Sciences, University of Milan, 20129 Milan, Italy2
Received 28 February 2003/ Returned for modification 28 May 2003/ Accepted 3 September 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(ER) is undetectable in the lung, and this has left many unanswered questions about the mechanism of estrogen action in this organ. Here we show, both in vivo and in vitro, that ERß is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type mice and mice with an inactivated ERß gene (ERß-/-) revealed decreased numbers of alveoli in adult female ERß-/- mice and findings suggesting deficient alveolar formation as well as evidence of surfactant accumulation. Platelet-derived growth factor A (PDGF-A) and granulocyte-macrophage colony-stimulating factor (GM-CSF), key regulators of alveolar formation and surfactant homeostasis, respectively, were decreased in lungs of adult female ERß-/- mice, and direct transcriptional regulation of these genes by ERß was demonstrated. This suggests that estrogens act via ERß in the lung to modify PDGF-A and GM-CSF expression. These results provide a potential molecular mechanism for the gender differences in alveolar structure observed in the adult lung and establish ERß as a previously unknown regulator of postnatal lung development and homeostasis. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although previous data suggest that estrogens might be important in lung development, physiology, and carcinogenesis, there is very little information about estrogen receptor-dependent functions in the lung. This is most likely related to the absence of estrogen receptor alpha (ER) in this tissue, which, for many years, was considered to be the only estrogen receptor. To better understand the role of estrogen in the lung, we have investigated the expression and physiological role of ERß (16) in the lung. In this paper, we show that ERß is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type (WT) and ERß-/- female mice indicate that this receptor modulates alveolar structure and surfactant homeostasis. Analysis of gene expression in WT and ERß-/- female mice and studies of transcriptional regulation show that platelet-derived growth factor A (PDGF-A), which plays a pivotal role in alveolar formation (4, 18), and granulocyte-macrophage colony-stimulating factor (GM-CSF), a key regulator of surfactant homeostasis (9, 38), are both controlled at the transcriptional level by estrogens via ERß in the lung. This provides a mechanism for the modulation of alveolar structure and surfactant homeostasis by estrogen.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Fixation and tissue preparation. Animals were killed through cervical dislocation, and anterior chest walls were removed. A cannula was inserted into the trachea and tied firmly in place. The trachea and lungs were infused with 4% paraformaldehyde (pH 7.4) at 20 cm H2O pressure and maintained at this pressure for 5 min or removed without intratracheal infusion. The lungs were subsequently kept in fixative overnight at 4°C. After fixation, the lungs were dehydrated through a graded series of ethanol. Finally, the right and left lungs were separated and placed into individual cassettes and embedded in paraffin. The central portions of the blocks were sectioned at 5-µm intervals, and the sections were mounted on glass slides, deparaffinized, and hydrated for staining.
Immunohistochemistry and immunofluorescence.
The cellular presence of ER and ß was detected by standard immunohistochemistry procedures as described by Patrone et al. (26) with some modifications. Briefly, for detection of ER, the rabbit polyclonal antibody MC20 from Santa Cruz Biotechnology (Santa Cruz, Calif.) was used, and for ERß, the chicken polyclonal ERß 503 immunoglobulin Y (33, 40), made by immunization with ERß 503, was used. ERß 503 is human ERß1, modified in its ligand-binding domain (LBD) by insertion of the rat 18-amino-acid sequence described in reference 24. The production and characterization of this ERß-specific antibody have previously been described (33, 40). After deparaffinization and rehydration, the lung section was boiled in 10 mM citrate buffer for antigen retrieval. The cooled sections were incubated in 0.5% H2O2 to quench endogenous peroxidase. To block unspecific binding of secondary antibodies, sections were incubated in blocking solution (5% normal goat serum). Primary antibody 503 was added (1:500 dilution in blocking solution, incubated overnight at 4°C). The ER antibody MC20 (Santa Cruz Biotechnology) was diluted 1:500, and antibodies against surfactant apoprotein A (SP-A) and the intracellular proform of surfactant apoprotein C (proSP-C) (N-19 and M-20, respectively, from Santa Cruz Biotechnology) were diluted 1:200 in blocking solution. After several washes, the Vectastain ABC kit (Vector Laboratories, Burlingame, Calif.) was used for visualization. All slices were slightly counterstained with Mayer's hematoxylin and mounted. Control experiments including incubation without primary antibodies, as well as studies with preadsorbed antibody, were all negative. Sections for immunofluorescence were deparaffinized and rehydrated. To block unspecific binding of secondary antibodies, sections were incubated in blocking solution (0.1 M lysine). Primary antibody against smooth muscle -actin (clone 1A4; Sigma, St. Louis, Mo.) was used at a 1:500 dilution. For visualization, a secondary fluorescein isothiocyanate-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.) was used at a 1:100 dilution. After counterstaining with 4',6'-diamidinio-2-phenylindole (DAPI), slides were mounted and examined with a Zeiss Axioplan 2 microscope with filters for fluorescein isothiocyanate and DAPI.
Protein extraction and Western blot analysis. All tissue handling was done at 4°C. Tissue samples were homogenized for a few seconds with a Polytron PT3100 in a buffer containing 600 mM Tris · HCl, 1 mM EDTA (pH 7.4) and protease inhibitor mixture tablets (Roche Molecular Biochemicals, Mannheim, Germany), and the homogenates were centrifuged for 1 h at 50,000 x g. To load equal amounts of protein for Western blot analysis, the protein content of the supernatant fractions was measured by a Bio-Rad (Hercules, Calif.) protein assay with bovine serum albumin as the standard. For ERß detection, samples were precipitated with trichloroacetic acid (TCA), and the precipitate was washed with methanol. Samples were placed on dry ice for 30 min, and the proteins were recovered by centrifugation. Pellets were then dissolved in sodium dodecyl sulfate (SDS) sample buffer and loaded on the gel. For SP-A and Clara cell secretory protein (CCSP), equal amounts of protein (20 µg) were loaded. Proteins were resolved on SDS-10, 12, and 15% polyacrylamide gels for ERß, SP-A, and CCSP, respectively. Transfer to polyvinylidene difluoride membranes was done by electroblotting in Tris-glycine buffer. The membranes were checked for equal transfer by Ponceau staining. The membranes were blocked in 10% skimmed milk in phosphate-buffered saline (PBS) buffer-0.1% NP-40. Incubation with antibodies was done at dilutions of 1:3,000 for ERß LBD (33, 40), 1:100 for SP-A (Santa Cruz Biotechnology), and 1:3,000 for CCSP (22) in the same buffer as used for the blocking reaction. The ERß LBD antibody is a rabbit polyclonal antibody prepared by using the LBD of human ERß1 (amino acids 320 to 527). The production and characterization of this ERß-specific antibody are described in references 33 and 40. All incubations were performed overnight at 4°C. After washing with PBS buffer-0.1% NP-40, horseradish peroxidase-coupled secondary antibodies (1:10,000; Santa Cruz Biotechnology) were added for 2 h at room temperature. After washing with PBS buffer-0.1% NP-40, the signals were visualized by using the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
N-terminal sequencing of proteins. To obtain sufficient ERß for N-terminal amino acid sequencing, cytosol from 10 g of lung was prepared in 50 ml of the Tris-EDTA buffer described above. This was diluted 10-fold with 20 mM sodium phosphate buffer, pH 7.4, to reduce the ionic concentration. Heparin-Sepharose (1 ml) was added, and the mixture was gently rotated for 1 h at 5°C. Heparin-Sepharose was recovered by centrifugation and washed 5 times with 20 mM sodium phosphate buffer. Proteins were eluted with 1 M NaCl, precipitated with 10% TCA, washed with methanol, and resolved on SDS gels in 6 lanes. Proteins were transferred to polyvinylidene difluoride membranes, a strip was cut from one lane for detection of ERß by Western blotting, and the rest of the membrane was stained with Coomassie brilliant blue. Protein bands corresponding to those reacting with the LBD antibody were cut from the membrane, and N-terminal sequencing was performed with an Applied Biosystems 473A protein sequencer.
Sucrose gradient sedimentation.
Tissues, frozen in liquid nitrogen, were pulverized in a dismembrator (Braun, Kronberg, Germany) in 10 mM Tris-HCl, pH 7.5, 1.5 mM EDTA, and 5 mM sodium molybdate. Cytosol was obtained by centrifugation of the homogenate at 204,000 x g. Cytosols were incubated for 3 h at 0°C with 10 nM tritiated estradiol in the presence or absence of excess radio-inert estradiol (50 nM), and the bound and unbound steroids were separated with Dextran-coated charcoal. Sucrose density gradients (10 to 30% [wt/vol] sucrose) were prepared in buffer containing 10 mM Tris-HCl, 1.5 mM EDTA, 1 mM -monothioglycerol (Sigma), and 10 mM KCl. Samples of 200 µl were layered on 3.5-ml gradients and centrifuged at 4°C for 16 h at 300,000 x g. Successive 100-µl fractions were collected from the bottom by paraffin oil displacement, by using a collector of our own design, and assayed for radioactivity by liquid scintillation counting. For ERß detection, samples were precipitated with TCA, and the precipitate was washed with methanol. Samples were placed on dry ice for 30 min, and the proteins were recovered by centrifugation. Pellets were then dissolved in SDS sample buffer and resolved by SDS-polyacrylamide gel electrophoresis by using 4 to 20% gradient gels.
Electrophoretic mobility shift assay. Estrogen receptor DNA binding was measured in nuclear extracts from primary mouse Clara cells. Primary Clara cells were isolated as described by Oreffo et al. (25). From these cells, nuclear proteins were prepared and electrophoretic mobility shift assays were performed essentially as described by Cassel et al. (6). Briefly, nuclear proteins were incubated with a labeled duplexed ERE (12) (sequence, 5'-GGG TAG AGG TCA CTG TGA CCT CTC GA-3') in binding buffer (100 mM KCl, 10 mM Tris-HCl [pH 7.5], 2 mM dithiothreitol, 5% glycerol, and 0.9 µM estradiol) with or without anti-estrogen receptor antibodies (503 and LBD, see above) (33, 40). In some experiments, unlabeled duplexed oligonucleotides were included as competitors: either the ERE-containing oligonucleotide as described above or an oligonucleotide carrying a single nucleotide substitution in one half site that has been described to disrupt DNA binding of estrogen receptors (34) (sequence [the mutated nucleotide is indicated by a lowercase letter], 5'-GGG TAG AaG TCA CTG TGA CCT CTC GA-3').
Transgenic mice. The transgenic mice carrying the luciferase reporter gene under the transcriptional control of an estrogen response element in front of a herpes simplex virus thymidine kinase promoter have been described elsewhere (7). Heterozygous male mice (2 months old) were injected subcutaneously with 50 µg of E2/kg of body weight or 250 µg of hydroxytamoxifen or vehicle (vegetable oil)/kg as control. At the indicated time points, the animals were sacrificed and luciferase activity was assayed as described.
Morphometry. Sections from lungs fixed by intratracheal inflation at constant pressure as described above were chosen at random and stained with hematoxylin and eosin. From these sections, randomly selected microscopic fields were photographed at x10 magnification with a Zeiss Axioplan 2 microscope. Five fields were analyzed per animal. The pictures were enlarged uniformly and used for morphometric analysis. All alveoli in each field were counted. The number of cells was determined by counting the number of stained nuclei. All pictures were counted by two independent observers who were unaware of the genotype of the animals. Means and standard deviations were calculated, and statistical comparisons were performed by unpaired Student's t test.
Quantitative reverse transcription-PCR. cDNA was synthesized by using the SuperScript first-strand synthesis system for reverse transcription-PCR (Life Technologies, Paisley, United Kingdom) according to the instructions of the manufacturer. For real-time quantitative reverse transcription-PCR analysis, predeveloped TaqMan assay reagents for mouse GM-CSF mRNA and 18S rRNA (Applied Biosystems) were used according to the instructions of the manufacturer. For PDGF-A mRNA, the sequences of the primers used were 5'-GGT CCA CCA CCG CAG TGT-3' (upper) and 5'-GGA CCT CTT TCA ATT TTG GCT TC-3' (lower), and they were used together with the SYBR Green PCR master mix kit (Applied Biosystems) according to the instructions of the manufacturer. The specificity of the PCR product was ensured by agarose gel electrophoresis in conjunction with melting curve analysis by using the Dissociation Curves software (Applied Biosystems) according to the instructions of the manufacturer. All analyses were carried out on an ABI PRISM 7700 sequence detector (Applied Biosystems).
Transient transfection studies. A549 cells were routinely cultured in Dulbecco's minimal essential medium (GibcoBRL, Paisley, United Kingdom) supplemented with 10% fetal bovine serum, 1% L-glutamine, 100 U of penicillin/ml, and 100 µg of streptomycin/ml. Twenty-four hours before transfection, cells were seeded in 12-well plates. Transient transfections were carried out as previously described (27). Briefly, the Lipofectamine Plus reagent (GibcoBRL) was used in serum, phenol red, and antibiotic-free media according to the instructions of the manufacturer. The 1.8-kb PDGF-A promoter-luciferase reporter gene construct and the 1.6-kb GM-CSF promoter-luciferase reporter gene construct were kind gifts from David M. Kaetzel (Department of Molecular and Biomedical Pharmacology, College of Medicine, University of Kentucky, Lexington, Ky.) and Peter Cockerill (Division of Human Immunology, Hanson Centre For Cancer Research, Institute for Medical and Veterinary Science, Adelaide, Australia), respectively. Each well received 200 ng of the reporter plasmid and 5 ng of pSG5-ERß (expressing the mouse ERß) (28), or the parental vector, as indicated. Twenty nanograms of cytomegalovirus-ß-galactosidase plasmid (constitutively expressing ß-galactosidase) was included as a control for transfection efficiency. Serum-containing medium with the addition of hormones (10 nM 17ß-estradiol, 250 nM ICI 182,780) as indicated was added 3 h posttransfection, and the cells were incubated for 24 h before harvest. Data are presented as inductions (n-fold) of luciferase activity corrected for the internal standard and represent the means ± standard deviations of the results from three independent experiments performed in duplicate. The activity of the luciferase reporter transfected without estrogen receptor-expressing plasmid and without hormone treatment was arbitrarily set to 1.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
staining could be detected (Fig. 1A, lower panel; mouse mammary gland inset as positive control). No differences with regard to ER staining were observed when sections from male and female lungs were compared (data not shown). Western blotting with lung extracts confirmed the expression of ERß (Fig. 1B, lane 2). In contrast, lung extracts from knockout mice lacking ERß (14) (Fig. 1B, lane 3) exhibited no immunoreactivity, corroborating the specificity of the antibody. N-terminal sequence analysis of the bands from SDS gels further confirmed ERß expression and showed that the doublet around 65 kDa corresponds to the two 530- and 549-amino-acid ERß isoforms (10). These data are in agreement with previous analyses of estrogen receptor mRNA in the rat lung by reverse transcription-PCR (15). They show that ERß is highly expressed in the adult mouse lung and also indicate that ERß is the predominant pulmonary estrogen receptor.
|
immunoreactivity and estrogen binding were detected in an 8S peak. In the lung, no ER immunoreactivity or 8S estradiol-binding peak was detected (Fig. 2A and data not shown). DNA binding was analyzed in electrophoretic mobility shift assays with an oligonucleotide containing a consensus ERE (12). A shift was observed when incubations were performed with nuclear extracts from isolated murine Clara cells (Fig. 3A, lane 2). When antibodies directed against the LBD of ERß or the entire protein (503) were included (Fig. 3A, lanes 3 and 4, respectively), the shift was clearly diminished, and in the case of the LBD antibody, a supershift appeared, together indicating that the shift contains ERß. The specificity of the shifted complex was demonstrated as these bands were efficiently abolished by competition with unlabeled homologous oligonucleotide (Fig. 3B, lanes 2 and 3) while no competition was observed upon inclusion of unlabeled oligonucleotide carrying a single nucleotide substitution described to disrupt DNA binding by estrogen receptors (34) (Fig. 3B, lanes 4 to 5). These results corroborate the finding that ERß is the major estrogen receptor expressed in the lung and demonstrate that it is functional with regard to ligand binding and DNA binding in in vitro assays.
|
|
, in the lung we conclude that the activation of the ERE-luciferase reporter gene in the lungs of these mice is most likely mediated by ERß. Together, these in vitro and in vivo experiments show that the lung contains functional ERß and is estrogen responsive, suggesting ERß as a mediator of estrogen effects in the lung.
|
|
|
-actin is a specific marker for these cells. To investigate whether these specialized mesenchymal cells were affected in ERß-/- mice, we stained WT and ERß-/- lungs for smooth muscle -actin. The staining pattern for these cells was changed in accordance with the altered alveolar structure. In the WT lung, stained cells were more evenly distributed compared to the staining pattern in the ERß-/- lung (Fig. 5B), indicating that the positioning of these cells is affected in lungs from ERß-/- mice. This suggests that a deficiency in alveolar formation underlies the differences in alveolar number. Together, these results indicate that the lack of ERß renders the female lung unresponsive to estrogen during sexual maturation and thus the normal increase in alveolar number does not occur in the lungs of female ERß-/- mice. Surfactant accumulation in mice lacking ERb. Histological examination of lungs from 1-year-old mice revealed amorphous, acellular, lightly eosinophilic material present inside the alveolar spaces of the female ERß-/- mice (Fig. 6A). This material stained positive for SP-A, one of the major surfactant proteins (17) (Fig. 6B), indicating accumulation of surfactant components. As a marker for the surfactant-producing alveolar type II cells, an antibody specific for proSP-C (17) was used to stain serial sections. However, no differences were noted in the number, or staining intensity, of the type II cells (Fig. 6B). Also, the proSP-C specific antibody failed to stain the accumulated material. The reactivity of the accumulated material with the antibody against SP-A, together with the absence of reactivity for the intracellular proSP-C, suggests that the material represents extracellular accumulation of surfactant inside the alveolar spaces. Accumulation was observed in four of five 1-year-old female ERß-/- mice investigated. In contrast, no evidence of surfactant accumulation was observed in 1-year-old WT female or male mice or in ERß-/- male mice (five animals examined per group). As demonstrated above, ERß is expressed at high levels in both bronchiolar and alveolar epithelial cells in the lung. Thus, we also analyzed a marker for bronchiolar epithelial cells, the CCSP (37), to investigate whether loss of ERß affected bronchiolar epithelial cell function as well. However, no differences in the levels or patterns of expression were observed (data not shown). These results suggest that alveolar homeostasis is affected in female lungs lacking ERß, resulting in accumulation of surfactant components.
|
-actin) (4, 18). GM-CSF, on the other hand, is critical in the regulation of lung surfactant. Disruption of the gene for GM-CSF in mice results in severe abnormalities in alveolar homeostasis with gross accumulation of surfactant (9, 38). We therefore compared the expression of these two signaling molecules in the lungs of WT and ERß-/- female mice. For this purpose, PDGF-A and GM-CSF expression were analyzed by quantitative real-time reverse transcription-PCR. The results in Table 2 show that both PDGF-A and GM-CSF mRNAs were significantly lower in lungs from female ERß-/- mice than in WT littermates. In male mice, no differences were detected between WT and ERß-/- mice. PDGF-A mRNA levels in male mice were similar to the levels in ERß-/- female mice, in accordance with the similarity in alveolar number between the male and female ERß-/- mouse lungs. With regard to GM-CSF expression, male lungs instead exhibited mRNA levels similar to those of female WT lungs, in agreement with the absence of surfactant accumulation in the male WT and ERß-/- lungs. In light of the central role of these factors in regulation of alveolar structure and surfactant homeostasis, these results suggest that the phenotypic alterations observed in female ERß-/- mice may be related to diminished PDGF-A and GM-CSF signaling in the lungs.
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Alveolar formation (or alveologenesis) occurs postnatally by the formation of alveolar septa. Formation of these septa is dependent on a specialized subset of mesenchymal cells that also deposit elastin, the molecule providing elasticity to the lung (18, 30). Adult virgin female rats and mice have a larger number of alveoli, smaller in size, than males. These differences probably exist to meet the metabolic demands of reproduction (20). They are first seen after the animals have reached sexual maturity and seem to be mediated mainly by estrogens (19). Our morphometric analysis shows that this increase in alveolar number does not occur in the female ERß-/- mice. It seems likely that the absence of ERß renders the lung unresponsive to the elevated circulating estrogen levels in the sexually mature female. In males, no differences in alveolar structure were detected between WT and ERß-/- mice. An explanation for this sexual dimorphism is that at sexual maturity females have higher levels of circulating estrogens than males. Furthermore, aromatase, the enzyme converting androgen to estrogen, is lacking in the male mouse lung (36), making it unlikely that there would be any substantial local production of estrogens in the male lung. In addition, recently presented results from the mice carrying the ERE-luciferase reporter gene reveal that whereas transcriptional activation can occur independent of hormone in some organs, this does not occur in the lung (8). Together, this supports the notion that the presence of circulating estrogens acting via ERß in the sexually mature female is the major determinant of the observed lung phenotype.
The extracellular signaling molecule PDGF-A is a key regulator of alveologenesis, as demonstrated in mice carrying a targeted disruption of the PDGF-A gene. PDGF-A-/- mice exhibit complete failure of alveolar septation and loss of elastin expression and die postnatally due to pulmonary problems (4). Further analysis of these mice suggests that PDGF-A from the lung epithelium is crucial for the proliferation, migration, and elastin deposition of the mesenchymal cells central for alveologenesis. In the absence of PDGF-A, these mesenchymal cells do not take their correct positions and fail to deposit elastin, resulting in failure of alveolar formation (18). In light of these observations, our findings of reduced PDGF-A expression and changed positioning of the mesenchymal cells in the ERß-/- female lung, together with transcriptional regulation of the PDGF-A promoter via ERß in lung epithelial cells, provide a mechanistic explanation for the decreased number of alveoli in ERß-/- female mice. That the lung phenotype of female ERß-/- mice is less severe than that of PDGF-A-/- is to be expected, as estrogens have their main role in alveolar development after sexual maturation and serve to induce the increase in alveolar number observed at this time. This is in line with the study by Massaro et al. (20), in which estrogen is proposed to cause the increase in alveolar number in female rodents after sexual maturity. Our results provide a potential mechanism for this effect, and we speculate that estrogens act via ERß in lung epithelial cells to directly modify PDGF-A expression and thereby influence alveologenesis (Fig. 8). In conclusion, our data suggest that, in the absence of ERß, estrogen-dependent up-regulation of PDGF-A will not occur and, therefore, mesenchymal cells will not be stimulated to form additional alveoli, resulting in the loss of the estrogen-dependent increase in alveolar number occurring in the sexually mature female.
|
Taken together, our results provide new mechanistic insights regarding estrogen action in the lung. We have formulated a speculative model shown in Fig. 8, where we propose that estrogen acts through ERß in the lung epithelium and influences the transcription of PDGF-A and GM-CSF. This can give further insight into the effects of estrogen on lung carcinogenesis, since PDGF-A is highly mitogenic for a large number of different cell types in vitro (3) and GM-CSF has been suggested to stimulate proliferation of alveolar type II cells in in vivo mouse models (31). Our data thus provide new information that could help in understanding the gender differences in lung cancer. In the human population, women are more susceptible than males to the deleterious effects of tobacco smoking, are more prone to develop chronic obstructive pulmonary disease, and incur a higher risk of lung cancer (29, 41). There also appears to be a sexual dimorphism regarding types of lung cancer (13, 35). The reasons for these differences are unknown, but estrogens are likely to play a major role, since in animal models, there are estrogen-dependent sex differences in susceptibility towards tobacco-associated lung carcinogens (23). Furthermore, epidemiological studies suggest that hormone replacement therapy with estrogen is associated with a higher risk of lung cancer in postmenopausal women (1, 39). In the present study, we show that estrogen directly regulates the PDGF-A and GM-CSF promoters via ERß in lung cells. Our demonstration of direct estrogen regulation of these potent growth factors in the lungs provides new vistas for the investigation of the mechanisms underlying the observed gender differences in lung cancer.
In conclusion, the data presented in this paper give new mechanistic insights regarding estrogen action in the lung, as summarized in Fig. 8, including an understanding of the observed gender differences in postnatal lung development and structure. This provides a basis for further studies aimed at understanding the sex differences in common and severe lung disorders such as chronic obstructive pulmonary disease and lung cancer. Perhaps increased knowledge in this field may also help uncover new possibilities for treatment of these diseases.
|
ACKNOWLEDGMENTS |
|---|
We thank Peter Cockerill and David M. Kaetzel for reagents needed for this study. We are grateful for the skilled technical assistance provided by Lena Nordlund-Möller and Christina Thulin and for the very valuable suggestions and help from Gil-Jin Shim, Shigehira Saji, Zhang Weihua, Sari Mäkelä, and Tove Berg.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Ballard, P. L. 1989. Hormonal regulation of pulmonary surfactant. Endocr. Rev. 10:165-181.[Medline]
3. Betsholtz, C., and E. W. Raines. 1997. Platelet-derived growth factor: a key regulator of connective tissue cells in embryogenesis and pathogenesis. Kidney Int. 51:1361-1369.[Medline]
4. Bostrom, H., K. Willetts, M. Pekny, P. Leveen, P. Lindahl, H. Hedstrand, M. Pekna, M. Hellstrom, S. Gebre-Medhin, M. Schalling, M. Nilsson, S. Kurland, J. Tornell, J. K. Heath, and C. Betsholtz. 1996. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 85:863-873.[CrossRef][Medline]
5. Breeze, R. G., and E. B. Wheeldon. 1977. The cells of the pulmonary airways. Am. Rev. Respir. Dis. 116:705-777.[Medline]
6. Cassel, T. N., L. Norlund-Möller, O. Andersson, J.-Å. Gustafsson, and M. Nord. 2000. C/EBPalpha and C/EBPdelta activate the Clara cell secretory protein gene through interaction with two adjacent C/EBP-bindng sites. Am. J. Respir. Cell Mol. Biol. 22:469-480.
7. Ciana, P., G. Di Luccio, S. Belcredito, G. Pollio, E. Vegeto, L. Tatangelo, C. Tiveron, and A. Maggi. 2001. Engineering of a mouse for the in vivo profiling of estrogen receptor activity. Mol. Endocrinol. 15:1104-1113.
8. Ciana, P., M. Raviscioni, P. Mussi, E. Vegeto, I. Que, M. G. Parker, C. Lowik, and A. Maggi. 2003. In vivo imaging of transcriptionally active estrogen receptors. Nat. Med. 9:82-86.[CrossRef][Medline]
9. Dranoff, G., A. D. Crawford, M. Sadelain, B. Ream, A. Rashid, R. T. Bronson, G. R. Dickersin, C. J. Bachurski, E. L. Mark, J. A. Whitsett, and R. C. Mulliugan. 1994. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264:713-716.
10. Fuqua, S. A., R. Schiff, I. Parra, W. E. Friedrichs, J. L. Su, D. D. McKee, K. Slentz-Kesler, L. B. Moore, T. M. Willson, and J. T. Moore. 1999. Expression of wild-type estrogen receptor beta and variant isoforms in human breast cancer. Cancer Res. 59:5425-5428.
11. Gray, K., B. Eitzman, K. Raszmann, T. Steed, A. Geboff, J. McLachlan, and M. Bidwell. 1995. Coordinate regulation by diethylstilbestrol of the platelet-derived growth factor-A (PDGF-A) and -B chains and the PDGF receptor alpha- and beta-subunits in the mouse uterus and vagina: potential mediators of estrogen action. Endocrinology 136:2325-2340.[Abstract]
12. Klein-Hitpass, L., G. U. Ryffel, E. Heitlinger, and A. C. Cato. 1988. A 13 bp palindrome is a functional estrogen responsive element and interacts specifically with estrogen receptor. Nucleic Acids Res. 16:647-663.
13. Kmietowicz, Z. 1998. Women at double risk of small cell lung cancer. BMJ 317:1614.
14. Krege, J. H., J. B. Hodgin, J. F. Couse, E. Enmark, M. Warner, J. F. Mahler, M. Sar, K. S. Korach, J. A. Gustafsson, and O. Smithies. 1998. Generation and reproductive phenotypes of mice lacking estrogen receptor beta. Proc. Natl. Acad. Sci. USA 95:15677-15682.
15. Kuiper, G. G., B. Carlsson, K. Grandien, E. Enmark, J. Haggblad, S. Nilsson, and J. A. Gustafsson. 1997. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863-870.
16. Kuiper, G. G. J. M., E. Enmark, M. Pelto-Huikko, S. Nilsson, and J.-Å. Gustafsson. 1996. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. USA 93:5925-5930.
17. Kuroki, Y., and D. R. Voelker. 1994. Pulmonary surfactant proteins. J. Biol. Chem. 269:25943-25946.
18. Lindahl, P., L. Karlsson, M. Hellstrom, S. Gebre-Medhin, K. Willetts, J. K. Heath, and C. Betsholtz. 1997. Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development. Development 124:3943-3953.[Abstract]
19. Massaro, G. D., J. P. Mortola, and D. Massaro. 1996. Estrogen modulates the dimensions of the lung's gas-exchange surface area and alveoli in female rats. Am. J. Physiol. 270:L110-L114.
20. Massaro, G. D., J. P. Mortola, and D. Massaro. 1995. Sexual dimorphism in the architecture of the lung's gas-exchange region. Proc. Natl. Acad. Sci. USA 92:1105-1107.
21. Mendelson, C. R., P. C. MacDonald, and J. M. Johnston. 1980. Estrogen binding in human fetal lung tissue cytosol. Endocrinology 106:368-374.[Abstract]
22. Nord, M., M. Låg, T. N. Cassel, M. Randmark, R. Becher, H. J. Barnes, P. E. Schwarze, J.-Å. Gustafsson, and J. Lund. 1998. Regulation of CCSP (PCB-BP/Uteroglobin) expression in primary cultures of lung cells - involvement of C/EBP. DNA Cell Biol. 17:481-492.[Medline]
23. Noronha, R. F., and C. M. Goodall. 1984. The effects of estrogen on single dose dimethylnitrosamine carcinogenesis in male inbred Crl/CDF rats. Carcinogenesis 5:1003-1007.
24. Ogawa, S., S. Inoue, T. Watanabe, A. Orimo, T. Hosoi, Y. Ouchi, and M. Muramatsu. 1998. Molecular cloning and characterization of human estrogen receptor betacx: a potential inhibitor of estrogen action in human. Nucleic Acids Res. 26:3505-3512.
25. Oreffo, V. I., A. Morgan, and R. J. Richards. 1990. Isolation of Clara cells from the mouse lung. Environ. Health Perspect. 85:51-64.[Medline]
26. Patrone, C., S. Andersson, L. Korhonen, and D. Lindholm. 1999. Estrogen receptor-dependent regulation of sensory neuron survival in developing dorsal root ganglion. Proc. Natl. Acad. Sci. USA 96:10905-10910.
27. Pettersson, K., F. Delaunay, and J. A. Gustafsson. 2000. Estrogen receptor beta acts as a dominant regulator of estrogen signaling. Oncogene 19:4970-4978.[CrossRef][Medline]
28. Pettersson, K., K. Grandien, G. G. Kuiper, and J. A. Gustafsson. 1997. Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha. Mol. Endocrinol. 11:1486-1496.
29. Prescott, E., A. M. Bjerg, P. K. Andersen, P. Lange, and J. Vestbo. 1997. Gender difference in smoking effects on lung function and risk of hospitalization for COPD: results from a Danish longitudinal population study. Eur. Respir. J. 10:822-827.[Abstract]
30. Prodhan, P., and T. B. Kinane. 2002. Developmental paradigms in terminal lung development. Bioessays 24:1052-1059.[CrossRef][Medline]
31. Reed, J. A., and J. A. Whitsett. 1998. Granulocyte-macrophage colony-stimulating factor and pulmonary surfactant homeostasis. Proc. Assoc. Am. Physicians 110:321-332.[Medline]
32. Robertson, S. A., G. Mayrhofer, and R. F. Seamark. 1996. Ovarian steroid hormones regulate granulocyte-macrophage colony-stimulating factor synthesis by uterine epithelial cells in the mouse. Biol. Reprod. 54:183-196.[Abstract]
33. Saji, S., E. V. Jensen, S. Nilsson, T. Rylander, M. Warner, and J. A. Gustafsson. 2000. Estrogen receptors alpha and beta in the rodent mammary gland. Proc. Natl. Acad. Sci. USA 97:337-342.
34. Schwabe, J. W., L. Chapman, J. T. Finch, and D. Rhodes. 1993. The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75:567-578.[CrossRef][Medline]
35. Sekine, I., Y. Nishiwaki, T. Yokose, K. Nagai, K. Suzuki, and T. Kodama. 1999. Young lung cancer patients in Japan: different characteristics between the sexes. Ann. Thorac. Surg. 67:1451-1455.
36. Simpson, E. R., Y. Zhao, V. R. Agarwal, M. D. Michael, S. E. Bulun, M. M. Hinshelwood, S. Graham-Lorence, T. Sun, C. R. Fisher, K. Qin, and C. R. Mendelson. 1997. Aromatase expression in health and disease. Recent Prog. Horm. Res. 52:185-213.
37. Singh, G., and S. L. Katyal. 2000. Clara cell proteins. Ann. N. Y. Acad. Sci. 923:43-58.[Medline]
38. Stanley, E., G. J. Lieschke, D. Grail, D. Metcalf, G. Hodgson, J. A. Gall, D. W. Maher, J. Cebon, V. Sinickas, and A. R. Dunn. 1994. Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc. Natl. Acad. Sci. USA 91:5592-5596.
39. Taioli, E., and E. L. Wynder. 1994. Re: Endocrine factors and adenocarcinoma of the lung in women. J. Natl. Cancer Inst. 86:869-870.
40. Weihua, Z., S. Makela, L. C. Andersson, S. Salmi, S. Saji, J. I. Webster, E. V. Jensen, S. Nilsson, M. Warner, and J. A. Gustafsson. 2001. A role for estrogen receptor beta in the regulation of growth of the ventral prostate. Proc. Natl. Acad. Sci. USA 98:6330-6335.
41. Zang, E. A., and E. L. Wynder. 1996. Differences in lung cancer risk between men and women: examination of the evidence. J. Natl. Cancer Inst. 88:183-192.
This article has been cited by other articles:
| ||||||||||||||||||||||||||||
