Am J Physiol Lung Cell Mol Physiol 293: L222-L228, 2007.
First published April 20, 2007; doi:10.1152/ajplung.00384.2006
1040-0605/07 $8.00
Estrogen receptor-
regulates pulmonary alveolar loss and regeneration in female mice: morphometric and gene expression studies
Donald Massaro,1,3
Linda Biadasz Clerch,2 and
Gloria DeCarlo Massaro2
Lung Regeneration Laboratory, Departments of 1Medicine and 2Pediatrics, and 3Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, District of Columbia
Submitted 28 September 2006
; accepted in final form 14 April 2007
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ABSTRACT
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Pulmonary alveoli, especially in females, are estrogen-responsive structures: ovariectomy in wild-type (WT) adult mice results in alveolar loss, and estradiol replacement induces alveolar regeneration. Furthermore, estrogen receptor (ER)-
and ER-
are required for the developmental formation of a full complement of alveoli in female mice. We now show ovariectomy resulted in alveolar loss in adult ER-–/– mice but not in adult ER-–/– mice. Estradiol treatment of ovariectomized ER-–/– mice induced alveolar regeneration. In ovariectomized WT mice, estradiol treatment resulted, within 1 h, in RNA-level gene expression supportive of processes needed to form an alveolar septum, e.g., cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion. Among these processes, protein expression supporting angiogenesis and cell replication was elevated 1 and 3 h, respectively, after estradiol treatment; similar findings were not present in either mutant. We conclude: 1) loss of signaling via ER- is not required for postovariectomy-induced alveolar loss or estradiol-induced regeneration; this indicates ER- is key for estrogen-related alveolar loss and regeneration in adult female mice; 2) taken together with prior work showing that developmental formation of a full complement of alveoli requires ER- and ER-, the present findings indicate the developmental and regenerative formation of alveoli are regulated differently, i.e., signaling for alveolar regeneration is not merely a recapitulation of signaling for developmental alveologenesis; and 3) the timing of estradiol-induced gene expression in lung supportive of processes required to form a septum differs between ovariectomized WT and ER-–/– mice.
mutant mice; ovariectomy; hormone replacement therapy; estrogen signaling
STUDIES OF FEMALE RODENTS and women indicate pulmonary gas-exchange units (alveoli) are estrogen-dependent and -responsive structures (14, 17, 19, 23) and provide evidence this responsiveness plays a role in age-related loss of lung function (2, 21, 22) and, less conclusively, in the development and clinical course of chronic obstructive pulmonary disease (COPD) (1, 18, 27). Prior work showed 1) estrogen receptor (ER)- and ER-, the only known mammalian ERs (8), and estrogen, are, respectively, required for the developmentally regulated formation of a full complement of pulmonary alveoli in female mice (14, 22) and rats (17), 2) ovariectomy in adult wild-type (WT) mice results in loss of alveoli (15), and 3) after alveolar loss in adult WT ovariectomized mice, treatment with estradiol induces alveolar regeneration (15). Now, using ER-–/– and ER-–/– mice, we examined the role of these ERs in the alveolar architectural response to ovariectomy and to estradiol treatment after ovariectomy. We also compared the effect, and the rapidity of the effect, of estradiol on gene expression in the lung of ovariectomized WT, ER-–/–, and ER-–/– mice. We selected for analysis genes for which expression would be supportive of biological processes required to convert a flat segment of alveolus into a fold (septum), thereby increasing alveolar surface area, e.g., genes supporting cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion (10, 11).
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MATERIALS AND METHODS
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Animals and experimental manipulations.
Adult bilateral sham ovariectomized and bilateral ovariectomized ER-–/– and ER-–/– adult mice, on a background of C57BL/6, were obtained from Taconic Farms. WT C57BL/6J mice were obtained from The Jackson Laboratories. All mice were allowed ad libitum access to Rodent Chow 5001 and tap water and were housed four or five per cage on a 12:12-h light-dark cycle. We received mice 1 wk after surgery and began studies 1 mo after surgery. Some mice were injected subcutaneously with sesame oil, the vehicle for estradiol, or with equivolume (0.5 µl/g body mass) estradiol (10 µg/kg body mass) once and killed 1 or 3 h later; other mice were treated daily for 3 wk; all mice were killed by exsanguination produced by cutting large abdominal vessels after achieving a surgical level of anesthesia (failure to withdraw from a toe pinch) with xylazine (
10 mg/kg) plus ketamine (75 mg/kg). All procedures were approved by the Georgetown University Animal Care and Use Committee and comply with American Physiological Society Guiding Principles on the Care and Use of Animals.
Morphometric studies.
We anesthetized mice, intubated the trachea, punctured the diaphragm from below, and instilled glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, into the trachea at a transpulmonary pressure of 20 cmH2O. The trachea was ligated, and the lungs were removed from the thorax and fixed as previously described (16). Serial sections were cut from three blocks per mouse at about 0.8-µm thickness to a depth of 150–250 µm. Alveolar spaces were identified (16), and the selector method (4) was used to select alveoli for analysis. The point-sample intercepts method (5) was used to determine the volume of an average alveolus, the number of alveoli was calculated (16), and alveolar surface area was determined by point and intersection counting (30).
Real-time PCR.
RT-PCR was used to measure the relative amount of RNA. TRIzol reagent (Invitrogen) was used to isolate total RNA from mouse lung tissue, followed by cleanup with RNeasy mini kit (Qiagen). A two-step RT-PCR was performed. In step one, cDNA was reverse-transcribed from total RNA using random primers from the High-Capacity cDNA Archive Kit (Applied Biosystems, AB). In step two, PCR products were synthesized using PCR master mix (AB) with AmpErase uracil-N-glycosylase (UNG) and AmpliTaq Gold polymerase. We monitored the exponential increase of PCR products by following the increase of fluorescence of the reporter dye. Sequence-specific primers (unlabeled) and TaqMan minor groove binder 6-FAM dye-labeled probes were obtained from AB. Supplementary Table 1 (available in the data supplement online at the AJP-Lung Cellular and Molecular Physiology web site) contains the AB assay ID for each mRNA measured. We used TaqMan 18s rRNA, VIC-labeled, primer-limited, endogenous control (AB) to normalize the amount of cDNA added to the reaction; all reactions were performed in an AB 7300 RT-PCR instrument. Standard curves for 18s rRNA and the gene of interest were used for quantitation.
DNA synthesis by lung.
DNA synthesis by lung slices was measured as previously described in detail (6, 9). Briefly, we incubated lung slices in Krebs-Ringer bicarbonate (KRB) solution that was previously gassed for 1 h with 95% O2-5% CO2, to which had been added 5.5 mM glucose and serum concentrations of amino acids (20). We then added [3H]thymidine plus sufficient nonradioactive thymidine to achieve a final medium concentration of 15 µM thymidine (6). This concentration of thymidine was used to eliminate possible differences in the pool size of endogenous thymidine (6). We sliced excised lungs using a McIlwain Tissue Chopper at a setting of 1.0 mm (13). The slices were placed in KRB with glucose, amino acids, and [3H]thymidine and incubated for 1 h at 37°C, shaking at 120 oscillations/min while being gassed with 95% O2-5% CO2. After 1 h of incubation, the slices were removed from the incubation mixture and homogenized with a Polytron homogenizer set at full speed. The homogenate and the medium were handled as previously described (6, 9). We counted radioactivity in the cold trichloroacetic acid (TCA) insoluble material that had been made soluble in hot 5% TCA and in medium 10% TCA soluble material and calculated the rate of incorporation of thymidine into DNA, i.e., the rate of DNA synthesis by lung slices.
Western blot and ELISA measurements.
Western blot and ELISA assays were done as previously described (15). We homogenized lung tissue at 4°C in lysis buffer containing 24 mM Tris (pH 7.3), 0.5 mM EDTA, 40 mM Triton X-100, 1.0 mM DTT, and protease inhibitor cocktail (Calbiochem). Homogenates were centrifuged at 10,000 g at 4°C for 1 h, and the protein concentration of the supernatant fluid was measured spectrophotometrically with Coomassie Plus protein assay reagent (Pierce) using bovine serum albumin as standard. Proteins in the 10,000 g supernatant fluid were separated by electrophoresis in 4–20% gradient SDS-PAGE gels (Bio-Rad) and then transferred onto nitrocellulose Hybond ECL membranes (Amersham). The membranes were incubated for 2 h at room temperature in 1% nonfat milk in Tris-buffered saline-Tween (TBS-T), 0.1% Tween 20, 20 mM Tris (pH 7.6), and 137 mM NaCl, followed by an incubation overnight at 4°C with the following primary antibodies: anti-cyclin D3 monoclonal (Cell Signaling Technology) at a dilution of 1:3,000, anti-matrix metalloproteinase-3 (MMP-3) polyclonal (AnaSpec) at a dilution of 1:1,000, or anti-dihydropyrimidase protein-3 (Affinity Reagents) at a dilution of 1:1,000. The membranes were first washed once for 15 min and then three times for 5 min with TBS-T and incubated for 1 h at room temperature with goat anti-mouse (1:3,000) or goat anti-rabbit (1:3,000) from Bio-Rad in 1% nonfat milk in TBS-T. Membranes were washed in TBS-T followed by detection of the immunoreactive protein with an ECL kit (Amersham). The membranes were then probed with rabbit anti-galectin-1 antibody, which acted as an internal standard. Galectin protein bands were visualized by ECL with goat anti-rabbit secondary antibody (Bio-Rad). The intensity of the immunoreactive protein band was quantitated by laser densitometry (Molecular Dynamics) using ImageQuant software. Cyclin D3, MMP-3 densities, and dihydropyrimidase protein were expressed as relative densitometry units per galectin-1. ELISA for vascular endothelial growth factor A (VEGF) protein was performed using Quantikine mouse VEGF immunoassay kit (R&D Systems). Lung homogenates were diluted 20-fold with calibrator diluent RD5T provided in the kit. The protocol followed was as described in the manufacturer's instructions. VEGF concentrations were expressed as VEGF protein per total lung protein.
Statistical analysis.
The mean and standard deviation were calculated for each group. A one-way analysis of variance (ANOVA) was used for comparison of more than two groups. An unpaired two-tailed t-test analysis was performed following the ANOVA or when only two groups were being compared. The Bonferroni adjustment was applied when one mean was compared with more than one other mean. We used the StatMost 32 program for the statistical analysis.
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RESULTS
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Morphometric studies.
Body mass, body length, and lung volume were not different from the same parameters in sham ovariectomized and ovariectomized ER-–/– mice (Fig. 1). The volume of an average alveolus was slightly (7%) smaller, and the number of alveoli 6% larger, in ovariectomized ER-–/– mice compared with sham ovariectomized ER-–/– mice, but alveolar surface area did not differ between groups (Fig. 2). The lack of a detectable intergroup difference of alveolar surface area, in light of the intergroup difference of the volume of an average alveolus, reflects the small intergroup difference in surface area and the fact that area changes to the power 2, whereas volume changes to the power 3. If the alveolar response of ER-–/– mice reflects that of WT mice, which is the assumption underlying the use of mutants, it indicates the postovariectomy decline of estrogen signaling via ER- is responsible for postovariectomy alveolar loss. Put differently, in the absence of ER-, i.e., in ER-–/– mice, ovariectomy, and hence loss of estrogen signaling, did not result in loss of alveoli (Fig. 2). Because alveolar loss did not occur in ovariectomized ER-–/– mice, it was pointless to test for alveolar regeneration.
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Fig. 1. Ovariectomy (Ovx) in adult estrogen receptor (ER)-–/– mice did not alter body mass or length or lung volume. Means ± 1 SD are given. Numbers beneath the error bars represent the number of mice.
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Ovariectomy in ER-–/– mice did not affect body mass or lung volume, but ovariectomized ER-–/– mice were slightly longer than sham ovariectomized ER-–/– mice (Fig. 3). Estradiol treatment of ovariectomized ER- mice did not influence body mass or body length. Neither ovariectomy nor ovariectomy plus estradiol treatment affected lung volume in ER-–/– mice (Fig. 3). Ovariectomized ER-–/– mice had larger and fewer alveoli, and a smaller alveolar surface area, than sham-ovariectomized ER-–/– mice (Fig. 4). Estradiol treatment of ER-–/– mice abrogated the effect of ovariectomy on the volume of an average alveolus, the number of alveoli, and alveolar surface area (Fig. 4).
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Fig. 3. Ovariectomy in adult ER-–/– mice resulted in a slight increase of body length; none of the other parameters were altered by ovariectomy or by treatment with estradiol (Estro). Means ± 1 SD are given. Numbers beneath the error bars represent the number of mice.
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Gene expression in lungs of ovariectomized WT and mutant mice treated with estradiol.
Within 1 h of treating ovariectomized WT mice with estradiol, RNA-level gene expression supportive of cell replication (Table 1), angiogenesis (Table 2), extracellular matrix remodeling (Table 3), and guided cell motion (Table 4) were present. Protein level expression supportive of cell replication (cyclin D3) or guided cell motion (dihydropyrimidase) was not different between ovariectomized WT mice treated with vehicle or estradiol and killed 1 h later (Table 5). Expression of VEGF protein and MMP-3 protein were elevated 1 h after treatment of ovariectomized WT mice with estradiol (Table 5). Three hours after treatment, cyclin D3 protein expression was higher in lungs of ovariectomized estradiol-treated mice than in ovariectomized vehicle-treated WT mice (Table 5). However, this was not yet reflected in a detectable increase of DNA synthesis by lung slices. By 3 h, expression of VEGF and MMP-3 were not different between groups (Table 5). Unlike treatment of ovariectomized WT mice with estradiol, which resulted in a rapid onset of gene expression supportive of processes needed to form a septum, treatment of ovariectomized ER-–/– and ER-–/– mice with estradiol did not increase expression of any of the genes tested (Tables 6
–9).
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Table 1. Cell replication-related gene expression identified by RT-PCR in lung of wild-type ovariectomized mice killed 1 h after injection of vehicle or estradiol
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Table 2. Angiogenesis-related gene expression in lung identified by RT-PCR 1 h after treating ovariectomized wild-type mice with vehicle or estradiol
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Table 3. Extracellular matrix-related gene expression in lungs of ovariectomized wild-type mice 1 h after treatment with vehicle or estradiol
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Table 4. Cell motion and guidance-related gene expression in lungs of ovariectomized wild-type mice 1 h after treatment with vehicle or estradiol
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Table 5. Protein expression in lung of wild-type ovariectomized mice killed 1 or 3 h after injection of vehicle or estradiol
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Table 6. Cell replication-related gene expression in lung of ovariectomized ER-–/– mice 1 h after treatment with vehicle or estradiol
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Table 7. Cell replication-related gene expression in lung of ovariectomized ER-–/– mice 1 h after treatment with vehicle or estradiol
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Table 8. Cell replication-related gene expression in lung of ovariectomized ER-–/– mice 3 h after treatment with vehicle or estradiol
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Table 9. Protein expression in lung of ER-–/– ovariectomized mice killed 1 or 3 h after injection of vehicle or estradiol
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The absence in ovariectomized ER-–/– of estradiol-induced expression of genes supportive of processes required to form a septum was expected because, in the absence of ER-, ovariectomy did not result in alveolar loss. However, in ovariectomized ER-–/– mice, estradiol did induce alveolar regeneration. Therefore, we were surprised at the complete lack of evidence, among the genes tested, of expression supportive of process required to form an alveolus. We do not have information that sheds light on this, we think, interesting finding.
DNA synthesis by lung slices.
To determine if changes at the RNA and protein level supporting cell replication effected cell replication, we measured [3H]thymidine incorporation into acid-insoluble material by lung slices from WT female mice. We did not detect differences of DNA synthesis between ovariectomized WT mice treated with vehicle or estradiol 3 h after treatment (ovariectomy + vehicle = 56.4 ± 7.9 picomoles of thymidine incorporated per milligram of DNA per hour, N = 5; ovariectomy + estradiol = 59.5 ± 8.0 picomoles of thymidine incorporated per milligram of DNA per hour, N = 5), indicating the changes of gene expression had not yet been translated into a detectable alteration in cell replication in the lung. This differs from another model of alveolar regeneration, that occurring after allowing calorie restricted mice ad libitum access to food. In the latter, the onset of detectable cell replication, as evidenced by increased DNA synthesis by lung slices, was present within 3 h of allowing calorie restricted mice ad libitum access to food (15).
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DISCUSSION
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ERs, ovariectomy, and developmental and regenerative alveologenesis.
Ovariectomy in WT mice results in loss of 40% of alveoli, whereas ovariectomy in ER-–/– mice is followed by a slight (6%) but statistically significant increase in the number of alveoli. This contrast demonstrates that, in the absence of ER-, the loss of ovarian hormone signaling, presumably estrogen, does not cause loss of alveoli as it does in WT mice and, conversely, the abrogation of ovarian hormone signaling via ER- is responsible for the postovariectomy loss of alveoli in WT mice. Because postovariectomy alveolar loss occurs in ER-–/– mice albeit to a lesser degree than in WT mice (Table 10), and the absence of ER- does not prevent postovariectomy estradiol-induced alveolar regeneration (10), we conclude ER- does not have a role, or has only a minor role, in alveolar architectural maintenance and regeneration in adult female mice.
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Table 10. Summary: genotype, developmental alveolus formation, postovariectomy alveolar loss, and postovariectomy response to estradiol
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During developmental alveologenesis in female mice, the absence of ER- or ER- results, respectively, in 28% or 38% fewer alveoli than are found in female WT mice (Table 10). Thus both ERs are required for the developmental formation of a full complement of alveoli in female mice. By contrast, the presence of ER-, but not ER-, is required for estradiol-induced postovariectomy alveolar regeneration. Thus signaling for developmental and postovariectomy estradiol-induced alveologenesis are not the same, i.e., signaling for postovariectomy alveologenesis is not merely a recapitulation of signaling for developmental alveologenesis. Beyond estrogen signaling for alveologenesis and regeneration, this finding demonstrates the need for caution before assuming information for alveologenesis and models of alveolar regeneration are interchangeable (15, 29).
The difference in ER signaling between developmental and regenerative alveologenesis, the latter requiring ER- but not ER- (Fig. 2 and 4), taken with the much lower expression of ER- than ER- in the lung (3, 7), suggests ER- may be present in only a few cells in the lung. This suggests ER-, and hence its role in estrogen-induced regenerative alveologenesis, may be effected because ER- is present in intra-alveolar stem cells. Alternatively, ER--containing cells may not be alveolar stem cells but may be estrogen-sensing cells, which then act in a paracrine or endocrine manner to stimulate pulmonary alveolar stem cells in the alveolus or bone marrow, respectively.
Menopause and lung function.
Lung function studies indicate estrogen plays a role in alveolar loss and, less conclusively, in alveolar regeneration in healthy women. Pulmonary diffusing capacity, an index of alveolar gas exchange capacity and alveolar surface area (25), decreases in never-smoker men at a rate of about 6% per decade; women never-smokers lose diffusing capacity at a rate of 2% per decade before, but about 6% per decade after, menopause (21). Postmenopausal women receiving estrogen have a higher forced expiratory flow rate than same-age women not receiving estrogen, an effect not explained by lower rates of smoking or by other health factors (27). The highest rate at which gas can be voluntarily exhaled from the lung (forced timed vital capacity) is determined by resistance to airflow in the conducting airways of the lung (bronchi and bronchioles) and by lung tissue elastic recoil (26). High resistance in the small conducting airways, due to premature or excessive closure of these airways, can be the result of loss of the tethering effect of alveolar attachments caused by diminished developmental alveologenesis (16) or by alveolar destruction (28). More direct evidence of a salutary effect of estrogen on the lung is that, when postmenopausal women are treated with estrogen, their forced expiratory flow rate increases (22). The possibility that ovarian hormones other than estrogen exert an alveolar maintaining and regenerative effect cannot presently be excluded. However, the data in this and prior reports (2, 14, 17, 19, 22) strongly support the notion that estrogen is the ovarian hormone responsible for alveolar maintenance and postovariectomy, or menopausal alveolar loss, and that this effect is conserved from mice to women.
Estrogen and lung disease.
Beyond the role of estrogen in the lung of healthy women, low concentrations of estrogen may play a role in lung disease, in particular, COPD. For the same degree of COPD, women age 45–60 reach that level of disease having smoked fewer cigarettes (18). About 75% of aged never-smokers with COPD are women (1); this does not reflect that women outliving men is shown by the standardized mortality rate, which is almost twice higher in 56.6-yr-old and older women than men with COPD (27).
The prevalence of chronic progressive mountain disease (Monge's disease), which is characterized by a progressive rise of hemoglobin concentration, pulmonary hypertension, and heart failure (8), increases in native Peruvians after menopause (12). This may be at least partly related to postmenopause diminished O2 uptake as suggested by the accelerated loss of lung diffusing capacity following menopause (21) and by the evidence that failed alveogenesis in ER-–/– mice results in hypoxia (19).
In summary, based on our findings (Ref. 14 and present paper) and those of others (2, 21, 22), we suggest estrogen and ER- play a major role in alveolar architectural stability and in alveolar regeneration after surgical or age-related loss of estrogen and that a low concentration of estrogen associated with aging has a deleterious effect on the development (1) and progression (18, 27) of COPD.
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GRANTS
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D. Massaro is Cohen Professor at Georgetown University. We thank Zofia Opalka and Emma Alexander for expert technical assistance.
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ACKNOWLEDGMENTS
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Supported by National Heart, Lung, and Blood Institute Grants HL-37666, HL-20366, and HL-073558.
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FOOTNOTES
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Address for reprint requests and other correspondence: D. Massaro, Lung Regeneration Laboratory, Box 571481, Georgetown Univ. School of Medicine, 3900 Reservoir Rd. NW, Washington, DC 20057-1481 (e-mail: massarod{at}georgetown.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Copyright © 2007 by the American Physiological Society.
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ABSTRACT
MATERIALS AND METHODS
a) and more alveoli (Na) without a difference in alveolar surface area (Sa). Means ± 1 SD are given. Numbers beneath the error bars represent the number of mice.