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. 2006 May 2;103(18):7165-9.
doi: 10.1073/pnas.0602194103. Epub 2006 Apr 24.

Lung dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ERbeta-/-) mice

Andrea Morani  1 , Rodrigo P A Barros, Otabek Imamov, Kjell Hultenby, Anders Arner, Margaret Warner, Jan-Ake Gustafsson
Affiliations

Lung dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ERbeta-/-) mice

Andrea Morani et al. Proc Natl Acad Sci U S A. .

Abstract

Estrogen receptor beta (ERbeta) is highly expressed in both type I and II pneumocytes as well as bronchiolar epithelial cells. ERalpha is not detectable in the adult lung. Lungs of adult female ERbeta knockout (ERbeta-/-) mice have already been reported to have fewer alveoli and reduced elastic recoil. In this article, we report that, by 5 months of age, there are large areas of unexpanded alveoli in lungs of both male and female ERbeta-/- mice. There is increased staining for collagen and, by EM, abnormal clusters of collagen fibers are seen in the alveolar septa of ERbeta-/- mice. Immunohistochemical analysis and Western blotting with lung membrane fractions of ERbeta-/- mice revealed down-regulation of caveolin-1, increased expression of membrane type-1 metalloproteinase, matrix metalloproteinase 2 (active form), and tissue inhibitors of metalloproteinases 2. Hypoxia, measured by immunohistochemical analysis for hypoxia-inducible factor 1alpha and chemical adducts (with Hypoxyprobe), was evident in the heart, ventral prostate, periovarian sac, kidney, liver, and brain of ERbeta-/- mice under resting conditions. Furthermore, both male and female adult ERbeta-/- mice were reluctant to run on a treadmill and tissue hypoxia became very pronounced after exercise. We conclude that ERbeta is necessary for the maintenance of the extracellular matrix composition in the lung and loss of ERbeta leads to abnormal lung structure and systemic hypoxia. Systemic hypoxia may be responsible for the reported left and right heart ventricular hypertrophy and systemic hypertension in ERbeta-/- mice.

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Conflict of interest statement

Conflict of interest statement: In addition to the partial funding of this study from Karo Bio AB, J.-Å.G. is a cofounder, deputy board member, stockholder, and consultant of Karo Bio AB.

Figures

Fig. 1.
Fig. 1.
Hematoxylin/eosin staining of lung from 1-year-old WT and ERβ−/− mice. There is normal alveolar structure in WT lung sections (A), whereas areas of collapsed alveoli are evident in the ERβ−/− lung sections (B). (C) Azan staining shows blue-stained collagenous extracellular material in the alveolar septa of WT lung section. (D) Intense blue-stained accumulation of ECM and thick alveolar septa in ERβ−/− lung. (E) Immunoblotting of lung membrane fraction shows decreased ratio of inactive 72-kDa MMP2 form to the 62-kDa MMP2 active form and increased expression of MT1-MMP and TIMP2 in ERβ−/− mice. (Magnification: A and B, ×10; C and D, ×20.)
Fig. 2.
Fig. 2.
EM of alveolar septa of WT and ERβ−/− lung sections. Accumulation of collagen fibers are evident in ERβ−/− lung septa (B and D) in longitudinal (arrows) and transversal (arrowheads) positions of the lung sections. In WT lung, collagen appears to be distributed in smaller and scattered fibers (A and C; arrows) than in ERβ−/− lung.
Fig. 3.
Fig. 3.
Immunofluorscence staining for caveolin-1 shows down-regulation of this protein in the lungs of ERβ−/− mice (B) compared with WT mice (A). (Magnification: ×20.)
Fig. 4.
Fig. 4.
Immunohistochemical detection of HIF-1α in heart, ventral prostate, and ovarian sac. There is no nuclear staining in the cardiomyocytes of WT mice (A). Several cells are positively stained in the nucleus in the heart section of ERβ−/− mice (B). Very few weakly stained epithelial cell nuclei are found in the ventral prostate of WT mice (C), whereas almost the whole prostatic epithelium of ERβ−/− mice is intensively stained (D). There are very strong positive signals in many cells of the periovarian sac in the ERβ−/− mice (F) but only weakly stained cells in WT sections (E). There were no HIF-1α-positive cells in the ovary of either WT or ERβ−/− mice. (Magnification: A–D, ×20; E and F, ×40.)
Fig. 5.
Fig. 5.
Positive and negative controls for determination of the specificity of Hypoxyprobe. A generally stronger and more widely distributed positive signal is evident in the kidney sections of mice exposed to prolonged hypoxia (B) compared with mice exposed to normal air (A). (Magnification: ×10.)
Fig. 6.
Fig. 6.
Immunohistochemical detection of Hypoxyprobe chemical adducts. Liver and kidney are hypoxic organs. Hypoxia has been demonstrated in hepatocytes close to the central vein (CV) areas (31) . (A) In hepatocytes, there is a gradual increase in cytoplasmic staining from the portal veins (PV) toward the CV. (B) There is very strong nuclear and cytoplasmic staining in the hepatocytes of ERβ−/− mice. (C) Positive cytoplasmic staining is seen in many tubular epithelial cells of the kidney of WT mice. (D) Stronger cytoplasmic staining and presence of some areas of nuclear staining in the kidney of ERβ−/− mice. (E) No staining in the whole-brain section of WT mice (here represented as a supraopticus area). (F) Presence of some positively stained neurons in the brain of ERβ−/− mice. (Magnification: A and B, ×10; C–F, ×20; Inset, ×40.)
Fig. 7.
Fig. 7.
Immunohistochemical detection of Hypoxyprobe chemical adducts in mice killed after physical strain on a treadmill (speed, 10 m/min). (A) A weak cytoplasmic signal is evident in the hepatocytes close to the central vein (CV) in WT mice. PV, portal veins. (B) Extended regions of strong cytoplasmic and nuclear staining in the liver of ERβ−/− mice. (C) Some tubular epithelial cells are stained in both cytoplasm and nucleus of WT kidney, whereas most cells show only cytoplasmic signal. (D) Almost the entire epithelial tubular cell of the kidney of ERβ−/− mice shows strong positive signals in both cytoplasm and nucleus. (E) There are very few stained neurons in brain section of WT mice. (F) There are many areas with stained neurons in the section of ERβ−/− mice. (Magnification: A and B, ×10; C–F, ×20; Inset, ×40.)
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