Targeted skin overexpression of the mineralocorticoid receptor in mice causes epidermal atrophy, premature skin barrier formation, eye abnormalities, and alopecia

Abstract

The mineralocorticoid receptor (MR) is a transcription factor of the nuclear receptor family, activation of which by aldosterone enhances salt reabsorption in the kidney. The MR is also expressed in nonclassical aldosterone target cells (brain, heart, and skin), in which its functions are incompletely understood. To explore the functional importance of MR in mammalian skin, we have generated a conditional doxycycline-inducible model of MR overexpression, resulting in double-transgenic (DT) mice [keratin 5-tTa/tetO-human MR (hMR)], targeting the human MR specifically to keratinocytes of the epidermis and hair follicle (HF). Expression of hMR throughout gestation resulted in early postnatal death that could be prevented by antagonizing MR signaling. DT mice exhibited premature epidermal barrier formation at embryonic day 16.5, reduced HF density and epidermal atrophy, increased keratinocyte apoptosis at embryonic day 18.5, and premature eye opening. When hMR expression was initiated after birth to overcome mortality, DT mice developed progressive alopecia and HF cysts, starting 4 months after hMR induction, preceded by dystrophy and cycling abnormalities of pelage HF. In contrast, interfollicular epidermis, vibrissae, and footpad sweat glands in DT mice were normal. This new mouse model reveals novel biological roles of MR signaling and offers an instructive tool for dissecting nonclassical functions of MR signaling in epidermal, hair follicle, and ocular physiology.

Figure 1

Conditional hMR expression in the skin. A: hMR mRNA expression [as determined by reverse transcription (RT)-PCR] in dorsal skin of adult double-transgenic DT (K5-tTA/tetO-hMR) mice: hMR is expressed in the absence of DOX, whereas 15-day DOX administration (provided in the food) fully suppressed hMR expression. β2-Microglobulin was used as internal standard (different mice in each lane). B: hMR expression (RT-PCR) in the skin of five monotransgenic (control) embryos and five DT embryos (E18.5). The transgene was detected only in DT mice. 18S signal was used as internal standard. C: Western blot of the mouse GR in the skin of three control and three DT embryos (E18.5). Quantification of signal (normalized to Ponceau staining) showed similar GR expression in control mice (arbitrary units: 1.17 ± 0.07) and hMR-overexpressing DT mice (1.08 ± 0.11), n = 5 embryos in each group.

Figure 2

Immunolocalization of MR in the skin. Paraformaldehyde-fixed sections of the skin of 9-week-old mice (4 weeks after DOX withdrawal) and of E18.5 DT embryos were immunostained with monoclonal anti-MR antibodies. A: Skin from control (a) and DT (b) mice (9 weeks old) immunostained with the 2D6 antibody (endogenous mMR) have comparable epidermal (arrow) and follicular (asterisk) mMR expression over both cytoplasm and nuclei. In c, the 2D6 antibody has been substituted by the unrelated UPC10 mouse antiserum over DT skin (showing low nonspecific signal). B: Immunolocalization of the hMR with the 6G1 antibody (labeling the endogenous MR and the transgenic hMR) in the skin of 9-week-old control (a, without hMR overexpression) and DT (b and c) mice. Cytoplasmic staining was comparable in control (a) and DT (b and c) epidermis. Nuclear signal with 6G1 was found in epidermis (arrow), sebaceous gland (sg), and hair follicle (asterisk) of DT mice (not in control mice). In d, the 6G1 antibody has been substituted by the unrelated UPC10 mouse antiserum over DT skin (showing low nonspecific signal). C: Immunolocalization of the hMR in the skin of E18.5 DT embryos. Nuclear labeling with the 6G1 antibody is apparent over several epidermal layers (arrow) and in hair follicle (asterisk). Bar = 50 μm.

Figure 3

Conditional hMR expression in the skin during gestation provokes perinatal mortality. A: Number of mice alive at postnatal day 10 according to their genotype: wild-type (WT), monotransgenic tTA or hMR (K5-tTA or tetO-hMR), and DT (K5-tTA/tetO-hMR) pups. A major deficiency of DT mice was observed at day 10, attesting for their earlier mortality. B: DT mice recovered at E18.5 (ie, 1 day before birth), at postnatal day 1 (D1), and at day 10 (D10); results are expressed as the percentage of DT mice among offspring. At E18.5, the number of DT mice was close to that expected, ie, 25% (according to Mendelian distribution), whereas DT mice were all dead at the end of day 1, indicating perinatal mortality of DT mice. Administration of DOX or the MR antagonist potassium canrenoate during gestation prevented the mortality of DT pups, as seen in surviving treated DT pups at day 10. Numbers above bars indicate the number of DT versus total mice in each series. *P < 0.05; ***P < 0.001; χ² test.

Figure 4

Phenotype of MR-expressing embryos. Expression of hMR during development impairs skin and eye formation at E18.5 and leads to skin atrophy at birth. A: E18.5 DT embryos (a′) presented with a shiny erythematous skin and open eyes compared with their control littermates (a). Low-magnification view of the eye (bar = 250 μm) of E18.5 control (b) and DT (b′) embryos (hematoxylin-eosin staining): At this stage of development the eyelids are fully formed and fused in control embryos, whereas in DT mice, they are poorly developed and do not cover the eye, leading to the eye-open phenotype (also note the thin epidermis in DT mice). High-magnification view (bar = 50 μm) of the anterior eye of control (c) and DT (c′) embryos: eyelids (el) cover the cornea of controls only (in c); the corneal epithelium (arrow) is partly absent in DT (c′) embryos; the anterior chamber [between the corneal stroma (cs) and the lens (**)] is obliterated in DT embryos. White arrowhead, corneal endothelium. B: Skin of the skull of control (a) and DT (a′) E18.5 embryos. The morphology of hair follicle (asterisk) is comparable in control and DT embryos. Bar = 50 μm; **, epidermis; ***, dermis. C: Abdominal skin of control (a) and DT (a′) E18.5 embryos illustrating the flat and thinner epidermis and the reduction in the density of hair follicles (HF) in DT mice; arrow, epidermis; bar = 40 μm. D: Quantification of the epidermis thickness in abdominal skin of control and E18.5 DT embryos (n = 5 controls and 6 DT embryos; average number of measurements, 268 per embryo) and hair follicle (HF) density per millimeter of epidermis (n = 5 controls and 5 DT embryos; 528 HF counted in controls, 220 in DT mice). *P < 0.05; **P < 0.001. E: Skin covering the ear in control (a and b) and DT (a′ and b′) E18.5 embryos at low (a and a′) and high (b and b′) magnification. *, stratum corneum; **, epidermis; ***, dermis; arrowhead, basal layer of keratinocytes. Note the epidermal hypoplasia with the reduction of the granular layer (just below the stratum corneum) in DT mice. Bar = 50 μm. F: Skin of control (a and b) and DT (a′ and b′) mice at birth (5 to 10 minutes after birth). Abdominal (a and a′) and dorsal (b and b′) skin is atrophic in DT mice, with intraepidermal zones of rupture (arrow) and loosely adhering stratum corneum (a′ and b′). The arrow denotes a split right above basal layer of keratinocytes in DT epidermis. Bar = 50 μm.

Figure 5

Expression of hMR during development leads to increased apoptosis at E18.5 and accelerated skin barrier formation at E16.5. A: Proliferation and apoptosis in the skin of E18.5 embryos. TUNEL assay showed an increased number of apoptotic cells (TUNEL-positive red nuclei, arrow) in the epidermis of DT mice compared with their control littermates (bar = 50 μm). Ki 67 staining showed no difference in cell proliferation between controls and DT mice (bar = 40 mm). B: Toluidine blue test of epidermal permeability in E16.5 embryos (Hardman test). Most control embryos had a highly permeable skin (extensive blue staining), whereas DT embryos have large portions of the skin that are impermeable to toluidine blue (pink aspect), indicating that expression of hMR during development accelerates skin barrier formation at E16.5. C: Hardman scoring (see Materials and Methods) of the degree of epidermal impermeability, from completely permeable blue skin (score 1) to completely impermeable pink skin (score 8). E16.5 DT embryos (pink bars) have higher impermeability score than controls (blue bars). P < 0.01, χ² test.

Figure 6

Cutaneous phenotype of adult control and DT mice. Postnatal expression of hMR results in alopecia and hair follicle cysts. Mice were fed with DOX food from the onset of gestation until the age of 5 weeks, and then DOX was withdrawn to allow MR expression. A: An adult 8-month-old DT mouse (left) with extensive alopecia compared with its control littermate (right). B: Ki 67 staining of the skin of adult (6 months) control and DT mice, showing the increased proliferation in the multilayered hair follicle cysts (white asterisk) in DT mice. Asterisk indicates HF in control and dilated hair follicle cysts in DT mice. Bar = 50 μm. C–H: Consecutive biopsies of murine skin at different time intervals after DOX withdrawal (on week 5 after birth), illustrating the progressive appearance of hair follicle abnormalities and cystic hair follicle degeneration. Lag time after DOX withdrawal: C, 2 weeks; D, 4 weeks; E, 9 weeks; F, 12 weeks; G, 16 weeks; and H, 22 weeks. Bars: 200 μm (in H, c); 50 μm (in all other panels). C: Two weeks after DOX withdrawal, no abnormalities are visible in DT skin. D: Four weeks after DOX withdrawal, control mice show telogen hair follicles, whereas DT skin shows mature anagen hair follicles, as indicated by their characteristic morphology, location (deep in subcutis), and pigmentation (Supplemental Fig. S2 for further evidence of anagen, available at http://ajp.amjpathol.org), suggesting an abnormality in hair follicle cycling. E: Nine weeks after DOX withdrawal, many microscopic fields of DT skin exhibited signs of severe hair follicle dystrophy [as evident from the presence of ectopic melanin granules, melanin incontinence (arrowheads), and malformed inner (white asterisk) and outer (arrow) root sheaths]. This was seen only very rarely and, when present, in a more discrete manner in control skin of this age group. F: Twelve weeks after DOX withdrawal, the skin of DT mice continued to show signs of hair follicle dystrophy, as evidenced by dilated hair canal (asterisk in a) and abnormal hair follicle (HF) architecture and pigmentation (b) as well as of relative asynchrony of hair follicle cycling, as indicated by the simultaneous presence of telogen and anagen hair follicles next to each other (b and c; *, telogen HF; **, anagen HF; arrowhead, dystrophic anagen HF; double arrowhead, dystrophic telogen/catagen HF). G: Sixteen weeks after DOX withdrawal, dermal cysts of HF were apparent in several DT mice, not in controls. H: Histology of the skin 22 weeks after DOX withdrawal in control [a, sebaceous glands (sg)] and DT (b–d) mice. Cysts of sebaceous glands (b, arrow) and of hair follicle (*, c and d) in DT mice are shown. See Table 1 for comments.