Targeted Skin Overexpression of the Mineralocorticoid Receptor in Mice Causes Epidermal Atrophy, Premature Skin Barrier Formation, Eye Abnormalities, and Alopecia
2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.060991
ISSN1525-2191
AutoresYannis Sainte–Marie, A. Toulon, Ralf Paus, E. Maubec, Aïcha Cherfa, M Grossin, V. Descamps, Maud Clemessy, Jean-Marie Gasc, Michel Peuchmaur, Adam B. Glick, Nicolette Farman, Frédéric Jaisser,
Tópico(s)Skin Protection and Aging
ResumoThe 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. 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. The skin forms an epithelial barrier that protects the body from environmental damage. This barrier is generated by the epidermis and its constituent cells, most of which are keratinocytes, organized into constantly renewing layers. The basal layer of epidermal keratinocytes is highly proliferative and gives rise to suprabasal layers committed to terminal differentiation that migrate to the surface to produce the stratum corneum, the external cornified layer.1Blanpain C Fuchs E Epidermal stem cells of the skin.Annu Rev Cell Dev Biol. 2006; 22: 339-373Crossref PubMed Scopus (598) Google Scholar The epidermis also gives rise to skin appendages, such as hair follicles, the epithelial component of which is principally formed by keratinocytes and the development (and growth) of which is a highly controlled dynamic process.2Schmidt-Ullrich R Paus R Molecular principles of hair follicle induction and morphogenesis.Bioessays. 2005; 27: 247-261Crossref PubMed Scopus (406) Google Scholar, 3Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1157) Google Scholar Epidermal and hair follicle keratinocytes are highly susceptible to regulation by nuclear receptors.4Alonso LC Rosenfield RL Molecular genetic and endocrine mechanisms of hair growth.Horm Res. 2003; 60: 1-13Crossref PubMed Scopus (50) Google Scholar, 5Calléja C Messaddeq N Chapellier B Yang H Krezel W Li M Metzger D Mascrez B Ohta K Kagechika H Endo Y Mark M Ghyselinck NB Chambon P Genetic and pharmacological evidence that a retinoic acid cannot be the RXR-activating ligand in mouse epidermis keratinocytes.Genes Dev. 2006; 20: 1525-1538Crossref PubMed Scopus (107) Google Scholar, 6Icre G Wahli W Michalik L Functions of the peroxisome proliferator-activated receptor (PPAR) α and β in skin homeostasis, epithelial repair, and morphogenesis.J Investig Dermatol Symp Proc. 2006; 11: 30-35Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 7Ohnemus U Uenalan M Inzunza J Gustafsson JA Paus R The hair follicle as an estrogen target and source.Endocr Rev. 2006; 27: 677-706Crossref PubMed Scopus (150) Google Scholar, 8Bikle DD Elalieh H Chang S Xie Z Sundberg JP Development and progression of alopecia in the vitamin D receptor null mouse.J Cell Physiol. 2006; 207: 340-353Crossref PubMed Scopus (74) Google Scholar, 9Radoja N Stojadinovic O Waseem A Tomic-Canic M Milisavljevic V Teebor S Blumenberg M Thyroid hormones and gamma interferon specifically increase K15 keratin gene transcription.Mol Cell Biol. 2004; 24: 3168-3179Crossref PubMed Scopus (34) Google Scholar Nuclear receptors are transcription factors that regulate various cell functions throughout the body. The nuclear receptor superfamily includes the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), sex steroid and thyroid hormone receptors, vitamin D and retinoic acid receptors, peroxisome proliferator-activated receptors, and numerous orphan receptors.10Lu NZ Wardell SE Burnstein KL Defranco D Fuller PJ Giguere V Hochberg RB McKay L Renoir JM Weigel NL Wilson EM McDonnell DP Cidlowski JA International Union of Pharmacology. LXV. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors.Pharmacol Rev. 2006; 58: 782-797Crossref PubMed Scopus (308) Google Scholar In the skin, members of this family prominently participate in the control of epidermal and hair follicle development, differentiation, and remodeling, and key ligands of nuclear receptors are potent modulators of keratinocyte growth.4Alonso LC Rosenfield RL Molecular genetic and endocrine mechanisms of hair growth.Horm Res. 2003; 60: 1-13Crossref PubMed Scopus (50) Google Scholar, 5Calléja C Messaddeq N Chapellier B Yang H Krezel W Li M Metzger D Mascrez B Ohta K Kagechika H Endo Y Mark M Ghyselinck NB Chambon P Genetic and pharmacological evidence that a retinoic acid cannot be the RXR-activating ligand in mouse epidermis keratinocytes.Genes Dev. 2006; 20: 1525-1538Crossref PubMed Scopus (107) Google Scholar, 6Icre G Wahli W Michalik L Functions of the peroxisome proliferator-activated receptor (PPAR) α and β in skin homeostasis, epithelial repair, and morphogenesis.J Investig Dermatol Symp Proc. 2006; 11: 30-35Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 7Ohnemus U Uenalan M Inzunza J Gustafsson JA Paus R The hair follicle as an estrogen target and source.Endocr Rev. 2006; 27: 677-706Crossref PubMed Scopus (150) Google Scholar, 8Bikle DD Elalieh H Chang S Xie Z Sundberg JP Development and progression of alopecia in the vitamin D receptor null mouse.J Cell Physiol. 2006; 207: 340-353Crossref PubMed Scopus (74) Google Scholar, 9Radoja N Stojadinovic O Waseem A Tomic-Canic M Milisavljevic V Teebor S Blumenberg M Thyroid hormones and gamma interferon specifically increase K15 keratin gene transcription.Mol Cell Biol. 2004; 24: 3168-3179Crossref PubMed Scopus (34) Google Scholar For example, thyroid hormone stimulates epidermal proliferation and hair growth,11Safer JD Crawford TM Holick MF Topical thyroid hormone accelerates wound healing in mice.Endocrinology. 2005; 146: 4425-4430Crossref PubMed Scopus (74) Google Scholar whereas vitamin D derivatives, retinoids, and glucocorticoids inhibit keratinocyte proliferation, which is exploited for treating chronic hyperproliferative skin diseases like psoriasis.12Pèrez A Chen TC Turner A Raab R Bhawan J Poche P Holick MF Efficacy and safety of topical calcitriol (1,25-dihydroxyvitamin d3) for the treatment of psoriasis.Br J Dermatol. 1996; 134: 238-246Crossref PubMed Scopus (78) Google Scholar The potency of glucocorticoids as inhibitors of keratinocyte proliferation is mirrored by their cutaneous side effects (eg, epidermal and dermal atrophy, resulting in thin and fragile skin).13Schäcke H Docke WD Asadullah K Mechanisms involved in the side effects of glucocorticoids.Pharmacol Ther. 2002; 96: 23-43Crossref PubMed Scopus (1413) Google Scholar The MR is crucial for body fluid homeostasis. On binding the mineralocorticoid hormone aldosterone, the renal MR activates transcription of several genes that ultimately up-regulate renal sodium reabsorption, thus contributing to regulation of extracellular fluid volume and blood pressure.14Farman N Rafestin-Oblin ME Multiple aspects of mineralocorticoid selectivity.Am J Physiol. 2001; 280: F181-F192Google Scholar At variance with its closest homolog, the ubiquitous GR, the MR is expressed in a more limited number of target cells. In addition to the classical mineralocorticoid-sensitive epithelial tissues (the distal parts of the renal tubule, the colonic epithelium, and the excretory ducts of sweat and salivary glands), the MR is also expressed in some nonepithelial cells (neurons and cardiomyocytes) in which its functions are not fully understood.14Farman N Rafestin-Oblin ME Multiple aspects of mineralocorticoid selectivity.Am J Physiol. 2001; 280: F181-F192Google Scholar Interestingly, the MR was also found (mRNA and protein) in human epidermis and hair follicles.15Kenouch S Lombes M Delahaye F Eugene E Bonvalet JP Farman N Human skin as target for aldosterone: coexpression of mineralocorticoid receptors and 11 β-hydroxysteroid dehydrogenase.J Clin Endocrinol Metab. 1994; 79: 1334-1341PubMed Google Scholar High levels of MR transcripts were also reported in murine skin,16Bookout AL Jeong Y Downes M Yu RT Evans RM Mangelsdorf DJ Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network.Cell. 2006; 126: 789-799Abstract Full Text Full Text PDF PubMed Scopus (825) Google Scholar where—due to absence of sweat glands—MR is unlikely to play a major role in fluid homeostasis. Some clinical observations point to the possibility that the aldosterone-MR system may be involved in epidermal and/or hair growth abnormalities. An association was noted between baldness and coronary diseases and/or hypertension, classical outcomes of states of hyperaldosteronism.17Lotufo PA Chae CU Ajani UA Hennekens CH Manson JE Male pattern baldness and coronary heart disease: the Physicians' Health Study.Arch Intern Med. 2000; 160: 165-171Crossref PubMed Scopus (128) Google Scholar, 18Matilainen VA Makinen PK Keinanen-Kiukaanniemi SM Early onset of androgenetic alopecia associated with early severe coronary heart disease: a population-based, case-control study.J Cardiovasc Risk. 2001; 8: 147-151Crossref PubMed Scopus (58) Google Scholar The MR antagonist spironolactone is frequently used to treat hirsutism and occasionally androgenetic alopecia, which has been classically attributed to the antagonism of androgen receptors by spironolactone and its interference with steroidogenesis.4Alonso LC Rosenfield RL Molecular genetic and endocrine mechanisms of hair growth.Horm Res. 2003; 60: 1-13Crossref PubMed Scopus (50) Google Scholar, 19Moghetti P Tosi F Tosti A Negri C Misciali C Perrone F Caputo M Muggeo M Castello R Comparison of spironolactone, flutamide, and finasteride efficacy in the treatment of hirsutism: a randomized, double blind, placebo-controlled trial.J Clin Endocrinol Metab. 2000; 85: 89-94Crossref PubMed Scopus (224) Google Scholar, 20Sinclair R Chronic telogen effluvium: a study of 5 patients over 7 years.J Am Acad Dermatol. 2005; 52: 12-16Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Mammalian skin also displays classical downstream effectors of the aldosterone-MR signaling cascade. The epithelial sodium channel ENaC involved in aldosterone-dependent sodium reabsorption14Farman N Rafestin-Oblin ME Multiple aspects of mineralocorticoid selectivity.Am J Physiol. 2001; 280: F181-F192Google Scholar, 21Rossier BC Pradervand S Schild L Hummler E Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors.Annu Rev Physiol. 2002; 64: 877-897Crossref PubMed Scopus (326) Google Scholar is also expressed in human and rat epidermal keratinocytes and hair follicle22Brouard M Casado M Djelidi S Barrandon Y Farman N Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation.J Cell Sci. 1999; 112: 3343-3352PubMed Google Scholar, 23Roudier-Pujol C Rochat A Escoubet B Eugene E Barrandon Y Bonvalet JP Farman N Differential expression of epithelial sodium channel subunit mRNAs in rat skin.J Cell Sci. 1996; 109: 379-385PubMed Google Scholar; genetic disruption of the ENaC α-subunit in mice leads to epidermal hyperplasia and abnormal epidermal terminal differentiation.24Mauro T Guitard M Behne M Oda Y Crumrine D Komuves L Rassner U Elias PM Hummler E The ENaC channel is required for normal epidermal differentiation.J Invest Dermatol. 2002; 118: 589-594Crossref PubMed Scopus (52) Google Scholar Moreover, genetic disruption of the ENaC-activating serine-protease CAP1 also leads to severe impairment of skin barrier permeability.25Leyvraz C Charles RP Rubera I Guitard M Rotman S Breiden B Sandhoff K Hummler E The epidermal barrier function is dependent on the serine protease CAP1/Prss8.J Cell Biol. 2005; 170: 487-496Crossref PubMed Scopus (238) Google Scholar However, the actual functions of MR in skin physiology and pathology are still completely obscure. To investigate the role of the MR in the skin, we have generated a conditional transgenic model to control MR expression26Ouvrard-Pascaud A Sainte-Marie Y Benitah JP Perrier R Soukaseum C Cat AN Royer A Le Quang K Charpentier F Demolombe S Mechta-Grigoriou F Beggah AT Maison-Blanche P Oblin ME Delcayre C Fishman GI Farman N Escoubet B Jaisser F Conditional mineralocorticoid receptor expression in the heart leads to life-threatening arrhythmias.Circulation. 2005; 111: 3025-3033Crossref PubMed Scopus (227) Google Scholar selectively in keratinocytes (and not in other aldosterone target cells) by use of the keratin 5 (K5) promoter.27Diamond I Owolabi T Marco M Lam C Glick A Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter.J Invest Dermatol. 2000; 115: 788-794Crossref PubMed Scopus (170) Google Scholar Because transgene expression is inducible, it permits either embryonic or postnatal expression of the MR. Whereas embryonic MR expression led to early postnatal death preceded by epidermal atrophy and apoptosis, postnatal MR expression resulted in progressive alopecia and formation of hair follicle cysts without alterations of interfollicular epidermis and keratinocyte-derived appendages. This study conforms to the standards of INSERM (the French National Institute of Health) regarding the care and use of laboratory animals and was performed in accordance with European Union Council Directives (86/609/EEC). Skin conditional expression of human MR (hMR) in double-transgenic (DT) mice was obtained by crossing the previously characterized tetO-hMR mice L2 line26Ouvrard-Pascaud A Sainte-Marie Y Benitah JP Perrier R Soukaseum C Cat AN Royer A Le Quang K Charpentier F Demolombe S Mechta-Grigoriou F Beggah AT Maison-Blanche P Oblin ME Delcayre C Fishman GI Farman N Escoubet B Jaisser F Conditional mineralocorticoid receptor expression in the heart leads to life-threatening arrhythmias.Circulation. 2005; 111: 3025-3033Crossref PubMed Scopus (227) Google Scholar with the K5-tTA transactivator mouse strain derived from founder 1216, kindly provided by Dr. A. Glick.27Diamond I Owolabi T Marco M Lam C Glick A Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter.J Invest Dermatol. 2000; 115: 788-794Crossref PubMed Scopus (170) Google Scholar Both strains were crossed on B6D2 genetic background. Genotypes were determined by polymerase chain reaction (PCR) co-amplification of hMR (forward, 5′-AACTAAgTgTCCCAACAATT-3′, and reverse, 5′-gAAAAgAAAATAAgAggAgACAATgg-3′) or tTA (forward, 5′-AACAACCCgTAAACTCgCCCAgAAg-3′, and reverse, 5′-CgCAACCTAAAgTAAAATgCCCCAC-3′) and actin (forward, 5′-gTgTTAgACACTgTggACATgg-3′, and reverse, 5′-gAgAgAgCCATACCAAgAATgg-3′) from tail genomic DNA using recombinant Taq (Invitrogen, Cergy-Pontoise, France) in conditions described by the supplier. Heterozygous tetO-hMR and K5-tTA were crossed to produce DT mice and study genotype distribution at different ages. In some experimental series, the hMR strain was bred to homozygosity and used to produce DT mice. Monotransgenic tetO-hMR, K5-tTA, and wild-type mice did not differ in skin phenotype, indicating that the observed phenotype of DT (K5-tTA/tetO-hMR) mice was not due to the position of transgene insertion into the genome or to tTA transactivator expression. Monotransgenic mice were therefore used as controls of their DT littermates. Dorsal skin tissues or organs were frozen in liquid nitrogen. Total RNA was isolated by mechanical disruption in TRIzol (Invitrogen) directly for embryos' skin or after grinding under liquid nitrogen in a mortar (Biospec, Bartesville, OK) for adult skin. RNAs from organs were further purified using cationic resin column from RNeasy kit (Qiagen, Courtaboeuf, France). First-strand cDNA were synthesized after DNase I treatment (DNAfree; Ambion, Huntingdon, UK) using 1 μg of total RNA, random hexamers (Amersham, Saclay, France), and Superscript II reverse transcriptase (Invitrogen). Transgene expression was analyzed as previously described26Ouvrard-Pascaud A Sainte-Marie Y Benitah JP Perrier R Soukaseum C Cat AN Royer A Le Quang K Charpentier F Demolombe S Mechta-Grigoriou F Beggah AT Maison-Blanche P Oblin ME Delcayre C Fishman GI Farman N Escoubet B Jaisser F Conditional mineralocorticoid receptor expression in the heart leads to life-threatening arrhythmias.Circulation. 2005; 111: 3025-3033Crossref PubMed Scopus (227) Google Scholar using β2-microglobulin or 18S as internal controls (β2-microglobulin: forward, 5′-TTCTATATCCTGGCTCACACTGAA-3′, and reverse, 5′-CACATGTCTCGATCCCAGTAGA-3′; and 18S: forward, 5′-CCCTGCCCTTTGTACACACC-3′, and reverse, 5′-CGATCCGAGGGCCTCATCA-3′). Transcripts levels of mouse MR, human MR, ENaC subunits, Gpx3, Dusp10, and glyceraldehyde-3-phosphate dehydrogenase expression were analyzed by real-time PCR performed in a iCycler iQ apparatus (Bio-Rad Laboratories LifeSciences, Marnes La Coquette, France) with SYBR Green I detection. PCR was performed in triplicate for each sample using a qPCR Core kit for SYBR Green I (Eurogentec, Seraing, Belgium) containing 300 nmol/L of specific primers and 3 μl of diluted template cDNA in a 25-μl total volume. Serial dilutions of pooled cDNA were used in each experiment to assess PCR efficiency. Expression of hMR relative to mouse MR (mMR) was estimated on the same run and with the same fluorescence threshold by Δcycle threshold calculation after assessment that PCR efficiency was 100%; results were adjusted to amplicon length. The following primers were used. mMR: forward, 5′-TCACATTTTTAACATGTGACGGC-3′, and reverse, 5′-CTTAGTCAGCTCAGGCTTGCC-3′; hMR: forward, 5′-TGCTTCTACACCTTCCGAGAGTC-3′, and reverse, 5′-ACACCAGCCACCACCTTCTGATAG-3′; αENaC: forward, 5′-CGGAGTTGCTAAACTCAACATC-3′, and reverse, 5′-TGGAGACCAGTACCGGCT-3′; βENaC: forward, 5′-ATGTGGTTCCTGCTTACGCTG-3′, and reverse, 5′-GTCCTGGTGGTGTTGCTGTG-3′; γENaC: forward, 5′-CCAAAGCCAGCAAATAAACAAA-3′, and reverse, 5′-GCGGCGGGCAATAATAGAGA-3′; Gpx3: forward, 5′-AAACAGGAGCCAGGCGAGAACT-3′, and reverse, 5′-CCCgTTCACATCTCCTTTCTCAAA-3′; and Dusp10: forward, 5′-AGCCACAGACAGCAACAAAC-3′, and reverse, 5′-CATCAAGTAGGCGATGACGA-3′. The reference gene was glyceraldehyde-3-phosphate dehydrogenase: forward, 5′-AATGGTGAAGGTCGGTGTG-3′, and reverse, 5′-GAAGATGGTGATGGGCTTCC-3′. Mice were fed standard chow (A04; Scientific Animal Food Engineering, Epinay sur Orge, France) and drank tap water ad libitum. When required, food containing 1g/kg doxycycline (DOX; Sigma-Aldrich, La Verpilliere, France) was provided to mothers from mating until weaning of their offsprings. The MR antagonist potassium canrenoate (Sigma-Aldrich) was provided in the drinking water during pregnancy to yield 40 mg kg−1 day−1. Skin biopsies matched for age and body sites or embryos (head and dorsal skin) were fixed in 4% phosphate-buffered paraformaldehyde and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin. Parafin-embedded sections were deparaffinized in xylene, hydrated in a graded alcohol series, and washed in phosphate-buffered saline (PBS). Ki 67 histochemistry and terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL) assay were performed as previously described.28Saadi-Kheddouci S Berrebi D Romagnolo B Cluzeaud F Peuchmaur M Kahn A Vandewalle A Perret C Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the β-catenin gene.Oncogene. 2001; 20: 5972-5981Crossref PubMed Scopus (205) Google Scholar In brief, Ki 67 histochemistry was done after antigen retrieval by heating in citrate buffer and inactivation of endogenous peroxidase by 1% H2O2 treatment. Slides were successively incubated with blocking agent (1/10; Power Block; Biogenex, San Ramon, CA), with rabbit anti-Ki 67 antibody (1/400; Novacastra, Newcastle on Tyne, UK), with goat anti-rabbit antibody (1/100; Santa Cruz, Heidelberg, Germany) and finally with peroxidase-conjugated rabbit anti-goat antibody (1/100; Dako, Trappes, France). Ki 67 antigen was revealed with 3,3′-diaminobenzidine tetrahydrochloride (Dako), and sections were counterstained with hematoxylin. For TUNEL assay, slides were treated with 0.05% pepsin (Sigma-Aldrich); elongation of the DNA fragments was achieved using digoxigenin coupled-11-dUTP (Enzo, Farmingdale, NY) and 0.5 U of TdT (Amersham) for 60 minutes at 37°C in a humid incubator. The reaction was stopped and after washing in 10 mmol/L Tris-HCl, digoxigenin was detected with alkaline phosphatase-conjugated anti-digoxigenin antibodies (1/200; Enzo). Cleaved caspase 3 was detected using a polyclonal antibody (1/100; Cell Signaling, Ozyme, Saint Quentin en Yvelines, France) that was revealed using an avidin-biotin kit. Immunohistochemistry of the MR was performed on paraformaldehyde-fixed skin sections from mice 1 month after DOX withdrawal (allowing hMR expression in DT mice) and in embryonic day (E) 18.5 DT embryos. Two monoclonal antibodies29Gomez-Sanchez CE de Rodriguez AF Romero DG Estess J Warden MP Gomez-Sanchez MT Gomez-Sanchez EP Development of a panel of monoclonal antibodies against the mineralocorticoid receptor.Endocrinology. 2006; 147: 1343-1348Crossref PubMed Scopus (155) Google Scholar (kindly provided by C. Gomez-Sanchez, Division of Endocrinology, University of Mississippi Medical Center, Jackson, MS) were used: 2D6 (1/100), which was raised against a peptide that differs by three amino acids between mouse and human MR and labels essentially the endogenous mMR (not hMR), and 6G1 antibody (1/100), which was raised against a peptide entirely homologous between mouse and human MRs, and labels the endogenous MR and the transgenic hMR. Paraffin-embedded sections were deparaffinized and hydrated, and the nonspecific signal was reduced by the use of the MOM kit (Mouse On Mouse; Vector, AbCys, Paris, France) according to the manufacturer's recommendations. Sections were incubated for 30 minutes with MR-specific antibodies and rinsed in PBS, followed by a streptavidin-based detection system. Amplification of the signal was obtained with the TSA kit (Tyramide Signal Amplification; Perkin Elmer, Courtaboeuf, France) according to the manufacturer's recommendations. The mouse IgG UPC10 (1/5000; Sigma-Aldrich) was used as a nonimmune control. Immunofluorescence was performed on 10-μm cryostat sections of unfixed frozen skin samples. Sections were postfixed for 20 minutes at room temperature with methanol (10 minutes) and permeabilized with 0.25% Nonidet P-40. Primary antibodies (incubated overnight at 4°C) were as follows: anti-K1 (1/500), anti-K10 (1/100), anti-loricrine (1/250), anti-filaggrin (1/2000), kindly provided by E. Hummler (Institut de Pharmacologie, Université de Lausanne, Lausanne, Switzerland),25Leyvraz C Charles RP Rubera I Guitard M Rotman S Breiden B Sandhoff K Hummler E The epidermal barrier function is dependent on the serine protease CAP1/Prss8.J Cell Biol. 2005; 170: 487-496Crossref PubMed Scopus (238) Google Scholar anti-ZO1 (1/100; Zymed; Invitrogen), and anti-occludin (1/100; Zymed); secondary antibody was Cy3-goat anti-rabbit (1/200; Jackson ImmunoResearch Laboratories, West Grove, PA). Nuclei were labeled with Sytox green (Molecular Probes, Eugene, OR) for 5 minutes in the dark. Fluorescence was examined under confocal microscope (Zeiss Meta LSM 510; Mannheim, Germany). The expression of the mouse glucocorticoid receptor mGR was measured on skin from E18.5 embryos by Western blot, using the M20 anti-GR antibody (1/1000; Santa Cruz) as described by the supplier. K5-tTA and tetO-hMR mice were mated during the night, and presence of vaginal plug next morning was considered as gestational day 0.5. Embryos were collected by cesarean section at 14.5, 16.5, or 18.5 gestational days around 11:00 AM. Newborn pups from three litters were collected just after birth. E16.5 embryos were processed as described previously30Hardman MJ Sisi P Banbury DN Byrne C Patterned acquisition of skin barrier function during development.Development. 1998; 125: 1541-1552Crossref PubMed Google Scholar for toluidine blue staining assay. In brief, embryos were successively immersed under shaking in 100% methanol for 1 minute in PBS and then in 0.1% toluidine blue in PBS for 3 minutes and rinsed several times in PBS until excess blue was removed. Treated embryos were photographed by digital camera (Nikon, Melville, NY), and the tail was cut off for genotyping. The different patterns of skin barrier permeability described for control mice between embryonic days 16 and 1730Hardman MJ Sisi P Banbury DN Byrne C Patterned acquisition of skin barrier function during development.Development. 1998; 125: 1541-1552Crossref PubMed Google Scholar were used to score (1 to 8) and to compare semiquantitatively barrier formation status in DT and control embryos. In this series, DOX treatment was provided throughout gestation and interrupted at birth. Pups were wax-depilated at postnatal day 21 as described previously31Müller-Röver S Handjiski B van der Veen C Eichmuller S Foitzik K McKay IA Stenn KS Paus R A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Crossref PubMed Google Scholar; skin samples were collected either at day 6 or at day 14 after depilation for histological examination (hematoxylin and eosin staining). The NIH Image J software (Bethesda, MD) was used to measure epidermis thickness and hair follicle density per millimeter of epidermis in E18.5 skin sections. Data are provided as mean values and SEM. Comparison between control and DT mice was done using Student's t-test. Genotype distributions were compared for each condition (age and treatment) using χ2 test to expected Mendelian distribution considering theoretical size as 25% of total number of mice. Barrier permeability score distributions were analyzed using χ2 test (results were grouped into two classes, scores 1 to 4 and scores 5 to 8, and compared). The DT mice (K5-tTa/tetO-hMR) express the hMR in keratin 5-expressing epithelia, including the basal layer of epidermal keratinocytes and the outer root sheath of the hair follicle.27Diamond I Owolabi T Marco M Lam C Glick A Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter.J Invest Dermatol. 2000; 115: 788-794Crossref PubMed Scopus (170) Google Scholar As shown in Figure 1A, administration of DOX to double-transgenic mice results in complete extinction of transgene expression in DT skin. In the absence of DOX, the expression of hMR is detectable only in DT mice (Figure 1B). A real-time PCR comparison of endogenous mMR and exogenous hMR mRNA levels showed an apparent ratio of hMR over mMR of 6.97 ± 1.00 in E18.5 DT mice (n = 4) and of 1.72 ± 0.40 in adult DT mice (n = 3); this ratio is only an estimate because the efficiency of reverse transcription may differ between mMR and hMR mRNA. Figure 1C shows that mGR levels (immunoblot analysis) were similar between the skin of E18.5 control and hMR-overexpressing DT mice. Thus, MR overexpression is not associated with obvious changes in GR protein levels. No significant differences were found in the expression (real-time PCR) of a glucocorticoid-sensitive gene (Gpx332Tuckermann JP Reichardt HM Arribas R Richter KH Schutz G Angel P The DNA binding-independent function of the glucocortic
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