Year: 2015
Type: article
Abstract: The importance of hormonal regulation in skin is evidenced by numerous endocrine abnormalities with cutaneous manifestations, including disorders of the epidermal barrier and hair (1,s1). Hormone action is mediated by nuclear receptor superfamily members, ligand-activated transcription factors that integrate multiple cellular processes, including the glucocorticoid (GC) receptor (GR or Nr3c1) and the mineralocorticoid receptor (MR or Nr3c2) 2. Both proteins are structurally and functionally related, and upon GC binding, recognize the same hormone responsive elements to transcriptionally regulate target genes 3-5. GC access to GR and MR is modulated by two enzymes, 11β-hydroxysteroid dehydrogenases type I and II (HSD11B1/HSD11B2), which mediate interconversion between inactive and active GCs. Human and mouse skin can synthesize GCs and key enzymes of steroid synthesis, acting as a functional hypothalamo–pituitary–adrenal axis to secure epidermal homeostasis (6,s2,s3). While our previous work demonstrated that GR is required for epidermal development and homeostasis (7, 8,s4), much less is known regarding MR in this tissue 9. Remarkably, transgenic mice with keratinocyte-specific overexpression of either MR or GR (s5,s6) showed strong phenotypical similarities at birth including atrophic skin, reduced hair follicle number and impaired epidermal maturation. We have analysed the following: (i) MR expression during mouse skin development; (ii) the consequences of MR inactivation in developing mouse skin; and (iii) the relative expression of HSD11B1/HSD11B2 during this process. MR−/− mice were generated using generalized CRE-mediated recombination by intercrossing K5-cre mice (s7) and MRloxP/loxP mice (s8). Skin samples were collected from at least three individuals of each genotype and age and analysed as reported 8. Mean value ± SD was calculated and statistical significance assessed using the Student's t-test 8. We analysed the relative expression of Nr3c2 by RT-QPCR in WT embryonic (E) 18.5 and adult mouse skin in the telogen (8 weeks old) and anagen (5 weeks old) phases of the hair cycle. Nr3c2 levels were 17-fold higher in telogen relative to anagen skin, suggesting a role for MR in the resting phase of the hair cycle; however, Nr3c2 was most abundant in E18.5 skin, fivefold higher than in telogen adult skin (Fig. 1a). Assessment of Nr3c2 during skin development revealed a peak in expression at E16.5 which decreased thereafter (E18.5 and postnatal day 0 (P0); Fig. 1b). These findings were paralleled by changes in MR protein expression in developing and adult skin (Fig. 1c). Nr3c1 levels also peaked in E16.5 skin, but unlike Nr3c2 did not decrease at later time points (Figure S1). Krt77 and Sprr2d are controls known to be expressed differentially during epidermal development (Figure S1). To evaluate the consequences of MR loss-of-function in developing skin, we generated MR−/− mice (Fig. 1d) using generalized CRE-mediated recombination in MRloxP/loxP mice (Appendix S1, s7,s8). MR−/− mice died perinatally around P10 similar to MRnull/null mice (s9), presumably due to renal loss of sodium and water (not shown). Epidermal permeability assays in MR+/− and MR−/− E17.5 embryos showed no major changes in the pattern of barrier formation among genotypes (Fig. 1e). However, analysis of haematoxylin/eosin-stained skin sections showed statistically significant increases of epidermal thickness in MR−/− relative to MR+/− embryos (Fig. 1e,f). Altered differentiation, with a more restricted expression of keratin (K)5 and abnormal expression patterns of K10 and loricrin (LOR), was also detected in MR−/− embryos and quantitated as minor but statistically significant changes of per cent positive layers (Fig. 1e,g). These alterations resolved spontaneously with age as MR−/− skin had a similar appearance as MR+/− or WT littermates at P0 and P4 (Fig. 2 and data not shown). We assessed stratum corneum lipids by Nile red staining and quantitated epidermal thickness and loricrin staining in postnatal skin samples but found no changes among genotypes (Fig. 2a and Figure S2). MR−/− epidermal proliferation was unchanged at P0, but statistically significant increases were detected at P4 (Fig. 2b), relative to controls; however, we cannot exclude the possibility that the MR−/− postnatal renal defects indirectly affect proliferation in this tissue. To understand whether the transient role of MR in epidermal development is due to alterations in GC availability, we assessed Hsd11b1 and Hsd11b2 in skin at distinct stages and observed pronounced peaks in expression for both genes at E18.5 (Fig. 2c). In adult skin, the expression of Hsd11b1 decreased twofold and that of Hsd11b2 more than 30-fold relative to E18.5. Immunostaining confirmed the restricted expression of HSD11B2 to the upper epidermal layers with statistically significant increases at P0 (Fig. 2d and Figure S3a). Importantly, HSD11B2 epidermal expression was similar in MR+/+ versus MR−/− mice but decreased significantly in GR−/− skin (Fig. 2e and Figure S3b). The α-subunit of the amiloride-sensitive epithelial Na channel (ENaCα/Scnn1a), necessary for epidermal homeostasis, is a transcriptional target of both MR and GR (s10). We assessed whether the absence of MR affected ENaCα but found no differences in P0 skin (Figure S4), similar to that reported in colon and kidney of MR−/− newborn mice (s11). The fact that Nr3c1 levels are unchanged in MR−/− skin suggests that GR expression is not negatively affected by loss of MR (Figure S4). Our findings indicate that MR plays a role in regulating epidermal differentiation at late embryonic stages. The transient expression peak of MR at E16.5, coinciding with low levels of HSD11B2, argues for a role of GC-MR complexes in epidermal maturation. The fact that HSD11B2 was virtually absent in GR−/− skin but normally expressed in MR−/− skin, together with its reported induction by dexamethasone in human keratinocytes (s12), suggests that this enzyme is regulated by GR but not MR and may represent a mechanism to modulate GC-dependent actions locally. The hypothalamo–pituitary–adrenal axis becomes active around E15.5 ensuring proper control of circulating GC and aldosterone levels, which are normally high perinatally (s13). MR and GR expression peaks shortly afterwards, at E16.5. MR expression decreases at later developmental time points while that for GR remains constant. This observation together with our phenotypic data supports the hypothesis that MR plays a transient role in skin development. While GR−/− late embryos featured defective epidermal differentiation with virtually absent stratum corneum and impaired barrier function (s4), the skin phenotype of MR−/− embryos was relatively milder suggesting functional compensation by GR in the perinatal period. We thank Jose Nieto for expert technical help. JB, EC and VMG are recipients of FPI, FPU (MINECO) and JAE-INTRO (CSIC) fellowships. We thank COST ADMIRE BM-1301 for support for dissemination. JB generated the mouse model; JB, EC, LMS and VMG analysed skin samples. PP designed the experiments and wrote the study. All authors read and approved the manuscript. This work was supported by the Spanish Ministerio de Ciencia e Innovación (grants SAF2011-28115 and SAF2014-59474-R). The authors have declared no conflict of interests. Figure S1. Relative mRNA levels of Nr3c1, Krt77, and Sprr2d in embryonic (E15.5–E18.5) and newborn (P0) mouse skin. Figure S2. Quantitation of epidermal thickness (a) and loricrin immunostaining (b) in MR+/− and MR−/− P0 and P4 mice (n = 6 per genotype). Figure S3. Quantitation of epidermal HSD11B2 immunostaining A. Figure S4. Relative mRNA levels of the α-subunit of the amiloride-sensitive epithelial Na channel (ENaCα/Scnn1a) and Nr3c1 in MR−/− versus MR+/+ P0 skin (n = 5 per genotype). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Source: Experimental Dermatology
DOI: 10.1111/exd.12822
Language: English
Topic: Hair Growth and Disorders
Subfield: Urology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W1961288059