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Centro de Investigación Príncipe Felipe (P.B., A.S., P.P.), E-46013 Valencia, Spain; Department of Veterinary Clinical Sciences (A.B., J.L.C.), Veterinary Faculty, University of Santiago de Compostela, E-27002 Lugo, Spain; Molecular Biology of Tissue-Specific Hormone Action (K.B., J.T.), Leibniz Institute for Age Research, Fritz Lipmann Institute, D-07745 Jena, Germany; Department of Molecular Biology of the Cell I (J.T., G.S.), German Cancer Research Center, D-69120, Heidelberg, Germany; and Instituto de Biomedicina de Valencia Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Cientificas (P.P.), E-46010 Valencia, Spain
Address all correspondence and requests for reprints to: Paloma Pérez, Centro de Investigación Príncipe Felipe, Valencia, Avenida Autopista del Saler 16, Camino de las Moreras, E-46013 Valencia, Spain. E-mail: pperez{at}cipf.es.
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Introduction |
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Although glucocorticoid (GC) analogs are widely prescribed as the treatment of choice in many cutaneous disorders (6), the role of GCs in skin development has not been completely deciphered (3). In some reports, GCs have been shown to accelerate epidermal barrier formation as seen by either GC injections in utero (7) or, conversely, by showing that CRH-deficient newborn mice, which exhibited GC deficiency, had delayed maturation of the SC (8); moreover, supplementation of GCs in these mice fully rescued skin phenotype (8). In experiments performed in adult mouse skin, short-term topical and systemic GC treatment disturbed epidermal barrier competence (9). These results highlight that the responses to GCs depend on the stages in development (fetal vs. adult) and also on the use of physiological vs. pharmacological doses of GCs.
Because GC effects are mediated through the glucocorticoid receptor (GR), studying the impact of the gain and loss of function of GR in skin development and function through genetically modified mice constitutes a relevant issue from the basic and clinical perspective. GR acts through the so-called genomic and nongenomic actions exerting pleiotropic roles in many tissues, including skin (reviewed in Ref. 10). GR belongs to the superfamily of steroid nuclear receptors and is a ligand-dependent transcription factor. In the absence of ligand, GR resides in the cytoplasm associated with chaperones such as heat shock protein 90, in an inactive form. Upon ligand binding, GR dissociates from cytoplasmic complexes, dimerizes, and translocates to the nucleus, where it can then regulate gene transcription by binding to positive and negative glucocorticoid response elements (GREs). GR can regulate gene expression through DNA-binding-dependent and -independent mechanisms. The former requires ligand-induced dimerization of GR and binding to specific GREs, whereas the latter does not require DNA binding of GR but rather is mediated through interference with other transcription factors, such as nuclear factor-
B or activator protein-1 (AP-1) (11). Nongenomic actions of GR have been demonstrated and are mediated through physical interaction of the receptor at the plasma membrane with p85
/phosphatidylinositol 3-kinase, which, in turn, modulates acutely transforming retrovirus AKT8 in rodent T cell lymphoma activity (12).
To decipher the role of GR in skin development, we have evaluated the skin architecture of two mouse models in which GR has been functionally inactivated: the knockout GR–/– mice (13, 14) and the knock-in mice carrying the point mutation A458 in the D-loop of GR, which impairs dimerization-induced DNA binding of the GR, thus resulting in a mutant protein that displays defective DNA-binding-dependent transcription (GRdim/dim) (15). GR–/– mice die perinatally, whereas GRdim/dim animals are viable. Altogether, our analyses demonstrate that GR is required for proper epidermal differentiation, which is in part mediated by caspase-14 processing during mouse embryogenesis. In addition, GR regulated keratinocyte growth and apoptosis in a cell-autonomous manner, as shown by the observed increased proliferation and cell death in cultured mouse primary keratinocytes (MPKs) from GR–/– mice. GR controlled skin homeostasis, at least partially, through antagonistic modulation of ERK function both in vivo and in vitro. Moreover, and given that the epidermis of GRdim/dim embryos appeared normal, our data strongly support the idea that DNA-binding-independent actions of GR are sufficient to mediate epidermal development during embryogenesis.
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Materials and Methods |
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Mice were housed in microisolated boxes in which the air is filtered in both directions, allowing maximal isolation. Animal experimentation was always conducted with accepted standards of humane animal care in our registered animal facility (CV-46007, Centro de Investigación Príncipe Felipe). Experiments were performed in accordance with the Principles of Laboratory Animal Care (National Institutes of Health Publication no. 85-23, revised 1985) and with the current Spanish and European normative that governs research with animals (Real Decreto 1201/2005, B.O.E. 252, October 10, 2005, and Convenio Europeo 1-2-3, March 18, 1986).
Antibodies
The antibodies used included rabbit polyclonal antibodies to GR (sc-1004), ERK (sc-154), c-jun-N-terminal kinase (JNK) (sc-474), phospho-(p-)c-jun (sc-822), β-catenin (sc-7199), c-myc (sc-788), and caspase-14 (sc-5628) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against c-jun (no. 9165), p-ERK (Thr202/Tyr204; no. 4376), and p-JNK (Thr183/Tyr185; no. 9251) were purchased from Cell Signaling Technology Inc. (Beverley, MA). E-cadherin (610181), plakoglobin (610253), and desmoglein-1 (610273) antibodies were from BD Transduction Laboratories (BD Biosciences, San Jose, CA). An antibody against actin (A-2066; Sigma Chemical Co., St. Louis, MO) was used for loading control. Secondary peroxidase-conjugated antirabbit antibody was from Amersham (Aylesbury, UK), and secondary peroxidase-conjugated antimouse antibody and biotin-conjugated antirabbit or antimouse antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Antibodies against keratin K5 (PRB-160P), K6 (PRB-169P), K10 (PRB-159P), loricrin (PRB-145P), filaggrin (PRB-417P), and involucrin were from Covance (Babco, Berkeley, CA).
Histological and immunohistochemical analysis
Whole embryos and skin samples were fixed in 4% paraformaldehyde or 70% ethanol and embedded in paraffin. Consecutive 3- to 4-µm-thick sections were obtained. For histopathology, sections were stained with hematoxylin and eosin (H&E). Before immunostaining, paraffin sections were dewaxed and microwaved in 10 mM citrate solution. For immunohistochemistry, paraffin sections were blocked with 5% fetal bovine serum and then incubated with the primary antibody for at least 1 h. Slides were washed three times with PBS and then incubated with conjugated secondary antibodies for 1 h. Finally, the reaction was visualized with the avidin-biotin complex (ABC) kit from Dako (Vectastain Elite; Vector Laboratories, Inc., Burlingame, CA) using diaminobenzidine as chromogenic substrate for peroxidase. Slides were mounted and analyzed by light microscopy (Leica DM RXA2), and microphotographs were taken at x40 magnification unless otherwise indicated.
In vivo epidermal bromodeoxyuridine (BrdU) labeling
Epithelial cell proliferation was measured by ip injection of BrdU (130 µg/g body weight) from Roche (Indianapolis, IN) into pregnant female mice 1 h before they were killed. BrdU incorporation was detected by immunohistochemistry of paraffin-embedded sections using a mouse anti-BrdU monoclonal antibody (Biotest; Roche). The number of BrdU-positive cells and the number of total cells was determined per 200 µm of interfollicular epithelium in each section. Experiments were performed in at least five individuals of each genotype, and differences were assessed by using the t test, with statistical significance when P < 0.05.
Morphometric analysis
Determination of the average number of HFs, HF length, and epidermal width was performed using 4-µm-thick skin paraffin sections from 16.5 and 18.5 dpc embryos stained with H&E and counting at least 10 individual fields of 1 mm per slide using the software MetaMorph (Premier Offline 7.0; Molecular Devices, Downingtown, PA). Experiments were performed in at least five individuals of each genotype, and differences were assessed by using the t test, with statistical significance when P < 0.05.
Permeability barrier assays
The 18.5 dpc embryos were collected, euthanized, and submerged in methanol for 3 min. The embryos were placed in a 0.1% solution of toluidine blue dye in PBS for 2 min, washed several times in PBS, dried, and photographed.
Transmission electron microscopy studies
Skin samples were spread on the bottom of a 12-well plate, washed in PBS, and fixed in 2.5% glutaraldehyde/2.5% paraformaldehyde/PBS (pH 7.2) for 15 h at 20 C. Skin samples were 1 h postfixed with 1% OsO4/PBS in the dark at 4 C followed by dehydration in an ascending water/acetone series and embedded in AGAR 100 epoxy resin (Agar Scientific, Stansted, UK). The resin was allowed to polymerize for 2 d at 60 C in flat embedding molds. Sections were obtained by an ultramicrotome (Reichert Ultracut S; Leica, Wetzlar, Germany) and stained with 2% uranyl acetate and lead citrate at various concentrations. Sections were examined with a transmission electron microscope (CEM 902A; Zeiss, Oberkochen, Germany) at an acceleration voltage amounting to 80 kV.
MPK isolation, culture, and treatments
The 18.5 dpc embryos were killed by CO2 asphyxiation, their bodies were rinsed in 70% ethanol, and limbs and tail were removed; tails were used for PCR genotyping. Skin was peeled off, rinsed in PBS, and then incubated in 0.25% trypsin at 4 C overnight. The epidermis was separated from dermis with forceps, minced, and homogenized in complete low-calcium medium by vigorous shaking for 10 min at 4 C. The filtered solution contained MPKs, which were collected by centrifugation. MPKs were pooled (two mice of each genotype), and 106 cells were plated into one 35-mm diameter collagen-coated petri dish (BD Biosciences) and cultured at 37 C in standard medium. After 24 h, the medium was replaced with complete low-calcium medium, and cells were grown until subconfluency. Cells were refed every other day. The composition of standard medium was essential MEM (BioWhitakker, Inc., Walkersville, MD), supplemented with 4% fetal calf serum (BioWhitakker) plus 0.6 mM CaCl2 and antibiotics. To prepare low-calcium medium, fetal calf serum was depleted of divalent cations by treatment with Chelex deionizing resin (Bio-Rad, Hempstead, UK) and supplemented with CaCl2 to a final concentration of approximately 0.05 mM. Epidermal growth factor (Sigma) (10 ng/ml) and antibiotics were added to growth medium.
Dexamethasone (Dex, 1µM) (Sigma) or vehicle was added for the indicated times to confluent wild-type (wt) MPKs that had been incubated in charcoal-stripped serum overnight to deplete steroid hormones. Cells were then washed in PBS, and either total RNA or whole-cell extracts were isolated to check caspase-14 mRNA levels by semiquantitative RT-PCR using primers previously described (16) and caspase-14 and filaggrin expression by immunoblotting. Experiments were performed in triplicate and mean value ± SD estimated.
Phorbol 12-myristate 13-acetate (PMA) (Sigma) or vehicle was added to subconfluent MPKs at a final concentration of 100 ng/ml for 1 h. After this time, cells were either fixed for ERK1/2 and p-ERK1/2 immunolocalization or washed with cold PBS and then lysed to prepare whole-cell extracts. Experiments were performed in triplicate and mean value ± SD estimated.
In vitro differentiation of MPKs
To assess in vitro differentiation, equal numbers of MPKs were plated and grown in coverslips to confluency under low-calcium (0.05 mM) conditions and then shifted to high calcium (1.2 mM) for 24 or 48 h. Differentiation of wt and GR–/– keratinocytes was assessed by phase contrast, and expression of K10 and involucrin, as markers of early and late differentiation, was examined by immunofluorescence.
BrdU immunofluorescence
To determine BrdU incorporation in MPKs, cells were seeded on glass coverslips until subconfluency and then incubated with 3 µl BrdU (Biotest, 1 µg/µl; Roche) for 3 h at 37 C. After fixation, cells were permeabilized, treated with 2 N HCl for 30 min, and then blocked and incubated with anti-BrdU antibody (Biotest; Roche) followed by antimouse-fluorescein isothiocyanate. Coverslips were mounted with polyvinyl-alcohol-1,4-diazabicyclo[2.2.2]octane (Fluka, Sigma-Aldrich Chemie, GmbH, Buchs, Switzerland) containing 4',6-diamidino-2-phenylindole (DAPI), and samples were visualized under a fluorescence microscope (Leica DM RXA2). Positive nuclei were counted (at least 500 cells, four coverslips per genotype) in five independent experiments and percentages of BrdU-positive cells ± SD determined. Routinely, controls of staining specificity were performed by using preimmune serum and the secondary antibodies, which gave no signal (not shown).
Analysis of apoptosis in tissue sections and cultured keratinocytes
To detect individual apoptotic cells in paraffin-embedded tissue sections and MPKs, the In Situ Cell Death Detection kit (Roche) was used, following the manufacturer’s recommendation. Paraffin sections immersed in 0.1 M citrate buffer (pH 6) were microwave-irradiated for 5 min and then rinsed with PBS before the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction. Four 16.5 dpc embryos of each genotype were examined.
MPKs isolated from GR–/– and GR+/+ embryos (four of each genotype) were seeded on glass coverslips until subconfluency and treated as indicated. Cells were fixed and permeabilized and incubated with the TUNEL reaction mixture for 1 h at 37 C in the dark. Sections were mounted with DAPI and samples visualized under a fluorescence microscope (Leica DM RXA2) to determine the percentages of apoptotic cells. As a positive control, wt MPKs were treated with vehicle or TNF- (100 mg/ml) plus cycloheximide (CHX, 1 µg/ml; Sigma) for 16 h, as previously described (17). When indicated, the ERK inhibitor PD098059 (50 µM; Calbiochem, San Diego, CA) was added for 16 h. Quantitation of single apoptotic cells represents the mean value ± SD estimated obtained from three different experiments with MPKs using three replicates for each experimental condition. Differences were assessed by using the t test, with statistical significance when P < 0.05.
Immunoblotting
Whole-cell extracts (20 µg) were prepared as previously described (15), boiled in Laemmli buffer, separated on 10% SDS-PAGE, and then transferred to nitrocellulose filters (Hybond ECL; Amersham). Filters were blocked with 5% nonfat dry milk in PBS/0.1% Tween 20 at 4 C overnight, washed three times in PBS/0.1% Tween 20, and incubated with the indicated antibodies. After washing, membranes were incubated with a peroxidase-conjugated secondary antibody (Amersham), washed again, and analyzed using the enhanced chemiluminescence method (ECL; Amersham), according to the manufacturer’s recommendations. The membranes were also stained with Ponceau S (Sigma) to verify equal protein loading and transfer.
Specific bands were scanned by using ImageQuant (Amersham), quantitated with Quantity One Software (Bio-Rad), and represented as graph bars. Values for control samples (basal protein levels in wt skin) were arbitrarily set as 1, and the values in GR–/– are expressed as relative to wt. Experiments were performed in at least three individuals of each genotype, and differences were assessed by using the t test, with statistical significance when P < 0.05.
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Results |
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). The epidermis of wt late embryos is organized as stratified epithelia composed of a single basal cell layer and six to eight suprabasal cell layers, including the SL, the GL containing keratohyalin granules, and the outermost layer, the SC. At 16.5 dpc, epidermal maturation is characterized by the appearance of the GL and SC, key to the formation of a competent skin barrier, which is established at 18.5 dpc. In sharp contrast to wt late embryos, 16.5 and 18.5 dpc GR–/– epidermis was highly immature and featured only three to four suprabasal layers with an abnormally high number of apoptotic keratinocytes (arrows) in the lower suprabasal layers (Fig. 1, compare C and F with A and D). Apoptotic keratinocytes were further identified by TUNEL staining in the suprabasal layers of the GR–/– skin sections. In contrast, wt skin displayed nonapoptotic cells or few scattered apoptotic cells (Fig. 1G and data not shown). Moreover, both the GL and the SC were barely detected. However, GR+/– skin appeared indistinguishable from wt (Fig. 1, B and E).
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). Filaggrin is specifically expressed in the GL of the epidermis, and loricrin and involucrin constitute the major cornified envelope precursors. Remarkably, the expression of all these markers was either residual or absent in GR–/– mice, in contrast to GR+/– and wt littermates (Fig. 2).
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). We next quantitated the number of anagenic HF as well as HF length in dorsal skin of GR–/–, GR+/–, and GR+/+ mice by morphometric analysis (Table 1). Surprisingly, neither the density nor the average length of HF was significantly changed, as assessed by H&E staining (Table 1).
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, A–F) and found an abnormal morphology of keratinocytes in the BL as well as abnormally differentiated suprabasal keratinocytes. Remarkably, the GL and SC were poorly differentiated, and intercellular spaces were increased in GR–/– epidermis (Fig. 3B, asterisks). Keratohyalin granules appeared rudimentary with decreased electron density (Fig. 3D, arrows, compare with C), and the SC showed parakeratotic squames with increased electron density and contained cellular debris, a feature of incomplete cellular death (Fig. 3D, asterisks, compare with C). Desmosomes (indicated by arrows) could be detected in all epidermal layers of GR+/+ mice (Fig. 3E), whereas only few desmosome-like structures could be located in suprabasal layers of GR–/– embryo skin (Fig. 3F). Given that epidermal terminal differentiation was severely impaired in GR–/– mice, we evaluated the functionality of the permeability barrier in 18.5 dpc embryos by toluidine blue staining (Fig. 3G). As expected, epidermal maturation was complete in wt embryos at this stage and, consequently, epidermal regions that have acquired a barrier appeared as white. In contrast, some regions around the paws, forelimbs, chin, and neck of GR–/– embryos were visualized as blue, thus evidencing a compromised skin barrier function (Fig. 3G). Overall, our results indicate that GR is required for keratinocyte terminal differentiation, and thus skin barrier competence, during embryogenesis.
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, top). This correlated with the appearance of mature filaggrin at 16.5 and 18.5 dpc, as detected by immunoblotting (Fig. 4A, bottom, arrowhead). Unprocessed and incompletely processed profilaggrin is indicated by a bracket. In contrast, caspase-14 processing was not detected in 16.5 dpc or 18.5 dpc GR null epidermises with undetectable levels of filaggrin at 16.5 dpc and only minor levels at 18.5 dpc (Fig. 4A). In addition, caspase-3 was not activated during fetal epidermis development in wt or GR null mice (Fig. 4B).
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, C–E). Our results showed that Dex, at a concentration inducing MPK differentiation, produced a 2-fold increase in the procaspase-14 protein levels without eliciting its processing (Fig. 4D). We also checked whether Dex regulated caspase-14 at the mRNA level and found no differences in caspase-14A and -14B transcripts (Fig. 4E). In agreement with this, both caspase-14 mRNA forms were similar in GR–/– skin compared with GR+/+ (Fig. 4E).
GR inhibits keratinocyte growth in a cell-autonomous manner
The high immaturity of GR–/– epidermis suggested a defective switch between proliferation and differentiation. We observed that immunolocalization of K5, normally restricted to the proliferative single BL (Fig. 5A, arrows), was abnormally found in suprabasal layers of GR–/– skin (Fig. 5B, arrowheads). This finding prompted us to examine whether these K5-positive keratinocytes were indeed proliferating by assessing in vivo BrdU incorporation (Fig. 5, C–E). In contrast to control mouse skin, in which all positive nuclei were located in the epidermal BL or in the outer root sheath of HFs (Fig. 5C, thin and thick arrows, respectively), we observed some BrdU-positive cells in the first suprabasal layer of GR null skin (Fig. 5D, arrowheads), a typical feature of hyperproliferative skin disorders. However, and despite abnormal localization of proliferating keratinocytes, we did not find statistically significant changes in the proliferation of interfollicular GR–/– keratinocytes as compared with wt epidermis (Fig. 5E, 15.3 vs. 10.8%; P > 0.05). These findings were somehow unexpected because we and others previously reported that overexpression of GR exerts an antiproliferative role in keratinocytes in vivo (18, 19).
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and data not shown). In sharp contrast, GR–/– keratinocytes formed colonies, consisting of cells with heterogeneous size and morphology, where bigger cells were located at the borders (Fig. 5F, thin arrows) and smaller rounded cells at the center (Fig. 5F, thick arrows), with many cells exhibiting apoptotic features (Fig. 5F, arrowheads; see also Fig. 8).
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), we found a robust increase (28.12%) in the proliferation rate of GR–/– cells as compared with wt keratinocytes (12.47%). Collectively, and despite that GR–/– epidermis showed no overall changes in proliferation, our data indicate that GR regulates keratinocyte growth in a cell-autonomous manner.
In vitro differentiation of GR–/– keratinocytes
The lack of differentiation of GR–/– keratinocytes in vivo (Figs. 1–3) could be due to an intrinsic defect of these cells or to an inappropriate calcium supply, which is required for normal differentiation of keratinocytes. To examine this, we cultured MPKs isolated from GR–/– and wt littermates and analyzed their responses to an elevated concentration of calcium (Fig. 6). As reported (20), high calcium induced evident changes in wt keratinocyte morphology, which appeared as flat and enlarged cells, with numerous cell-cell contacts, and formed epithelial sheets as differentiation proceeded (Fig. 6, GR+/+ Ca2+ 24 and 48 h). Surprisingly, the presence of high calcium in GR–/– MPKs caused similar morphological changes compared with wt keratinocytes. Moreover, the augmented expression of keratins that are markers of differentiation, normally induced in wt MPKs by high-calcium treatment, was similarly induced in cultured GR–/– keratinocytes (Fig. 6). Given that epidermal differentiation is severely impaired in GR–/– embryos (Figs. 1–3) but cultured GR–/– keratinocytes differentiate upon high calcium, our data suggest that a defect in the formation of the calcium stimulatory system of GR–/– skin exists, which can be overcome in cell culture by adding calcium.
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). Despite ERK1/2 protein levels being unaltered, p-ERK1/2 (Thr202/Tyr204, which leads to its activation) was increased an average 3.5-fold in GR–/– skin compared with controls (Fig. 7B). Immunostaining showed a marked augmentation of both cytosolic and nuclear p-ERK staining in GR–/– epidermal keratinocytes (Fig. 7C, p-ERK). In contrast to control epidermis, where p-ERK predominantly localized to GL nuclei, p-ERK was detected throughout all suprabasal layers. However, JNK and p-JNK protein levels were unchanged in GR-deficient skin with no major changes in their cellular distribution (Fig. 7, A and C). Moreover, phosphorylation of c-jun on residues S63/S73 as well as total c-jun protein levels remained unchanged in GR–/– epidermis relative to wt (Fig. 7, A and C).
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, GR+/+). In GR–/– MPKs, expression of total ERK was similar to that in controls; however, the majority of cells showed a positive nuclear signal for p-ERK (Fig. 8A, GR–/–). The enhanced activation of ERK in cultured keratinocytes from GR–/– mice under nonstimulated conditions was confirmed by immunoblotting using whole-cell extracts (Fig. 8B). Quantitation of specific bands revealed an average 3.2-fold increase of p-ERK levels in GR–/– MPKs compared with wt keratinocytes. When MPKs were treated with PMA, a known inducer of ERK activity, we observed a similar increase of p-ERK in both GR+/+ and GR–/– MPKs (Fig. 8C). Collectively, our data support a key role for GR in controlling keratinocyte function through inhibition of the MAPK/AP-1 signaling pathway. To further demonstrate the contribution of ERK activation in the impaired keratinocyte function of GR–/– mice, we analyzed the apoptotic rate of cultured keratinocytes and the consequences of adding the pharmacological ERK inhibitor PD098059, which has been used extensively as a selective inhibitor of the activation of the ERK1/2 kinase, MEK-1, to block ERK1/2 phosphorylation and activation in keratinocytes and other cell types (23, 24).
Cultured keratinocytes were grown in the absence or presence of PD098059 (50 µM), and apoptosis was evaluated by TUNEL immunostaining in wt and GR–/– MPKs and quantitated (Fig. 8D). Basal apoptotic rate in wt keratinocytes (6%) was reduced in the presence of PD98059 (3%). In contrast to control cells, GR–/– MPKs exhibited an increase of apoptosis of approximately 16%. Moreover, inhibition of ERK reduced the augmented apoptosis of GR–/– keratinocytes (10%). As a positive control for apoptosis, we treated wt keratinocytes with TNF- plus CHX for 16 h, which augmented apoptosis by approximately 6-fold (30%). Our results with pharmacological inhibitors of ERK activity provide evidence that the increased apoptosis rate in GR–/– keratinocytes is, at least partially, linked to augmented ERK activation in these cells, in agreement with previous reports (25).
To further investigate the mechanisms underlying GR function in skin development, we used GRdim/dim mice carrying a point mutation that impairs dimerization-induced DNA binding of the GR (15). Our histological analysis revealed no differences between GRdim/dim, GR+/dim, and wt littermates in either epidermal or HF formation (Fig. 9A). In addition, we found no differences in the expression and localization of the markers of keratinocyte proliferation and differentiation K5, K10, and loricrin (Fig. 9B). Collectively, data obtained from the analysis of GRdim/dim embryos along with those regarding the role of ERK in GR–/– skin support the idea that GR regulates epidermal formation during embryogenesis most likely through DNA-binding-independent actions.
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and 8). Although many key processes that are impaired in the GRdim/dim mice (15, 27) are most likely due to defective DNA-binding-dependent transactivation of GR, neither A458T nor other GR dimerization mutants are globally deficient in transactivating via GR at promoters, as demonstrated by several reports (28, 29). This implies that the mechanisms through which GR controls keratinocyte function could depend on nontranscriptional events, on transrepression, or on transactivation of genes with a more complex GRE configuration that can be regulated in the GRdim/dim mice, as occurs with the PNMT gene (28, 29).
Skin barrier acquisition starts at 16.5 dpc on the dorsal surface and spreads laterally to the ventral surface in a patterned fashion (3). Our present findings demonstrate that GR is required for epidermal terminal differentiation and skin barrier function (Figs. 1–3). However, cultured GR–/– MPKs differentiated in vitro upon high calcium, thus supporting the hypothesis that a defect in the formation of the calcium stimulatory system of GR–/– skin exists that can be overcome in cell culture by adding calcium.
A recent report has identified the transcriptomic profile of cultured human keratinocytes in response to GCs (19). In this work, the authors propose that GCs may have a dual effect on epidermal differentiation by promoting the late stages of terminal differentiation and, at the same time, inhibiting the early stages. In particular, several genes encoding for enzymes involved in SC formation were identified as GC regulated, such as transglutaminase 1, filaggrin, and corneodesmosin (19). These findings are in agreement with our in vivo model.
On the other hand, the ultrastructure of GR–/– embryo skin revealed a reduced number of desmosomes (Fig. 3). Previous reports indicated that GCs up-regulate several adhesion molecules, including markers of adherens junctions (E-cadherin), desmosomes (desmoglein-1), or both (plakoglobin) (19). We examined the expression of these markers in GR-deficient skin and found reduced levels of these molecules compared with controls (not shown).
Remarkably, GR knockout embryos exhibit increased levels of ACTH and corticosterone as a compensatory mechanism for the GR loss of function (13). Our results indicate that excess steroid hormones correlate with defective epidermal maturation rather than cause these defects. Accordingly, impaired proliferation and apoptosis as well as constitutively augmented ERK activity was found in GR–/– keratinocytes, which suggests that the observed effects are independent of increased hormone levels.
Another relevant finding of this study reveals that GR loss of function correlates with the lack of processing of caspase-14, which prevents the formation of mature filaggrin and thus the appearance of SC providing mammals with a competent epidermal barrier function. Our results show that ligand-activated GR regulates caspase-14 expression in cultured keratinocytes at the posttranscriptional level. The effect of Dex on caspase-14 expression occurred at concentrations inducing keratinocyte differentiation. Noteworthy, the regulation of caspase-14 in vivo was also posttranscriptional because no changes in caspase-14 transcripts were found in GR–/– skin compared with GR+/+ mice (Fig. 4). The importance of caspase-14 as a GR target is supported by the recent findings in caspase-14–/– mice. These mice exhibited reduced skin-hydration levels and increased water loss and an altered profilaggrin processing pattern that could explain the observed phenotype (30). However, other proteolytic enzymes of this family, such as caspase-1 and -4 have been described as GC targets in keratinocytes (19).
Remarkably, and despite the reported antiproliferative role of GR, GR null mice did not show overall increased epidermal proliferation in vivo (Fig. 5). However, we observed abnormal proliferating suprabasal keratinocytes in GR–/– skin that indicated a defective switch between proliferation and differentiation. When MPKs were cultured, an increased proliferation rate of GR–/– keratinocytes compared with wt was apparent (Fig. 5), thus demonstrating that GR regulates keratinocyte proliferation in a cell-autonomous manner.
GR modulates ERK activity to regulate keratinocyte function
Because the MAPK/AP-1 signaling pathway plays a relevant role in keratinocyte biology (reviewed in Ref. 31), and given that GR inhibits MAPK function in several cell types, we analyzed whether ERK and JNK activities were altered in GR-deficient keratinocytes. We found an increase in p-ERK1/2 but not JNK activity in GR–/– keratinocytes by immunoblotting and immunostaining techniques (Figs. 6 and 7). It is assumed that ERK1 and ERK2 isoforms play unique roles because ERK1-deficient mice are viable, whereas ERK2 knockout mice are embryonic lethal (32, 33). However, one should also consider that a threshold of total ERK activity may be required, at least in skin, because in this tissue, ERK2 cannot compensate for the loss of ERK1 (33). Given that both p-ERK1/2 isoforms were augmented in the epidermis as a consequence of GR loss of function, our data suggest that the hormone receptor modulates both activities. Recent work has highlighted the relevance of ERK1 in skin homeostasis and skin tumor development (25). ERK1-deficient mice showed reduced proliferation of basal keratinocytes upon 12-O-tetradecamoyl-phorbol-13-acetate treatment and resistance to development of skin papillomas induced by 7,12-dimethylbenz(a)anthracene-TPA protocol (25). Worth noting, the delayed tumor appearance and reduced tumor number and size found in ERK1 null mice was also reported in K5-GR transgenic mice that were subjected to different protocols of tumor formation (34, 35). Collectively, the existing data argue in favor of antagonistic functions of GR and ERK in keratinocyte proliferation.
On the other hand, ERK1 has also been implicated in modulating the proliferation/apoptosis balance, based on the increased epidermal thickness and the resistance of ERK1-deficient MPKs to apoptosis. In wt skin, p-ERK was predominantly localized to nuclei in the granular layer (Fig. 7), which would fit with the proposed role of ERK in keratinocyte apoptosis. In contrast, p-ERK was detected throughout all suprabasal layers in GR–/– skin, and remarkably, most of p-ERK staining was detected in GR–/– keratinocytes exhibiting apoptotic features (Fig. 7). In accord with this, the phenotype of MPKs from GR null mice (increased growth and apoptotic features; Figs. 5 and 8) was the opposite of that found in ERK null primary keratinocytes (reduced growth and resistance to apoptotic signals) (25). The fact that constitutive ERK activation was found in normally growing keratinocytes from GR–/– mice by immunofluorescence and immunoblotting (Fig. 8) further supports a role for GR in controlling ERK function. Given that GR–/– MPKs exhibited increased apoptosis, which was partially reduced by a selective ERK inhibitor, as demonstrated by TUNEL assays, our data reinforce that GR plays a role in keratinocyte apoptosis through regulation of the ERK function (Fig. 8, D and E). Our results are in agreement with the reduced apoptosis response of ERK–/– keratinocytes both in vivo in total skin and in culture (25).
Because the role of ERK2 has not been yet defined, it cannot be ruled out that the observed changes in this isoform modulate other aspects of keratinocyte function, such as differentiation. In this regard, previous studies in primary human keratinocytes have shown that sustained activity of ERK suppresses and ERK inhibition induces differentiation (36).
Surprisingly, we did not detect major changes in either the protein levels or cellular distribution of JNK, p-JNK, c-jun, and p-c-jun in GR-deficient skin relative to wt (Fig. 6). This was rather unexpected given the reported negative cross-talk between GR and JNK in many cell types, including keratinocytes (11, 22). A recent work by Gazel and co-workers (37) has examined the transcriptomic profile of human keratinocytes upon pharmacological inhibition of the JNK pathway. The outcome of this study is that JNK does not directly regulate many cell-cycle and proliferation genes but rather modulates proliferation as a consequence of keratinocyte differentiation.
We previously reported that GR-mediated repression of AP-1 target genes in skin upon phorbol ester treatment occurred through the DNA-binding-independent function of GR (38). However, and despite that many AP-1 target genes in the epidermis include members of the cytokeratin gene family that are repressed by GR (22, 39), we did not observe changes in the level of these transcripts (not shown). It is feasible that GRs play differential roles during epidermal development compared with stress responses in adult individuals. Overall, our present findings demonstrate that GR is required for epidermal terminal differentiation and skin barrier competence during embryo development.
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Disclosure Statement: The authors have nothing to disclose.
First Published Online November 26, 2007
Abbreviations: AP-1, Activator protein-1; BL, basal layer; BrdU, bromodeoxyuridine; CHX, cycloheximide; DAPI, 4',6-diamidino-2-phenylindole; Dex, dexamethasone; dpc, days post conception; GC, glucocorticoid; GL, granular layer; GR, glucocorticoid receptor; GRE, glucocorticoid response element; H&E, hematoxylin and eosin; HF, hair follicle; JNK, c-jun-N-terminal kinase; MPK, mouse primary keratinocyte; p-, phospho-; PMA, phorbol 12-myristate 13-acetate; SC, strarum corneum; SL, spinous layer; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; wt, wild type.
Received June 18, 2007.
Accepted for publication November 13, 2007.
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B or activator protein-1: molecular mechanisms for gene repression. Endocr Rev 24:488–522
induces apoptosis in keratinocytes and impairs the AKT/PTEN signaling pathway. Exp Cell Res 313:742–752[CrossRef][Medline] pathway, and wound healing along with promotion of terminal differentiation. J Biol Chem 282:4021–4034
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