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Glucocorticoid Receptor Is Required for Skin Barrier Competence

Pilar Bayo, Ana Sanchis, Ana Bravo, Jose Luis Cascallana, Katrin Buder, Jan Tuckermann, Günther Schütz and Paloma Pérez

To investigate the contribution of the glucocorticoid receptor (GR) in skin development and the mechanisms underlying this function, we have analyzed two mouse models in which GR has been functionally inactivated: the knockout GR−/− mice and the dimerization mutant GRdim/dim that mediates defective DNA binding-dependent transcription. Because GR null mice die perinatally, we evaluated skin architecture of late embryos by histological, immunohistochemical, and electron microscopy studies. Loss of function of GR resulted in incomplete epidermal stratification with dramatically abnormal differentiation of GR−/−, but not GR+/− embryos, as demonstrated by the lack of loricrin, filaggrin, and involucrin markers. Skin sections of GR−/− embryos revealed edematous basal and lower spinous cells, and electron micrographs showed increased intercellular spaces between keratinocytes and reduced number of desmosomes. The absent terminal differentiation in GR−/− embryos correlated with an impaired activation of caspase-14, which is required for the processing of profilaggrin into filaggrin at late embryo stages. Accordingly, the skin barrier competence was severely compromised in GR−/− embryos. Cultured mouse primary keratinocytes from GR−/− mice formed colonies with cells of heterogeneous size and morphology that showed increased growth and apoptosis, indicating that GR regulates these processes in a cell-autonomous manner. The activity of ERK1/2 was constitutively augmented in GR−/− skin and mouse primary keratinocytes relative to wild type, which suggests that GR modulates skin homeostasis, at least partially, by antagonizing ERK function. Moreover, the epidermis of GR+/dim and GRdim/dim embryos appeared normal, thus suggesting that DNA-binding-independent actions of GR are sufficient to mediate epidermal and hair follicle development during embryogenesis.

IN MAMMALS, THE EPIDERMIS is a stratified epithelium that acts as a barrier preserving the organism from dehydration, uncontrolled thermoregulation, and potentially environmental damage. The acquisition of a competent barrier occurs during embryonic development and it requires a correct balance between proliferation, differentiation and controlled apoptosis. To exert this key function, the epidermis must be able to self renew under both homeostatic and injury conditions by maintaining a population of proliferative keratinocytes in the basal layer (BL) and hair follicles (HFs). The process of differentiation implies that basal keratinocytes cease to proliferate, lose adherence to the basement membrane, and migrate to outer layers called spinous layer (SL), granular layer (GL), and stratum corneum (SC). During mouse skin development and as basal cells move outward, gene expression of basal keratinocytes, such as keratin K5, is repressed and switches toward differentiation-specific markers, including keratins K1 and K10. Epidermal terminal differentiation is a tightly regulated process that ends up in the conversion of viable keratinocytes into dead, flattened squames of the SC, a process that represents a form of programmed cell death. In the mouse, the first suprabasal layer of the epidermis is formed around embryonic 14.5–15.5 d post conception (dpc), and the number of epidermal cell layers increases, with the SC being formed at 18.5 dpc. Alterations in the processes of keratinocyte proliferation and differentiation during fetal development may lead to a disturbed epidermal barrier, which can cause skin disorders of keratinization and cornification. Recent findings demonstrated that the nonapoptotic caspase-14 plays a role in epidermal homeostasis because its activation at late stages of epidermal development in utero is required for terminal keratinocyte differentiation, contrary to classical caspases involved in apoptosis, such as caspase-3.

Although glucocorticoid (GC) analogs are widely prescribed as the treatment of choice in many cutaneous disorders, the role of GCs in skin development has not been completely deciphered. In some reports, GCs have been shown to accelerate epidermal barrier formation as seen by either GC injections in utero or, conversely, by showing that CRH-deficient newborn mice, which exhibited GC deficiency, had delayed maturation of the SC; moreover, supplementation of GCs in these mice fully rescued skin phenotype. In experiments performed in adult mouse skin, short-term topical and systemic GC treatment disturbed epidermal barrier competence. 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-κB or activator protein-1 (AP-1). Nongenomic actions of GR have been demonstrated and are mediated through physical interaction of the receptor at the plasma membrane with p85α/phosphatidylinositol 3-kinase, which, in turn, modulates acutely transforming retrovirus AKT8 in rodent T cell lymphoma activity.

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 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). 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.