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Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor ¹

PALOMA PÉREZ, ANGUSTIAS PAGE, ANA BRAVO^*, MARCELA DEL RÍO, IRMA GIMÉNEZ-CONTI, IRINA BUDUNOVA, THOMAS J. SLAGA and JOSÉ L. JORCANO²

Project on Cell and Molecular Biology and Gene Therapy, CIEMAT, Av. Complutense 22, E-28040 Madrid, Spain;
^* Department of Animal Pathology, Veterinary Faculty, University of Santiago de Compostela, E-27002 Lugo, Spain;
Science Park, University of Texas M.D. Anderson Cancer Center, Smithville Texas 78957, USA; and
AMC Cancer Research Center, Denver, Colorado 80214, USA

²Correspondence: Project on Cell and Molecular Biology and Gene Therapy, CIEMAT, Av. Complutense 22, E-28040 Madrid, Spain. E-mail: jl.jorcano{at}ciemat.es

SPECIFIC AIMS

We studied the role of the glucocorticoid receptor (GR) in skin by generating transgenic mice that overexpress this protein, which mediates the biological effects of the glucocorticoids (GCs), under the control of the keratin K5 promoter. K5-GR mice allowed us to study developmental and pathophysiological aspects of GR function in skin and other tissues and to investigate the molecular mechanisms underlying the antiproliferative and anti-inflammatory effect of glucocorticoids in skin in vivo, focusing on the negative interference between GR and nuclear factor B (NF-B).

PRINCIPAL FINDINGS

1. Altered skin development in K5-GR transgenic mice
We generated five founder lines with the K5-GR construct and carried out our studies in lines 72 and 285, as they presented the highest transgene expression levels. K5-GR mice overexpressed the glucocorticoid receptor in the basal cells of epidermis, hair follicles, and other stratified epithelia by means of the keratin K5 promoter. K5-GR mice exhibited a variable degree of phenotype severity according to transgene expression levels, yet the phenotype was heterogeneous within a single litter (Fig. 1 ). Transgenic mice of line 72 died several hours after birth, whereas line 285 animals survived to adulthood and were fertile. However, homozygous mice of line 285 also died perinatally. At 18.5 d.p.c., all transgenic mice showed reduced embryonic growth paralleling the severity of the phenotype, abnormally smooth and thin skin, and incomplete closure of the upper/lower eyelid (Fig. 1A , B ). Skin defects were variable sized, ranging from epidermal hypoplasia and underdeveloped dysplastic hair follicles (Fig. 1, C -E ) to a complete absence of all epidermal layers. In the most affected animals, skin was absent at the cranial region overlapping the anterior fontanella and at the umbilical region (Fig. 1A , B ). At 16.5 d.p.c., skin of control head sections appeared as an organized stratified epithelium, in contrast to transgenic head sections, consisting of a monolayer with flattened cells resembling an immature epithelium, although positive for K5 staining. At 18.5 d.p.c, K5 staining progressively decreased and disappeared at the region overlapping the anterior fontanella of transgenic mice; at birth, this epidermal region was necrotic. These results indicate that GR plays a developmental role in epidermal proliferation/differentiation. Impaired hair follicle formation was further confirmed by scanning electron microscopy and observed in transgenic skin grafted onto immunodeficient SCID recipient mice.

View larger version (91K): [in this window] [in a new window]   Figure 1. Phenotype of 18.5 d.p.c. K5-GR transgenic mice. A) Control mouse (left) and two transgenic mice of line 72 with intermediate (center) and severe phenotype (right). B) Absent skin is associated with the incomplete closure of the fontanella and dilated scalp veins (center) and acrania and exencephaly (right). C–E) Hematoxylin/eosin staining of newborn control (C) and transgenic (D, E) mice. Notice epidermal hypoplasia (D) and disorganized or missing epidermis (E) in newborn transgenic mice. Arrowheads in panels C, D point to the basal layer; the arrows in panel E point to the epidermal/dermal border. Bar = 25 µm.

2. GR overexpression severely affects the development of other ectodermal-derived tissues
Epithelia other than the epidermis, such as the cornea and oral epithelia, were also severely affected in transgenic mice, as they are also targeted by means of the keratin K5 promoter. As a consequence, these mice presented alterations in tissues and structures underlying the affected epithelia. K5-GR mice exhibited an extensive area at the anterior region of the head with defects not only in the skin, but also the dura and cranium, leading in some cases to acrania with exencephaly (Fig. 1A , right). All together, these features of congenital dysplasia affecting one or more ectodermal-derived structures and their accessory appendages strikingly resembles the clinical findings in the human syndrome previously described as ectodermal dysplasia (ED), which includes aplasia cutis congenita (ACC).

3. Impaired proliferative and inflammatory responses to TPA in adult transgenic skin
Glucocorticoids play an important anti-inflammatory role in skin; since this effect is mediated through GR/NF-B interference in other cell types, we investigated whether this mechanism was also relevant in skin. TPA and other tumor promoters elicit hyperplasia and inflammation in skin and strongly induce NF-B activity in mouse skin. To further analyze the role of GR in skin function, we investigated the response of K5-GR mice to topical TPA treatment and found a strongly decreased proliferative response, as demonstrated by its dramatically reduced hyperplasia and BrdU labeling index of basal cells. We also measured the mRNA levels of several proinflammatory cytokines in the back skin of acetone-treated (untreated, U), TPA-treated (T), and dexamethasone-treated (D) adult transgenic and nontransgenic mice by Northern blot (Fig. 2A ). The endogenous mRNA levels corresponding to interleukin 1 (IL-1), IL-6, and tumor necrosis factor (TNF-) in untreated transgenic skin were almost undetectable compared with controls. In TPA-treated control skin, the mRNA levels of these cytokines were highly increased compared with acetone-treated samples. In contrast, the TPA response was abolished in TPA-treated transgenic skin. Topical application of dexamethasone elicited a marked decrease in the indicated mRNA levels in control skin. Given that the cytokine genes analyzed are regulated by NF-B, our results suggest that GR overexpression in basal keratinocytes interferes with the transcriptional activity of this factor. To confirm this, we prepared nuclear skin extracts from newborn control and transgenic mice and assayed their B binding activity by electrophoretic mobility shift assay (EMSA) (Fig. 2B ).

View larger version (54K): [in this window] [in a new window]   Figure 2. Proinflammatory cytokine transcriptional regulation is abrogated in transgenic skin due to GR/NF-B interference. A) Northern blot. Total RNA from control (C) and transgenic (Tg) adult back skin either treated with vehicle (untreated, U), TPA (T, 18 h after topical application), or dexamethasone (D, 24 h after topical application) was hybridized with the probes indicated. B) EMSA using nuclear skin extracts from newborn control (C) and transgenic (lines 72 and 285) mice and a B-labeled probe. B complexes are indicated at the left. C) Northern blot using total RNA from newborn control (C) and transgenic (lines 72 and 285) mouse skin was hybridized with and an IB (upper panel) or 7S probe (lower panel).

In control skin extracts, we detected basal B binding activity that was dramatically reduced in skin extracts from transgenic line 285 and almost undetectable in transgenic line 72 (Fig. 2B ). This basal B binding activity in newborn mouse skin consisted of p50/p65 heterodimers and p50 homodimers as identified by supershift assays. These findings indicate that overexpressed GR negatively interfered with basal B binding activity in transgenic skin. In some cell types, glucocorticoids can increase IB mRNA levels; however, we did not detect changes of this transcript either in transgenic skin or in dexamethasone-treated control skin (Fig. 2C and data not shown). According to our data, the anti-inflammatory action of glucocorticoid in skin is due at least in part to the interference between GR and NF-B, leading to a strong reduction in the B binding activity. We are currently using our transgenic mouse model to gain further insight into the molecular mechanisms responsible for the antiproliferative, anti-inflammatory, and antitumor role of GR in skin.

CONCLUSIONS AND SIGNIFICANCE

Many morphogenetic processes depend on glucocorticoid signaling; GR and its natural ligand must therefore be expressed properly in order to render a normal embryo. GCs are known to act as potent teratogens; accordingly, K5-GR mice exhibit dramatic multiple defects affecting skin and other tissues. The affected tissues were targeted for transgene expression driven by the keratin K5 promoter and embryologically derived from superficial ectoderm. Phenotypic abnormalities include thin, smooth skin and sparse hair growth in the whole embryo, and an absence of skin localized at the cranium and at the umbilical region. We also observed dramatic ophthalmological, palatal, and dental defects. These traits strikingly resemble the abnormalities and the characteristic phenotypic variability reported in the human disorder ED, and in particular, ACC (Fig. 3 ). ACC is a complex disease characterized by absence of localized or widespread skin areas at birth and is associated with malformations that affect diverse ectodermal derivatives. Remarkably, acrania has previously been considered a severe form of ACC in humans, and we have detected delayed ossification of the supraoccipital bone or complete absence of the cranium in the most extremely affected mice. Our data suggest that deregulated GR expression may be involved in ED/ACC; thus, K5-GR mice could be useful in unraveling the molecular basis and providing a diagnostic tool for this complex human disorder.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic. Study of glucocorticoid receptor functions in skin and other ectodermal-derived tissues by using K5-GR transgenic mice. The effects of GCs are mediated by GR through mechanisms that are DNA binding-dependent (left) and DNA binding-independent (right).

So far, a detailed study of GR function in skin has not been feasible, since GR null mice die perinatally. Homozygous mice carrying a DNA binding defective mutant of GR (GR dim) were viable and showed no overt skin phenotype. K5-GR mice allow us to address the role of GR in the development and pathology of skin and other tissues in which GCs are widely used as therapeutic drugs. These mice also permit the study of the effect of both DNA-dependent and -independent mechanisms of GR action on these processes. We have observed an augmented transcriptional regulation of the metallothionein gene Mt-1 (DNA binding-dependent mechanism; not shown), as well as a negative interference between GR and NF-B (DNA binding-independent mechanism). We do not have an explanation for the constitutive nuclear localization of GR in the absence of added ligand; however, in some cell types, overexpression of this protein promotes its transfer to the nucleus even without hormonal stimulus.

The present study underscores the antiproliferative and anti-inflammatory roles of GR in the epidermis: 1) constitutive expression of GR leads to decreased epidermal proliferation in newborn mice and even produces complete absence of skin; 2) the hyperplastic and inflammatory responses of adult transgenic skin to topical TPA treatment are dramatically impaired; 3) in vivo, the anti-inflammatory action of GR is most likely due to a negative interference between GR and NF-B, since the proinflammatory cytokines IL-1, IL-6, and TNF- lack glucocorticoid response elements but contain B-responsive elements in their promoters. Mechanistically, GR/NF-B antagonism in mouse skin involves a marked decreased in B binding activity without up-regulating the inhibitor IB. GR/NF-B cross-talk appears to be a relevant issue also in skin tumorigenesis. We have previously demonstrated that constitutive NF-B activation parallels the loss of GR function during two-stage mouse skin carcinogenesis protocol. K5-GR mice will provide an invaluable tool for studying the molecular mechanisms underlying the antitumor activity of GR during skin tumor promotion.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0772fje ; to cite this article, use FASEB J. (July 24, 2001) 10.1096/fj.00-0772fje

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