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Human Molecular Genetics Advance Access originally published online on January 6, 2006
Human Molecular Genetics 2006 15(4):531-542; doi:10.1093/hmg/ddi470
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© The Author 2006. Published by Oxford University Press. All rights reserved.
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Skin lesion development in a mouse model of incontinentia pigmenti is triggered by NEMO deficiency in epidermal keratinocytes and requires TNF signaling

Arianna Nenci1, Marion Huth1, Alfred Funteh2, Marc Schmidt-Supprian3, Wilhelm Bloch4, Daniel Metzger5, Pierre Chambon5, Klaus Rajewsky3, Thomas Krieg2, Ingo Haase2 and Manolis Pasparakis1,*

1European Molecular Biology Laboratory, Mouse Biology Unit, via Ramarini 32, 00016 Monterotondo-Scalo (Rome), Italy, 2Department of Dermatology, Center for Molecular Medicine, University of Cologne (CMMC), Joseph-Stelzmann-Strasse 9, 50924 Cologne, Germany, 3CBR Institute for Biomedical Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA, 4Abteilung für Molekulare und Zelluläre Sportmedizin, Deutsche Sporthochschule Köln, IG I, Carl-Diem-Weg 6, D-50933 Köln, Germany and 5Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS, INSERM, ULP, and Institut Clinique de la Souris (ICS), BP 10142-67404, ILLKIRCH, C.U. de Strasbourg, France

* To whom correspondence should be addressed. Tel: +39 0690091222/90091271; Fax: +39 0690091272; Email: pasparakis{at}embl-monterotondo.it

Received October 13, 2005; Revised December 7, 2005; Accepted December 31, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
NF-{kappa}B essential modulator (NEMO), the regulatory subunit of the I{kappa}B kinase, is essential for NF-{kappa}B activation. Mutations disrupting the X-linked NEMO gene cause incontinentia pigmenti (IP), a human genetic disease characterized by male embryonic lethality and by a complex pathology affecting primarily the skin in heterozygous females. The cellular and molecular mechanisms leading to skin lesion pathogenesis in IP patients remain elusive. Here we used epidermis-specific deletion of NEMO in mice to investigate the mechanisms causing the skin pathology in IP. NEMO deletion completely inhibited NF-{kappa}B activation and sensitized keratinocytes to tumor necrosis factor (TNF)-induced death but did not affect epidermal development. Keratinocyte-restricted NEMO deletion, either constitutive or induced in adult skin, caused inflammatory skin lesions, identifying the NEMO-deficient keratinocyte as the initiating cell type that triggers the skin pathology in IP. Furthermore, genetic ablation of tumor necrosis factor receptor 1 (TNFRI) rescued the skin phenotype demonstrating that TNF signaling is essential for skin lesion pathogenesis in IP. These results identify the NEMO-deficient keratinocyte as a potent initiator of skin inflammation and provide novel insights into the mechanism leading to the pathogenesis of IP.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The NF-{kappa}B transcription factors control the expression of many genes with important functions in inflammation, immune responses, cell proliferation, survival and apoptosis (1Go,2Go). In resting cells, NF-{kappa}B dimers are kept inactive by association with inhibitory proteins belonging to the I{kappa}B family. NF-{kappa}B activation is induced by the I{kappa}B kinase (IKK) complex consisting of the IKK1(IKK{alpha}) and IKK2(IKKß) catalytic subunits and the NF-{kappa}B essential modulator (NEMO)/IKK{gamma} regulatory protein (3Go–5Go). Upon activation by a variety of stimuli, including proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) and bacterial lipopolysaccharide (LPS), the IKK phosphorylates I{kappa}B proteins at specific serine residues targeting them for polyubiquitination and proteasome-mediated degradation, thus releasing NF-{kappa}B, which then accumulates in the nucleus and activates its target genes (6Go,7Go). IKK2 and NEMO are essential for NF-{kappa}B activation by proinflammatory signals via the classical activation pathway, whereas IKK1 is required for the alternative NF-{kappa}B activation pathway controlling p100 processing (8Go).

Several studies have suggested that the NF-{kappa}B pathway is involved in the regulation of epidermal homeostasis (9Go,10Go). Inhibition of NF-{kappa}B activation in epidermal keratinocytes either by transgenic overexpression of a mutant non-degradable I{kappa}B{alpha} or by knockout of the p65 NF-{kappa}B subunit lead to epidermal hyperplasia, suggesting a growth-regulatory role for NF-{kappa}B in keratinocytes (10Go–15Go). Furthermore, Dajee et al. (13Go) showed that inhibition of NF-{kappa}B in combination with expression of oncogenic Ras in human keratinocytes transplanted on the skin of severe combined immunodeficiency (SCID) mice caused the development of invasive tumors resembling squamous cell carcinoma. In a different study, overexpression of I{kappa}B in the epidermis of transgenic mice caused an inflammatory hyperproliferative epidermal phenotype leading to the development of squamous cell carcinomas (16Go,17Go). In this case, however, blockade of TNF signaling inhibited both epidermal hyperplasia and tumor formation, suggesting that the development of squamous cell carcinomas in this model depends on a TNF-induced inflammatory response (18Go). Mice with epidermis-specific deletion of IKK2 display an inflammatory skin phenotype characterized by expression of proinflammatory cytokines and chemokines, infiltration of immune cells, epidermal hyperplasia and deregulated expression of epidermal differentiation markers (19Go). Also in this case, genetic ablation of TNF signaling rescued the skin phenotype demonstrating that the epidermal hyperplasia is a secondary effect of the inflammatory response.

In humans, mutations disrupting the X-linked gene encoding NEMO cause incontinentia pigmenti (IP), a genetic disease characterized by male embryonic lethality and by the development of multiple cutaneous, neurological and ophthalmological abnormalities in heterozygous females (20Go). The skin lesions of IP patients develop in four successive, sometimes overlapping, characteristic stages, starting with the appearance of an inflammatory vesicular rash shortly after birth (21Go,22Go). This initial inflammatory phase is followed by the formation of verrucous lesions during the first weeks of life, which disappear shortly as the disease moves into the third stage marked by the appearance of skin areas displaying hyperpigmentation. During the fourth stage and usually around puberty, the hyperpigmented areas disappear leaving behind patches of atrophic hypopigmented skin (21Go,22Go). Targeted disruption of the gene encoding NEMO causes male embryonic lethality and the appearance of transient inflammatory skin lesions in heterozygous females, a phenotype that shares many of the characteristics of IP in humans (23Go,24Go). Although many of the heterozygous NEMO knockout mice die at the peak of the disease, some of them recover showing healing of the skin lesions and survive to adulthood displaying an apparently normal skin (23Go,24Go). The mosaic expression of NEMO due to random X-chromosome inactivation in heterozygous females is thought to be critical for the development of the skin phenotype in both the human patients and in the mouse model. The identification of NEMO mutations as the cause of human IP, in combination with the results obtained from the NEMO knockout mouse, leads to the proposal of a new model for the pathogenesis of this disease. According to this model, the cell autonomous hyperproliferation of NEMO knockout keratinocytes, possibly in combination with the spontaneous necrotic cell death of some NEMO-deficient keratinocytes, triggers the expression of proinflammatory mediators by the neighboring wild-type keratinocytes resulting in the development of the skin lesions. During the inflammatory phase of the disease, proinflammatory cytokines such as TNF released by the invading immune cells are thought to kill NEMO-deficient keratinocytes, which are then replaced by healthy wild-type keratinocytes resulting in the healing of skin lesions.

Although these studies provided important information for the understanding of the pathogenesis of the skin lesions in IP, several questions regarding the mechanism triggering this disease remain unresolved. Both IP patients and heterozygous NEMO-deficient mice show mosaic presence of NEMO-deficient and wild-type cells in many different cell types. It is, therefore, not clear whether the skin lesions are triggered by the presence of NEMO-deficient keratinocytes in the epidermis or by an abnormal inflammatory response caused by the lack of NEMO in non-epidermal cell types. In addition, it is not known whether the interaction between NEMO-deficient and wild-type keratinocytes is essential for the initiation and progression of the skin lesions. Furthermore, it is not clear why the skin lesions appear shortly after birth both in humans and in the mouse model. This early postnatal initiation of the phenotype may be caused by the exposure of the skin to environmental stimuli, or alternatively, developmental processes occurring in the skin at this stage may be involved.

We have used Cre/loxP-mediated gene targeting in mice to investigate in vivo the function of NEMO in the epidermis and to address experimentally the cellular and molecular mechanisms that are involved in the pathogenesis of the skin lesions in IP. We show here that NEMO deletion in epidermal keratinocytes blocks NF-{kappa}B activation in response to proinflammatory signals, but it does not cause cell autonomous hyperproliferation or differentiation defects. We also show that epidermis-restricted NEMO deletion is sufficient to trigger inflammatory skin lesions and that the mosaic presence of NEMO-deficient and wild-type keratinocytes in the epidermis is not necessary for disease development. Furthermore, we demonstrate that an inflammatory response requiring TNF receptor I signaling is essential for the pathogenesis of the skin lesions, suggesting that TNF-mediated inflammation is an obligatory component of the disease. Finally, inducible deletion of NEMO in the epidermis of adult mice was sufficient to trigger the development of skin lesions, suggesting that disease initiation is not related to developmental processes occurring shortly after birth but is a direct and immediate consequence of the inhibition of NEMO-dependent NF-{kappa}B activation in epidermal keratinocytes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
NEMO-deficient epidermal keratinocytes do not activate NF-{kappa}B and are highly sensitive to TNF-induced death
To investigate the function of NEMO in the epidermis, we generated mice with epidermal keratinocyte-restricted NEMO deletion, by crossing mice expressing Cre recombinase under the control of the human keratin 14 promoter (K14-Cre) (25Go) with mice carrying loxP-flanked (floxed) NEMO alleles (23Go). In contrast to control cells, primary NEMO-deficient keratinocytes failed to activate NF-{kappa}B upon stimulation with IL-1, TNF or 12-O-Tetradecanoylphorbol 13-acetate (TPA) (Fig. 1B and data not shown). Furthermore, NEMO knockout keratinocytes showed impaired induction of NF-{kappa}B-dependent genes upon stimulation with TNF (Fig. 1C and data not shown). As NF-{kappa}B-dependent gene expression is known to be important for the protection of cells from TNF-induced cytotoxicity, we investigated the sensitivity of NEMO-deficient keratinocytes to TNF. In accordance to the complete lack of NF-{kappa}B activation, NEMO-deficient keratinocytes were extremely sensitive to TNF-induced cell death (Fig. 1D). These results show that NEMO knockout causes complete inhibition of NF-{kappa}B activation in epidermal keratinocytes and renders them sensitive to TNF-induced death.


Figure 4701
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  		Figure 1. Deletion of NEMO in keratinocytes impairs NF-{kappa}B activation and confers sensitivity to TNF-induced cell death. (A) Western blot analysis of NEMO, IKK1, IKK2 and actin expression in protein extracts from P0 epidermis and from primary keratinocytes of control and NEMOEKO mice. Extracts from wild-type and NEMO-deficient mouse embryonic fibroblasts (MEFs) are used as controls. (B) Primary keratinocytes isolated from wild-type and NEMOEKO mice were stimulated with IL-1ß (10 ng/ml) for the indicated time points. Phosphorylation and degradation of I{kappa}B{alpha} was assayed by western blot analysis of cytoplasmic extracts, and NF-{kappa}B DNA-binding activity was measured by gel mobility shift analysis of nuclear extracts. (C) Real-time PCR analysis of I{kappa}B{alpha} expression in primary keratinocytes isolated from control and NEMOEKO mice after treatment with TNF (20 ng/ml) for the indicated time points. Results are shown as the mean and SE of triplicate samples. (D) Sensitivity to TNF-induced cytotoxicity. Primary keratinocytes isolated from control and NEMOEKO mice were left untreated or stimulated with TNF (20 ng/ml) for 6 h.

 
NEMO deletion restricted to keratinocytes causes skin lesions
For the generation of epidermis-specific NEMO knockouts, we used a breeding scheme where K14Cre transgenic males were crossed with females homozygous for the NEMO-floxed locus. As the NEMO gene is X-linked, all male progeny were hemizygous (FL/Y) and all female progeny were heterozygous (FL/+) for the NEMO-floxed allele. Male progeny carrying the K14Cre transgene showed complete absence of NEMO in all epidermal keratinocytes (NEMOEKO), as shown by western blot analysis of extracts from isolated epidermis or cultured primary keratinocytes (Fig. 1A). In contrast, the epidermis of heterozygous females expressing K14Cre (NEMOEHET) is expected to contain both NEMO-deficient and NEMO-expressing cells in a mosaic pattern due to random X-chromosome inactivation. Using this genetic approach, we were able to investigate the skin phenotype of mice showing either complete or mosaic deletion of NEMO in epidermal keratinocytes.

NEMOEKO and NEMOEHET mice were born at the expected ratio and were macroscopically indistinguishable from their K14Cre-negative control littermates until postnatal day 2. At this stage, NEMOEKO pups showed a characteristic lack of skin pigmentation, which in NEMOEHET mice appeared only in patches presumably at areas of the skin containing NEMO-deficient keratinocytes. The lack of pigmentation became more obvious at P3 and P4, and at P5 both NEMOEKO and NEMOEHET mice displayed a skin phenotype characterized by the presence of red and inflamed areas with scaling, especially around the neck (Fig. 2A). The NEMOEKO mice rapidly developed severe skin lesions and failed to survive further than postnatal day 6. NEMOEHET mice developed skin lesions in a patchy fashion and most of them died between P7 and P10. Some NEMOEHET mice showing mild skin phenotype with less and smaller affected skin areas, presumably due to the presence of only a small percentage of NEMO-deficient keratinocytes in their epidermis, survived to adulthood and showed healing of the skin lesions similar to heterozygous NEMO knockout mice (23Go). These results show that deletion of NEMO in the epidermis, either in a complete or in a mosaic fashion, causes skin lesions, suggesting that the skin manifestations of IP are the result of NEMO deficiency in epidermal keratinocytes.


Figure 4702
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  		Figure 2. Skin phenotype of mice with epidermis-specific deletion of NEMO. (A) Epidermis-specific NEMO knockout (EKO), heterozygous (EHET) and control mice at 3, 4, 5 and 6 days after birth. (B) Skin sections from NEMOEKO, NEMOEHET and control mice at P0, P3 and P5 stained with hematoxylin/eosin. Scale bar: 40 µm.

 
Inflammatory skin disease in mice with epidermis-restricted NEMO deletion
To characterize the skin lesions that develop in NEMOEKO and NEMOEHET mice, we performed detailed immunohistological analysis of skin sections from these animals. Histological examination of skin samples taken from mice at P0 and P3 did not show epidermal hyperplasia or signs of skin inflammation, however at P5 the skin of both NEMOEKO and NEMOEHET mice displayed thickening of the epidermis, hyperkeratosis, loss of the granular layer and the presence of infiltrating mononuclear and polymorphonuclear cells in the dermis (Fig. 2B and data not shown). In addition, a small number of dyskeratotic basal keratinocytes were observed in the NEMOEKO but not in control skin. Examination of skin samples from mice at P0 using electron microscopy did not reveal ultrastructural abnormalities in cell–cell contacts, cytoskeletal organization and cell organelles in the NEMOEKO skin, but readily detected the presence of a small number of dead basal keratinocytes in knockout but not in control epidermis.

Immunohistochemical analysis using antibodies against different epidermal differentiation markers confirmed that the skin of NEMOEKO and NEMOEHET mice at P0 shows a normal morphology with keratin 14 expressed in the basal layer, keratin 10 in the suprabasal layers and loricrin and filaggrin in the terminally differentiated upper layers of the epidermis (Fig. 3 and data not shown). These results demonstrate that NEMO-mediated NF-{kappa}B activation is not required for the normal formation and differentiation of the epidermis. However, as early as 3 days after birth the epidermis of both NEMOEKO and NEMOEHET mice showed upregulation of keratin 14 in the suprabasal layers, which was even more pronounced at P5 and was accompanied by the loss of keratin 10 and loricrin expression (Fig. 3). Furthermore, the skin of NEMOEKO and NEMOEHET mice showed upregulation of keratin 6, a marker of inflamed epidermis, at P5 (Fig. 4B). Thus, the skin lesions developing after P3 in both NEMOEKO and NEMOEHET mice are characterized by abnormal expression of epidermal differentiation markers.


Figure 4703
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  		Figure 3. Differentiation marker expression in mice with epidermis-specific deletion of NEMO. Skin sections from NEMOEKO, NEMOEHET and control mice at P0, P3 and P5 were immunostained for epidermal differentiation markers. Keratin14 (K14) is shown in green, Keratin 10 (K10) is shown in red and loricrin is shown in green. Blue staining shows nuclei. Scale bars: 100 µm.

 

Figure 4704
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  		Figure 4. Skin inflammation in mice with epidermis-specific deletion of NEMO. (A) Sections of NEMOEKO, NEMOEHET and control skins at P0, P3 and P5 were stained with antibodies recognizing T-cells (CD3), granulocytes (GR1) and macrophages (F4/80) (green signal). Nuclei are stained in red. Scale bar: 40 µm. (B) Staining of skin section from NEMOEKO and control mice at P5 with antibodies against keratin 6 (green). Nuclei are stained in blue. Scale bar: 40 µm. (C) RNase protection assay showing expression of IL-1{alpha}, IL-1ß, interleukin-1 receptor antagonist (IL-1RA) and interleukin 18/interferon-gamma-inducing factor (IL-18/IGIF) in the epidermis of two different NEMOEKO (ko) and two control (ct) mice at P0 and at P2. (D) Real-time PCR analysis of TNF expression in total skin isolated from two control and two NEMOEKO mice at P0, P3 and P4, respectively. Results are shown as the percentage of the mean relative to ubiquitin and error bars indicate SE of triplicate samples.

 
The presence of infiltrating inflammatory cells in the dermis and the upregulation of keratin 6 in the epidermis of NEMOEKO and NEMOEHET skin samples suggested that an inflammatory response may be implicated in the development of skin lesions in these mice. To further investigate the potential contribution of inflammatory cells to the pathogenesis of the skin lesions in NEMOEKO and NEMOEHET mice, we stained skin sections with antibodies for T lymphocytes (CD3), granulocytes (Gr-1) and macrophages (F4/80). These experiments showed large numbers of granulocytes and a few T lymphocytes infiltrating the dermis of NEMOEKO and NEMOEHET mice after P3, whereas the number of macrophages was also increased at this stage (Fig. 4A). Furthermore, analysis of proinflammatory cytokine expression in the skin of these mice showed upregulation of TNF, IL-1{alpha} and IL-1ß at P3, P4 and P5 (Fig. 4D and Supplementary Material, Fig. S1). IL-1ß and to a lesser extent IL-1{alpha} were upregulated as early as P2 in the NEMO-deficient epidermis (Fig. 4C).

NEMO-deficient keratinocytes do not show spontaneous hyperproliferation in vitro or in vivo
Inhibition of NF-{kappa}B activation in epidermal keratinocytes by overexpression of a dominant negative mutant I{kappa}B{alpha} or knockout of p65/RelA has been reported to cause cell autonomous hyperproliferation of epidermal keratinocytes (11Go,12Go). On the basis of the assumption that inhibition of NF-{kappa}B in keratinocytes results in impaired growth arrest and increased proliferation, it has been hypothesized that the skin phenotype in the NEMO heterozygous mice and in human IP patients is triggered by the hyperproliferation of NEMO-deficient keratinocytes (23Go,24Go). To test experimentally this hypothesis, we measured the proliferation of NEMO-deficient keratinocytes in vivo and in vitro.

Measurement of bromodeoxyuridine (BrdU) incorporation revealed that NEMO-deficient keratinocytes do not show hyperproliferation in culture (Fig. 5A). To investigate whether NEMO-deficient keratinocytes show increased proliferation in vivo, we used immunostaining with antibodies against Ki67 and proliferating cell nuclear antigen (PCNA), two markers exposed in proliferating cells (26Go), and also measured BrdU incorporation. Staining for Ki67 showed that NEMO-deficient keratinocytes do not display increased proliferation at P0 (Fig. 5D), suggesting that also in vivo the deletion of NEMO does not lead to spontaneous keratinocyte hyperproliferation. At P5, we detected increased numbers of Ki67-positive keratinocytes in NEMOEKO skin compared with controls (Fig. 5D). However, the fact that NEMO-deficient keratinocytes show increased proliferation only after P3 suggests that this hyperproliferation is secondary to the inflammatory response developing in the skin. Analysis of BrdU incorporation at E18.5 revealed reduced numbers of BrdU-positive basal keratinocytes in NEMOEKO skin compared with control embryos, showing that the NEMO-deficient epidermis contains less basal keratinocytes in the S phase of the cell cycle (Fig. 5B and C). Staining with PCNA revealed a surprising picture with the majority of basal cells in the NEMOEKO epidermis staining positive, in contrast to control skin that contained less PCNA-positive basal keratinocytes (Fig. 5B and C). Taken together, these results suggest that basal keratinocytes in the skin of NEMOEKO mice enter the cell cycle but delay to progress from G1 to the S phase resulting in less proliferation than control keratinocytes. This finding is consistent with a similar cell-cycle defect observed in epidermal keratinocytes from rel-a–/–c-rel–/–tnf–/– mice, which also showed a delay in G1 progression (27Go). Therefore, inhibition of NF-{kappa}B activation in epidermal keratinocytes either at the level of the IKK by NEMO knockout or by deletion of both p65/RelA and c-Rel does not result in cell autonomous hyperproliferation, but on the contrary it causes a delay in G1 transition and reduced proliferation.


Figure 4705
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  		Figure 5. In vitro and in vivo analyses of keratinocyte proliferation and apoptosis. (A) Primary keratinocytes from NEMOEKO and control mice were labeled with BrdU for the indicated times. BrdU-positive cells were counted from four different fields (≥500 cells/field). Results are shown as the percentage of BrdU-positive cells±SEM. (B) Quantitation of BrdU+ and PCNA+ basal keratinocytes from NEMOEKO and control mice (n=3) at E18.5. At least 200 basal cells were counted per mouse. Results are presented as percentage of positive cells±SEM. (C) Immunohistochemical staining of control and NEMOEKO E18.5 skin sections for PCNA expression and BrdU incorporation (red). Nuclei are stained in blue. Scale bars: 100 µm. (D) Analysis of cell proliferation by staining for Ki67 (red) in the skin of NEMOEKO and control mice at P0, P3 and P5. Nuclei are stained in blue. Scale bar: 100 µm. (E) TUNEL staining for the detection of apoptotic cells (green) on skin sections from control and NEMOEKO mice at E18.5, P0, P3 and P5. Nuclei are stained in blue. Scale bar: 100 µm.

 
Increased apoptosis in the epidermis of NEMOEKO mice
To investigate whether NEMO-deficient keratinocytes show increased apoptosis in vivo, we performed dUTP nick end labeling (TUNEL) staining on skin samples derived from mice at various stages between E18.5 and P5. This analysis revealed the presence of small numbers of apoptotic basal keratinocytes in the epidermis of NEMOEKO mice already at E18.5 and P0, whereas in control mice we only detected occasional TUNEL-positive cells in the suprabasal layers (Fig. 5E). This finding is consistent with our electron microscopy examination results showing the presence of dead basal keratinocytes in NEMOEKO but not in control epidermis at P0 (data not shown). TUNEL-positive cell clusters were also occasionally found around hair follicles in the skin of NEMOEKO mice at P0 but not in littermate controls (data not shown). Analysis of skin samples from mice at P5 revealed the presence of numerous apoptotic keratinocytes in NEMOEKO and to a lesser extent in NEMOEHET mice (Fig. 5E and data not shown). The increased apoptotic death of NEMO knockout keratinocytes at this late stage of the disease is presumably caused by the increased expression of TNF from the infiltrating immune cells.

Activation of STAT3 and JNK in the epidermis of NEMOEKO mice
Activation of STAT3 in epidermal keratinocytes has been shown to correlate with skin inflammation in human psoriatic skin, and expression of activated STAT3 in the epidermis of transgenic mice caused a psoriasis-like phenotype (28Go).To investigate whether STAT3 is activated in the epidermis of NEMOEKO mice, we performed immunostaining with an antibody specific for phosphorylated STAT3. These experiments revealed increased phosphorylation of STAT3 in the epidermis of NEMOEKO mice at P5 but not at P0 (Fig. 6). A recent study suggested that in p65/RelA-deficient keratinocytes, TNFRI signaling leads to cell autonomous hyperproliferation by inducing increased activation of JNK (11Go). We, therefore, investigated JNK activity in the epidermis of NEMOEKO mice and in cultured NEMO-deficient keratinocytes using immunostaining with an antibody specific for phosphorylated JNK. We could not detect differences in JNK activation between wild-type and mutant epidermis at P0, however at P5 we observed increased JNK phosphorylation in epidermal cells in NEMOEKO skin (Fig. 6). The increased activation of JNK and STAT3 is only detected after P3, suggesting that it is a secondary event induced by the expression of proinflammatory mediators in the skin at this stage.


Figure 4706
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  		Figure 6. Activation of STAT3 and JNK in the epidermis of NEMOEKO mice. Sections from NEMOEKO and control mice at P0 and P5 were immunostained with antibodies recognizing phosphorylated STAT3 or phosphorylated JNK. Scale bar: 100 µm.

 
TNF receptor I signaling is essential but B and T lymphocytes are dispensable for the development of skin inflammation in mice with epidermis-restricted NEMO deletion
Gene expression analysis revealed upregulation of TNF and IL-1 in the skin of NEMOEKO and NEMOEHET mice, suggesting that these potent proinflammatory cytokines play a role in the pathogenesis of the inflammatory skin disease. To investigate the function of TNF in the development of the skin lesions in epidermis-specific NEMO knockout mice, we bred these mice into a TNFRI-deficient background. Remarkably, elimination of TNFRI rescued the inflammatory skin phenotype in both NEMOEKO and NEMOEHET mice. Double NEMOEKO/TNFRI knockout mice often displayed transient mild flaking in skin patches at the age of P9–P10, which disappeared shortly and the mice developed into adulthood without showing skin lesions (Fig. 7B). Histological examination of skin samples from NEMOEKO/TNFRI knockout mice at P7 revealed a normal picture with no signs of skin inflammation (Fig. 7A). These results demonstrate that TNF signaling through TNF receptor I is essential for the development of skin inflammation in this model and suggest that a similar mechanism may be involved in the pathogenesis of skin lesions in human IP patients. Later in life and usually between 4 and 6 months of age, most of the NEMOEKO/TNFRI-deficient mice developed skin lesions appearing as flaky, hairless ulcerated plaques particularly around the neck and in areas where the skin is subjected to mechanical stress. Therefore, TNF signaling is required for the pathogenesis of the skin disease early after birth but is not essential for secondary skin lesion development in the adult NEMOEKO/TNFRI-deficient mice. Interestingly, apoptotic basal keratinocytes as well as upregulation of IL-1ß and (to a lesser extent) IL-1{alpha} was observed in the skin of double NEMOEKO/TNFRI-deficient mice (Supplementary Material, Fig. S1 and data not shown). This finding shows that the presence of dying basal keratinocytes and the induction of IL-1ß in NEMO-deficient epidermal keratinocytes occurs independent of the TNF-mediated response that is essential for the development of the skin disease.


Figure 4707
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  		Figure 7. TNFRI signaling is required for the development of the skin phenotype in NEMOEKO mice. (A) Analysis of skin samples from NEMOEKO/TNFRI–/– and control mice at P7. Sections were stained with hematoxylin/eosin (H/E) or with antibodies against the indicated differentiation (K14, K10, loricrin and K6) or immune cell markers (Gr-1). Nuclei are stained in blue. Scale bars: 100 µm. (B) Double NEMOEKO/TNFRI knockout mice at P9 and at 12 weeks of age.

 
To evaluate whether B and T lymphocytes are involved in the pathogenesis of the skin phenotype in NEMOEKO mice, we bred them into a RAG1-deficient background lacking B and T cells (29Go). Double NEMOEKO/RAG1-deficient mice showed a delay in the onset of the skin disease by 24–48 h, but ultimately developed skin lesions similar to NEMOEKO mice and died between P7 and P8 (data not shown). These results demonstrate that B and T cells might participate in the early initiation of the skin disease but they are not essential for the development of the skin lesions.

NEMO ablation in epidermal keratinocytes of adult mice causes inflammatory skin lesions
Deletion of NEMO in keratinocytes of NEMOEKO mice occurs already in utero and is complete at P0 (Fig. 1A) (25Go), however the inflammatory skin phenotype develops only 2–3 days after birth. It is unclear why the skin lesions do not appear at earlier stages. It is possible that contact of the skin with environmental factors after birth may trigger the disease. Alternatively, developmental processes occurring in the newborn mouse skin could be implicated in triggering the skin phenotype. If the latter were true, deletion of NEMO in epidermal keratinocytes from adult mice would not induce the skin disease. To investigate this possibility, we used the K14-Cre-ERT2 transgenic line expressing a tamoxifen-inducible fusion protein between the Cre recombinase and a mutated ligand-binding domain of the human estrogen receptor {alpha} in epidermal keratinocytes under the control of the human keratin 14 promoter (30Go). Young adult mice carrying loxP-flanked NEMO alleles and the K14-Cre-ERT2 transgene did not show significant deletion of NEMO in the skin and did not develop skin lesions (Fig. 8A and data not shown). Application of tamoxifen on the skin of adult NEMOFL/K14-Cre-ERT2 mice readily induced deletion of NEMO (Fig. 8A). At the end of the 5-day period of tamoxifen treatment, the skin of the NEMOFL/K14-Cre-ERT2 mice appeared red, hard and inflexible indicating the development of skin lesions (data not shown). Histological analysis of skin samples from these areas showed distinct pathological features including upregulation of keratin 14 and downregulation of keratin 10 and loricrin in the suprabasal layers of the epidermis, expression of keratin 6 in interfollicular epidermis, and infiltration of the dermis with large numbers of granulocytes and macrophages (Fig. 8C and D and data not shown). Furthermore, IL-1ß expression was rapidly induced in the skin of these mice after tamoxifen application (Fig. 8B). The skin lesions developed similarly in both hemizygous (nemoFL/Y) male and heterozygous female NemoFL/+ mice carrying the K14-Cre-ERT2 transgene upon induction with tamoxifen, but did not develop upon treatment of these mice with the vehicle [(dimethyl sulfoxide (DMSO)] or upon tamoxifen treatment of mice not carrying the K14-Cre-ERT2 transgene. Mice carrying NEMO-floxed alleles and the K14-Cre-ERT2 transgene spontaneously developed signs of skin lesions at the age of 4–5 months, suggesting that as the mice age the K14-Cre-ERT2 transgene becomes ‘leaky’ and induces NEMO deletion in the absence of induction with tamoxifen. These results demonstrate that deletion of NEMO in epidermal keratinocytes of adult mice causes inflammatory skin lesions similar to the phenotype observed in NEMOEKO and NEMOEHET mice, suggesting that the development of the skin disease is not related to developmental processes occurring in the skin shortly after birth.


Figure 4708
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  		Figure 8. Inducible deletion of NEMO in the epidermis of adult mice causes inflammatory skin lesions. (A) PCR analysis of DNA isolated from the skin of K14-CreERT2-positive (Cre+) or -negative (Cre–) mice treated with DMSO or tamoxifen. Deletion is detected by the presence of a 644 bp band (Del). A 446-bp band indicates the floxed allele and a 301-bp band the wild-type allele. C1 and C2 are control tail DNA samples taken from a NEMOEKO and NEMOHET mouse, respectively. (B) RNase protection assay showing expression of IL-1{alpha}, IL-1ß, IL-1RA and IL-18 in the skin of a control (Cre–) and a Cre+ mouse after treatment with tamoxifen. (C, D) Sections of skin biopsies taken 2 days after tamoxifen treatment of Cre– and Cre+ mice were immunostained for the indicated epidermal differentiation markers (K14, K10, K6, Loricrin) and with antibodies recognizing macrophages (F4/80), granulocytes (Gr-1). Nuclei are stained in red.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The role of NF-{kappa}B signaling in the epidermis remains controversial. Several studies suggest that inhibition of NF-{kappa}B activation causes increased keratinocyte proliferation and epidermal hyperplasia (11Go,12Go,14Go). Others and we have shown that inhibition of NF-{kappa}B signaling in the epidermis triggers inflammatory skin lesions, suggesting that the primary role of NF-{kappa}B in the epidermis is to regulate mechanisms that control immune homeostasis in the skin (18Go,19Go,27Go). The complex skin pathology caused by the mosaic disruption of the NEMO gene in female IP patients reflects the multifaceted role of NF-{kappa}B signaling in the regulation of skin homeostasis also in humans. Understanding the function of NF-{kappa}B in the skin is closely linked with the elucidation of the mechanisms leading to the pathogenesis of the skin disease in IP. The nature of the cell type that is responsible for the initiation of the skin lesions in IP patients has remained elusive. Furthermore, the development of inflammatory skin lesions by mosaic disruption of NEMO in these patients has been puzzling, as it is difficult to understand how inhibition of NF-{kappa}B could trigger an inflammatory response. For this reason, it was proposed that the presence of wild-type keratinocytes side by side with NEMO knockout keratinocytes might be important for the induction of skin inflammation (24Go). Using epidermis-restricted deletion of NEMO, we show here that the cell type responsible for initiation of the skin lesions is the epidermal keratinocyte. Furthermore, we show that the mosaic presence of NEMO-deficient and wild-type keratinocytes is not essential for the development of the inflammatory skin disease. The results obtained in our conditional mouse model strongly suggest that also in human IP patients the skin pathology is caused by the presence of NEMO-deficient keratinocytes in the epidermis and that the simultaneous mosaic presence of wild-type keratinocytes is not needed for disease induction.

Complete inhibition of NF-{kappa}B by NEMO deletion did not affect the normal differentiation and formation of the epidermis, demonstrating that NF-{kappa}B activation is not essential for this process. Because of the controversial role of NF-{kappa}B in regulating keratinocyte growth, we undertook a detailed analysis of the proliferation of NEMO-deficient keratinocytes. Examination of keratinocyte proliferation in vivo at E18.5 and P0 and also in vitro in primary cultures showed that NEMO-deficient keratinocytes do not proliferate more than control cells. Furthermore, the majority of NEMO knockout basal keratinocytes stained positive for PCNA, a marker upregulated during the late G1 and S phases of the cell cycle. These results indicate that NEMO-deficient keratinocytes show a delay in G1–S progression and are in agreement with a recent study showing a similar cell-cycle defect in the epidermis of mice lacking both RelA/p65 and cRel (27Go). NEMO-deficient epidermis showed increased numbers of proliferating keratinocytes during later stages at P3 and P5, but this hyperplasia was a secondary defect induced by the underlying inflammatory response. Taken together, our results demonstrate that complete inhibition of IKK-mediated NF-{kappa}B activation does not cause cell autonomous keratinocyte hyperproliferation but rather leads to a delay in cell-cycle progression.

Both in IP patients and in mice with epidermis-specific NEMO knockout, an inflammatory response seems to play a predominant role in the development of the skin lesions. In NEMOEKO mice, the inflammatory response is characterized by the expression of proinflammatory mediators and the infiltration of granulocytes, macrophages and T-cells in the skin shortly after birth. One of the earliest signs of skin inflammation in NEMOEKO mice is the upregulation of IL-1ß expression in the NEMO-deficient epidermis, detected already at P1–P2. We did not find increased IL-1ß expression in mutant epidermis at P0 or in cultured primary NEMO-deficient keratinocytes, suggesting that the upregulation of IL-1ß is not the result of a cell-intrinsic defect, but it is rather a secondary response to as-yet unidentified extrinsic factors. Although the cause of IL-1ß upregulation in the NEMO-deficient epidermis remains unclear, we hypothesized that the expression of this potent proinflammatory cytokine could be critical for the development of skin inflammation. IL-1ß signaling can induce the expression of multiple proinflammatory mediators and chemokines in fibroblasts, macrophages and endothelial cells (31Go), therefore it could be pivotal for the recruitment of immune cells into the skin of the NEMOEKO mice. Furthermore, double NEMOEKO/RAG1-deficient mice developed the skin disease with similar severity as NEMOEKO mice, showing that T or B cells are not essential for disease pathogenesis in this model. In contrast, breeding into a TNFRI-deficient background rescued the skin phenotype of NEMOEKO mice, with double NEMOEKO/TNFRI-deficient mice developing into adulthood without showing skin lesions. The results from our genetic experiments in this mouse model of IP suggest that TNFRI signaling is critical, whereas the presence of T lymphocytes is probably dispensable for the pathogenesis of the early inflammatory skin lesions in IP patients. Although double NEMOEKO/TNFRI-deficient mice were rescued from early skin disease, all such mice developed skin lesions later in life at the age of 4–6 months. The re-appearance of inflammatory skin lesions has been extensively described in adult IP patients, where the disease seems to be triggered by febrile illnesses. Our results show that TNFRI signaling is not essential for the induction of this second phase of inflammatory skin lesions in adult skin in our mouse model and suggest that the early and late lesions may be caused by different mechanisms.

Apoptosis of keratinocytes is a prominent feature of the skin disease in both human IP and in our mouse model. NEMO-deficient keratinocytes undergo massive apoptosis at late stages of the disease in NEMOEKO and NEMOEHET mice, most probably being killed by the increased expression of TNF in the skin at this stage. The killing of NEMO-deficient cells and their subsequent replacement with wild-type keratinocytes is thought to explain the healing of the skin lesions in the IP patients and in the few heterozygous NEMO-deficient mice that survive the initial phase of inflammation (23Go). Consistent with this hypothesis, we also observed that a small number of NEMOEHET mice show healing of the skin lesions after P8–P10 and survive to adulthood, whereas no NEMOEKO mouse survived further than P6. Furthermore, the NEMOEHET mice that survived into adulthood showed reduced disease severity with less involved skin areas already at the early stages of lesion development, suggesting that in these mice only a small fraction of epidermal keratinocytes were NEMO-deficient. In order to confirm whether the clearance of the epidermis from NEMO-deficient cells through apoptosis is responsible for the healing of the disease, we have tried to use immunostaining to distinguish NEMO-deficient from wild-type keratinocytes in the NEMOEHET mice at different stages, and in particular in adult mice after the disappearance of the lesions. Although we have used many commercially available and also our own anti-NEMO antibodies, we were never able to detect endogenous levels of NEMO by immunostaining of mouse tissue sections. Thus, this hypothesis remains to be formally proven. However, the fact that none of the NEMOEKO mice survived after P6 demonstrates that the presence of wild-type keratinocytes is essential for skin lesion healing, further supporting that the replacement of NEMO knockout keratinocytes with wild-type ones is essential for recovery. In addition to the large number of apoptotic cells found in the epidermis after P3, we consistently observed the presence of small numbers of apoptotic cells either in clusters scattered around hair follicles or as single cells in the basal layer of the epidermis of NEMOEKO mice in early stages from E18.5 to P2. These results suggest that some NEMO-deficient keratinocytes undergo apoptosis before the initiation of the inflammatory response. At present, we do not know the nature of the signals that induce apoptosis in NEMO-deficient keratinocytes at these early stages, however this spontaneous death of mutant keratinocytes may be implicated in the initiation of the skin lesions.

Analysis of STAT3 and JNK phosphorylation showed increased activation of both these pathways in the epidermis of NEMOEKO mice at P5, however at P0 there was no difference between mutant and control mice. STAT3 has been suggested to be specifically activated in psoriasis-like skin diseases and is proposed to provide the link for the interaction between keratinocytes and T-cells in this disease (28Go). Such a mechanism can be excluded in our model as T-cells are not required for disease pathogenesis. Therefore, the increased STAT3 activity in the epidermis of NEMOEKO mice is most probably a consequence of the inflammatory hyperproliferative disease and not an initiating factor. Increased JNK activation has been proposed to induce cell-autonomous hyperproliferation in p65/RelA-deficient keratinocytes in a TNFRI-dependent manner (11Go). We found increased JNK activity in the epidermis of NEMOEKO mice at P3 and P5, but not in earlier stages before the initiation of skin inflammation. Increased JNK activity upon TNF stimulation has been shown to induce apoptosis in NF-{kappa}B-deficient cells (11Go). In the epidermis of NEMOEKO mice at P3 and P5, we detected increased apoptosis and also increased proliferation of keratinocytes, with both events being the consequence of the underlying inflammatory response. Our findings strongly suggest that the increased JNK phosphorylation in the epidermis of NEMOEKO mice is a consequence of the inflammatory response and is implicated in mediating TNF-induced apoptosis of NEMO-deficient keratinocytes at this stage.

Deletion of NEMO in the epidermis of NEMOEKO mice is already complete before birth, however the skin lesions appear in the first few days after birth. Early postnatal appearance of the inflammatory lesions is also described for human IP patients, suggesting that disease initiation may correlate with events occurring shortly after birth. Although it seems plausible to speculate that the contact of the skin with the environment and in particular with microbial factors may be involved in disease initiation, we were thus far unable to affect lesion development by keeping the mice in animal facilities with different health status or by treating the mice with local antimicrobial agents. Alternatively, the development of the skin phenotype shortly after birth may be related to developmental processes occurring in the newborn skin. To investigate this hypothesis, we analyzed lesion formation upon inducible NEMO deletion in the epidermis of adult mice. These experiments showed that inactivation of NEMO in the adult epidermis caused an inflammatory skin phenotype that is similar to the skin lesions developing in the NEMOEKO mice. Therefore, the initiation of the inflammatory skin disease does not seem to be related to developmental processes.

The skin disease developing in mice with epidermis-specific deletion of NEMO shares similarities with the skin phenotype that develops in mice with epidermis-specific deletion of IKK2 (19Go). In both models, inhibition of IKK-mediated NF-{kappa}B activation in keratinocytes leads to TNF-mediated inflammatory skin disease, resulting in secondary epidermal hyperplasia and differentiation defects. An important difference between the two models is that apoptosis of keratinocytes is a prominent feature of the skin disease in NEMOEKO but not in IKK2EKO mice. This difference can be explained by the complete inhibition of NF-{kappa}B caused by NEMO deletion, in contrast to IKK2 deletion that allows some residual NF-{kappa}B activation presumably mediated by IKK1. The results from these two genetic models show that IKK2- and NEMO-mediated NF-{kappa}B activation in the epidermis is not required for the regulation of epidermal differentiation and growth inhibition, but is important for the maintenance of immune homeostasis in the skin.

We have taken a genetic approach using conditional gene targeting in a mouse model to investigate the cellular and molecular mechanisms that are implicated in the pathogenesis of inflammatory skin lesions in IP. Our results suggest that lack of NEMO-mediated NF-{kappa}B activation in epidermal keratinocytes is the primary defect triggering the development of skin lesions in IP. It remains to be tested whether the role of the NF-{kappa}B-deficient epidermal keratinocyte as a potent initiator of skin inflammation is also relevant for the pathogenesis of other inflammatory skin diseases in addition to IP. Furthermore, a TNF-mediated inflammatory response is crucial for disease pathogenesis in the mouse model, indicating that TNF signaling may also be important in the human disease. Identifying the cellular targets of TNF will be important to further elucidate the mechanisms leading to skin lesion development. For this task, new genetic models allowing the cell-specific manipulation of TNF signaling in combination with NEMO epidermis knockout will be required. Taken together, our results highlight the important role of NEMO-mediated NF-{kappa}B activation in the epidermis and provide new insights into the mechanisms causing the skin manifestations of IP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mice
Epidermis-specific NEMO knockout mice were generated by crossing mice carrying loxP-flanked NEMO alleles (23Go) to K14-Cre transgenic mice expressing Cre under the control of the human keratin 14 promoter (25Go), or to K14-Cre-ERT2 mice expressing a tamoxifen-inducible fusion protein between the Cre recombinase and a mutated ligand-binding domain of the human estrogen receptor {alpha} in epidermal keratinocytes under the control of the human keratin 14 promoter (30Go). TNFRI-deficient mice (32Go) (provided by K. Pfeffer) and RAG1 knockout mice (29Go) were crossed to K14-Cre/NEMOFL mice to generate double knockouts. All mice used in this study were backcrossed into the C57Bl/6 genetic background for at least five generations. Littermates lacking the Cre transgenes were used as controls in all experiments.

Western blotting and electromobility shift assay
Cytoplasmic extracts from cultured keratinocytes were prepared as described (23Go), electrophoresed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 10%) and transferred to Immobilon P membranes (Millipore). The membranes were blocked with 5% milk and probed using rabbit polyclonal antibodies against NEMO (33Go), I{kappa}B{alpha} (Santa-Cruz), mouse monoclonal antibodies against IKK1 (Imgenex), IKK2 (Imgenex), phospho-I{kappa}B{alpha} (Cell Signaling) and goat polyclonal antibody against actin (Santa-Cruz). Membranes were then incubated with secondary antibodies IgG-HRP conjugates (Amersham, Chemicon) and developed using ECL Supersignal (Pierce).

Electromobility shift assay of nuclear extracts from cultured keratinocytes was performed as described (23Go).

Tamoxifen-inducible deletion of NEMO in the epidermis of adult mice
To induce deletion of NEMO in the epidermis of adult mice, NEMOFL mice were crossed with K14Cre-ERT2 mice (34Go). Deletion was induced at 8 weeks of age by application of 100 µg of tamoxifen dissolved in DMSO, or DMSO alone as control, on a 1-cm2 patch of shaved skin for 5 consecutive days. Mice were sacrificed 2 days after the last day of treatment.

Histological analysis and immunohistochemistry
Five-micrometer thick paraffin sections were stained with hematoxylin and eosin (Sigma). Immunohistochemistry was performed on 5-µm thick paraffin or cryostat sections using polyclonal antibodies against mouse loricrin, K10, K6 (all purchased from Covance), or monoclonal antibodies against mouse K14 (Neomarkers), rat anti-mouse CD3 (Chemicon), Ly-6G (Gr-1) (BD Biosciences), F4/80 (Serotec), phospho-STAT3 (Cell Signaling), phospho-JNK (Promega), Ki67 (Dako) and PCNA (Biosource). We used secondary antibodies coupled to Alexa 488 or 594 (Molecular Probes). Sections were counterstained with ToProIII (Molecular Probes) or DAPI (Sigma) to visualize nuclei. For in vivo BrdU proliferation assays, we injected intraperitoneally pregnant females with BrdU (100 µg/g; Sigma) and dissected embryos after 3 h. Skin sections were stained with monoclonal antibodies against BrdU (Chemicon). When required, sections were processed in an antigen retriever apparatus (Pickcell Lab). Terminal deoxynucleotidyltransferase-mediated TUNEL staining of paraffin skin sections was performed according to the manufacturer's instructions (Apoptosis Detection System, Promega). Fluorescent stainings were analyzed using a Leica TCS NT upright confocal laser scanning microscope or a Leica DM RHC microscope.

Keratinocyte proliferation and TNF-induced apoptosis
Primary epidermal keratinocytes were isolated from the skin of newborn mice and cultured in minimal calcium medium (0.05 mM CaCl2; low calcium medium) supplemented as described (35Go). For measurement of proliferation, keratinocytes were plated in two-well permanox chamber slides and were grown to about 50% confluence. Cells were labeled with BrdU (1:1000 dilution BrdU labeling and detection kit II; Roche) for 3, 5 and 15 h at 37°C. BrdU incorporation was detected by immunofluorescence using anti-BrdU mouse monoclonal antibody (Roche) followed by secondary antibody coupled to Alexa 488 (Molecular Probes). For the detection of TNF-induced apoptosis, primary keratinocytes were grown in 96-well plates. Cells were treated with 20 ng/ml murine TNF for 6 h and cell viability was determined by the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl-)2,5-diphenyltetrazolium bromide] (Sigma) assay. Ten microliters of an MTT solution (1.25 mg/ml) was added to each well. After 2 h incubation, cells were lysed by adding 150 µl of isopropyl-HCl solution (600 µl of HCl/100 ml isopropanol) for 15 min. Absorbance was determined with an automated ELISA reader at 570 nm.

Gene expression analysis
Total RNA from primary keratinocytes, epidermis or total skin was isolated using Trizol (Invitrogen) and analyzed by real-time polymerase chain reaction (PCR) with SyBr Green (Finnzymes) or by RNase protection assay, respectively. For RT–PCR primers used for I{kappa}B{alpha} are 5'-CGAAGAGAAGCCGCTGACCAT-3' and 5'-CTCATCCTCGCTCTCGGGTAG-3' and for TNF are 5'-CACGCTCTTCTGTCTACTGAACTTCG-3' and 5'-GGCTGGGTAGAGAATGGATGAACACC-3'. All values were normalized to the level of ubiquitin mRNA. For RNase protection assay, total RNA was hybridized with RNA probes using a Riboquant multi-probe RPA system (BD Bioscience), following the manufacturer's instruction.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Emerald Perlas for technical assistance. This work was supported by grant QLG1-CT-1999-00202 from the European Union to M.P. ‘Funding to pay the Open Access publication charges for this article was provided by EMBL.’

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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