Skin lesion development in a mouse model of incontinentia pigmenti

Skin lesion development in a mouse model ofincontinentia pigmenti is triggered by NEMOdeficiency in epidermal keratinocytes andrequires 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 fur Molekulare und Zellulare Sportmedizin, Deutsche

Sporthochschule Koln, IG I, Carl-Diem-Weg 6, D-50933 Koln, Germany and 5Institut de Genetique et de Biologie

Moleculaire et Cellulaire (IGBMC), CNRS, INSERM, ULP, and Institut Clinique de la Souris (ICS), BP 10142-67404,

ILLKIRCH, C.U. de Strasbourg, France

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

NF-kB essential modulator (NEMO), the regulatory subunit of the IkB kinase, is essential for NF-kB activation.Mutations disrupting the X-linked NEMO gene cause incontinentia pigmenti (IP), a human genetic diseasecharacterized by male embryonic lethality and by a complex pathology affecting primarily the skin in hetero-zygous females. The cellular and molecular mechanisms leading to skin lesion pathogenesis in IP patientsremain elusive. Here we used epidermis-specific deletion of NEMO in mice to investigate the mechanismscausing the skin pathology in IP. NEMO deletion completely inhibited NF-kB activation and sensitized kera-tinocytes 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 inflammatoryskin lesions, identifying the NEMO-deficient keratinocyte as the initiating cell type that triggers the skinpathology in IP. Furthermore, genetic ablation of tumor necrosis factor receptor 1 (TNFRI) rescuedthe skin phenotype demonstrating that TNF signaling is essential for skin lesion pathogenesis in IP. Theseresults identify the NEMO-deficient keratinocyte as a potent initiator of skin inflammation and providenovel insights into the mechanism leading to the pathogenesis of IP.

INTRODUCTION

The NF-kB transcription factors control the expression ofmany genes with important functions in inflammation,immune responses, cell proliferation, survival and apoptosis(1,2). In resting cells, NF-kB dimers are kept inactive by

association with inhibitory proteins belonging to the IkBfamily. NF-kB activation is induced by the IkB kinase(IKK) complex consisting of the IKK1(IKKa) andIKK2(IKKb) catalytic subunits and the NF-kB essentialmodulator (NEMO)/IKKg regulatory protein (3–5). Uponactivation by a variety of stimuli, including proinflammatory

# The Author 2006. Published by Oxford University Press. All rights reserved.The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version ofthis article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as theoriginal place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as aderivative work this must be clearly indicated. For commercial re-use, please contact: [email protected]

*To whom correspondence should be addressed. Tel: þ39 0690091222/90091271; Fax: þ39 0690091272; Email: [email protected]

Human Molecular Genetics, 2006, Vol. 15, No. 4 531–542doi:10.1093/hmg/ddi470Advance Access published on January 6, 2006

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cytokines such as tumor necrosis factor (TNF) and interleukin-1(IL-1) and bacterial lipopolysaccharide (LPS), the IKK phos-phorylates IkB proteins at specific serine residues targetingthem for polyubiquitination and proteasome-mediated degra-dation, thus releasing NF-kB, which then accumulates in thenucleus and activates its target genes (6,7). IKK2 andNEMO are essential for NF-kB activation by proinflammatorysignals via the classical activation pathway, whereas IKK1 isrequired for the alternative NF-kB activation pathwaycontrolling p100 processing (8).

Several studies have suggested that the NF-kB pathway isinvolved in the regulation of epidermal homeostasis (9,10).Inhibition of NF-kB activation in epidermal keratinocyteseither by transgenic overexpression of a mutant non-degradable IkBa or by knockout of the p65 NF-kB subunitlead to epidermal hyperplasia, suggesting a growth-regulatoryrole for NF-kB in keratinocytes (10–15). Furthermore, Dajeeet al. (13) showed that inhibition of NF-kB in combinationwith expression of oncogenic Ras in human keratinocytestransplanted on the skin of severe combined immuno-deficiency (SCID) mice caused the development of invasivetumors resembling squamous cell carcinoma. In a differentstudy, overexpression of IkB in the epidermis of transgenicmice caused an inflammatory hyperproliferative epidermalphenotype leading to the development of squamous cell carci-nomas (16,17). In this case, however, blockade of TNF signal-ing inhibited both epidermal hyperplasia and tumor formation,suggesting that the development of squamous cell carcinomasin this model depends on a TNF-induced inflammatoryresponse (18). Mice with epidermis-specific deletion ofIKK2 display an inflammatory skin phenotype characterizedby expression of proinflammatory cytokines and chemokines,infiltration of immune cells, epidermal hyperplasia andderegulated expression of epidermal differentiation markers(19). Also in this case, genetic ablation of TNF signalingrescued the skin phenotype demonstrating that the epidermalhyperplasia is a secondary effect of the inflammatory response.

In humans, mutations disrupting the X-linked gene encod-ing NEMO cause incontinentia pigmenti (IP), a geneticdisease characterized by male embryonic lethality and by thedevelopment of multiple cutaneous, neurological and ophthal-mological abnormalities in heterozygous females (20). Theskin lesions of IP patients develop in four successive,sometimes overlapping, characteristic stages, starting withthe appearance of an inflammatory vesicular rash shortlyafter birth (21,22). This initial inflammatory phase is followedby the formation of verrucous lesions during the first weeks oflife, which disappear shortly as the disease moves into thethird stage marked by the appearance of skin areas displayinghyperpigmentation. During the fourth stage and usuallyaround puberty, the hyperpigmented areas disappear leavingbehind patches of atrophic hypopigmented skin (21,22).Targeted disruption of the gene encoding NEMO causesmale embryonic lethality and the appearance of transientinflammatory skin lesions in heterozygous females, a pheno-type that shares many of the characteristics of IP in humans(23,24). Although many of the heterozygous NEMO knockoutmice die at the peak of the disease, some of them recovershowing healing of the skin lesions and survive to adulthooddisplaying an apparently normal skin (23,24). The mosaic

expression of NEMO due to random X-chromosome inacti-vation in heterozygous females is thought to be critical forthe development of the skin phenotype in both the humanpatients and in the mouse model. The identification ofNEMO mutations as the cause of human IP, in combinationwith the results obtained from the NEMO knockout mouse,leads to the proposal of a new model for the pathogenesisof this disease. According to this model, the cell autonomoushyperproliferation of NEMO knockout keratinocytes, possiblyin combination with the spontaneous necrotic cell death ofsome NEMO-deficient keratinocytes, triggers the expressionof proinflammatory mediators by the neighboring wild-typekeratinocytes resulting in the development of the skin lesions.During the inflammatory phase of the disease, proinflam-matory cytokines such as TNF released by the invadingimmune cells are thought to kill NEMO-deficient keratino-cytes, which are then replaced by healthy wild-type keratino-cytes resulting in the healing of skin lesions.

Although these studies provided important information forthe understanding of the pathogenesis of the skin lesions inIP, several questions regarding the mechanism triggering thisdisease remain unresolved. Both IP patients and heterozygousNEMO-deficient mice show mosaic presence of NEMO-deficient and wild-type cells in many different cell types. Itis, therefore, not clear whether the skin lesions are triggeredby the presence of NEMO-deficient keratinocytes in theepidermis or by an abnormal inflammatory response causedby 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 theinitiation and progression of the skin lesions. Furthermore, itis not clear why the skin lesions appear shortly after birthboth in humans and in the mouse model. This early postnatalinitiation of the phenotype may be caused by the exposure ofthe skin to environmental stimuli, or alternatively, develop-mental processes occurring in the skin at this stage may beinvolved.

We have used Cre/loxP-mediated gene targeting in miceto investigate in vivo the function of NEMO in the epidermisand to address experimentally the cellular and molecularmechanisms that are involved in the pathogenesis of theskin lesions in IP. We show here that NEMO deletion inepidermal keratinocytes blocks NF-kB activation in responseto proinflammatory signals, but it does not cause cellautonomous hyperproliferation or differentiation defects. Wealso show that epidermis-restricted NEMO deletion issufficient to trigger inflammatory skin lesions and that themosaic presence of NEMO-deficient and wild-type keratino-cytes in the epidermis is not necessary for disease develop-ment. Furthermore, we demonstrate that an inflammatoryresponse requiring TNF receptor I signaling is essentialfor the pathogenesis of the skin lesions, suggesting thatTNF-mediated inflammation is an obligatory component ofthe disease. Finally, inducible deletion of NEMO in theepidermis of adult mice was sufficient to trigger the develop-ment of skin lesions, suggesting that disease initiation is notrelated to developmental processes occurring shortly afterbirth but is a direct and immediate consequence of theinhibition of NEMO-dependent NF-kB activation in epidermalkeratinocytes.

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RESULTS

NEMO-deficient epidermal keratinocytes do not activateNF-kB and are highly sensitive to TNF-induced death

To investigate the function of NEMO in the epidermis, wegenerated mice with epidermal keratinocyte-restrictedNEMO deletion, by crossing mice expressing Cre recombi-nase under the control of the human keratin 14 promoter(K14-Cre) (25) with mice carrying loxP-flanked (floxed)NEMO alleles (23). In contrast to control cells, primaryNEMO-deficient keratinocytes failed to activate NF-kB uponstimulation with IL-1, TNF or 12-O-Tetradecanoylphorbol13-acetate (TPA) (Fig. 1B and data not shown). Furthermore,NEMO knockout keratinocytes showed impaired induction ofNF-kB-dependent genes upon stimulation with TNF (Fig. 1Cand data not shown). As NF-kB-dependent gene expression isknown to be important for the protection of cells from TNF-induced cytotoxicity, we investigated the sensitivity ofNEMO-deficient keratinocytes to TNF. In accordance to thecomplete lack of NF-kB activation, NEMO-deficient keratino-cytes were extremely sensitive to TNF-induced cell death(Fig. 1D). These results show that NEMO knockout causescomplete inhibition of NF-kB activation in epidermal kerati-nocytes and renders them sensitive to TNF-induced death.

NEMO deletion restricted to keratinocytescauses skin lesions

For the generation of epidermis-specific NEMO knockouts, weused a breeding scheme where K14Cre transgenic males werecrossed with females homozygous for the NEMO-floxedlocus. As the NEMO gene is X-linked, all male progenywere hemizygous (FL/Y) and all female progeny were hetero-zygous (FL/þ) for the NEMO-floxed allele. Male progenycarrying the K14Cre transgene showed complete absence ofNEMO in all epidermal keratinocytes (NEMOEKO), asshown by western blot analysis of extracts from isolatedepidermis or cultured primary keratinocytes (Fig. 1A). Incontrast, the epidermis of heterozygous females expressingK14Cre (NEMOEHET) is expected to contain both NEMO-deficient and NEMO-expressing cells in a mosaic patterndue to random X-chromosome inactivation. Using thisgenetic approach, we were able to investigate the skin pheno-type of mice showing either complete or mosaic deletion ofNEMO in epidermal keratinocytes.

NEMOEKO and NEMOEHET mice were born at the expectedratio and were macroscopically indistinguishable from theirK14Cre-negative control littermates until postnatal day 2. Atthis stage, NEMOEKO pups showed a characteristic lack ofskin pigmentation, which in NEMOEHET mice appeared onlyin patches presumably at areas of the skin containingNEMO-deficient keratinocytes. The lack of pigmentationbecame more obvious at P3 and P4, and at P5 bothNEMOEKO and NEMOEHET mice displayed a skin phenotypecharacterized by the presence of red and inflamed areas withscaling, especially around the neck (Fig. 2A). TheNEMOEKO mice rapidly developed severe skin lesions andfailed to survive further than postnatal day 6. NEMOEHET

mice developed skin lesions in a patchy fashion and most ofthem died between P7 and P10. Some NEMOEHET mice

showing mild skin phenotype with less and smaller affectedskin areas, presumably due to the presence of only a small per-centage of NEMO-deficient keratinocytes in their epidermis,survived to adulthood and showed healing of the skinlesions similar to heterozygous NEMO knockout mice (23).These results show that deletion of NEMO in the epidermis,either in a complete or in a mosaic fashion, causes skinlesions, suggesting that the skin manifestations of IP are theresult of NEMO deficiency in epidermal keratinocytes.

Inflammatory skin disease in mice withepidermis-restricted NEMO deletion

To characterize the skin lesions that develop in NEMOEKO andNEMOEHET mice, we performed detailed immunohistologicalanalysis of skin sections from these animals. Histologicalexamination of skin samples taken from mice at P0 and P3did not show epidermal hyperplasia or signs of skin inflam-mation, however at P5 the skin of both NEMOEKO andNEMOEHET mice displayed thickening of the epidermis,hyperkeratosis, loss of the granular layer and the presence of

Figure 1. Deletion of NEMO in keratinocytes impairs NF-kB activation andconfers sensitivity to TNF-induced cell death. (A) Western blot analysis ofNEMO, IKK1, IKK2 and actin expression in protein extracts from P0 epider-mis and from primary keratinocytes of control and NEMOEKO mice. Extractsfrom wild-type and NEMO-deficient mouse embryonic fibroblasts (MEFs) areused as controls. (B) Primary keratinocytes isolated from wild-type andNEMOEKO mice were stimulated with IL-1b (10 ng/ml) for the indicatedtime points. Phosphorylation and degradation of IkBa was assayed bywestern blot analysis of cytoplasmic extracts, and NF-kB DNA-bindingactivity was measured by gel mobility shift analysis of nuclear extracts. (C)Real-time PCR analysis of IkBa expression in primary keratinocytes isolatedfrom control and NEMOEKO mice after treatment with TNF (20 ng/ml) for theindicated time points. Results are shown as the mean and SE of triplicatesamples. (D) Sensitivity to TNF-induced cytotoxicity. Primary keratinocytesisolated from control and NEMOEKO mice were left untreated or stimulatedwith TNF (20 ng/ml) for 6 h.

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infiltrating mononuclear and polymorphonuclear cells in thedermis (Fig. 2B and data not shown). In addition, a smallnumber of dyskeratotic basal keratinocytes were observed inthe NEMOEKO but not in control skin. Examination of skinsamples from mice at P0 using electron microscopy did notreveal ultrastructural abnormalities in cell–cell contacts, cyto-skeletal organization and cell organelles in the NEMOEKO

skin, but readily detected the presence of a small number ofdead basal keratinocytes in knockout but not in controlepidermis.

Immunohistochemical analysis using antibodies againstdifferent epidermal differentiation markers confirmed thatthe skin of NEMOEKO and NEMOEHET mice at P0 shows anormal morphology with keratin 14 expressed in the basallayer, keratin 10 in the suprabasal layers and loricrin and filag-grin in the terminally differentiated upper layers of the epider-mis (Fig. 3 and data not shown). These results demonstratethat NEMO-mediated NF-kB activation is not required forthe normal formation and differentiation of the epidermis.However, as early as 3 days after birth the epidermis of bothNEMOEKO and NEMOEHET mice showed upregulation ofkeratin 14 in the suprabasal layers, which was even morepronounced at P5 and was accompanied by the loss ofkeratin 10 and loricrin expression (Fig. 3). Furthermore,the skin of NEMOEKO and NEMOEHET mice showedupregulation of keratin 6, a marker of inflamed epidermis, at

P5 (Fig. 4B). Thus, the skin lesions developing after P3 inboth NEMOEKO and NEMOEHET mice are characterized byabnormal expression of epidermal differentiation markers.

The presence of infiltrating inflammatory cells in the dermisand the upregulation of keratin 6 in the epidermis ofNEMOEKO and NEMOEHET skin samples suggested that aninflammatory response may be implicated in the developmentof skin lesions in these mice. To further investigate the poten-tial contribution of inflammatory cells to the pathogenesis ofthe skin lesions in NEMOEKO and NEMOEHET mice, westained skin sections with antibodies for T lymphocytes(CD3), granulocytes (Gr-1) and macrophages (F4/80). Theseexperiments showed large numbers of granulocytes and afew T lymphocytes infiltrating the dermis of NEMOEKO andNEMOEHET mice after P3, whereas the number of macro-phages was also increased at this stage (Fig. 4A). Furthermore,analysis of proinflammatory cytokine expression in theskin of these mice showed upregulation of TNF, IL-1aand IL-1b at P3, P4 and P5 (Fig. 4D and SupplementaryMaterial, Fig. S1). IL-1b and to a lesser extent IL-1a wereupregulated as early as P2 in the NEMO-deficient epidermis(Fig. 4C).

NEMO-deficient keratinocytes do not show spontaneoushyperproliferation in vitro or in vivo

Inhibition of NF-kB activation in epidermal keratinocytes byoverexpression of a dominant negative mutant IkBa or knock-out of p65/RelA has been reported to cause cell autonomoushyperproliferation of epidermal keratinocytes (11,12). On thebasis of the assumption that inhibition of NF-kB in keratino-cytes results in impaired growth arrest and increased prolifer-ation, it has been hypothesized that the skin phenotype in theNEMO heterozygous mice and in human IP patients istriggered by the hyperproliferation of NEMO-deficient kerati-nocytes (23,24). To test experimentally this hypothesis, wemeasured the proliferation of NEMO-deficient keratinocytesin vivo and in vitro.

Measurement of bromodeoxyuridine (BrdU) incorporationrevealed that NEMO-deficient keratinocytes do not showhyperproliferation in culture (Fig. 5A). To investigatewhether NEMO-deficient keratinocytes show increased pro-liferation in vivo, we used immunostaining with antibodiesagainst Ki67 and proliferating cell nuclear antigen (PCNA),two markers exposed in proliferating cells (26), and alsomeasured BrdU incorporation. Staining for Ki67 showed thatNEMO-deficient keratinocytes do not display increased pro-liferation at P0 (Fig. 5D), suggesting that also in vivo the del-etion of NEMO does not lead to spontaneous keratinocytehyperproliferation. At P5, we detected increased numbers ofKi67-positive keratinocytes in NEMOEKO skin comparedwith controls (Fig. 5D). However, the fact that NEMO-deficient keratinocytes show increased proliferation onlyafter P3 suggests that this hyperproliferation is secondary tothe inflammatory response developing in the skin. Analysisof BrdU incorporation at E18.5 revealed reduced numbers ofBrdU-positive basal keratinocytes in NEMOEKO skin com-pared with control embryos, showing that the NEMO-deficientepidermis contains less basal keratinocytes in the S phase of thecell cycle (Fig. 5B and C). Staining with PCNA revealed a

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


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surprising picture with the majority of basal cells in theNEMOEKO epidermis staining positive, in contrast to controlskin that contained less PCNA-positive basal keratinocytes(Fig. 5B and C). Taken together, these results suggest thatbasal keratinocytes in the skin of NEMOEKO mice enter thecell cycle but delay to progress from G1 to the S phase result-ing in less proliferation than control keratinocytes. Thisfinding is consistent with a similar cell-cycle defect observedin epidermal keratinocytes from rel-a2/2c-rel2/2tnf2/2 mice,which also showed a delay in G1 progression (27). Therefore,inhibition of NF-kB activation in epidermal keratinocyteseither at the level of the IKK by NEMO knockout or by del-etion of both p65/RelA and c-Rel does not result in cell auton-omous hyperproliferation, but on the contrary it causes a delayin G1 transition and reduced proliferation.

Increased apoptosis in the epidermis of NEMOEKO mice

To investigate whether NEMO-deficient keratinocytes showincreased apoptosis in vivo, we performed dUTP nick endlabeling (TUNEL) staining on skin samples derived frommice at various stages between E18.5 and P5. This analysisrevealed the presence of small numbers of apoptotic basalkeratinocytes in the epidermis of NEMOEKO mice already atE18.5 and P0, whereas in control mice we only detectedoccasional TUNEL-positive cells in the suprabasal layers(Fig. 5E). This finding is consistent with our electronmicroscopy examination results showing the presence ofdead basal keratinocytes in NEMOEKO but not in control epi-dermis at P0 (data not shown). TUNEL-positive cell clusterswere also occasionally found around hair follicles in theskin 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 inNEMOEKO and to a lesser extent in NEMOEHET mice(Fig. 5E and data not shown). The increased apoptotic deathof NEMO knockout keratinocytes at this late stage of thedisease is presumably caused by the increased expression ofTNF from the infiltrating immune cells.

Activation of STAT3 and JNK in the epidermis ofNEMOEKO mice

Activation of STAT3 in epidermal keratinocytes has beenshown to correlate with skin inflammation in human psoriaticskin, and expression of activated STAT3 in the epidermis oftransgenic mice caused a psoriasis-like phenotype (28).Toinvestigate whether STAT3 is activated in the epidermis ofNEMOEKO mice, we performed immunostaining with an anti-body specific for phosphorylated STAT3. These experimentsrevealed increased phosphorylation of STAT3 in the epidermisof NEMOEKO mice at P5 but not at P0 (Fig. 6). A recent studysuggested that in p65/RelA-deficient keratinocytes, TNFRIsignaling leads to cell autonomous hyperproliferation byinducing increased activation of JNK (11). We, therefore,investigated JNK activity in the epidermis of NEMOEKO

mice and in cultured NEMO-deficient keratinocytes usingimmunostaining with an antibody specific for phosphorylatedJNK. We could not detect differences in JNK activationbetween wild-type and mutant epidermis at P0, however atP5 we observed increased JNK phosphorylation in epidermalcells in NEMOEKO skin (Fig. 6). The increased activation ofJNK and STAT3 is only detected after P3, suggesting that itis a secondary event induced by the expression of proinflam-matory mediators in the skin at this stage.

Figure 3. Differentiation marker expression in mice with epidermis-specific deletion of NEMO. Skin sections from NEMOEKO, NEMOEHET and control mice atP0, P3 and P5 were immunostained for epidermal differentiation markers. Keratin14 (K14) is shown in green, Keratin 10 (K10) is shown in red and loricrin isshown in green. Blue staining shows nuclei. Scale bars: 100 mm.


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TNF receptor I signaling is essential but B and Tlymphocytes are dispensable for the development ofskin inflammation in mice with epidermis-restrictedNEMO deletion

Gene expression analysis revealed upregulation of TNF andIL-1 in the skin of NEMOEKO and NEMOEHET mice,suggesting that these potent proinflammatory cytokines playa role in the pathogenesis of the inflammatory skin disease.To investigate the function of TNF in the development ofthe skin lesions in epidermis-specific NEMO knockout mice,we bred these mice into a TNFRI-deficient background.Remarkably, elimination of TNFRI rescued the inflammatoryskin phenotype in both NEMOEKO and NEMOEHET mice.Double NEMOEKO/TNFRI knockout mice often displayedtransient mild flaking in skin patches at the age of P9–P10,

which disappeared shortly and the mice developed intoadulthood without showing skin lesions (Fig. 7B). Histologicalexamination of skin samples from NEMOEKO/TNFRI knock-out mice at P7 revealed a normal picture with no signs ofskin inflammation (Fig. 7A). These results demonstrate thatTNF signaling through TNF receptor I is essential for thedevelopment of skin inflammation in this model and suggestthat a similar mechanism may be involved in the pathogenesisof skin lesions in human IP patients. Later in life and usuallybetween 4 and 6 months of age, most of the NEMOEKO/TNFRI-deficient mice developed skin lesions appearing asflaky, hairless ulcerated plaques particularly around the neckand in areas where the skin is subjected to mechanical

Figure 4. Skin inflammation in mice with epidermis-specific deletion ofNEMO. (A) Sections of NEMOEKO, NEMOEHET and control skins at P0, P3and 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 mm. (B) Staining of skin section from NEMOEKO and controlmice at P5 with antibodies against keratin 6 (green). Nuclei are stained inblue. Scale bar: 40 mm. (C) RNase protection assay showing expression ofIL-1a, IL-1b, interleukin-1 receptor antagonist (IL-1RA) and interleukin 18/interferon-gamma-inducing factor (IL-18/IGIF) in the epidermis of two differ-ent NEMOEKO (ko) and two control (ct) mice at P0 and at P2. (D) Real-timePCR analysis of TNF expression in total skin isolated from two control andtwo NEMOEKO mice at P0, P3 and P4, respectively. Results are shown asthe percentage of the mean relative to ubiquitin and error bars indicate SEof triplicate samples.

Figure 5. In vitro and in vivo analyses of keratinocyte proliferation andapoptosis. (A) Primary keratinocytes from NEMOEKO and control micewere labeled with BrdU for the indicated times. BrdU-positive cells werecounted from four different fields (�500 cells/field). Results are shown asthe percentage of BrdU-positive cells+ SEM. (B) Quantitation of BrdUþand PCNAþ basal keratinocytes from NEMOEKO and control mice (n ¼ 3)at E18.5. At least 200 basal cells were counted per mouse. Resultsare presented as percentage of positive cells+ SEM. (C) Immunohisto-chemical staining of control and NEMOEKO E18.5 skin sections for PCNAexpression and BrdU incorporation (red). Nuclei are stained in blue.Scale bars: 100 mm. (D) Analysis of cell proliferation by staining forKi67 (red) in the skin of NEMOEKO and control mice at P0, P3 and P5.Nuclei are stained in blue. Scale bar: 100 mm. (E) TUNEL staining for thedetection of apoptotic cells (green) on skin sections from control andNEMOEKO mice at E18.5, P0, P3 and P5. Nuclei are stained in blue. Scalebar: 100 mm.


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stress. Therefore, TNF signaling is required for the pathogen-esis of the skin disease early after birth but is not essential forsecondary skin lesion development in the adult NEMOEKO/TNFRI-deficient mice. Interestingly, apoptotic basal keratino-cytes as well as upregulation of IL-1b and (to a lesser extent)IL-1a was observed in the skin of double NEMOEKO/TNFRI-deficient mice (Supplementary Material, Fig. S1 and data notshown). This finding shows that the presence of dying basalkeratinocytes and the induction of IL-1b in NEMO-deficientepidermal keratinocytes occurs independent of the TNF-mediated response that is essential for the development ofthe skin disease.

To evaluate whether B and T lymphocytes are involved inthe pathogenesis of the skin phenotype in NEMOEKO mice,we bred them into a RAG1-deficient background lacking Band T cells (29). Double NEMOEKO/RAG1-deficient miceshowed 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). Theseresults demonstrate that B and T cells might participate inthe early initiation of the skin disease but they are not essentialfor the development of the skin lesions.

NEMO ablation in epidermal keratinocytes of adult micecauses inflammatory skin lesions

Deletion of NEMO in keratinocytes of NEMOEKO mice occursalready in utero and is complete at P0 (Fig. 1A) (25), howeverthe inflammatory skin phenotype develops only 2–3 days afterbirth. It is unclear why the skin lesions do not appear at earlierstages. It is possible that contact of the skin with environ-mental factors after birth may trigger the disease. Alterna-tively, developmental processes occurring in the newbornmouse skin could be implicated in triggering the skin pheno-type. If the latter were true, deletion of NEMO in epidermalkeratinocytes from adult mice would not induce the skindisease. To investigate this possibility, we used the K14-Cre-ERT2 transgenic line expressing a tamoxifen-induciblefusion protein between the Cre recombinase and a mutatedligand-binding domain of the human estrogen receptor a inepidermal keratinocytes under the control of the humankeratin 14 promoter (30). Young adult mice carrying loxP-flanked NEMO alleles and the K14-Cre-ERT2 transgenedid not show significant deletion of NEMO in the skin anddid not develop skin lesions (Fig. 8A and data not shown).

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

Figure 7. TNFRI signaling is required for the development of the skin pheno-type in NEMOEKO mice. (A) Analysis of skin samples from NEMOEKO/TNFRI2/2 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 inblue. Scale bars: 100 mm. (B) Double NEMOEKO/TNFRI knockout mice atP9 and at 12 weeks of age.


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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 skinof the NEMOFL/K14-Cre-ERT2 mice appeared red, hard andinflexible indicating the development of skin lesions (datanot shown). Histological analysis of skin samples from theseareas showed distinct pathological features including upregu-lation of keratin 14 and downregulation of keratin 10 andloricrin in the suprabasal layers of the epidermis, expressionof keratin 6 in interfollicular epidermis, and infiltration ofthe dermis with large numbers of granulocytes and macro-phages (Fig. 8C and D and data not shown). Furthermore,IL-1b expression was rapidly induced in the skin of thesemice after tamoxifen application (Fig. 8B). The skin lesions

developed similarly in both hemizygous (nemoFL/Y) maleand heterozygous female NemoFL/þ mice carrying theK14-Cre-ERT2 transgene upon induction with tamoxifen, butdid not develop upon treatment of these mice with thevehicle [(dimethyl sulfoxide (DMSO)] or upon tamoxifentreatment 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 atthe age of 4–5 months, suggesting that as the mice age theK14-Cre-ERT2 transgene becomes ‘leaky’ and induces NEMOdeletion in the absence of induction with tamoxifen. Theseresults demonstrate that deletion ofNEMO in epidermal keratino-cytes of adult mice causes inflammatory skin lesions similar to thephenotype observed in NEMOEKO and NEMOEHET mice, sug-gesting that the development of the skin disease is not related todevelopmental processes occurring in the skin shortly after birth.

DISCUSSION

The role of NF-kB signaling in the epidermis remains contro-versial. Several studies suggest that inhibition of NF-kBactivation causes increased keratinocyte proliferation andepidermal hyperplasia (11,12,14). Others and we haveshown that inhibition of NF-kB signaling in the epidermis trig-gers inflammatory skin lesions, suggesting that the primaryrole of NF-kB in the epidermis is to regulate mechanismsthat control immune homeostasis in the skin (18,19,27). Thecomplex skin pathology caused by the mosaic disruption ofthe NEMO gene in female IP patients reflects the multifacetedrole of NF-kB signaling in the regulation of skin homeostasisalso in humans. Understanding the function of NF-kB in theskin is closely linked with the elucidation of the mechanismsleading to the pathogenesis of the skin disease in IP. Thenature of the cell type that is responsible for the initiation ofthe skin lesions in IP patients has remained elusive. Further-more, the development of inflammatory skin lesions bymosaic disruption of NEMO in these patients has beenpuzzling, as it is difficult to understand how inhibition ofNF-kB could trigger an inflammatory response. For thisreason, it was proposed that the presence of wild-type keratino-cytes side by side with NEMO knockout keratinocytes mightbe important for the induction of skin inflammation (24).Using epidermis-restricted deletion of NEMO, we show herethat the cell type responsible for initiation of the skin lesionsis the epidermal keratinocyte. Furthermore, we show that themosaic presence of NEMO-deficient and wild-type keratino-cytes is not essential for the development of the inflammatoryskin disease. The results obtained in our conditional mousemodel strongly suggest that also in human IP patients theskin pathology is caused by the presence of NEMO-deficientkeratinocytes in the epidermis and that the simultaneousmosaic presence of wild-type keratinocytes is not needed fordisease induction.

Complete inhibition of NF-kB by NEMO deletion did notaffect the normal differentiation and formation of the epider-mis, demonstrating that NF-kB activation is not essential forthis process. Because of the controversial role of NF-kB inregulating keratinocyte growth, we undertook a detailed analy-sis of the proliferation of NEMO-deficient keratinocytes.

Figure 8. Inducible deletion of NEMO in the epidermis of adult mice causesinflammatory skin lesions. (A) PCR analysis of DNA isolated from the skin ofK14-CreERT2-positive (Creþ) or -negative (Cre2) mice treated with DMSOor tamoxifen. Deletion is detected by the presence of a 644 bp band (Del). A446-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 andNEMOHET mouse, respectively. (B) RNase protection assay showingexpression of IL-1a, IL-1b, IL-1RA and IL-18 in the skin of a control(Cre2) and a Creþ mouse after treatment with tamoxifen. (C, D) Sectionsof skin biopsies taken 2 days after tamoxifen treatment of Cre2 and Creþmice were immunostained for the indicated epidermal differentiationmarkers (K14, K10, K6, Loricrin) and with antibodies recognizing macro-phages (F4/80), granulocytes (Gr-1). Nuclei are stained in red.


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Examination of keratinocyte proliferation in vivo at E18.5 andP0 and also in vitro in primary cultures showed that NEMO-deficient keratinocytes do not proliferate more than controlcells. Furthermore, the majority of NEMO knockout basalkeratinocytes stained positive for PCNA, a marker upregulatedduring the late G1 and S phases of the cell cycle. These resultsindicate that NEMO-deficient keratinocytes show a delay inG1–S progression and are in agreement with a recent studyshowing a similar cell-cycle defect in the epidermis of micelacking both RelA/p65 and cRel (27). NEMO-deficient epider-mis showed increased numbers of proliferating keratinocytesduring later stages at P3 and P5, but this hyperplasia was asecondary defect induced by the underlying inflammatoryresponse. Taken together, our results demonstrate thatcomplete inhibition of IKK-mediated NF-kB activation doesnot cause cell autonomous keratinocyte hyperproliferationbut rather leads to a delay in cell-cycle progression.

Both in IP patients and in mice with epidermis-specificNEMO knockout, an inflammatory response seems to play apredominant role in the development of the skin lesions. InNEMOEKO mice, the inflammatory response is characterizedby the expression of proinflammatory mediators and the infil-tration of granulocytes, macrophages and T-cells in the skinshortly after birth. One of the earliest signs of skin inflam-mation in NEMOEKO mice is the upregulation of IL-1bexpression in the NEMO-deficient epidermis, detectedalready at P1–P2. We did not find increased IL-1b expressionin mutant epidermis at P0 or in cultured primary NEMO-deficient keratinocytes, suggesting that the upregulation ofIL-1b is not the result of a cell-intrinsic defect, but it israther a secondary response to as-yet unidentified extrinsicfactors. Although the cause of IL-1b upregulation in theNEMO-deficient epidermis remains unclear, we hypothesizedthat the expression of this potent proinflammatory cytokinecould be critical for the development of skin inflammation.IL-1b signaling can induce the expression of multipleproinflammatory mediators and chemokines in fibroblasts,macrophages and endothelial cells (31), therefore it could bepivotal for the recruitment of immune cells into the skin ofthe NEMOEKO mice. Furthermore, double NEMOEKO/RAG1-deficient mice developed the skin disease withsimilar severity as NEMOEKO mice, showing that T or Bcells are not essential for disease pathogenesis in this model.In contrast, breeding into a TNFRI-deficient backgroundrescued the skin phenotype of NEMOEKO mice, with doubleNEMOEKO/TNFRI-deficient mice developing into adulthoodwithout showing skin lesions. The results from our geneticexperiments in this mouse model of IP suggest that TNFRIsignaling is critical, whereas the presence of T lymphocytesis probably dispensable for the pathogenesis of the earlyinflammatory skin lesions in IP patients. Although doubleNEMOEKO/TNFRI-deficient mice were rescued from earlyskin disease, all such mice developed skin lesions later inlife at the age of 4–6 months. The re-appearance of inflam-matory skin lesions has been extensively described in adult IPpatients, where the disease seems to be triggered by febrile ill-nesses. Our results show that TNFRI signaling is not essentialfor the induction of this second phase of inflammatory skinlesions in adult skin in our mouse model and suggest that theearly and late lesions may be caused by different mechanisms.

Apoptosis of keratinocytes is a prominent feature of the skindisease in both human IP and in our mouse model. NEMO-deficient keratinocytes undergo massive apoptosis at latestages of the disease in NEMOEKO and NEMOEHET mice,most probably being killed by the increased expression ofTNF in the skin at this stage. The killing of NEMO-deficientcells and their subsequent replacement with wild-type kerati-nocytes is thought to explain the healing of the skin lesionsin the IP patients and in the few heterozygous NEMO-deficientmice that survive the initial phase of inflammation (23).Consistent with this hypothesis, we also observed that asmall number of NEMOEHET mice show healing of the skinlesions after P8–P10 and survive to adulthood, whereas noNEMOEKO mouse survived further than P6. Furthermore, theNEMOEHET mice that survived into adulthood showedreduced disease severity with less involved skin areasalready at the early stages of lesion development, suggestingthat in these mice only a small fraction of epidermal keratino-cytes were NEMO-deficient. In order to confirm whether theclearance of the epidermis from NEMO-deficient cellsthrough apoptosis is responsible for the healing of thedisease, we have tried to use immunostaining to distinguishNEMO-deficient from wild-type keratinocytes in theNEMOEHET mice at different stages, and in particular inadult mice after the disappearance of the lesions. Althoughwe have used many commercially available and also ourown anti-NEMO antibodies, we were never able to detectendogenous levels of NEMO by immunostaining of mousetissue sections. Thus, this hypothesis remains to be formallyproven. However, the fact that none of the NEMOEKO micesurvived after P6 demonstrates that the presence of wild-type keratinocytes is essential for skin lesion healing, furthersupporting that the replacement of NEMO knockout keratino-cytes with wild-type ones is essential for recovery. In additionto the large number of apoptotic cells found in the epidermisafter P3, we consistently observed the presence of smallnumbers of apoptotic cells either in clusters scattered aroundhair follicles or as single cells in the basal layer of the epider-mis of NEMOEKO mice in early stages from E18.5 to P2.These results suggest that some NEMO-deficient keratinocytesundergo apoptosis before the initiation of the inflammatoryresponse. At present, we do not know the nature of thesignals that induce apoptosis in NEMO-deficient keratinocytesat these early stages, however this spontaneous death ofmutant keratinocytes may be implicated in the initiation ofthe skin lesions.

Analysis of STAT3 and JNK phosphorylation showedincreased activation of both these pathways in the epidermisof NEMOEKO mice at P5, however at P0 there was no differ-ence between mutant and control mice. STAT3 has beensuggested to be specifically activated in psoriasis-like skin dis-eases and is proposed to provide the link for the interactionbetween keratinocytes and T-cells in this disease (28). Sucha mechanism can be excluded in our model as T-cells arenot required for disease pathogenesis. Therefore, the increasedSTAT3 activity in the epidermis of NEMOEKO mice is mostprobably a consequence of the inflammatory hyperproli-ferative disease and not an initiating factor. Increased JNKactivation has been proposed to induce cell-autonomoushyperproliferation in p65/RelA-deficient keratinocytes in a


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TNFRI-dependent manner (11). We found increased JNKactivity in the epidermis of NEMOEKO mice at P3 and P5,but not in earlier stages before the initiation of skin inflam-mation. Increased JNK activity upon TNF stimulation hasbeen shown to induce apoptosis in NF-kB-deficient cells(11). In the epidermis of NEMOEKO mice at P3 and P5, wedetected increased apoptosis and also increased proliferationof keratinocytes, with both events being the consequence ofthe underlying inflammatory response. Our findings stronglysuggest that the increased JNK phosphorylation in the epider-mis of NEMOEKO mice is a consequence of the inflammatoryresponse and is implicated in mediating TNF-inducedapoptosis of NEMO-deficient keratinocytes at this stage.

Deletion of NEMO in the epidermis of NEMOEKO mice isalready complete before birth, however the skin lesionsappear in the first few days after birth. Early postnatal appear-ance of the inflammatory lesions is also described for humanIP patients, suggesting that disease initiation may correlatewith events occurring shortly after birth. Although it seemsplausible to speculate that the contact of the skin with theenvironment and in particular with microbial factors may beinvolved in disease initiation, we were thus far unable toaffect lesion development by keeping the mice in animalfacilities with different health status or by treating the micewith local antimicrobial agents. Alternatively, the develop-ment of the skin phenotype shortly after birth may berelated to developmental processes occurring in the newbornskin. To investigate this hypothesis, we analyzed lesionformation upon inducible NEMO deletion in the epidermisof adult mice. These experiments showed that inactivationof NEMO in the adult epidermis caused an inflammatoryskin phenotype that is similar to the skin lesions developingin the NEMOEKO mice. Therefore, the initiation of theinflammatory skin disease does not seem to be related todevelopmental processes.

The skin disease developing in mice with epidermis-specificdeletion of NEMO shares similarities with the skin phenotypethat develops in mice with epidermis-specific deletion ofIKK2 (19). In both models, inhibition of IKK-mediatedNF-kB activation in keratinocytes leads to TNF-mediatedinflammatory skin disease, resulting in secondary epidermalhyperplasia and differentiation defects. An important differ-ence between the two models is that apoptosis of keratinocytesis a prominent feature of the skin disease in NEMOEKO but notin IKK2EKO mice. This difference can be explained by thecomplete inhibition of NF-kB caused by NEMO deletion, incontrast to IKK2 deletion that allows some residual NF-kBactivation presumably mediated by IKK1. The results fromthese two genetic models show that IKK2- and NEMO-mediated NF-kB activation in the epidermis is not requiredfor the regulation of epidermal differentiation and growthinhibition, but is important for the maintenance of immunehomeostasis in the skin.

We have taken a genetic approach using conditional genetargeting in a mouse model to investigate the cellular andmolecular mechanisms that are implicated in the pathogenesisof inflammatory skin lesions in IP. Our results suggest thatlack of NEMO-mediated NF-kB activation in epidermalkeratinocytes is the primary defect triggering the developmentof skin lesions in IP. It remains to be tested whether the role

of the NF-kB-deficient epidermal keratinocyte as a potentinitiator of skin inflammation is also relevant for the pathogen-esis of other inflammatory skin diseases in addition to IP.Furthermore, a TNF-mediated inflammatory response iscrucial for disease pathogenesis in the mouse model, indicatingthat TNF signaling may also be important in the human disease.Identifying the cellular targets of TNF will be important tofurther elucidate themechanisms leading to skin lesion develop-ment. For this task, new genetic models allowing the cell-specific manipulation of TNF signaling in combination withNEMO epidermis knockout will be required. Taken together,our results highlight the important role of NEMO-mediatedNF-kB activation in the epidermis and provide new insightsinto the mechanisms causing the skin manifestations of IP.

MATERIALS AND METHODS

Mice

Epidermis-specific NEMO knockout mice were generated bycrossing mice carrying loxP-flanked NEMO alleles (23) toK14-Cre transgenic mice expressing Cre under the controlof the human keratin 14 promoter (25), or to K14-Cre-ERT2

mice expressing a tamoxifen-inducible fusion protein betweenthe Cre recombinase and a mutated ligand-binding domain ofthe human estrogen receptor a in epidermal keratinocytesunder the control of the human keratin 14 promoter (30).TNFRI-deficient mice (32) (provided by K. Pfeffer) and RAG1knockout mice (29) were crossed to K14-Cre/NEMOFL mice togenerate double knockouts. All mice used in this study werebackcrossed into the C57Bl/6 genetic background for at leastfive generations. Littermates lacking the Cre transgenes wereused as controls in all experiments.

Western blotting and electromobility shift assay

Cytoplasmic extracts from cultured keratinocytes wereprepared as described (23), electrophoresed by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 10%) and transferred to Immobilon P membranes(Millipore). The membranes were blocked with 5% milk andprobed using rabbit polyclonal antibodies against NEMO(33), IkBa (Santa-Cruz), mouse monoclonal antibodiesagainst IKK1 (Imgenex), IKK2 (Imgenex), phospho-IkBa(Cell Signaling) and goat polyclonal antibody against actin(Santa-Cruz). Membranes were then incubated with secondaryantibodies IgG-HRP conjugates (Amersham, Chemicon) anddeveloped using ECL Supersignal (Pierce).

Electromobility shift assay of nuclear extracts from culturedkeratinocytes was performed as described (23).

Tamoxifen-inducible deletion of NEMO in theepidermis of adult mice

To induce deletion of NEMO in the epidermis of adult mice,NEMOFL mice were crossed with K14Cre-ERT2 mice (34).Deletion was induced at 8 weeks of age by application of100 mg of tamoxifen dissolved in DMSO, or DMSO alone ascontrol, on a 1-cm2 patch of shaved skin for 5 consecutivedays. Mice were sacrificed 2 days after the last day of treatment.


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Histological analysis and immunohistochemistry

Five-micrometer thick paraffin sections were stained withhematoxylin and eosin (Sigma). Immunohistochemistry wasperformed on 5-mm thick paraffin or cryostat sections usingpolyclonal antibodies against mouse loricrin, K10, K6 (allpurchased from Covance), or monoclonal antibodies againstmouse 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 antibodiescoupled to Alexa 488 or 594 (Molecular Probes). Sectionswere counterstained with ToProIII (Molecular Probes) orDAPI (Sigma) to visualize nuclei. For in vivo BrdU prolifer-ation assays, we injected intraperitoneally pregnant femaleswith BrdU (100 mg/g; Sigma) and dissected embryos after3 h. Skin sections were stained with monoclonal antibodiesagainst BrdU (Chemicon). When required, sections wereprocessed in an antigen retriever apparatus (Pickcell Lab).Terminal deoxynucleotidyltransferase-mediated TUNEL stain-ing of paraffin skin sections was performed according to themanufacturer’s instructions (Apoptosis Detection System,Promega). Fluorescent stainings were analyzed using a LeicaTCS NT upright confocal laser scanning microscope or aLeica DM RHC microscope.

Keratinocyte proliferation and TNF-induced apoptosis

Primary epidermal keratinocytes were isolated from the skin ofnewborn mice and cultured in minimal calcium medium(0.05 mM CaCl2; low calcium medium) supplemented asdescribed (35). For measurement of proliferation, keratinocyteswere plated in two-well permanox chamber slides and weregrown to about 50% confluence. Cells were labeled withBrdU (1:1000 dilution BrdU labeling and detection kit II;Roche) for 3, 5 and 15 h at 378C. BrdU incorporation wasdetected by immunofluorescence using anti-BrdU mousemonoclonal antibody (Roche) followed by secondary antibodycoupled to Alexa 488 (Molecular Probes). For the detection ofTNF-induced apoptosis, primary keratinocytes were grown in96-well plates. Cells were treated with 20 ng/ml murine TNFfor 6 h and cell viability was determined by the colorimetricMTT [3-(4,5-dimethylthiazol-2-yl-)2,5-diphenyltetrazoliumbromide] (Sigma) assay. Ten microliters of an MTT solution(1.25 mg/ml) was added to each well. After 2 h incuba-tion, cells were lysed by adding 150 ml of isopropyl-HClsolution (600 ml of HCl/100 ml isopropanol) for 15 min.Absorbance was determined with an automated ELISAreader at 570 nm.

Gene expression analysis

Total RNA from primary keratinocytes, epidermis or total skinwas isolated using Trizol (Invitrogen) and analyzed byreal-time polymerase chain reaction (PCR) with SyBr Green(Finnzymes) or by RNase protection assay, respectively. ForRT–PCR primers used for IkBa are 50-CGAAGAGAAGCCGCTGACCAT-30 and 50-CTCATCCTCGCTCTCGGGTAG-30 andfor TNF are 50-CACGCTCTTCTGTCTACTGAACTTCG-30

and 50-GGCTGGGTAGAGAATGGATGAACACC-30. All

values were normalized to the level of ubiquitin mRNA.For RNase protection assay, total RNA was hybridized withRNA probes using a Riboquant multi-probe RPA system(BD Bioscience), following the manufacturer’s instruction.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS

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

Conflict of Interest statement. None declared.

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Skin lesion development in a mouse model of incontinentia pigmenti

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