1 Max-Delbrück-Center of Molecular Medicine, Robert-Rössle Straße 10, 13092 Berlin, Germany
2 Max-Planck-Institute for Infection Biology, Schumannstraße 21/22, 10117 Berlin, Germany
*Author for correspondence (e-mail: rschmidt{at}mdc-berlin.de)
Accepted July 4, 2001
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SUMMARY |
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Key words: Cre-loxP, Hypohidrotic ectodermal dysplasia (HED), Skin disease, Immunity, Drug targeting, Mouse
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INTRODUCTION |
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The prevailing form of the IKK complex is an IKK-IKKß kinase heterodimer associated with IKK
/NEMO (Karin, 1999). Although the highly related IKK
and IKKß proteins are both able to phosphorylate I
B
in vitro, the physiological function of both isoforms differs grossly. Gene ablation disclosed that, similar to p65, IKKß is essential for NF-
B activation by pro-inflammatory stimuli and antagonizes tumor necrosis factor
(TNF
)- induced apoptosis (Li et al., 1999b; Li et al., 1999c; Tanaka et al., 1999). By contrast, IKK
is required for development of the epidermis and gene loss results in epidermal hyperproliferation (Hu et al., 1999; Li et al., 1999a; Takeda et al., 1999). This function, however, is completely independent of NF-
B and does not require the kinase activity of IKK
, as has been shown by complementation of IKK
-deficient keratinocytes (Hu et al., 2001).
Owing to the functional redundancy of NF-B/Rel subunits in the single or double knockouts and to embryonic lethality observed for RelA-, IKKß- or IKK
-deficient mice, the physiological role of NF-
B/Rel in the adult organism has remained widely obscure. We have examined the pathophysiological consequences of a systemic NF-
B/Rel suppression using a gene targeting approach by ubiquitous expression of a constitutively active I
B
mutant. Mice with suppressed NF-
B activity survive to adulthood and display macrophage dysfunction and alymphoplasia, the lack of secondary lymphoid organs. NF-
B suppression results in severe defects in the early steps of the development of epidermal appendices, including hair follicles, tear and sweat glands. In wild-type mice these structures reveal strong NF-
B transcriptional activity, as we demonstrate with ß-galactosidase reporter mice. This includes the multipotent stem cell-containing bulge region in hair follicles, which responds to morphogenic signals for hair follicle generation. NF-
B suppression results in strongly increased apoptosis in subregions of developing hair follicles. The epidermal phenotype is analogous to hypohidrotic (anhydrotic) ectodermal dysplasia (HED) in humans, and identical to phenotypes of Eda, Edar or crinkled (Cr) mice. The Eda and Edar genes are related to the TNF multigene family of ligands and receptors, respectively. Our data are consistent with the model that NF-
B is required for Edar to transmit Eda signals and to protect against apoptosis.
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MATERIALS AND METHODS |
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Electrophoretic mobility shift assay (EMSA) and western blotting
ES cells and mouse embryonal fibroblasts (MEFs) were washed with phosphate-buffered saline (PBS) and lysed in extraction buffer (20 mM Hepes (pH 7.0), 0.15 mM EDTA, 0.15 mM EGTA, 10 mM KCl, 0.15 mM spermidine, 1% NP40, 0.4 M NaCl, 10% glycerol and protease inhibitors). After 30 minutes on ice, extracts were centrifuged for 30 minutes at 86,000 g. Supernatants were used for western and EMSA analysis. Organs of mice were quick-frozen in liquid N2 and lysed by douncing in extraction buffer. ES cells were treated with 75 ng/ml phorbol ester (PMA) (Sigma) for 1 hour. MEFs were treated with 5 ng/ml IL1-ß (Promega) or 50 ng/ml TNF (Biomol) for the times indicated. EMSA was performed as described previously (Krappmann et al., 1996). The following antibodies were used for western blot analysis: p65 (#sc109, #sc8008, Santa Cruz) and I
B
(C-15, #sc203; C-21, #sc371, Santa Cruz). The polyclonal rabbit anti-ß-catenin antibody is directed against the C-terminal part of the protein.
Histology and in situ hybridization (ISH)
For liver analysis, embryos were removed and snap-frozen in OCT compound with subsequent storage at 80°C. Cryosections (10 µm) were made. TUNEL assays were performed using the ApopTag® Plus system from Intergen Company. For analysis of tissue from adult mice, tissue was removed from perfused mice (4% paraformaldehyde (PFA)) and fixed in 4% PFA for up to a week, depending on the size of the tissue. Tissue containing bone, such as the inner ear and feet was decalcified in 4% EDTA/1xPBS for a week at 4°C. After a series of dehydration steps in ethanol (30%-100%) the tissue was embedded in Technovit 7100 plastic (Heraeus Kulzer). Sections (5 µm) were cut and stained using the Hematoxylin (Delafields)-EosinY method. Pictures were taken with a Zeiss Axiophot camera.
Whole-mount in situ hybridization was performed as described (Huelsken et al., 2000). The single-stranded RNA probe was labeled with digoxigenin (DIG)-UTP according to the manufacturers instructions (Roche). The probe for downless used comprised nucleotides 302-1038 (AF160502). Subsequently embryos were embedded in Technovit 7100 and 12 µm sections were cut. Sections were counterstained with 0.01% pyroninG. Whole-mount X-Gal staining was performed as described (Schmidt-Ullrich et al., 1996).
Pathogen challenges and nitrate production in macrophages
Leishmania major V121 promastigotes were injected into the left hind footpad and thickness was recorded weekly using a caliper. The difference with the uninfected foot was plotted as the mean±s.d. Nitrite production was measured in culture supernatants of macrophages derived from bone marrow using DMEM/10% FCS/20% L929-conditioned medium. Cells were harvested after 6 days and cultured for a further 72 hours at 1,25x105 cells per cavity in 48-well plates in DMEM/10% FCS substituted with 100 U/ml IFN and increasing doses of TNF
. Nitrite was determined colorimetrically by the Griess reaction. Leishmania major antigens were produced by repeated freezing and thawing of the cells (five times). Lysate corresponding to 106 cells/ml was used to restimulate spleen cells.
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RESULTS |
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Impaired hair follicle development and absence of eccrine glands implicate NF-B as an essential component of the EDA/EDAR pathway
Animals which reached adulthood were small and thin, had shaggy fur, no hair on the tail and behind the ears, fewer vibrissae and slanted eyes (Fig. 1F). In wild-type mice, four hair types, monotrich, awl, auchenes and zigzag, are found, whereas in cIB
N mice only a monotrich-awl intermediate was detected (Fig. 2A). By contrast, uninduced cloxPI
B
N mice could not be distinguished phenotypically from wild-type mice. However, when mating cloxPI
B
N animals with deleter-Cre mice, which produce the Cre enzyme ubiquitously (Schwenk et al., 1995), the offspring had the same phenotype as cI
B
N mice (see Table 1 for phenotype summary).
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Although cIB
N mice opened the eyes only after 2.5-3 weeks after birth, the eye-bulb, conjunctiva and the cornea developed normally. However, cI
B
N mice revealed narrowed palpebral fissures and thickened margins of the eyelids, caused by a hyperproliferative epidermis of the eyelid margin (Fig. 2D). After about 1 month, mice developed conjunctivitis. Hyperproliferation of the corneal stroma, keratinization of the corneal epithelium, neoformation of blood vessels and granulocyte infiltration could be readily detected after 6 months (not shown). Histological sections disclosed a complete absence of Meibomian glands in all cI
B
N mice analyzed (Fig. 2D). The Meibomian glands produce the lipid layer of the tear film, preventing its evaporation. Insufficient development of the Harderian glands (data not shown; Table 1) may further facilitate evaporation. As a consequence, the eyes dehydrated with time and, owing to a reduced immune response (see below), the mice additionally acquired severe keratoconjunctivitis sicca, eventually resulting in blindness in all cases.
cIB
N mice did not show the hyperplasia of the suprabasal squamous layer of the epidermis observed in ikk
and
knockout or K14-mI
B
models (Kaufman and Fuchs, 2000). However, a requirement of NF-
B for hair follicle and exocrine gland formation is supported by blue staining of these structures in mice expressing a NF-
B responsive ß-galactosidase ((Ig
)3conalacZ) transgene (Schmidt-Ullrich et al., 1996) (Fig. 3A-F; data not shown). These mice did not show any X-Gal staining in the epidermal layers (Fig. 3). Embryos revealed ß-galactosidase activity as early as E12 for vibrissae and E15 for pelage hair (R. S.-U., unpublished). In vibrissal and pelage follicles of adult (Ig
)3conalacZ mice, strong X-Gal staining was detected in the matrix (Fig. 3B,C). Vibrissal follicles also showed ß-galactosidase activity in the hair shaft (Fig. 3C) and in the bulge (Fig. 3D). Similarly, a discrete region of pelage hair follicles, likely to be the bulge, stained blue (Fig. 3A). This indicates a role for NF-
B in de novo hair follicle formation, as the bulge region contains multipotent stem cells, which respond to morphogenic signals to generate a new hair follicle after each hair cycle. X-Gal staining was also observed in hair follicles of the tail (Fig. 3E), in sweat glands (Fig. 3F), Harderian glands and Meibomian glands (data not shown).
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In situ hybridization (ISH) did not show any Edar expression in pelage follicles of cIB
N mice at E15.5 (Fig. 4A, dl-
N1), when compared with wild-type mice (Fig. 4A, dl-w.t.1). As expected, no placodes were seen in cI
B
N mice at this stage (Fig. 4A, dl-
N2), while placodes of wild-type mice were developing normally, showing expression of Edar (Fig. 4A, dl-w.t.2). However, developing vibrissal follicles are present in cI
B
N mice and do show Edar expression identical to wild-type animals (Fig. 4B) indicating that Edar is not regulated by NF-
B in vibrissae. Preliminary ISH data of E17.5 embryos also showed Edar expression in body hair follicles in cI
B
N mice (data not shown). At E15.5, Eda expression, which is found throughout the epidermis, was identical in wild-type and cI
B
N mice (data not shown). TUNEL assays showed an increased rate of apoptosis in many pelage (Fig. 4C,
N1) and vibrissal follicles (Fig. 4C,
N2). Taken together, these results demonstrate that NF-
B is essential for Eda and Edar signaling, and the presence of apoptosis implies that NF-
B also acts as a survival factor in the EDA/EDAR pathway. Our data demonstrate a requirement of NF-
B for epithelial-derived organogenesis. Thus, impaired NF-
B activation is a crucial event in anhydrotic ectodermal dysplasia.
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DISCUSSION |
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As predicted, cytokine-induced NF-B in ES cells and in fibroblasts of I
B
N-expressing mice was strongly suppressed, and as expected from p65-, IKKß- or
-deficient mice, cI
B
N animals revealed pronounced hepatocyte apoptosis. However, in contrast to these knockouts, cI
B
N mice survived, perhaps as residual NF-
B activity restricted apoptosis to a non-lethal level. Residual NF-
B activity in cI
B
N mice may also account for the absence of some of the phenotypes observed in NF-
B single or double knockouts. In particular, I
B
has limited affinity for RelB and therefore I
B
N should inhibit RelB complexes less sufficiently. Thus, multiorgan inflammation, observed in RelB-deficient mice (Weih et al., 1995), was not observed. In addition to early lethality, functional redundancy in the single or double NF-
B knockouts may have obscured important novel functions detected in this study.
NF-B activity is requisite for formation of secondary lymphoid organs and macrophage-dependent immunity
We found that NF-B activity is indispensable for formation of secondary lymphoid organs and for macrophage-dependent immunity. Alymphoplasia, the complete absence of peripheral lymph nodes and Peyers patches (PPs), has previously been observed in Aly/Aly mice, which have a mutation in Nik (Fagarasan et al., 2000; Shinkura et al., 1999), and in mice deficient in NIK (Yin et al., 2001), lymphotoxin ß (LTß) (Alimzhanov et al., 1997) or LTß receptor (Futterer et al., 1998; Rennert et al., 1998). But it has not been detected in any of the NF-
B single or double knockouts reported so far. p105/p100 double knockouts reveal severe structural defects of lymph nodes and thymus, while p100-deficient mice have disorganized B- and T-cell areas in spleen and lymph nodes (Franzoso et al., 1998; Franzoso et al., 1997). However, all these models do form lymph nodes, presumably owing to functional redundancy at an early stage of development.
Our data are complementary to two recent reports revealing that the LTß pathway activates NF-B via NIK (Matsushima et al., 2001; Yin et al., 2001). Both find that NF-
B transcriptional activity induced by LTß is impaired in NIK-deficient and NIK-mutant (Aly/Aly) fibroblasts. Yet, LTß-induced NF-
B DNA-binding activity is not impaired in NIK-deficient cells and the mechanism of action of NIK is not understood (Yin et al., 2001). In IKK
-/- fibroblasts, LTß also fails to induce I
B
phosphorylation (Matsushima et al., 2001) and an essential role of IKK
for lymphorganogenesis was suggested by the lack of PPs in the embryonic intestine of IKK
-/- mice (Matsushima et al., 2001). However, these studies did not present a physiological evidence for a requirement of NF-
B in lymphorganogenesis.
NF-B is required for the development of epidermal appendices
An overt defect in NF-B deficient mice is the thin hair, caused by the absence of normal hair types. We further noted a characteristic lack of hair behind the ears and on the tail. A detailed analysis revealed a lack of plicea digitalis and sweat glands in the foot pads and other pathological signs, such as delayed molar and incisor outgrowth, lack of Meibomian and Harderian glands, as well as defective hair follicle development in cI
B
N mice.
In humans, congenital deficiencies of Meibomian glands, sweat glands and hair have been reported for an autosomal or X-linked syndrome, hypohidrotic (or anhydrotic) ectodermal dysplasia, caused by a mutation in the EDAR or EDA genes, respectively (Headon and Overbeek, 1999; Monreal et al., 1999). Humans suffering from this genetic disorder display a phenotype analogous to that of the Edar (autosomal) and Eda (X-linked) mice (Monreal et al., 1999). Eda encodes a novel TNF-family protein and recently it has been shown that it is the ligand of the TNF receptor homolog EDAR (downless) (Headon and Overbeek, 1999; Kumar et al., 2001; Monreal et al., 1999; Yan et al., 2000). Thus, cIB
N mice present a further model for HED.
We have used a complementary transgenic mouse model, which allows detection of NF-B activity via an integrated NF-
B responsive ß-gal reporter (Ig
3conalacZ) (Schmidt-Ullrich et al., 1996). Abundant NF-
B activity was detected in these transgenic mice, specifically in hair follicles, Meibomian and Harderian glands, sweat glands, and mucous glands (Fig. 3 and data not shown), all of which are ablated or severely affected in mice with suppressed NF-
B and in Eda or Edar mice. NF-
B activity in developing hair follicles was seen as early as stage I of hair follicle development (E12.5-E15.5). These findings rule out the possibility that NF-
B acts indirectly by affecting gene expression in the mesenchyme or through other distant structures, and underscore the importance of NF-
B activity at an early stage in the development of epidermal appendices. In fact, hair follicles of vibrissae and pelage hair displayed strong NF-
B activity in the matrix and in the bulge region, the latter of which contains stem cells that regenerate hair follicles after each cycle. This may hint to a further role of NF-
B in progression through the hair cycle phases: anagen (active growth phase), catagen (regression) and telogen (resting phase).
Hair follicles and other epidermal appendices originate as ectodermal placodes that result from the interaction of ectoderm with the underlying mesenchyme. The stages of hair follicle development take place at different time points (Hardy, 1992). The EDAR pathway is essential for the initiation of the development, when the placode is formed, starting at E14-E16 (Headon and Overbeek, 1999), although there are regional variations in timing (e.g. body, tail, whiskers, limb skin) (Headon and Overbeek, 1999; Wilson et al., 1994). At this stage, which is inhibited in Eda and Edar mice and also in the cIB
N mice, the cells of the placode are committed to differentiate into an epidermal appendix, which can still give rise to a hair follicle or an exocrine gland. The second stage, starting at E17, which commits the placode to make a hair follicle (Hardy, 1992) and depends on Lef-1/Wnt signaling (DasGupta and Fuchs, 1999; Kishimoto et al., 2000), is not inhibited. Interestingly, Edar, Eda and cI
B
N mice do develop hair follicles at this stage, but only the awl type. As mentioned above, the coat of mice consists of four hair types: monotrichs or guard hairs, awls, zigzags and the rare auchenes. The three main types, monotriches, awl and zigzag have been traced to three different waves of hair follicle development in embryonic skin. The formation of monotrich and zigzag follicles (and possibly auchenes) is initiated at E14 and depends on EDA/EDAR. These hair types are not present in either Eda and Edar mice (Vielkind and Hardy, 1996) or in cI
B
N mice. However, awl hairs do not develop until E16 and perhaps depend only on Wnt signaling. This is the reason why Eda, Edar and cI
B
N mice exclusively have awl-type hairs in their coat.
EDA expression was unchanged throughout the epidermis in cIB
N mice (data not shown) and thus did not require NF-
B activity. At day E15 body hair follicle formation was suppressed in cI
B
N mice and no EDAR-expressing placodes could be detected. However, EDAR was expressed in vibrissal follicles, which showed abundant NF-
B activity in the ß-galactosidase stain. This may suggest that NF-
B is required downstream, not upstream, of Edar. However, for body hair follicles, we may not exclude the possibility that the upregulation of EDAR at the site of placode formation around E14 could in part be induced by NF-
B. But at E13, when EDAR is still expressed evenly throughout the basal layer of the epidermis (Headon and Overbeek, 1999), no NF-
B activity can be detected in the same cell layer of (Ig
)3conalacZ mice (data not shown). This suggests that EDAR expression is not regulated by NF-
B.
As a further intriguing finding, suppression of NF-B (at E17) resulted in marked accumulation of apoptotic cells in hair follicles of pelage hair and vibrissae, indicating that NF-
B acts downstream of Edar to inhibit apoptosis. Thus, NF-
B may also play an important role in the homeostasis of hair follicles. In line with this, EDAR contains in its intracellular domain a death domain (Kumar et al., 2001). Similar to TNF-R1, overexpressed EDAR activates NF-
B and JNK but involving a different interaction with signaling molecules, since it does not appear to sequester TRADD, FADD or TRAF2 in the same way as TNF-R1. Furthermore, when overexpressed, EDAR induces apoptosis in a caspase-independent manner (Kumar et al., 2001).
Intriguingly, cIB
N mice did not show the hyperplasia of the suprabasal squamous and cornified layers of the epidermis observed in IKK
-/- mice. The IKK
-deficient epidermis is thickened, owing to hyperproliferation of basal layer cells and blocked keratinocyte differentiation (Hu et al., 1999; Li et al., 1999a; Takeda et al., 1999). Very similarly, the epidermis of transgenic mice expressing a keratin 14 promoter-driven mouse I
B
mutant produced a hyperplastic epithelial basal layer (Seitz et al., 1998). It was therefore originally assumed that IKK
was responsible for NF-
B activation in keratinocytes via an unknown inducing signal. However, as has recently been shown, hyperproliferation and blocked differentiation in the IKK
-/- epidermis can be rescued by kinase-inactive IKK
mutants. Furthermore, the skin function of IKK
is completely independent of NF-
B activity and involves the production of a soluble factor that induces keratinocyte differentiation (Hu et al., 2001). We also observed the hair alterations of cI
B
N mice, except for lack of tail hair, in identical form in offspring of cloxPI
B
N matings with animals expressing Cre under control of a keratin 14 promoter, but without the thickened epidermis seen by Seitz et al. (R. S.-U., unpublished). Furthermore, we did not detect any ß-galactosidase activity in the epidermal layers, indicating that NF-
B is not activated in the suprabasal layer. The reason underlying this discrepancy must await further analysis and may be related to different expression levels of the super-repressor.
IKK knockout mice are yet another example of IKK involvement in skin development. The gene for IKK
is located on the X chromosome and, hence, heterozygous female animals deficient in IKK
present a patchy skin phenotype. These mice manifest changes in skin pigmentation, hyperproliferation of the suprabasal and cornified layers of the epidermis and granulocyte infiltrations in the epidermis, also observed in individuals with incontinentia pigmenti (IP) (Makris et al., 2000; Schmidt-Supprian et al., 2000; Smahi et al., 2000). The alterations in pigmentation and granulocyte infiltration were not observed in IKK
-/- mice (Hu et al., 1999; Li et al., 1999a; Takeda et al., 1999). Nevertheless, IKK
and IKK
both seem to affect the differentiation of keratinocytes (Kaufman and Fuchs, 2000).
Three recent reports associate particular mutations of the X-linked IKK gene with IP- and HED-like symptoms in a subset of male individuals (Aradhya et al., 2001; Doffinger et al., 2001; Zonana et al., 2000). These individuals apparently have reduced, but not absent NF-
B activity (Aradhya et al., 2001; Doffinger et al., 2001). Usually female individuals with mutations in the IKK
locus suffer from IP; males do not survive (Smahi et al., 2000). However, the skin phenotype of cI
B
N mice does not resemble that of female mice with heterozygous IKK
gene ablation. Yet, the typical defects of footpad and tail skin, disturbed tooth, exocrine gland and hair follicle development observed in cI
B
N mice are identical to the phenotypes of Eda and Edar mice (Gruneberg, 1971) (see Table 1). In summary, IKK
and IKK
are essential for epidermal keratinocyte differentiation, in part involving NF-
B-independent mechanisms. The upstream signals of this process are yet unknown. Development of the epidermal appendices, such as hair follicles and eccrine glands is in need of EDA/EDAR and NF-
B, possibly via the IKK complex.
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ACKNOWLEDGMENTS |
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