Alain Israël

Complementation Cloning of NEMO, a Component of the IκB Kinase Complex Essential for NF-κB Activation

Abstract We have characterized a flat cellular variant of HTLV-1 Tax-transformed rat fibroblasts, 5R, which is unresponsive to all tested NF-κB activating stimuli, and we report here its genetic complementation. The recovered full-length cDNA encodes a 48 kDa protein, NEMO ( N F-κB E ssential MO dulator), which contains a putative leucine zipper motif. This protein is absent from 5R cells, is part of the high molecular weight IκB kinase complex, and is required for its formation. In vitro, NEMO can homodimerize and directly interacts with IKK-2. The NEMO cDNA was also able to complement another NF-κB–unresponsive cell line, 1.3E2, in which the protein is also absent, allowing us to demonstrate that this factor is required not only for Tax but also for LPS, PMA, and IL-1 stimulation of NF-κB activity.

A novel proteolytic cleavage involved in Notch signaling : the role of the disintegrin-metalloprotease TACE

The Notch1 receptor is presented at the cell membrane as a heterodimer after constitutive processing by a furin-like convertase. Ligand binding induces the proteolytic release of Notch intracellular domain by a gamma-secretase-like activity. This domain translocates to the nucleus and interacts with the DNA-binding protein CSL, resulting in transcriptional activation of target genes. Here we show that an additional processing event occurs in the extracellular part of the receptor, preceding cleavage by the gamma-secretase-like activity. Purification of the activity accounting for this cleavage in vitro shows that it is due to TACE (TNFalpha-converting enzyme), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. Furthermore, experiments carried out on TACE-/- bone marrow-derived monocytic precursor cells suggest that this metalloprotease plays a prominent role in the activation of the Notch pathway.

The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination.

NF-κB transcription factors have key roles in inflammation, immune response, oncogenesis and protection against apoptosis. In most cells, these factors are kept inactive in the cytoplasm through association with IκB inhibitors. After stimulation by various reagents, IκB is phosphorylated by the IκB kinase (IKK) complex and degraded by the proteasome, allowing NF-κB to translocate to the nucleus and activate its target genes. Here we report that CYLD, a tumour suppressor that is mutated in familial cylindromatosis, interacts with NEMO, the regulatory subunit of IKK. CYLD also interacts directly with tumour-necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), an adaptor molecule involved in signalling by members of the family of TNF/nerve growth factor receptors. CYLD has deubiquitinating activity that is directed towards non-K48-linked polyubiquitin chains, and negatively modulates TRAF-mediated activation of IKK, strengthening the notion that ubiquitination is involved in IKK activation by TRAFs and suggesting that CYLD functions in this process. Truncations of CYLD found in cylindromatosis result in reduced enzymatic activity, indicating a link between impaired deubiquitination of CYLD substrates and human pathophysiology.

X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling.

The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IκB kinase) complex, which is essential for NF-κB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-κB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-κB through the NEMO protein, indicating that EDA results from impaired NF-κB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1β, IL-18, TNFα and CD154. We thus report for the first time that impaired but not abolished NF-κB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.

The DNA binding subunit of NF-κB is identical to factor KBF1 and homologous to the rel oncogene product

The major determinant in the transcriptional control of class I genes of the major histocompatibility complex is an enhancer sequence located around -170 from the transcription start site, which binds a factor named KBF1. We have isolated a complementary cDNA coding for KBF1 and identified the DNA binding and dimerization domain of the protein. Because KBF1 and the transcription factor NF-kappa B bind to similar sequences, we investigated the relationship between these two molecules. It appeared that KBF1 is, by all criteria used, identical to the 50 kd DNA binding subunit of NF-kappa B. KBF1 (and therefore p50) also displays extensive amino acid sequence homology with the v-rel oncogene and the Drosophila maternal morphogen dorsal. In vitro experiments suggest functional homologies between KBF1 and v-rel.

Genomic rearrangement in NEMO impairs NF-κB activation and is a cause of incontinentia pigmenti: The International Incontinentia Pigmenti (IP) Consortium

Familial incontinentia pigmenti (IP; MIM 308310) is a genodermatosis that segregates as an X-linked dominant disorder and is usually lethal prenatally in males. In affected females it causes highly variable abnormalities of the skin, hair, nails, teeth, eyes and central nervous system. The prominent skin signs occur in four classic cutaneous stages: perinatal inflammatory vesicles, verrucous patches, a distinctive pattern of hyperpigmentation and dermal scarring1. Cells expressing the mutated X chromosome are eliminated selectively around the time of birth, so females with IP exhibit extremely skewed X-inactivation2. The reasons for cell death in females and in utero lethality in males are unknown. The locus for IP has been linked genetically to the factor VIII gene in Xq28 (ref. 3). The gene for NEMO (NF-κB essential modulator)/IKKγ (IκB kinase-γ) has been mapped to a position 200 kilobases proximal to the factor VIII locus4. NEMO is required for the activation of the transcription factor NF-κB and is therefore central to many immune, inflammatory and apoptotic pathways5,6,7,8,9. Here we show that most cases of IP are due to mutations of this locus and that a new genomic rearrangement accounts for 80% of new mutations. As a consequence, NF-κB activation is defective in IP cells.Familial incontinentia pigmenti (IP; MIM 308310) is a genodermatosis that segregates as an X-linked dominant disorder and is usually lethal prenatally in males. In affected females it causes highly variable abnormalities of the skin, hair, nails, teeth, eyes and central nervous system. The prominent skin signs occur in four classic cutaneous stages: perinatal inflammatory vesicles, verrucous patches, a distinctive pattern of hyperpigmentation and dermal scarring. Cells expressing the mutated X chromosome are eliminated selectively around the time of birth, so females with IP exhibit extremely skewed X-inactivation. The reasons for cell death in females and in utero lethality in males are unknown. The locus for IP has been linked genetically to the factor VIII gene in Xq28 (ref. 3). The gene for NEMO (NF-κB essential modulator)/IKKγ (IκB kinase-γ) has been mapped to a position 200 kilobases proximal to the factor VIII locus. NEMO is required for the activation of the transcription factor NF-κB and is therefore central to many immune, inflammatory and apoptotic pathways. Here we show that most cases of IP are due to mutations of this locus and that a new genomic rearrangement accounts for 80% of new mutations. As a consequence, NF-κB activation is defective in IP cells.

The IKK complex: an integrator of all signals that activate NF-κB?

Abstract The NF-κB family of transcription factors plays a crucial role in the immune, inflammatory and apoptotic responses. These proteins are normally found in the cytoplasm, retained by interaction with an inhibitory molecule called IκB. Activation of the NF-κB signalling cascade results in phosphorylation and degradation of IκB, allowing nuclear translocation of the NF-κB complexes. The recent identification of a high-molecular-weight complex containing two kinases and a regulatory subunit has led to a flurry of new results that shed light on some of the most complex mechanisms contributing to the exquisite regulation of NF-κB activity.

TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2

The IκB kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKKγ) regulatory subunit, phosphorylates IκB proteins, targeting them for degradation and thus inducing activation of NF-κB (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-κB activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-κB. Previous studies suggested that NF-κB has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or IκBα, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-κB in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-κB activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, αβ T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-κB activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.

NEMO/IKKγ-Deficient Mice Model Incontinentia Pigmenti

Disruption of the X-linked gene encoding NF-kappa B essential modulator (NEMO) produces male embryonic lethality, completely blocks NF-kappa B activation by proinflammatory cytokines, and interferes with the generation and/or persistence of lymphocytes. Heterozygous female mice develop patchy skin lesions with massive granulocyte infiltration and hyperproliferation and increased apoptosis of keratinocytes. Diseased animals present severe growth retardation and early mortality. Surviving mice recover almost completely, presumably through clearing the skin of NEMO-deficient keratinocytes. Male lethality and strikingly similar skin lesions in heterozygous females are hallmarks of the human genetic disorder incontinentia pigmenti (IP). Together with the recent discovery that mutations in the human NEMO gene cause IP, our results indicate that we have created a mouse model for that disease.

I kappa B epsilon, a novel member of the IκB family, controls RelA and cRel NF‐κB activity

We have isolated a human cDNA which encodes a novel IκB family member using a yeast two‐hybrid screen for proteins able to interact with the p52 subunit of the transcription factor NF‐κB. The protein is found in many cell types and its expression is up‐regulated following NF‐κB activation and during myelopoiesis. Consistent with its proposed role as an IκB molecule, IκB‐ϵ is able to inhibit NF‐κB‐directed transactivation via cytoplasmic retention of rel proteins. IκB‐ϵ translation initiates from an internal ATG codon to give rise to a protein of 45 kDa, which exists as multiple phosphorylated isoforms in resting cells. Unlike the other inhibitors, it is found almost exclusively in complexes containing RelA and/or cRel. Upon activation, IκB‐ϵ protein is degraded with slow kinetics by a proteasome‐dependent mechanism. Similarly to IκB‐α and IκB‐β, IκB‐ϵ contains multiple ankyrin repeats and two conserved serines which are necessary for signal‐induced degradation of the molecule. A unique lysine residue located N‐terminal of the serines appears to be not strictly required for degradation. Unlike IκB‐α and IκB‐β, IκB‐ϵ does not contain a C‐terminal PEST‐like sequence. IκB‐ϵ would, therefore, appear to regulate a late, transient activation of a subset of genes, regulated by RelA/cRel NF‐κB complexes, distinct from those regulated by other IκB proteins.