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  • Defective nuclear IKKaa function in patients with ectodermal dysplasia with immune deficiency

    Stephane T. Temmerman, … , Ralph Shapiro, Ashish Jain

    J Clin Invest. 2012;122(1):315-326. https://doi.org/10.1172/JCI42534.

    Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-kB essential modulator (NEMO; also known as IkB kinase g subunit [IKKg]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-kB proteins, resulting in defective expression of NF-kB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-kB. However, cells deficient in full-length NEMO were defective in expression of NF- kB–regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-kB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKa-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-kB heterodimers RelA and cRel to the promoter. Expression of a super- active form of IKKa restored IL-12 production in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKa and offer new insights into the mechanisms underlying diminished NF-kB signaling in patients with EDI.

    Research Article Immunology

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  • Research article

    The Journal of Clinical Investigation      http://www.jci.org      Volume 122      Number 1      January 2012  315

    Defective nuclear IKKα function in patients with ectodermal dysplasia

    with immune deficiency Stephane T. Temmerman,1 Chi A. Ma,1 Yongge Zhao,1 Jeffrey Keenan,1 Ivona Aksentijevich,2

    Margaret Fessler,1 Margaret R. Brown,3 Alan Knutsen,4 Ralph Shapiro,5 and Ashish Jain1

    1Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), 2Genetics and Genomics Branch, National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS), and 3Department of Laboratory Medicine, Clinical Center, NIH, Bethesda,

    Maryland, USA. 4Pediatric Allergy and Immunology, St. Louis University, St. Louis, Missouri, USA. 5Midwest Immunology Clinic, Minneapolis, Minnesota, USA.

    Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB–regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 pro- duction in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.

    Introduction Ectodermal dysplasia with immune deficiency (EDI) is an X-linked  immunological and developmental disorder caused by mutations  in the gene encoding NF-κB essential modulator (NEMO; also  known as IκB kinase γ subunit [IKKγ]). NEMO is a regulatory  subunit of the IκB kinase (IKK) complex, which also contains 2  structurally related catalytic subunits, IKKα and IKKβ (1). The  IKK complex tightly controls the classical activation pathway of  the NF-κB/Rel transcription factor family by promoting phos- phorylation  and  degradation  of  cytosolic  IκBs  (2).  Although  IKKα and IKKβ have a high degree of amino acid homology, these  kinases have distinct functional properties (3): IKKβ is primarily  cytoplasmic, whereas IKKα is present in both the cytoplasm and  the nucleus (4). Gene-targeting studies in mice revealed that IKKβ  and NEMO are critical for NF-κB activation in response to proin- flammatory stimuli and microbial products. However, IKKα is not  essential for cytokine-induced IκB degradation and primarily con- trols NF-κB–mediated gene expression by 2 mechanisms. The first  involves a NEMO-independent NF-κB signaling pathway that is  critical for lymphoid organ development and B cell maturation (5,  6). Following activation by NF-κB–inducing kinase (NIK), IKKα  homodimers process NF-κB2 to produce the p52 subunit. The NF- κB p52 subunit then associates with another NF-κB family mem- ber, RelB, forming a dimer that migrates to the nucleus and stimu- lates target gene expression (7). This alternative NF-κB signaling  pathway is activated by certain members of the TNF family, but  not by pattern recognition receptors such as TLR4 (8). A second 

    hypothesis involves a nucleosomal function for IKKα in response  to proinflammatory stimuli. Recent reports demonstrate that  IKKα is recruited to NF-κB–regulated promoters and phosphory- lates specific residues of histone H3 upon activation by cytokines  (9–12). However, the precise mechanism by which nuclear IKKα  activity is regulated is not known.

    The  gene  encoding  NEMO  lies  on  the  X  chromosome,  and  depending upon their severity, alterations in the NEMO gene lead  to 2 allelic diseases: incontinentia pigmenti (IP) and EDI (13). IP  corresponds to a complete loss of NEMO function and is typi- cally lethal in utero in males. Female carriers of NEMO mutations  exhibit abnormalities of teeth, hair, skin, nails, and the central ner- vous system. The severity of such abnormalities in females can be  highly variable and depends on lyonization of the X chromosome  in individual cells. EDI results from hypomorphic coding muta- tions of NEMO that partially impair, but do not abolish, IKK com- plex–dependent phosphorylation and subsequent degradation of  cytoplasmic inhibitors of NF-κB (14, 15). Male patients with EDI  exhibit abnormal development of ectoderm-derived structures and  suffer from recurrent pyogenic infections involving deep-seated  organs (16). We have previously demonstrated that B lymphocytes  and APCs of individuals with a C417 mutation in the zinc finger  domain of NEMO show a profound defect in CD40-mediated IκBα  degradation that results in reduced NF-κB nuclear translocation  and binding activity (15). In such patients, B cells stimulated with  CD40 ligand lack both class-switch recombination and somatic  hypermutation in the variable region of immunoglobulin (17).  Furthermore, DCs fail to upregulate costimulatory molecules,  secrete proinflammatory cytokines such as IL-12, or induce allo- geneic T cell proliferation in response to CD40 stimulation (18).

    Conflict of interest: The authors have declared that no conflict of interest exists.

    Citation for this article: J Clin Invest. 2012;122(1):315–326. doi:10.1172/JCI42534.

  • research article

    316 The Journal of Clinical Investigation      http://www.jci.org      Volume 122      Number 1      January 2012

    In this study, we report 2 patients with typical features of EDI who  fail to express normal levels of full-length NEMO, but do not have  mutations in the coding r

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