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 (IkappaB kinase) complex, which is essential for NF-kappaB 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-kappaB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-kappaB through the NEMO protein, indicating that EDA results from impaired NF-kappaB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1beta, IL-18, TNFalpha and CD154. We thus report for the first time that impaired but not abolished NF-kappaB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.

Inhibition of the NF-kappaB survival pathway via caspase-dependent cleavage of the IKK complex scaffold protein and NF-kappaB essential modulator NEMO.

Apoptosis is mediated by cysteine-dependent, aspartate-directed proteases of the caspase family that proteolyse strategic intracellular substrates to induce cell suicide. We describe here that engagement of apoptotic processes by Fas triggering or by staurosporine stimulation leads to the caspase-dependent inactivation of the nuclear factor kappa B (NF-kappaB) pathway after cleavage of IKK1 (IkappaB kinase 1) and NEMO (NF-kappaB essential modulator), which are needed to transduce NF-kappaB activation signals. In this study, we have analyzed in more detail, the role of NEMO cleavage, as NEMO, but not IKK1, is important for the pro-survival actions of NF-kappaB. We demonstrate that NEMO is cleaved after Asp355 to remove the last 64 C-terminal amino acids. This short form was unable to rescue NF-kappaB activation by tumor necrosis factor-alpha (TNF-alpha) when transfected in NEMO-deficient cells. Consequently, inactivation of NEMO resulted in an inhibition of the expression of antiapoptotic NF-kappaB-target genes coding for caspase inhibitors (cIAP-1, cIAP-2) or adaptors of the TNF receptor family. NEMO-deficient Jurkat cells transiently expressing a non-cleavable mutant of NEMO were less sensitive to TNF-alpha-induced apoptosis. Therefore, downmodulation of NF-kappaB activation via the proteolytic cleavage of NEMO could represent an amplification loop for apoptosis.

Interaction of a liver-specific nuclear factor with the fibrinogen and alpha 1-antitrypsin promoters.

The orderly and sequential activation of genes during development is hypothesized to be related to the selective expression of groups of regulatory proteins acting primarily at the level of transcription. A nuclear protein was found in hepatocytes, but not other cell types, that binds to a sequence required for hepatocyte-specific transcription of the gene for the beta chain of fibrinogen. This protein, hepatocyte nuclear factor 1 (HNF1), also interacts with homologous sequences required for optimal promoter function of the genes for the alpha chain of fibrinogen and alpha 1-antitrypsin. The promoter or enhancer regions for several viral and cellular genes not expressed in the liver did not compete for this binding. The restricted expression of HNF1 and its selective interaction with the control regions of several liver-specific genes indicate that it is involved in developmentally regulated gene expression in the liver.

Mutations in the NF-kappaB signaling pathway: implications for human disease.

The nuclear factor-kappa B (NF-kappaB) signaling pathway is a multi-component pathway that regulates the expression of hundreds of genes that are involved in diverse and key cellular and organismal processes, including cell proliferation, cell survival, the cellular stress response, innate immunity and inflammation. Not surprisingly, mis-regulation of the NF-kappaB pathway, either by mutation or epigenetic mechanisms, is involved in many human and animal diseases, especially ones associated with chronic inflammation, immunodeficiency or cancer. This review describes human diseases in which mutations in the components of the core NF-kappaB signaling pathway have been implicated and discusses the molecular mechanisms by which these alterations in NF-kappaB signaling are likely to contribute to the disease pathology. These mutations can be germline or somatic and include gene amplification (e.g., REL), point mutations and deletions (REL, NFKB2, IKBA, CYLD, NEMO) and chromosomal translocations (BCL-3). In addition, human genetic diseases are briefly described wherein mutations affect protein modifiers or transducers of NF-kappaB signaling or disrupt NF-kappaB-binding sites in promoters/enhancers.

Autosomal dominant anhidrotic ectodermal dysplasias at the EDARADD locus.

Anhidrotic ectodermal dysplasia (EDA) is a disorder of ectodermal differentiation characterized by sparse hair, abnormal or missing teeth, and inability to sweat. X-linked EDA is the most common form, caused by mutations in the EDA gene, which encodes ectodysplasin, a member of the tumor necrosis factor (TNF) family. Autosomal dominant and recessive forms of EDA have been also described and are accounted for by two genes. Mutations in EDAR, encoding a TNF receptor (EDAR) cause both dominant and recessive forms. In addition, mutations in a recently identified gene, EDARADD, encoding EDAR-associated death domain (EDARADD) have been shown to cause autosomal recessive EDA. Here, we report a large Moroccan family with an autosomal dominant EDA. We mapped the disease gene to chromosome 1q42.2-q43, and identified a novel missense mutation in the EDARADD gene (c.335T>G, p.Leu112Arg). Thus, the EDARADD gene accounts for both recessive and dominant EDA. EDAR is activated by its ligand, ectodysplasin, and uses EDARADD to build an intracellular complex and activate nuclear factor kappa B (NF-kB). We compared the functional consequences of the dominant (p.Leu112Arg) and recessive mutation (p.Glu142Lys), which both occurred in the death domain (DD) of EDARADD. We demonstrated that the p.Leu112Arg mutation completely abrogated NF-kB activation, whereas the p.Glu142Lys retained the ability to significantly activate the NF-kB pathway. The p.Leu112Arg mutation is probably a dominant negative form as its cotransfection impaired the wild-type EDARADD's ability to activate NF-kB. Our results confirm that NF-kB activation is impaired in EDA and support the role of EDARADD DD as a downstream effector of EDAR signaling.

NF-kappaB-related genetic diseases.

The recent identification of genetic diseases (incontinentia pigmenti, anhidrotic ectodermal dysplasia with immunodeficiency and cylindromatosis) resulting from mutations affecting components of the nuclear factor-kappaB (NF-kappaB) signaling pathway provides a unique opportunity to understand the function of NF-kappaB in vivo. Besides confirming the importance of NF-kappaB in innate and acquired immunity or bone mass control, analysis of these diseases has uncovered new critical roles played by this transcription factor in the development and homeostasis of the epidermis and the proper function of lymphatic vessels. In addition, the identified mutations will help understanding at the molecular level how NF-kappaB is activated in response to cell stimulation.

The human beta fibrinogen promoter contains a hepatocyte nuclear factor 1-dependent interleukin-6-responsive element.

Acute-phase reactants are liver proteins whose synthesis is positively or negatively regulated during inflammation. The main mediators of this phenomenon are glucocorticoids and interleukin-6 (IL-6), a pleiotropic cytokine that also controls hematopoiesis. Functional analysis of several acute-phase reactant promoter regions has identified two major DNA motifs used by IL-6-regulated genes. The first one corresponds to a CTGG(G/A)AA sequence, and the other is a binding site for members of the C/EBP family of nuclear proteins. We have previously shown that the human beta fibrinogen (beta Fg) promoter contains an IL-6-responsive region, located between bp -150 and -67 (P. Huber, M. Laurent, and J. Dalmon, J. Biol. Chem. 265:5695-5701, 1990). In this study, using DNase I footprinting, mobility shift assays, and mutagenesis, we demonstrate that at least three subdomains of this region are necessary to observe a full response to IL-6. The most distal contains a CTGGGAA motif, and its mutation inhibits IL-6 stimulation. Another, which is able to interact with several distinct nuclear proteins, among them members of the C/EBP family, is dispensable for IL-6 induction but plays an important role in the constitutive expression of beta Fg. Finally, a proximal hepatocyte nuclear factor 1 binding site, already described as the major determinant of beta Fg tissue-specific expression, is also required for IL-6 stimulation. These results indicate a complex interplay between nuclear proteins within the beta Fg IL-6-responsive region and suggest a tight functional coupling between the tissue-specific and inducible elements.

Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli.

Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation.

Sp1, a CAAT-binding factor, and the adenovirus major late promoter transcription factor interact with functional regions of the gamma-fibrinogen promoter.

To study the factors which influence the coordinately and developmentally regulated expression of the three adjacent fibrinogen genes, we have defined the functional regions of the gamma-fibrinogen promoter and the proteins which bind to them. Using a series of 5' and internal deletion mutations, we found that sequences between 88 and 43 base pairs (bp) upstream of the gamma-fibrinogen transcription initiation site functioned in cis to direct properly initiated mRNA accumulation in transfected hepatocytes. The efficient function of these sequences was highly distance dependent, since transcriptional activity decreased by 92% when they were moved 32 bp upstream of the TATA box. We demonstrated that two known and one putative transcriptional factors interacted with this 47-bp sequence. The transcription factor Sp1 interacted with sequences between -51 and -46 as demonstrated by protection from DNase I digestion with the purified protein. Directly adjacent to the Sp1 site, between nucleotides -66 and -53, there was a sequence which bound a CAAT-binding factor. Finally, sequences just 5' to the CAAT factor-binding site interacted with the adenovirus major late transcriptional factor as previously demonstrated. Internal deletion mutations which disrupt these interactions diminished the activity of the promoter in vivo. One consequence of the interaction of these proteins is that a bend is placed in the DNA at or near their sites of interaction.

Down-regulation of NF-kappaB activity and NF-kappaB p65 subunit expression by ras and polyoma middle T oncogenes in human colonic Caco-2 cells.

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras or pp60c-src in human colonic cells is associated with alterations of the activity and expression of nuclear factor kappaB (NF-kappaB), a transcription factor suspected to participate in the development of cancer. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated Val-12 human Ha-ras gene or with the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines exhibited: (i) decreased constitutive NF-kappaB DNA-binding activity and NF-kappaB-mediated reporter gene expression, without alteration of their response to TNF-alpha for activation of these parameters; (ii) reduced NF-kappaB cytosolic stores along with a decreased p65 expression due, at least in part, to destabilization of p65 mRNA; (iii) a decrease in adhesion to extracellular matrix component-coated substrata which was partially corrected when stimulating NF-kappaB transcriptional activity with TNF-alpha. These results indicate that the tumorigenic progression induced by oncogenic p21ras or PyMT/pp60c-src in human colonic Caco-2 cells is associated with a down-regulation of p65 expression and NF-kappaB activity which could be responsible for the reduced adhesive properties of these cells after oncogene transfection.

Phosphorylation of ribosomal proteins S3, L1 and L24 during spherulation in Physarum polycephalum.

The phosphate content of ribosomal proteins S3, L1 and L24 has been determined in the course of spherulation of Physarum polycephalum. The major phosphoprotein, S3, was completely dephosphorylated after 4 h of differentiation. The phosphate content of L1 and L24 was not altered during the differentiation. The cellular level of ATP remained constant for at least 5 h. A 3-fold reduction of cyclic AMP concentration occurred in the first hour, followed by a slow increase to a final value of twice the level observed in growing cells. The results showed that the phosphorylation of ribosomal proteins is regulated by at least two different mechanisms and that the dephosphorylation of S3 is not induced by a lack of cellular ATP. Although cyclic AMP might trigger the dephosphorylation of S3, the phosphate content of this protein remained at a very low value even when the cellular concentration of cyclic AMP rose significantly. Since the polysome level remains constant during the first 24 h of spherulation, the phosphorylation of S3 is not necessary for active protein synthesis and the phosphorylation of L1 and L24 is not involved in ribosome inactivation, which occurs after 24 h.

NF-kappa B defects in humans: the NEMO/incontinentia pigmenti connection.

The components of the nuclear factor-kappaB (NF-kappaB) family of transcription factors are critical for regulating the response to immune challenges. Recently, a role for NF-kappaB in skin biology has been revealed. Within the cascade of proteins whose activities impinge upon the activation of NF-kappaB, the NEMO (NF-kappaB essential modulator)/IKKgamma protein is required for the activation of the IkappaB kinases, which in turn, promote the degradation of IkappaB proteins, leading to the derepression of NF-kappaB activity. Courtois and Israël discuss the role of NEMO/IKKgamma in normal physiological activation of NF-kappaB and the consequences of defective NF-kappaB activation, as an effect of NEMO/IKKgamma mutations, which can lead to incontinentia pigmenti, a disease marked by alopecia, tooth eruption, skin lesions, and changes in skin pigmentation.

Protein kinase C-alpha is an upstream activator of the IkappaB kinase complex in the TPA signal transduction pathway to NF-kappaB in U2OS cells.

Inactive nuclear factor kappaB (NF-kappaB) complexes are retained in the cytoplasm by binding to inhibitory proteins, such as IkappaBalpha. Various stimuli lead to phosphorylation and subsequent processing of IkappaBalpha in the 26S proteasome and import of the active NF-kappaB transcription factor into the nucleus. In agreement with our previous finding that p90(rsk1) is essential for TPA-induced activation of NF-kappaB in Adenovirus 5E1-transformed Baby Rat Kidney cells, we now report that the MEK/ERK/p90(rsk1) inhibitor U0126 efficiently blocks TPA-induced IkappaBalpha processing in these cells. However, in U2OS cells, the cytokine-inducible IkappaB kinase complex (IKK) is the essential component of the TPA signal transduction pathway. Activation of the IKK complex in response to TPA is mediated by PKC-alpha, since both the PKC inhibitor GF109203 and a catalytically inactive PKC-alpha mutant inhibit activation of endogenous IKK by TPA, but not by tumor necrosis factor-alpha (TNF-alpha). We conclude that IKK is an integrator of TNF-alpha and TPA signal transduction pathways in U2OS cells.

Mapping the DNA fragments produced by cleavage of EcoRI F fragment of adenovirus 2 with HaeIII, HpaII and AluI.

Hydrolysis of the EcoRI F fragment of the adenovirus 2 genome with HaeIII, HpaII and AluI restriction enzymes gives respectively 9, 11, and 11 fragments, the size of which ranges from 20 bp for the smallest HpaII k fragment to 585 for the largest AluI A fragment. The order of fragments was mainly deduced from partial hydrolysis analyses. The relative order of all restriction sites and the distance in nucleotides between them were obtained through secondary analyses of each restriction fragment by the two other enzymes. Position of the KpnI, HindIII, MboI and SmaI sites within the EcoRI F fragment were also reevaluated.

Nucleotide sequence of the EcoRI-F fragment of adenovirus 2 genome.

Using the DNA sequence method of Maxam and Gilbert the entire nucleotide sequence of the adenovirus 2 EcoRI-F fragment was determined. Information contained in that nucleotide sequence, which is 1743 base pairs long, is interpreted with respect to the mapping and processing of the three mRNAs partly encoded by the EcoRI-F fragment. A method to rapidly determine the cleavage site of restriction endonucleases is also reported.

Differentiation of murine pre-B cell line by an adjuvant muramyl peptide via NF-kappa B activation.

Muramyl dipeptide (MDP) induces NF-kappa B activation in the murine pre-B cell line 70Z/3, increases the expression of surface immunoglobulins, and potentiates the response to other inducers such as LPS or IL-1. In the present study we investigated whether NF-kappa B activation was related to the MDP-stimulated immunoglobulin expression. In a gel shift assay our results confirmed that MDP but not MDP(D,D), an adjuvant-inactive stereoisomer, could induce a kappa B-binding activity in 70Z/3 cells. The LPS or IL-1 induced NF-kappa B binding activity was increased in the presence of MDP but not of MDP(D,D). A mutant of the cell line called 1.3E2, defective in NF-kappa B activations by LPS, did not respond to MDP. The enhanced surface immunoglobulin expression induced in the wild type 70Z/3 cells by MDP alone or combined to LPS, IL-1 or IFN gamma was not obtained in this variant. The ability of various treatments to activate the kappa gene enhancer was quantitatively evaluated in cells transfected with a kappa-enhancer-luciferase expression plasmid. Treatment of transfected 70Z/3 cells with MDP resulted in a dose-dependent enhancement of luciferase activity, an additive effect to that induced by LPS or IL-1. Treatment of the defective variant transfected with the same construct did not result in luciferase expression after stimulation with the various agents. The transient transfection assays were used to compare the effectiveness of some MDP analogs. Two adjuvant-active compounds unable to enhance kappa light chain expression did not increase the basal response in the transfected 70Z/3 cells, indicating that NF-kappa B activation was not related to the adjuvant potency of MDP but correlated with the kappa induction.

High-performance liquid chromatographic determination of Maillard compounds in store-brand and name-brand ultra-high-temperature-treated cows' milk.

Furosine and furfural products of the Maillard reaction are used as specific indicators of the effect of heating treatments on milk quality. Their contents were measured in representative samples of store- and name-brand ultra-high-temperature-treated milks using RP-HPLC with UV detection. Furosine contents ranged from 40.32 to 50.67 and from 65.48 to 310.58 mg/100 g protein in name- and store-brand milks, respectively. Of the furfurals, only hydroxymethylfurfural was detected. The free hydroxymethylfurfural contents of store-brand milks ranged from 0.22 to 1.70 mg/100 g protein. Total hydroxymethylfurfural contents ranged from 0.29 to 0.41 and from 0.72 to 2.21 mg/100 g protein, for name- and store-brands, respectively.

Acute necrotizing ulcerative gingivitis. A transmission electron microscope study.

The purpose of this study was to provide additional evidence that in the lesions of acute necrotizing ulcerative gingivitis (ANUG), spirochetes and other microorganisms are capable of invading the nonnecrotic lamina propria of affected gingivae. Biopsies from eight patients with pathognomonic signs of ANUG were obtained under local anesthesia and processed for light and transmission electron microscopy. The results indicated that spirochetes and other bacteria are capable of penetrating the viable connective tissues of the lamina propria. The four zones of the ANUG lesion as first described by Listgarten were observed without difficulty. The maximum depth of bacterial infiltration into the lamina propria ranged from 155 to 400 microns from the nearest epithelial basal lamina. The histologic picture of the ANUG lesion did not always conform to the acute clinical manifestations.

[HSP70, an erythropoiesis regulator that determines the fate of erythroblasts between death and differentiation]. / HSP70 un régulateur de l'érythropoïèse qui détermine le destin des érythroblastes entre mort et différenciation.

Erythropoiesis is finely regulated by two major cytokines, stem cell factor (SCF) and erythropoietin (Epo). Decrease levels of Epo result in caspase activation and erythroid progenitors apoptosis. However, normal erythroid cell maturation requests caspase activation and cleavage of various caspase substrates, except the erythroid transcription factor GATA-1, that is protected by interaction with the chaperone HSP70 in the nucleus. Therefore, molecular abnormalities associated with decrease of HSP70 expression in the nucleus may result in ineffective erythropoiesis characterized by apoptosis and impaired maturation of erythroid precursors. These findings open new potential targeted therapies for erythroid disorders.