Truncation of C-mip (Tc-mip), a New Proximal Signaling Protein, Induces c-maf Th2 Transcription Factor and Cytoskeleton Reorganization

Several arguments suggest that minimal change nephrotic syndrome (MCNS) results from yet unknown systemic disorder of T cell function. By screening a cDNA library from T cell relapse, we identified a new pleckstrin homology (PH) domain-containing protein encoded by a gene located on chromosome 16q24. Two alternative transcripts were identified. The first species (c-mip) was expressed in fetal liver, kidney, and peripheral blood mononuclear cells (PBMCs), but weakly detected in PBMCs from MCNS patients. The second form (Tc-mip, standing for truncated c-maf inducing protein), corresponds to subtracted transcript and lacks the NH(2)-terminal PH domain. The expression of Tc-mip was restricted to fetal liver, thymus, and MCNS PBMCs where it was specifically recruited in CD4(+) T cells subset. Overexpression of Tc-mip in T cell Jurkat induced c-maf, transactivated the interleukin 4 gene and down-regulated the interferon γ expression, characteristic of a Th2 commitment. Moreover, the overexpression of Tc-mip induced Src phosphorylation, T cell clustering, and a cellular redistribution of the cytoskeleton-associated L-plastin, by a PI3 kinase independent pathway. Tc-mip represents therefore the first identified protein, which links proximal signaling to c-maf induction

Paradoxical embolism following thromboaspiration of an arteriovenous fistula thrombosis: a case report

<p>Abstract</p> <p>Introduction</p> <p>Paradoxical embolism is an increasingly reported cause of arterial embolism. Several embolic sources have been described, but thrombosis of an arteriovenous fistula as a paradoxical emboligenic source has not, to the best of our knowledge, been reported.</p> <p>Case presentation</p> <p>A 50-year-old Caucasian woman received a renal graft for primary hyperoxaluria. After transplantation, she was maintained on daily hemodialysis. Thrombosis of her arteriovenous fistula occurred two weeks post-transplantation and was treated by thromboaspiration, which was partially successful. During a hemodialysis session immediately following thromboaspiration, she developed a coma with tetraplegia requiring intensive cardiorespiratory resuscitation. Brain magnetic resonance imaging revealed various hyperdense areas in the vertebrobasilar territory resulting from bilateral occlusion of posterior cerebral arteries. Transesophageal echocardiographic examination showed a patent foramen ovale, while pulse echography of the arteriovenous fistula revealed the persistence of extensive clots that were probably the embolic source. A paradoxical embolus through a patent foramen ovale was suggested because of the proximity of the neurological event to the thrombectomy procedure.</p> <p>Conclusions</p> <p>The risk of paradoxical embolism in a hemodialyzed patient with a patent foramen ovale deserves consideration and requires careful evaluation in situations of arteriovenous fistula thrombosis.</p

Rôle de la protéine C-MIP dans la physiopathologie moléculaire du syndrome néphrotique à lésions glomérulaires minimes

Le syndrome néphrotique à lésions glomérulaires minimes (SNLGM) à rechutes est la forme la plus fréquente des néphropathies glomérulaires chez l enfant. Il est considéré comme une pathologie dysimmunitaire dans laquelle des perturbations lymphocytaires T entraîneraient la sécrétion d un facteur circulant dont l interaction avec les podocytes causerait une désorganisation de la barrière de filtration et une protéinurie massive. Malgré de nombreuses études moléculaires et des avancées scientifiques indiscutables sur les formes génétiques, la pathogénie du SNLGM à rechutes reste une énigme. Nous avons trouvé que c-mip (c-maf induced protein) est surexprimée dans les lymphocytes ainsi que dans les podocytes et les tubules distaux de malades en poussée du SNLGM. Nous avons montré que les souris transgéniques qui surexpriment c-mip dans les podocytes présentent une protéinurie massive et reproduisent des lésions histologiques comparables à celles des patients atteints de SNLGM. Nous avons montré, in vitro, que la protéine c-mip interagit avec Fyn, inhibe son activation et rompt la liaison de Fyn avec la néphrine. La signalisation podocytaire de la néphrine est ainsi rompue entraînant une désorganisation du cytosquelette et une proteinurie massive. D autre part, nous avons démontré que la surexpression de c-mip, in vitro dans les lymphocytes T, inhibe l activation des voies NF B et AP-1. La protéine c-mip immunoprécipite avec le complexe p50/p65/I Ba et empêche la dégradation de I Ba et par conséquent la libération du dimère NF B et sa translocation nucléaire. La protéine c-mip bloque la translocation nucléaire de phospho-ERK1/2 via le recrutement de la DAPK ce qui empêche le facteur de transcription AP-1 d activer ses gènes cibles. Nous avons démontré pour la première fois l implication d une même protéine au niveau de la signalisation des lymphocytes T et des podocytes, deux cellules clés touchées au cours du SNLGM. La protéine c-mip est dotée de fonctions répressives dont les conséquences fonctionnelles dans les lymphocytes T et les podocytes sont différentes et peuvent expliquer le phénotype bipolaire de la maladie.Minimal Change Nephrotic Syndrome (MCNS) with relapse is an acquired glomerular disease characterized by a heavy proteinuria without inflammatory lesions or cell infiltrations. The pathophysiology of this disease remains an enigma. We showed that c-mip (c-maf inducing protein) is up-regulated in both T cells and podocytes, during the active phase of MCNS. We generated c-mip transgenic mice using the nephrin promoter to restrict transgene expression to podocytes. c-mip transgenic mice developed morphological and biochemical alterations similar to MCNS disease. We showed that c-mip switches off the podocyte proximal signaling by preventing the interaction between Fyn and nephrin. We also showed that c-mip blocks NF B and AP-1 signaling in T cells. c-mip interacts with the p65/p50/I Ba complex and inhibits I Ba degradation resulting in p65/p50 cytosolic sequestration. c-mip blocks phospho-ERK1/2 nuclear translocation via DAPK recruitment. Cytosolic phospho-ERK1/2 fails to activate AP-1 and consequently the transcription of its target genes. We are the first to describe the implication of a single protein, c-mip, in the regulation of the signaling pathways of both podocytes and T cells. c-mip functions as a negative regulator, the impacts of which on podocytes and T cells are different and might explain the bipolarity of the disease.PARIS12-Bib. électronique (940280011) / SudocSudocFranceF

The Tumor Necrosis Factor α-dependent Activation of the Human Mediterranean Fever (MEFV) Promoter Is Mediated by a Synergistic Interaction between C/EBPβ and NFκB p65

International audienceMEFV is a gene expressed specifically in myeloid cells and whose mutations underlie an autosomal recessive auto-inflammatory disease, called familial Mediterranean fever (FMF), characterized by recurrent episodes of serosal inflammation. This gene, which encodes a protein with unclear physiological functions, has been shown to be up-regulated by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). However, the mechanism of this regulation is unknown, and the MEFV promoter is still to be characterized. Here, we show that 243 bp of the 5'-flanking region of the human MEFV gene are sufficient to direct high level expression of MEFV in TNFalpha-treated cells. The TNFalpha-induced expression of MEFV is dependent on both NFkappaB p65 and C/EBPbeta that bind to evolutionarily conserved sites located, in the human promoter, at positions -163 and -55, respectively. As shown by a series of transcription and gel shift assays performed with wild-type and mutated promoter sequences, these two transcription factors act differently on the TNFalpha-dependent transcription of MEFV: C/EBPbeta is the key regulatory factor required to confer cell responsiveness to TNFalpha, whereas NFkappaB p65 increases this response by means of a synergistic interaction with C/EBPbeta that is dependent on the integrity of the identified -55 C/EBP binding site. Given the phenotype of patients with FMF, this C/EBP-NFkappaB interaction may represent a key step in the control of an inflammatory response that is abnormally high in this disease. These data, which shed novel light on the pathophysiology of FMF, represent an unusual example of cross-talk between C/EBP and NFkappaB pathways in TNFalpha signaling

Immunopathogenesis of idiopathic nephrotic syndrome.

International audienceIdiopathic nephrotic syndrome is the most frequent glomerular disease in children. The mechanisms underlying its pathophysiology have been investigated by genetic, cellular and molecular approaches. While genetic analyses have provided new insights into disease pathogenesis through the discovery of several podocyte genes mutated in distinct forms of inherited nephrotic syndrome, the molecular bases of minimal change nephrotic syndrome and focal and segmental glomerulosclerosis with relapse remain unclear. The immune system seems to play a critical role in the active phase of this disease through disturbances involving several cell subsets, mainly T cells. The innate immune system may also contribute to the immune disorders. In this review, we discuss recent insights from the molecular and immunological findings and their significance in the context of the clinical course of the disease

Differential expression of NFRKB in MCNS.

<p><b>A</b>, RT-PCR analyses in six patients with MCNS during the relapse and remission phase. The top panel shows the quantification of PCR products, as determined using the Image Quant, version 1.11, analysis software, after normalization against the corresponding GAPDH mRNA values (▪, relapse; □, remission). These data are representative of two independent experiments. <b>B</b>, Western blots of PBMC protein lysates using anti-NFRKB antibody reveals a larger size of the protein (≈220 kDa), compared with the band visualized in jurkat cell lysates. The specificity was confirmed by the loss of the binding upon preincubation of antibody with specific peptides. These data are representative of three independent experiments. <b>C</b>, Western blot analysis of NFRKB abundance in PBMC of patients with MCNS relapse or remission and in normal subjects. These data are representative of three independent experiments. <b>D</b>, The relative abundance of NFRKB was assessed by quantifying the specific band after normalization with the corresponding actin, using the Image J software. The amount of NFRKB is significantly increased in relapse, as compared with remission or normal subjects (*p<0.05; ** p<0.01, unpaired t test).</p

Expression of NFRKB in Jurkat cells.

<p><b>A,</b> Western blot of total lysates using anti-NFRKB reveals a double band at the expected size. The specificity was attested by the loss of the binding upon preincubation of antibody with synthetic peptides corresponding to positions 543–557 and 1091–1106 within the primary structure of NFRKB. These data are representative of three independent experiments. <b>B, </b><b>Northern blot analysis of NFRKB.</b> Multiple tissue-Northern blot (Clontech Laboratories, Inc.) was hybridized with a full-length-probe and exposed to Phosphoimager Storm for 24 h. This experiment was repeated once.</p

Sequence of primers and PCR conditions.

<p>The underlined sequences correspond to attB-sites used to incorporate the PCR product into the gateway plasmids.</p

Post-translational modifications of NFRKB.

<p><b>A</b>, representative experiment of N-glycosidase-F traitement. Total protein lysates were purified from PBMC, treated with the N-glycosidase-F, then immunoblotted with anti-NFRKB antibody. Note that the band size is reduced by ∼20 kDa. <b>B</b>, Immunoprecipitation of NFRKB from PBMC protein lysates followed by immunoblotting with anti-sumo-1 or anti-sumo-2/3. These data are representative of two independent experiments.</p

NFRKB induces hypomethylation of genomic DNA.

<p>Genomic DNA was prepared from NFRKB-transfected HEK cells or empty vector and methylation assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030523#s4" target="_blank">Materials and Methods</a>. Statistical analyses were carried out on data from five independent experiments using the two-tailed <i>t</i>-test (*<i>p</i><0.05; unpaired <i>t</i>-test).</p