c-mip Down-Regulates NF-κB Activity and Promotes Apoptosis in Podocytes
2012; Elsevier BV; Volume: 180; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2012.02.008
ISSN1525-2191
AutoresVirginie Ory, Qingfeng Fan, Nabila Hamdaoui, Shaoyu Zhang, Dominique Desvaux, Vincent Audard, Marina Candelier, Laure‐Hélène Noël, Philippe Lang, Georges Guellaën, André Pawlak, Dil Sahali,
Tópico(s)Dermatological and Skeletal Disorders
ResumoThe mechanisms of podocyte disorders in cases of idiopathic nephrotic syndrome (INS) are complex and remain incompletely elucidated. The abnormal regulation of NF-κB may play a key role in the pathophysiology of these podocyte diseases, but at present, NF-κB has not been thoroughly investigated. In this study, we report that induction of c-mip in podocytes of patients with INS is associated with a down-regulation of RelA, a potent antiapoptotic factor that belongs to the NF-κB family. Overexpression of c-mip in differentiated podocytes promotes apoptosis by inducing caspase-3 activity and up-regulating the proapoptotic protein Bax, whereas the overall levels of the antiapoptotic protein Bcl-2 was concomitantly decreased. The associated overexpression of RelA prevented the proapoptotic effects of c-mip. In addition, the targeted induction of c-mip in podocytes in vivo inhibited the expression of the RelA protein and increased the Bax/Bcl-2 ratio. The expression of both c-mip and active caspase-3 increased in focal and segmental glomerulosclerosis biopsies, and both proteins displayed a close spatial relationship. These results suggest that alterations in NF-κB activity might result from the up-regulation of c-mip and are likely to contribute to podocyte disorders in cases of INS. The mechanisms of podocyte disorders in cases of idiopathic nephrotic syndrome (INS) are complex and remain incompletely elucidated. The abnormal regulation of NF-κB may play a key role in the pathophysiology of these podocyte diseases, but at present, NF-κB has not been thoroughly investigated. In this study, we report that induction of c-mip in podocytes of patients with INS is associated with a down-regulation of RelA, a potent antiapoptotic factor that belongs to the NF-κB family. Overexpression of c-mip in differentiated podocytes promotes apoptosis by inducing caspase-3 activity and up-regulating the proapoptotic protein Bax, whereas the overall levels of the antiapoptotic protein Bcl-2 was concomitantly decreased. The associated overexpression of RelA prevented the proapoptotic effects of c-mip. In addition, the targeted induction of c-mip in podocytes in vivo inhibited the expression of the RelA protein and increased the Bax/Bcl-2 ratio. The expression of both c-mip and active caspase-3 increased in focal and segmental glomerulosclerosis biopsies, and both proteins displayed a close spatial relationship. These results suggest that alterations in NF-κB activity might result from the up-regulation of c-mip and are likely to contribute to podocyte disorders in cases of INS. Idiopathic nephrotic syndrome (INS) defines several entities, including minimal change nephrotic syndrome (MCNS) and focal and segmental glomerulosclerosis (FSGS), which are considered to be prototypic podocyte diseases.1Mathieson P.W. Minimal change nephropathy and focal segmental glomerulosclerosis.Semin Immunopathol. 2007; 29: 415-426Crossref PubMed Scopus (46) Google Scholar Podocytes are terminally differentiated cells that line the outer aspect of the glomerular basement membrane and constitute the ultimate barrier to urinary protein loss by the formation and maintenance of the podocyte foot processes and the interposed slit diaphragm.2Tryggvason K. Patrakka J. Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria.N Engl J Med. 2006; 354: 1387-1401Crossref PubMed Scopus (444) Google Scholar Podocyte diseases might result from genetic defects of proteins playing a key structural and/or regulatory role in the integrity of the glomerular filtration barrier.3Shankland S.J. The podocyte's response to injury: role in proteinuria and glomerulosclerosis.Kidney Int. 2006; 69: 2131-2147Crossref PubMed Scopus (659) Google Scholar Regardless of the underlying cause, the early podocyte damages are characterized by ultrastructural alterations of the slit diaphragm. Cellular injuries may progress through a reversible stage to podocyte depletion and glomerulosclerosis, which commonly complicate the outcome of chronic glomerular diseases in INS. In acquired INS, the mechanisms preceding these changes remain to be elucidated. The NF-κB family of transcription factors plays a central role in many cellular processes through the regulation of genes involved in immunity, inflammation, cell proliferation, differentiation, and apoptosis. In mammalian cells, the NF-κB family consists of five members, NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), RelB, and cRel.4Shih V.F. Tsui R. Caldwell A. Hoffmann A. A single NF-kappa B system for both canonical and non-canonical signaling.Cell Res. 2011; 21: 86-102Crossref PubMed Scopus (323) Google Scholar The hallmark of the NF-κB family is the presence in all members of a Rel-homology domain at the NH2-teminus, responsible for DNA binding, dimerization, and association with the I-κB inhibitory proteins.5Zheng C. Yin Q. Wu H. Structural studies of NF-kappa B signaling.Cell Res. 2011; 21: 183-195Crossref PubMed Scopus (96) Google Scholar Among the NF-κB proteins, only RelA, RelB, and c-Rel have a COOH-terminus transactivation domain, which is lacking in p50 and p52, so that RelA, RelB, and c-Rel homo- or heterodimers function as transcriptional activators, whereas p50 and p52 homodimers function as repressors.4Shih V.F. Tsui R. Caldwell A. Hoffmann A. A single NF-kappa B system for both canonical and non-canonical signaling.Cell Res. 2011; 21: 86-102Crossref PubMed Scopus (323) Google Scholar Although in most cases, Rel proteins act as transcriptional activators, they may repress some target genes.6Fu T. Li P. Wang H. He Y. Luo D. Zhang A. Tong W. Zhang L. Liu B. Hu C. c-Rel is a transcriptional repressor of EPHB2 in colorectal cancer.J Pathol. 2009; 219: 103-113Crossref PubMed Scopus (15) Google Scholar, 7Ashburner B.P. Westerheide S.D. Baldwin Jr., A.S. The p65 (RelA) subunit of NF-kappa B interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression.Mol Cell Biol. 2001; 21: 7065-7077Crossref PubMed Scopus (625) Google Scholar Because of its implication in many biological processes and the adverse consequences of its dysregulation, the activity of NF-κB is tightly regulated at multiple translational and posttranslational levels. The most common regulatory mechanism relies on the actions of the inhibitory I-κB proteins.8Ferreiro D.U. Komives E.A. Molecular mechanisms of system control of NF-kappa B signaling by I-kappa B alpha.Biochemistry. 2010; 49: 1560-1567Crossref PubMed Scopus (104) Google Scholar In resting cells, I-κB protein binds to the nuclear localization site of the Rel-homology domain, preventing the nuclear translocation of NF-κB protein and thereby its transcriptional activity. On activation, I-κB protein is phosphorylated, which allows its ubiquitination and subsequent degradation by proteasome. We recently shown that c-mip interacts with RelA and prevents its dissociation from the NFκB/IκBα complexes, resulting in the inhibition of nuclear translocation of NFκB and stabilization of IκBα.9Kamal M. Valanciute A. Dahan K. Ory V. Pawlak A. Lang P. Guellaen G. Sahali D. C-mip interacts physically with RelA and inhibits nuclear factor kappa B activity.Mol Immunol. 2009; 46: 991-998Crossref PubMed Scopus (26) Google Scholar In human and experimental glomerular diseases, several mediators regulated by NF-κB such as cytokines and adhesion molecules have been reported to play pathogenic roles in inflammatory and proliferative glomerular diseases.10Mudge S.J. Paizis K. Auwardt R.B. Thomas R.J. Power D.A. Activation of nuclear factor-kappa B by podocytes in the autologous phase of passive Heymann nephritis.Kidney Int. 2001; 59: 923-931Crossref PubMed Scopus (45) Google Scholar In nonproliferative glomerular diseases characterized by heavy proteinuria, such as minimal change disease and membranous nephropathy, it has been suggested that the excess of proteins in the tubules and their subsequent degradation induce tubular expression of many proinflammatory genes through NF-κB activation.11Remuzzi G. Ruggenenti P. Benigni A. Understanding the nature of renal disease progression.Kidney Int. 1997; 51: 2-15Crossref PubMed Scopus (614) Google Scholar To our knowledge, the mechanisms of NF-κB regulation in the podocytes are little known. We recently reported that c-mip is overproduced in the podocytes during glomerular damages and interferes with signaling pathways playing a key role in podocyte function. Whether c-mip influences NF-κB activity has not been investigated. We report here that c-mip represses in vitro and in vivo NF-κB activity by down-regulating the expression of RelA. This effect was only observed in podocytes, whereas tubular cells displayed a contrasting increase of NF-κB activity. Moreover, c-mip increases the levels of Bax and enhances caspase-3 activity, but it reduces the Bcl-2 level, suggesting that c-mip exerts a proapoptotic function. The diagnosis of kidney disease was performed by renal biopsy, which was performed before the start of treatment. All patients had proteinuria above 3 g in 24 hours and severe hypoalbuminemia at the time of blood sampling. MCNS and FSGS were clinically classified as idiopathic in all cases. Control for c-mip screening includes adult patients with glomerular diseases (IgA, lupus nephritis) who exhibited a nephrotic syndrome. Normal renal samples were supplied by the hospital tissue bank (Platform of Biological Resources, Henri Mondor Hospital) from patients undergoing nephrectomy for polar kidney tumor. The expression plasmids for c-mip and RelA have been previously described.12Valanciute A. le Gouvello S. Solhonne B. Pawlak A. Grimbert P. Lyonnet L. Hue S. Lang P. Remy P. Salomon R. Bensman A. Guellaen G. Sahali D. NF-kappa B p65 antagonizes IL-4 induction by c-maf in minimal change nephrotic syndrome.J Immunol. 2004; 172: 688-698PubMed Google Scholar, 13Zhang S.Y. Kamal M. Dahan K. Pawlak A. Ory V. Desvaux D. Audard V. Candelier M. Mohamed F.B. Matignon M. Christov C. Decrouy X. Bernard V. Mangiapan G. Lang P. Guellaen G. Ronco P. Sahali D. c-mip impairs podocyte proximal signaling and induces heavy proteinuria.Sci Signal. 2010; 3: ra39PubMed Google Scholar Mouse total RNA was prepared from glomerular fractions isolated by graded sieving, using RNeasy kit (Qiagen, Courtaboeuf, France). Quantitative real-time RT-PCR (RT-qPCR) for RelA was performed using the oligonucleotides sense (5′-TGTTACCATCAGGGCAGATC-3′) and anti-sense (5′-CAGGGTACTCCATCAGCATG-3′). The samples (2 μL of the reverse transcription reaction mixture, corresponding to 20 ng of total RNA) were amplified in a 20-μL reaction mixture containing 0.5 mmol/L of each primer and 1× LightCycler DNA Master SYBR Green buffer (Roche Molecular Biochemical, Mannheim, Germany). RT-qPCR conditions include an initial denaturing step at 95°C for 15 minutes, followed by 40 cycles (denaturing: 95°C, 15 seconds; annealing: 59°C, 30 seconds; extension, 72°C, 35 seconds). Conditionally immortalized mouse podocytes have been described elsewhere.14Mundel P. Reiser J. Zuniga Mejia Borja A. Pavenstadt H. Davidson G.R. Kriz W. Zeller R. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (765) Google Scholar Before transfection, podocytes were maintained at 60% confluence, under permissive conditions (cells were cultured in RPMI 1640 medium containing 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 U/mL γ-INF, at 33°C). The full-length human c-mip was inserted into a pDEST40 plasmid (Invitrogen, Carlsbad, CA). Proliferating podocytes were transfected with c-mip expression plasmid (1 μg/106 cells) using the Amaxa system. Following transfection, cells were maintained in complete medium for 24 hours, then Geneticin (antibiotic G-418 sulfate; Invitrogen) was added at 400 μg/mL (100% lethal to untransfected cells). Stably transfected cells were selected in serial passages using Geneticin, then stored in liquid nitrogen or expanded under permissive conditions in the presence of Geneticin until they were in sufficient number. Podocyte clones were isolated by limiting dilution and stored in liquid nitrogen. For transient transfection, podocytes were transfected with c-mip expression plasmid (pDEST40) alone or cotransfected with empty vector or RelA, using the Nanofectin method according to the instructions provided by the manufacturer (PAA Laboratories, Pasching, Austria). In some experiments, podocytes were incubated with 2 μmol/L lactacystin (Sigma-Aldrich, St. Louis, MO) for 20 hours, and then protein lysates were prepared as previously described.13Zhang S.Y. Kamal M. Dahan K. Pawlak A. Ory V. Desvaux D. Audard V. Candelier M. Mohamed F.B. Matignon M. Christov C. Decrouy X. Bernard V. Mangiapan G. Lang P. Guellaen G. Ronco P. Sahali D. c-mip impairs podocyte proximal signaling and induces heavy proteinuria.Sci Signal. 2010; 3: ra39PubMed Google Scholar Caspase-3 activity was monitored in a quantitative assay using a fluorogenic substrate. Podocytes stably transfected with c-mip or expressing empty vector (∼200,000 cells) were grown on 60-mm dishes at 37°C for 96 hours. Floating cells were collected and centrifuged, and the pellet was lysed in 50 μL of lysis buffer containing 50 mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, 1% Nonidet P-40, 1 mmol/L EDTA (pH 8.0), 1 mmol/L dithiothreitol, 2 μg/mL leupeptin, 2 μg/mL aprotinin. Adherent cells were washed three times with cold PBS and lysed for 10 minutes on ice in 0.2 mL of lysis buffer. The lysates from adherent and floating cells were pooled and centrifuged, and the supernatants were collected. The activity of caspase recognizing the DEVD (Asp-Glu-Val-Asp) motif (DEVDase activity) was measured in 200 μL of assay buffer containing 100 mmol/L HEPES (pH 7.4), 10% sucrose, 10 mmol/L dithiothreitol, 500 μmol/L EDTA, 50 μg of protein, and 20 μmol/L N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (AC-DEVD-AFC) as fluorogenic substrate (Biomol, Tebu, France). After 3 hours at 37°C, the fluorescence of the reaction mixture was monitored every 30 minutes for 7 hours, using a spectrofluorometer (FL600 Fluorescence Microplate Reader; Biotek Instruments, Winooski, VT), with excitation and emission wavelengths of 400 and 530 nm, respectively. Paraffin-embedded kidney tissue sections were dewaxed, rehydrated, and then treated with proteinase K solution [10 μg/mL in 10 mmol/L Tris-HCl (ph7.4)] for 30 minutes at room temperature. The sections were rinsed twice with PBS and the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Mannheim, Germany).was used to label apoptotic cells. The sections were incubated with 50 μL of TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) reaction mixture for 60 minutes at 37°C in a humidified atmosphere in the dark. Terminal transferase was omitted for the negative control. As a positive control, one section was incubated with DNase I [2000 U/mL in 50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl2, and 1 mg/mL BSA] for 10 minutes at room temperature to induce DNA strand breaks, prior to the labeling procedures. The sections were rinsed three times with PBS, and then incubated with 50 μL of Converter-POD on the sample, for 30 minutes at 37°C. The sections were rinsed three times with PBS, and then 50 μL of diaminobenzidine substrate and incubated for several minutes at room temperature. Finally, the sections were rinsed with PBS, mounted under glass coverslips, and analyzed under a light microscope. For light microscopy, the kidney sections from wild-type and c-mip transgenic (Tg) mice were incubated for 16 hours in Dubosc-Brasil solution, and subsequently dehydrated, paraffin embedded, and stained with periodic acid-Schiff reagent (PAS). For immunohistochemistry study, antigen retrieval was performed by immersing the slides in boiling 0.01 mol/L citrate buffer in a 500-W microwave oven for 15 minutes. The endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 30 minutes. Slides were incubated with the blocking reagents consisting of the avidin–biotin solution for 30 minutes and the normal blocking serum for 20 minutes, and then incubated overnight with a specific polyclonal antibody. After washing with PBS, they were incubated with biotinylated goat anti-rabbit antibody. An avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Reagent; Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine (Sigma Biochemical, St. Louis, MO) as a chromogen were applied for visualization of the immunoreaction. Anti-RelA, anti–c-mip,13Zhang S.Y. Kamal M. Dahan K. Pawlak A. Ory V. Desvaux D. Audard V. Candelier M. Mohamed F.B. Matignon M. Christov C. Decrouy X. Bernard V. Mangiapan G. Lang P. Guellaen G. Ronco P. Sahali D. c-mip impairs podocyte proximal signaling and induces heavy proteinuria.Sci Signal. 2010; 3: ra39PubMed Google Scholar and anti-cleaved caspase-3 (Asp175) were used at a final concentration of 1:50, 1:200, and 1:30 dilution, respectively. Omission of the primary antibody was considered a negative control. Immunofluorescence studies on kidney tissues were performed with 4-μm-thick cryostat sections fixed in acetone for 10 minutes, air-dried 30 minutes at room temperature, and then kept in PBS for 3 minutes and blocked in 1% BSA-PBS. The sections were incubated with the indicated antibodies for 1 hour at room temperature, washed with PBS, and incubated with fluorescein isothiocyanate–conjugated or red fluorescent dye–conjugated secondary antibodies. For double fluorochrome labeling, the slides were simultaneously incubated with rabbit anti–c-mip antibody and mouse anti-nephrin antibody. After washing with PBS, the slides were simultaneously incubated with fluorescein isothiocyanate–conjugated goat anti-rabbit IgG and cyanine 3–conjugated sheep anti-mouse IgG. Sections were examined by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany) using red and green filters. The primary antibodies used in this study included anti-RelA, anti-Tie2, anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, CA), anti-cleaved caspase-3 (Asp 75), anti-Bax, (Cell Signaling Technology, Danvers, MA), anti–Bcl-2 (BD Transduction Laboratories; BD Biosciences, San Jose, CA), monoclonal anti-vimentin (Dako, Glostrup, Denmark), monoclonal anti-GAPDH (Abcam, Cambridge, UK), anti-actin (Sigma-Aldrich), and anti-nephrin (Progen, Heidelberg, Germany). The anti–c-mip polyclonal antibody has been previously described.15Audard V. Zhang S.Y. Copie-Bergman C. Rucker-Martin C. Ory V. Candelier M. Baia M. Lang P. Pawlak A. Sahali D. Occurrence of minimal change nephrotic syndrome in classical Hodgkin lymphoma is closely related to the induction of c-mip in Hodgkin-Reed Sternberg cells and podocytes.Blood. 2010; 115: 3756-3762Crossref PubMed Scopus (51) Google Scholar Western blot analysis was performed, using classical methods. The generation of c-mip transgenic mice has been previously described.13Zhang S.Y. Kamal M. Dahan K. Pawlak A. Ory V. Desvaux D. Audard V. Candelier M. Mohamed F.B. Matignon M. Christov C. Decrouy X. Bernard V. Mangiapan G. Lang P. Guellaen G. Ronco P. Sahali D. c-mip impairs podocyte proximal signaling and induces heavy proteinuria.Sci Signal. 2010; 3: ra39PubMed Google Scholar All experiments involving animals were conducted in accordance with French laws. All of the mice analyzed in this study were hemizygous males [Tg(+)] from the F8 to F12 generations. Morphological lesions were studied on 4-mm PAS-stained sections. Five kidney sections by mouse (n = 5 mice for each group) were analyzed. Between 20 and 30 glomeruli per kidney section were randomly selected. Statistical analyses of the data were performed using PRIZM 4 for Macintosh (GraphPad Software, La Jolla, CA). Unpaired or paired Student's t-tests were used. P values of less than 0.05 were considered significant. Although the role of NF-κB in podocytes is poorly understood, NF-κB likely mediates important functions including regulation of signaling pathways and podocyte survival.16Sanz A.B. Sanchez-Nino M.D. Ramos A.M. Moreno J.A. Santamaria B. Ruiz-Ortega M. Egido J. Ortiz A. NF-kappa B in renal inflammation.J Am Soc Nephrol. 2010; 21: 1254-1262Crossref PubMed Scopus (434) Google Scholar To investigate the possible influence of c-mip on NF-κB activity in podocytes of glomerular diseases, we studied the kidney biopsies of patients with INS. As previously reported, c-mip was clearly induced in kidney biopsies of patients with MCNS, but it was scarcely or not detected in normal human kidney.13Zhang S.Y. Kamal M. Dahan K. Pawlak A. Ory V. Desvaux D. Audard V. Candelier M. Mohamed F.B. Matignon M. Christov C. Decrouy X. Bernard V. Mangiapan G. Lang P. Guellaen G. Ronco P. Sahali D. c-mip impairs podocyte proximal signaling and induces heavy proteinuria.Sci Signal. 2010; 3: ra39PubMed Google Scholar The distribution pattern shows that c-mip was not confined to a cytoplasmic compartment, it was also visualized in nuclei (Figure 1A). By contrast, we did not detect c-mip in glomeruli of IgA nephropathy or proliferative lupus nephritis. Immunohistochemistry analysis on the same kidney specimens (5 normal specimens and 12 MCNS) using an anti-RelA antibody showed that RelA was clearly detected in normal podocytes, but it was dramatically altered in the podocytes of MCNS (Figure 1, A and B). Confocal microscopy analysis of normal glomeruli (Figure 2) shows that RelA colocalized partially with nephrin, but not with Tie2, suggesting that its expression is restricted to podocytes and did not overlap with vimentin but mostly colocalized with lamin-B, a marker of nuclear membrane. In some podocytes, RelA was also localized within the nucleus. By contrast, in glomeruli of patients with MCNS, the abundance of RelA was lower (Figure 2). These results suggest that induction of c-mip in podocytes is associated with a down-regulation of RelA.Figure 2Confocal microscopy analysis. Detection of RelA (green); nephrin, tie-2, vimentin, and lamin-B (red) in normal human kidney (NHK, left) and in kidney biopsy specimens from MCNS relapse (right). Note that RelA partially colocalized with nephrin and lamin, but not with vimentin and tie-2 in normal glomeruli. The expression of RelA was hardly detected in MCNS relapse. Scale bars: 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To understand the functional consequences of c-mip induction in podocytes, we generated transgenic mice expressing c-mip under nephrin promoter to restrict transgene expression to podocytes. c-mip transgenic mice develop heavy proteinuria with foot process effacement without inflammatory lesions or immune complex deposits.13Zhang S.Y. Kamal M. Dahan K. Pawlak A. Ory V. Desvaux D. Audard V. Candelier M. Mohamed F.B. Matignon M. Christov C. Decrouy X. Bernard V. Mangiapan G. Lang P. Guellaen G. Ronco P. Sahali D. c-mip impairs podocyte proximal signaling and induces heavy proteinuria.Sci Signal. 2010; 3: ra39PubMed Google Scholar The abundance of the RelA transcripts measured by quantitative RT-PCR did not significantly differ between wild-type and transgenic mice (Figure 3A). By contrast, Western blot analysis of glomerular protein lysates showed that RelA abundance was significantly decreased in c-mip transgenic mice, as compared with wild-type mice (Figure 3B), which suggests that c-mip affects only the stability of the RelA protein. Indeed, co-culture of c-mip–transfected podocytes in the presence of lactacystin, a potent proteasome inhibiter, prevented the decrease of endogenous RelA (Figure 3C), suggesting that c-mip targets RelA through proteasome-mediated degradation. On the basis of the studies performed in NF-κB–deficient mice, it is believed that NF-κB is a potent antiapoptotic transcription factor.17Gerondakis S. Grumont R. Gugasyan R. Wong L. Isomura I. Ho W. Banerjee A. Unravelling the complexities of the NF-kappa B signalling pathway using mouse knockout and transgenic models.Oncogene. 2006; 25: 6781-6799Crossref PubMed Scopus (241) Google Scholar Given our findings showing a down-regulation of RelA, which is essential for NF-κB activity, we sought to determine whether c-mip influences the signaling pathways involved in apoptosis. Apoptosis is defined by morphological features including loss of adhesion, cell shrinkage, and biochemical alterations such as up-regulation of Bax and caspase-3 activity. Bax belongs to the Bcl-2 family of proteins, which include both antiapoptotic (eg, Bcl-2 and Bcl-xl) and proapoptotic (eg, Bax, Bad) members.18Youle R.J. Strasser A. The BCL-2 protein family: opposing activities that mediate cell death.Nat Rev Mol Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3537) Google Scholar Caspase-3 is one of the final downstream effectors of the apoptotic process, and its activation is closely associated with mitochondrial dysfunction.19Wada T. Pippin J.W. Marshall C.B. Griffin S.V. Shankland S.J. Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins.J Am Soc Nephrol. 2005; 16: 2615-2625Crossref PubMed Scopus (161) Google Scholar To assess whether c-mip is a proapoptotic molecule, we first transiently transfected c-mip alone in podocyte cell line and analyzed the expression level of Bax, BclII, and caspase-3. Overexpression of c-mip induced an up-regulation of Bax and caspase-3, whereas BclII abundance decreased when compared with empty vector–transfected cells (Figure 4A). On the other hand, cotransfection of RelA antagonized the proapoptotic effects of c-mip. We then sought to find whether these changes are reproducible in differentiated podocytes stably transfected with c-mip or empty vector. We studied five independent c-mip stably transfected podocyte cell lines and two control clones stably transfected with empty vector. We performed quantitative Western blot analysis to measure the abundance of the Bcl-2 and Bax proteins that were normalized to GAPDH loading. As shown in Figure 4B, Bax was overproduced in c-mip–overexpressing podocytes, so that the Bax/Bcl-2 ratio was significantly increased (Figure 4B). Morphological analyses showed that empty vector–transfected cells displayed small morphological changes, whereas c-mip–overexpressing podocytes exhibited severe cell shrinkage and most cells had detached. To assess whether these morphological changes involve caspase-3 activation, we performed a caspase-3 activity assay on stable transfectant cells. Podocytes overexpressing c-mip exhibited an activation of caspase-3 in a time-dependent manner, whereas no significant change was detected in the empty vector–transfected podocytes (Figure 4C). The relevance of these findings was analyzed in c-mip transgenic mice. In this model, mice develop after 3 months of age progressive FSGS.13Zhang S.Y. Kamal M. Dahan K. Pawlak A. Ory V. Desvaux D. Audard V. Candelier M. Mohamed F.B. Matignon M. Christov C. Decrouy X. Bernard V. Mangiapan G. Lang P. Guellaen G. Ronco P. Sahali D. c-mip impairs podocyte proximal signaling and induces heavy proteinuria.Sci Signal. 2010; 3: ra39PubMed Google Scholar Histological analysis (Figure 5A) showed that the number of glomeruli displaying FSGS lesions was increased from 3 (4.0% ± 1.5%), 6 (7.5% ± 2.5%) to 12 months of age (23.0% ± 3.5%). Western blot analysis from glomerular extracts revealed increased Bax in c-mip Tg mice, whereas Bcl-2 was reduced, with a Bax/Bcl-2 ratio that was increased by 3.1 fold, as compared with wild-type mice (Figure 5B). We then used the TUNEL assay to detect DNA fragmentation in podocytes in vivo. In wild-type mice and in 3-month-old transgenic mice, TUNEL-positive nuclei were not detectable in glomeruli. However, in transgenic mice of 6 and 12 months of age, a number of podocytes became positive for TUNEL (Figure 6). Collectively, these results suggest that c-mip promotes apoptosis in vitro and in vivo.Figure 6c-mip promotes apoptosis in vivo. TUNEL assay on paraffin kidney sections of wild-type and c-mip transgenic mice at 3, 6, and 12 months of age. Note that TUNEL-positive cells were not detected in 3-month-old transgenic mice, whereas many podocytes became positive at 6 months of age. Scale bars: 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) FSGS lesions are characterized by dysregulation of podocyte function, leading to apoptosis. We have previously shown that c-mip abundance is increased in primary FSGS. Therefore, we investigated whether FSGS biopsy specimens exhibit active, cleaved caspase-3, a marker of cell apoptosis. Immunochemistry analysis was performed in seven FSGS biopsy specimens. Active caspase-3 was detected in all examined cases, to a variable degree, along the external side of some capillary loops, consistent with podocyte localization (Figure 7). The expression was decreased or disappeared in podocyte-depleted areas corresponding to severe FSGS lesions or glomerulosclerosis (Figure 7). Interestingly, c-mip was increased in glomeruli in a pattern similar to that observed with active caspase-3. The role of NF-κB in glomerular biology and in pathophysiology of podocyte disease remains little explored. In the present work, we provide evidence that i) increased expression of c-mip in podocytes of patients with INS is correlated with a down-regulation of RelA; ii) c-mip destabilizes endogenous RelA protein in vitro,
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