RESUMEN
BACKGROUND: The Wilms tumor 1 suppressor gene, WT1, is expressed throughout life in podocytes and is essential for their function. Downregulation of WT1 has been reported in podocyte diseases but the underlying mechanisms remain unclear. Podocyte injury is the hallmark of idiopathic nephrotic syndrome (INS), the most frequent glomerular disease in children and young adults. An increase in the abundance of Cmaf-inducing protein (CMIP) has been found to alter podocyte function, but it is not known whether CMIP affects WT1 expression. METHODS: Transcriptional and post-transcriptional regulation of WT1in the presence of CMIP was studied using transient transfection, mouse models, and siRNA handling. RESULTS: We showed that overproduction of CMIP in the podocyte was consistently associated with a downregulation of WT1 according to two mechanisms. We found that CMIP prevented the NF-kB-mediated transcriptional activation of WT1. We demonstrated that CMIP interacts directly with WT1 through its leucine-rich repeat domain. Overexpression of CMIP in the M15 cell line induced a downregulation of WT1, which was prevented by lactacystin, a potent proteasome inhibitor. We showed that CMIP exhibits an E3 ligase activity and targets WT1 to proteasome degradation. Intravenous injection of Cmip-siRNA specifically prevented the repression of Wt1 in lipopolysaccharides-induced proteinuria in mice. CONCLUSIONS: These data suggest that CMIP is a repressor of WT1 and might be a critical player in the pathophysiology of some podocyte diseases. Because WT1 is required for podocyte integrity, CMIP could be considered a therapeutic target in podocyte diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Síndrome Nefrótico/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas WT1/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Regulación hacia Abajo/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/metabolismo , Síndrome Nefrótico/metabolismo , Podocitos/citología , Podocitos/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Unión Proteica , Proteinuria/patología , Proteinuria/prevención & control , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/metabolismo , Activación Transcripcional , Proteínas WT1/genéticaRESUMEN
Transcription factors critical for the transition of normal breast epithelium to ductal carcinoma in situ (DCIS) and invasive breast cancer are not clearly defined. Here, we report that the expression of a subset of YAP-activated and YAP-repressed genes in normal mammary and early-stage breast cancer cells is dependent on the nuclear co-activator AIB1. Gene expression, sequential ChIP, and ChIP-seq analyses show that AIB1 and YAP converge upon TEAD for transcriptional activation and repression. We find that AIB1-YAP repression of genes at the 1q21.3 locus is mediated by AIB1-dependent recruitment of ANCO1, a tumor suppressor whose expression is progressively lost during breast cancer progression. Reducing ANCO1 reverts AIB1-YAP-dependent repression, increases cell size, and enhances YAP-driven aberrant 3D growth. Loss of endogenous ANCO1 occurs during DCIS xenograft progression, a pattern associated with poor prognosis in human breast cancer. We conclude that increased expression of AIB1-YAP co-activated targets coupled with a loss of normal ANCO1 repression is critical to patterns of gene expression that mediate malignant progression of early-stage breast cancer.
Asunto(s)
Neoplasias de la Mama , Coactivador 3 de Receptor Nuclear/genética , Proteínas Represoras/genética , Mama , Neoplasias de la Mama/genética , Humanos , Coactivador 3 de Receptor Nuclear/metabolismoRESUMEN
CDK4/6 inhibitors are used in the treatment of advanced estrogen receptor (ER)(+) breast cancer. Their efficacy in ER(-) and early-stage breast cancer is currently under investigation. Here, we show that palbociclib, a CDK4/6 inhibitor, can inhibit both progression of ductal carcinoma in situ (DCIS) and growth of invasive disease in both an ER(-) basal breast cancer model (MCFDCIS) and an ER(+) luminal model (MCF7 intraductal injection). In MCFDCIS cells, palbociclib repressed cell-cycle gene expression, inhibited proliferation, induced senescence, and normalized tumorspheres formed in Matrigel while the formation of acini by normal mammary epithelial cells (MCF10A) was not affected. Palbociclib treatment of mice with MCFDCIS tumors inhibited their malignant progression and reduced proliferation of invasive lesions. Transcriptomic analysis of the tumor and stromal cell compartments showed that cell cycle and senescence genes, and MUC16, an ovarian cancer biomarker gene, were repressed during treatment. Knockdown of MUC16 in MCFDCIS cells inhibited proliferation of invasive lesions but not progression of DCIS. After cessation of palbociclib treatment genes associated with differentiation, for example, P63, inflammation, IFNγ response, and antigen processing and presentation remained suppressed in the tumor and surrounding stroma. We conclude that palbociclib can prevent progression of DCIS and is antiproliferative in ER(-) invasive disease mediated in part via MUC16. Lasting effects of CDK4/6 inhibition after drug withdrawal on differentiation and the immune response could impact the approach to treatment of early-stage ER(-) breast cancer.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/uso terapéutico , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/uso terapéutico , Animales , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Estadificación de NeoplasiasRESUMEN
PURPOSE: Ductal carcinoma in situ (DCIS) is a pre-invasive lesion of the breast considered a precursor of invasive ductal carcinoma. This study aimed to determine whether activated PPARγ acts as a tumor suppressor in human DCIS progression. METHODS: We utilized the high-affinity PPARγ agonist, efatutazone, to activate endogenous PPARγ in a well-defined model for the progression of basal (triple negative) DCIS, MCFDCIS cells, cultured under 2D and 3D conditions. We studied the effects of activated PPARγ on DCIS progression in MCFDCIS xenograft and C3(1)/Tag transgenic mice treated with 30 mg/kg of efatutazone. RESULTS: In vitro, efatutazone did not alter the MCFDCIS cell proliferation but induced phenotypic and gene expression changes, indicating that activated PPARγ is able to differentiate MCFDCIS cells into more luminal and lactational-like cells. In addition, MCFDCIS tumorsphere formation in 3D was reduced by PPARγ activation. In vivo, efatutazone-treated MCFDCIS tumors exhibited fat deposition along with upregulation of PPARγ responsive genes in both epithelial and stromal compartments, suggesting features of milk-producing mammary epithelial cell differentiation. The efatutazone-treated lesions were less invasive with fewer CD44+/p63+ basal progenitor cells. PPARγ activation downregulated Akt phosphorylation in these tumors, although the ERK pathway remained unchanged. Similar trends in gene expression changes consistent with lactational and luminal cell differentiation were observed in the C3(1)/Tag mouse model after efatutazone treatment. CONCLUSIONS: Our data suggest that activation of the PPARγ pathway differentiates DCIS lesions and may be a useful approach to delay DCIS progression.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Carcinoma Intraductal no Infiltrante/tratamiento farmacológico , PPAR gamma/genética , Tiazolidinedionas/administración & dosificación , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. We have previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions -290/-274 and -57/-41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified in the mouse Cmip proximal promoter located at position -217/-206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1, including Denys-Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Podocitos/metabolismo , Proteínas WT1/metabolismo , Animales , Secuencia de Bases , Síndrome de Denys-Drash/metabolismo , Femenino , Síndrome de Frasier/metabolismo , Regulación de la Expresión Génica , Humanos , Riñón/embriología , Masculino , Ratones , Regiones Promotoras GenéticasRESUMEN
Mammary epithelial (ME) cells cultured under conventional conditions senesce after several passages. Here, we demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neu-induced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs) on irradiated fibroblasts in the presence of the Rho kinase inhibitor Y-27632. Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f+ ESA+ CD44+, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44+, CD49f+, and ESA+ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.
Asunto(s)
Células Epiteliales/citología , Glándulas Mamarias Animales/citología , Neoplasias Mamarias Experimentales/patología , Virus del Tumor Mamario del Ratón/metabolismo , Amidas/química , Animales , Transformación Celular Neoplásica/genética , Células Cultivadas , Técnicas de Cocultivo , Colágeno/química , Hibridación Genómica Comparativa , Combinación de Medicamentos , Inhibidores Enzimáticos/química , Transición Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Femenino , Fibroblastos/metabolismo , Laminina/química , Ratones , Ratones Transgénicos , Microscopía Confocal , Fenotipo , Proteoglicanos/química , Piridinas/química , Células Madre/citologíaRESUMEN
Renal toxicity constitutes a dose-limiting side effect of anticancer therapies targeting vascular endothelial growth factor (VEGF). In order to study this further, we followed up 29 patients receiving this treatment, who experienced proteinuria, hypertension, and/or renal insufficiency. Eight developed minimal change nephropathy/focal segmental glomerulopathy (MCN/FSG)-like lesions and 13 developed thrombotic microangiopathy (TMA). Patients receiving receptor tyrosine kinase inhibitors (RTKIs) mainly developed MCN/FSG-like lesions, whereas TMA complicated anti-VEGF therapy. There were no mutations in factor H, factor I, or membrane cofactor protein of the complement alternative pathway, while plasma ADAMTS13 activity persisted and anti-ADAMTS13 antibodies were undetectable in patients with TMA. Glomerular VEGF expression was undetectable in TMA and decreased in MCN/FSG. Glomeruli from patients with TMA displayed a high abundance of RelA in endothelial cells and in the podocyte nuclei, but c-mip was not detected. Conversely, MCN/FSG-like lesions exhibited a high abundance of c-mip, whereas RelA was scarcely detected. RelA binds in vivo to the c-mip promoter and prevents its transcriptional activation, whereas RelA knockdown releases c-mip activation. The RTKI sorafenib inhibited RelA activity, which then promoted c-mip expression. Thus, our results suggest that c-mip and RelA define two distinct types of renal damage associated with VEGF-targeted therapies.
Asunto(s)
Inhibidores de la Angiogénesis/efectos adversos , Proteínas Portadoras/metabolismo , Enfermedades Renales/inducido químicamente , Glomérulos Renales/efectos de los fármacos , Niacinamida/análogos & derivados , Compuestos de Fenilurea/efectos adversos , Inhibidores de Proteínas Quinasas/efectos adversos , Factor de Transcripción ReIA/metabolismo , Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Animales , Secuencia de Bases , Sitios de Unión , Biomarcadores/metabolismo , Proteínas Portadoras/genética , Estudios de Casos y Controles , Línea Celular , Femenino , Regulación de la Expresión Génica , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Glomeruloesclerosis Focal y Segmentaria/diagnóstico , Glomeruloesclerosis Focal y Segmentaria/enzimología , Humanos , Hipertensión/inducido químicamente , Hipertensión/diagnóstico , Hipertensión/enzimología , Enfermedades Renales/diagnóstico , Enfermedades Renales/enzimología , Glomérulos Renales/enzimología , Glomérulos Renales/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Datos de Secuencia Molecular , Nefrosis Lipoidea/inducido químicamente , Nefrosis Lipoidea/diagnóstico , Nefrosis Lipoidea/enzimología , Niacinamida/efectos adversos , Valor Predictivo de las Pruebas , Regiones Promotoras Genéticas , Proteinuria/inducido químicamente , Proteinuria/diagnóstico , Proteinuria/enzimología , Insuficiencia Renal/inducido químicamente , Insuficiencia Renal/diagnóstico , Insuficiencia Renal/enzimología , Sorafenib , Microangiopatías Trombóticas/inducido químicamente , Microangiopatías Trombóticas/diagnóstico , Microangiopatías Trombóticas/enzimología , Factor de Transcripción ReIA/deficiencia , Factor de Transcripción ReIA/genética , Transcripción Genética , Transfección , Factores de Crecimiento Endotelial Vascular/metabolismo , Adulto JovenRESUMEN
Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.
Asunto(s)
Proteínas Portadoras , Citocinas , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Glándulas Mamarias Animales/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Uniones Célula-Matriz/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Embarazo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/fisiología , FN-kappa B/metabolismo , Síndrome Nefrótico/metabolismo , Podocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adulto , Animales , Proteínas Portadoras/biosíntesis , Caspasa 3/metabolismo , Línea Celular , Regulación hacia Abajo/fisiología , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Síndrome Nefrótico/patología , Podocitos/patología , Factor de Transcripción ReIA/biosíntesis , Factor de Transcripción ReIA/genética , Regulación hacia Arriba/fisiologíaRESUMEN
We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.
Asunto(s)
Amidas/farmacología , Proliferación Celular/efectos de los fármacos , Reprogramación Celular/fisiología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Nutrientes/fisiología , Piridinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Mama/citología , Técnicas de Cultivo de Célula , Reprogramación Celular/efectos de los fármacos , Colágeno , Combinación de Medicamentos , Células Epiteliales/citología , Células Nutrientes/citología , Femenino , Humanos , Laminina , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Próstata/citología , Neoplasias de la Próstata/patología , Proteoglicanos , Trasplante HeterólogoRESUMEN
Idiopathic nephrotic syndrome comprises several podocyte diseases of unknown origin that affect the glomerular podocyte, which controls the permeability of the filtration barrier in the kidney to proteins. It is characterized by the daily loss of more than 3 g of protein in urine and the lack of inflammatory lesions or cell infiltration. We found that the abundance of c-mip (c-maf inducing protein) was increased in the podocytes of patients with various acquired idiopathic nephrotic syndromes in which the podocyte is the main target of injury. Mice engineered to have excessive c-mip in podocytes developed proteinuria without morphological alterations, inflammatory lesions, or cell infiltration. Excessive c-mip blocked podocyte signaling by preventing the interaction of the slit diaphragm transmembrane protein nephrin with the tyrosine kinase Fyn, thereby decreasing phosphorylation of nephrin in vitro and in vivo. Moreover, c-mip inhibited interactions between Fyn and the cytoskeletal regulator N-WASP (neural Wiskott-Aldrich syndrome protein) and between the adaptor protein Nck and nephrin, potentially accounting for cytoskeletal disorganization and the effacement of foot processes seen in idiopathic nephrotic syndromes. The intravenous injection of small interfering RNA targeting c-mip prevented lipopolysaccharide-induced proteinuria in mice. Together, these results identify c-mip as a key component in the molecular pathogenesis of acquired podocyte diseases.
Asunto(s)
Proteínas Portadoras/fisiología , Podocitos/fisiología , Proteinuria/fisiopatología , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Fosforilación , Podocitos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Interferencia de ARN , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
It is currently considered that idiopathic minimal change nephrotic syndrome is an immune-mediated glomerular disease. Its association with classical Hodgkin lymphoma minimal change nephrotic syndrome (cHL-MCNS) suggests a molecular link, which remains to be elucidated. We analyzed the expression of cmaf inducing protein (c-mip) in lymphomatous tissues and kidney biopsy samples of patients with cHL-MCNS (n = 8) and in lymphomatous tissues of patients with isolated cHL (n = 9). Because c-mip affects the regulatory loop involving Fyn, we investigated possible structural defects in this signaling pathway, using laser capture microdissection, reverse transcription polymerase chain reaction, and Western blotting. We found that c-mip was selectively expressed in Hodgkin and Reed-Sternberg (HRS) cells and podocytes of patients with cHL-MCNS but is undetectable in patients with isolated cHL. We demonstrated that c-mip was specifically involved in the negative regulation of early proximal signaling through its interaction with phosphoprotein associated with glycosphingolipid-enriched microdomains and Fyn. We showed that the up-regulation of c-mip in cHL-MCNS was associated with a possible Fyn defect in HRS cells and podocytes. Moreover, we showed that c-mip was up-regulated in Fyn-deficient podocytes. c-mip may be a useful marker of cHL-MCNS and its induction reflects the dysregulation of proximal signaling.
Asunto(s)
Proteínas Portadoras/metabolismo , Enfermedad de Hodgkin/complicaciones , Nefrosis Lipoidea/complicaciones , Podocitos/metabolismo , Células de Reed-Sternberg/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Western Blotting , Proteína Tirosina Quinasa CSK , Estudios de Casos y Controles , Células Cultivadas , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/metabolismo , Humanos , Hibridación in Situ , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Microdisección , Nefrosis Lipoidea/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Familia-src QuinasasRESUMEN
The fine regulation of NF-kappaB activity is crucial for both resting and stimulated cells and relies on complex balance between multiple activators and inhibitors. We report here that c-mip, a recently identified pleckstrin homology (PH) and leucine-rich repeat (LRR)-domain-containing protein, inactivates GSKbeta and interacts with RelA, a key member of the NF-kappaB family. We show that c-mip inhibits the degradation of I-kappaBalpha and impedes the dissociation of the NF-kappaB/I-kappaBalpha complexes. C-mip acts downstream signaling of classical NF-kappaB pathway and may represent one of the missing links in the control of NF-kappaB activity.