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2.
Nephron ; 138(1): 71-87, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-28965116

RESUMEN

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is considered a subset of the podocytopathies. The molecular pathogenesis of podocytopathy is still unknown. There has not been an experimental animal model of isolated podocytopathy induced by antibody in C57BL/6 strain, which is widely used as the genetic background. Nephrin is closely associated with the slit diaphragm of the glomerular podocyte, and has recently received attention as a potential therapeutic target. The function of nephrin, especially its role in FSGS development via podocytopathy, is being elucidated. We report our experience with a C57BL/6 FSGS model induced by polyclonal rabbit anti-mouse nephrin antibody (α-mNep Ab). METHODS: α-mNep Ab, which was generated by genetic immunization, was administered into C57BL/6 mice at once, intravenously. Urinary protein excretion, the development of glomerulosclerosis and the number of podocyte in mouse kidney were evaluated. RESULTS: The α-mNep Ab-induced FSGS was associated with massive proteinuria and nephrotic syndrome. In periodic acid-Schiff staining, FSGS was observed from day 7 after antibody injection. Podocyte numbers and podocyte marker (anti-Wilms tumor 1 and anti-synaptopodin)-positive areas were clearly decreased. These results suggest that this FSGS mouse model reliably reproduces the human nephrotic syndrome and FSGS. CONCLUSION: We succeeded in making the nephrotic syndrome model mice induced by α-mNep Ab using C57BL/6. This model may be useful for studying the mechanisms of podocytopathy.


Asunto(s)
Anticuerpos/inmunología , Glomeruloesclerosis Focal y Segmentaria/inmunología , Proteínas de la Membrana/inmunología , Podocitos/inmunología , Podocitos/patología , Animales , Peso Corporal , Femenino , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Glomeruloesclerosis Focal y Segmentaria/patología , Células HEK293 , Humanos , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Síndrome Nefrótico/genética , Síndrome Nefrótico/patología , Proteinuria/genética , Proteinuria/patología , Conejos , Urodinámica
3.
Mol Biol Cell ; 24(19): 3085-96, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23904271

RESUMEN

The X-linked gene Rnf12 encodes the ubiquitin ligase really interesting new gene (RING) finger LIM domain-interacting protein (RLIM)/RING finger protein 12 (Rnf12), which serves as a major sex-specific epigenetic regulator of female mouse nurturing tissues. Early during embryogenesis, RLIM/Rnf12 expressed from the maternal allele is crucial for the development of extraembryonic trophoblast cells. In contrast, in mammary glands of pregnant and lactating adult females RLIM/Rnf12 expressed from the paternal allele functions as a critical survival factor for milk-producing alveolar cells. Although RLIM/Rnf12 is detected mostly in the nucleus, little is known about how and in which cellular compartment(s) RLIM/Rnf12 mediates its biological functions. Here we demonstrate that RLIM/Rnf12 protein shuttles between nucleus and cytoplasm and this is regulated by phosphorylation of serine S214 located within its nuclear localization sequence. We show that shuttling is important for RLIM to exert its biological functions, as alveolar cell survival activity is inhibited in cells expressing shuttling-deficient nuclear or cytoplasmic RLIM/Rnf12. Thus regulated nucleocytoplasmic shuttling of RLIM/Rnf12 coordinates cellular compartments during mammary alveolar cell survival.


Asunto(s)
Transporte Activo de Núcleo Celular/genética , Supervivencia Celular/genética , Glándulas Mamarias Animales/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Núcleo Celular/genética , Desarrollo Embrionario , Epigénesis Genética/genética , Femenino , Células HeLa , Humanos , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Ratones , Fosforilación , Embarazo , Procesos de Determinación del Sexo , Ubiquitina-Proteína Ligasas/genética
4.
Hum Mol Genet ; 20(9): 1701-11, 2011 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-21300694

RESUMEN

Spinal muscular atrophy (SMA), an inherited disease of motor neuron dysfunction, results from insufficient levels of the survival motor neuron (SMN) protein. Movement of the SMN protein as granules within cultured axons suggests that the pathogenesis of SMA may involve defects in neuronal transport, yet the nature of axon transport vesicles remains enigmatic. Here we show that SMN directly binds to the α-subunit of the coat protein I (COPI) vesicle coat protein. The α-COP protein co-immunoprecipitates with SMN, small nuclear ribonucleoprotein-associated assembly factors and ß-actin mRNA. Although typically Golgi associated, in neuronal cells α-COP localizes to lamellipodia and growth cones and moves within the axon, with a subset of these granules traveling together with SMN. Depletion of α-COP resulted in mislocalization of SMN and actin at the leading edge at the lamellipodia. We propose that neurons utilize the Golgi-associated COPI vesicle to deliver cargoes necessary for motor neuron integrity and function.


Asunto(s)
Axones/metabolismo , Proteína Coat de Complejo I/metabolismo , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Línea Celular , Supervivencia Celular , Proteína Coat de Complejo I/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Neuronas Motoras/citología , Atrofia Muscular Espinal/genética , Unión Proteica , Transporte de Proteínas , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Vesículas Transportadoras/genética
5.
Dev Biol ; 349(2): 213-24, 2011 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-21056553

RESUMEN

The developmental activity of LIM homeodomain transcription factors (LIM-HDs) is critically controlled by LIM domain-interacting cofactors of LIM-HDs (CLIM, also known as NLI or LDB). CLIM cofactors associate with single-stranded DNA binding proteins (SSDPs, also known as SSBPs) thereby recruiting SSDP1 and/or SSDP2 to LIM-HD/CLIM complexes. Although evidence has been presented that SSDPs are important for the activity of specific LIM-HD/CLIM complexes, the developmental roles of SSDPs are unclear. We show that SSDP1a and SSDP1b mRNAs are widely expressed early during zebrafish development with conspicuous expression of SSDP1b in sensory trigeminal and Rohon-Beard neurons. SSDP1 and CLIM immunoreactivity co-localize in these neuronal cell types and in other structures. Over-expression of the N-terminal portion of SSDP1 (N-SSDP1), which contains the CLIM-interaction domain, increases endogenous CLIM protein levels in vivo and impairs the formation of eyes and midbrain-hindbrain boundary. In addition, manipulation of SSDP1 via N-SSDP1 over-expression or SSDP1b knock down impairs trigeminal and Rohon-Beard sensory axon growth. We show that N-SSDP1 is able to partially rescue the inhibition of axon growth induced by a dominant-negative form of CLIM (DN-CLIM). These results reveal specific functions of SSDP in neural patterning and sensory axon growth, in part due to the stabilization of LIM-HD/CLIM complexes.


Asunto(s)
Axones/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Neurogénesis/fisiología , Células Receptoras Sensoriales/fisiología , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Animales , Western Blotting , Diferenciación Celular/fisiología , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Inmunohistoquímica , Hibridación in Situ , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/metabolismo
6.
Nature ; 467(7318): 977-81, 2010 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-20962847

RESUMEN

Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.


Asunto(s)
Cromosomas de los Mamíferos/genética , Impresión Genómica , Madres , Proteínas Represoras/metabolismo , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Animales Congénicos , Blastocisto/metabolismo , Línea Celular , Pérdida del Embrión/genética , Padre , Femenino , Silenciador del Gen , Masculino , Ratones , Ratones Transgénicos , ARN Largo no Codificante , ARN no Traducido/genética , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas
7.
Cancer Res ; 69(1): 128-36, 2009 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19117995

RESUMEN

Mammary oncogenesis is profoundly influenced by signaling pathways controlled by estrogen receptor alpha (ERalpha). Although it is known that ERalpha exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HD) is critically regulated by cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain-interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERalpha cofactors that colocalize and interact with ERalpha in primary human breast tumors. We show that both cofactors associate with estrogen-responsive promoters and regulate the expression of endogenous ERalpha target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERalpha and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERalpha on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs but enhances transcriptional activation of endogenous ERalpha target genes. Results from a human breast cancer tissue microarray of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/progesterone receptor positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERalpha during the development of human breast cancer.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Receptor alfa de Estrógeno/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Neoplasias de la Mama/genética , Proteínas CCN de Señalización Intercelular , Catepsina D/biosíntesis , Catepsina D/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas con Dominio LIM , Presenilina-2/biosíntesis , Presenilina-2/genética , Receptores de Progesterona/biosíntesis , Proteínas Represoras/genética , Elementos de Respuesta/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Activación Transcripcional , Ubiquitina-Proteína Ligasas , Ubiquitinación
8.
Proc Natl Acad Sci U S A ; 104(38): 15000-5, 2007 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-17848518

RESUMEN

Complexes composed of multiple proteins regulate most cellular functions. However, our knowledge about the molecular mechanisms governing the assembly and dynamics of these complexes in cells remains limited. The in vivo activity of LIM homeodomain (LIM-HD) proteins, a class of transcription factors that regulates neuronal development, depends on the high-affinity association of their LIM domains with cofactor of LIM homeodomain proteins (LIM-HDs) (CLIM, also known as Ldb or NLI). CLIM cofactors recruit single-stranded DNA-binding protein 1 (SSDP1, also known as SSBP3), and this interaction is important for the activation of the LIM-HD/CLIM protein complex in vivo. Here, we identify a cascade of specific protein interactions that protect LIM-HD multiprotein complexes from proteasomal degradation. In this cascade, CLIM stabilizes LIM-HDs, and SSDP1 stabilizes CLIM. Furthermore, we show that stabilizing cofactors prevent binding of ubiquitin ligases to multiple protein interaction domains in LIM-HD recruited protein complexes. Together, our results indicate a combinatorial code that selects specific multiprotein complexes via proteasomal degradation in cells with broad implications for the assembly and specificity of multiprotein complexes.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Complejos Multiproteicos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/metabolismo , Células Cultivadas , Modelos Biológicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección
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