RESUMEN
Male infertility might be caused by genetic and/or environmental factors that impair spermatogenesis and epididymal sperm maturation. Here we report that heterozygous deletion of the nuclear receptor coactivator-5 (Ncoa5) gene resulted in decreased motility and progression of spermatozoa in the cauda epididymis, leading to infertility in male mice. Light microscopic and ultrastructural analysis revealed morphological defects in the spermatozoa collected from the cauda epididymis of Ncoa5+/- mice. Immunohistochemistry showed that interleukin-6 (IL-6) expression in epithelial cells of Ncoa5+/- epididymis was higher than wild type counterparts. Furthermore, heterozygous deletion of Il-6 gene in Ncoa5+/- male mice partially improved spermatozoa motility and moderately rescued infertility phenotype. Our results uncover a previously unknown physiological role of NCOA5 in the regulation of epididymal sperm maturation and suggest that NCOA5 deficiency could cause male infertility through increased IL-6 expression in epididymis.
Asunto(s)
Regulación de la Expresión Génica , Infertilidad Masculina , Interleucina-6/biosíntesis , Coactivadores de Receptor Nuclear/deficiencia , Motilidad Espermática/genética , Espermatozoides , Animales , Epidídimo/metabolismo , Epidídimo/patología , Haploinsuficiencia , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Interleucina-6/genética , Masculino , Ratones , Ratones Noqueados , Coactivadores de Receptor Nuclear/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patologíaRESUMEN
Hepatocellular carcinoma (HCC) is the common primary liver cancer and the third leading cause of cancer related mortality worldwide. It is generally thought that the estrogen-signaling pathway is not related to the development and progression of human HCC. However, accumulating evidences indicate the existence of a rapid estrogen signaling in HCC cells that is able to promote cell growth. However, the receptor that mediates the rapid estrogen signaling in HCC cells has not been established. Previously, our laboratory identified a variant of ER-α, ER-α36, and found that ER-α36 mediates the rapid estrogen signaling such as the activation of the MAPK/ERK signaling in breast carcinoma cells. Our current experiments studied the role of the rapid estrogen signaling mediated by ER-α36 in growth of HCC HepG2 and PLC/PRF/5 cells that highly express ER-α36 and found these cells were strongly responsive to the rapid estrogen signaling. Knockdown of ER-α36 expression in these HCC cells using the shRNA method attenuated their responsiveness to estrogen and destabilized EGFR protein. ER-α36 mediated estrogen-induced phosphorylation of Src and the MAPK/ERK as well as cyclin D1 expression. In addition, there existed an ER-α36/EGFR positive regulatory loop in HCC cells that was important for the maintenance and positive regulation of HCC tumorsphere cells. Our results thus indicated that the rapid estrogen receptor is mediated by ER-α36 in HCC cells through the EGFR/Src/ERK signaling pathway and suggested that the ER-α36/EGFR signaling loop is a potential target to develop novel therapeutic approaches for HCC treatment.
Asunto(s)
Carcinoma Hepatocelular/patología , Receptores ErbB/metabolismo , Neoplasias Hepáticas/patología , Coactivadores de Receptor Nuclear/metabolismo , Transducción de Señal , Proliferación Celular , Ciclina D1/metabolismo , Estrógenos/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Coactivadores de Receptor Nuclear/deficiencia , Coactivadores de Receptor Nuclear/genéticaRESUMEN
We recently reported that nuclear receptor coactivator 7 (Ncoa7) is a vacuolar proton pumping ATPase (V-ATPase) interacting protein whose function has not been defined. Ncoa7 is highly expressed in the kidney and partially colocalizes with the V-ATPase in collecting duct intercalated cells (ICs). Here, we hypothesized that targeted deletion of the Ncoa7 gene could affect V-ATPase activity in ICs in vivo. We tested this by analyzing the acid-base status, major electrolytes, and kidney morphology of Ncoa7 knockout (KO) mice. We found that Ncoa7 KO mice, similar to Atp6v1b1 KOs, did not develop severe distal renal tubular acidosis (dRTA), but they exhibited a persistently high urine pH and developed hypobicarbonatemia after acid loading with ammonium chloride. Conversely, they did not develop significant hyperbicarbonatemia and alkalemia after alkali loading with sodium bicarbonate. We also found that ICs were larger and with more developed apical microvilli in Ncoa7 KO compared with wild-type mice, a phenotype previously associated with metabolic acidosis. At the molecular level, the abundance of several V-ATPase subunits, carbonic anhydrase 2, and the anion exchanger 1 was significantly reduced in medullary ICs of Ncoa7 KO mice, suggesting that Ncoa7 is important for maintaining high levels of these proteins in the kidney. We conclude that Ncoa7 is involved in IC function and urine acidification in mice in vivo, likely through modulating the abundance of V-ATPase and other key acid-base regulators in the renal medulla. Consequently, mutations in the NCOA7 gene may also be involved in dRTA pathogenesis in humans.
Asunto(s)
Equilibrio Ácido-Base , Acidosis Tubular Renal/genética , Eliminación de Gen , Túbulos Renales/metabolismo , Coactivadores de Receptor Nuclear/genética , Acidosis Tubular Renal/patología , Acidosis Tubular Renal/fisiopatología , Acidosis Tubular Renal/orina , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Anhidrasa Carbónica II/genética , Anhidrasa Carbónica II/metabolismo , Predisposición Genética a la Enfermedad , Concentración de Iones de Hidrógeno , Túbulos Renales/patología , Túbulos Renales/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Coactivadores de Receptor Nuclear/deficiencia , Fenotipo , Orina/química , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Mice with a knockout of phospholipase C (PLC)-related inactive protein type 1 (PRIP1-/- mice) display anxiety-like behavior and altered γ-aminobutyric acid (GABA)A-receptor pharmacology. Here, we examined associations between anxiety and motor-function recovery in PRIP1-/- mice after a spinal cord injury (SCI) induced by a moderate contusion injury at the 10th thoracic level. Uninjured PRIP1-/- mice showed less distance than wild-type (WT) mice in the center 25% in an open field test (OFT), indicating anxiety-like behavior. Anxiety behavior increased in both WT and PRIP1-/- mice after SCI. WT and PRIP1-/- mice were completely paralyzed on day 1 after SCI, but gradually recovered until reaching a plateau at â¼4 weeks. After SCI, the PRIP1-/- mice had significantly greater motor dysfunction than the WT mice. In WT mice after SCI, the percentage of distance spent in the center 25% of the OFT was correlated with the OFT distance traveled and velocity, and with the reaction time in a plantar pressure-sensitivity mechanical test. In PRIP1-/- mice after SCI, the percentage of distance spent in the center 25% of the OFT was correlated with the OFT distance traveled and with the latency to fall in the rotarod test. Six weeks after SCI, ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) expressions were elevated at the lesion epicenter in PRIP1-/- mice, and spinal cord atrophy and demyelination were more severe than in WT mice. The axonal fiber development was also decreased in PRIP1-/- mice, consistent with the poor motor-function recovery after SCI in these mice.
Asunto(s)
Ansiedad/complicaciones , Recuperación de la Función , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/psicología , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Coactivadores de Receptor Nuclear/deficienciaRESUMEN
Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo, including organelles, proteins or intracellular pathogens, are targeted for selective autophagy is limited. Here we use quantitative proteomics to identify a cohort of novel and known autophagosome-enriched proteins in human cells, including cargo receptors. Like known cargo receptors, nuclear receptor coactivator 4 (NCOA4) was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo-receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species but is degraded via autophagy to release iron through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin led to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy), which is critical for iron homeostasis, and provides a resource for further dissection of autophagosomal cargo-receptor connectivity.
Asunto(s)
Autofagia , Ferritinas/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Fagosomas/metabolismo , Proteómica , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia , Disponibilidad Biológica , Ferritinas/química , Homeostasis , Humanos , Hierro/metabolismo , Lisosomas/metabolismo , Proteínas de Microfilamentos/metabolismo , Coactivadores de Receptor Nuclear/deficiencia , Coactivadores de Receptor Nuclear/genética , Unión Proteica , Transporte de Proteínas , Especificidad por SustratoRESUMEN
Hepatitis C Virus (HCV) is a serious global health problem, infecting almost 3% of the world's population. The lack of model systems for studying this virus limit research options in vaccine and therapeutic development, as well as for studying the pathogenesis of chronic HCV infection. Herein we make use of the liver-specific microRNA miR-122 to render mouse cell lines permissive to HCV replication in an attempt to develop additional model systems for the identification of new features of the virus and its life cycle. We have determined that some wild-type and knockout mouse cell lines--NCoA6 and PKR knockout embryonic fibroblasts--can be rendered permissive to transient HCV sub-genomic RNA replication upon addition of miR-122, but we did not observe replication of full-length HCV RNA in these cells. However, other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122, and in fact, different NCoA6 and PKR knockout cell line passages and isolates from the same mice demonstrated varying permissiveness phenotypes and eventually complete loss of permissiveness. When we tested knockdown of NCoA6 and PKR in Huh7.5 cells, we saw no substantial impact in sub-genomic HCV replication, which we would expect if these genes were inhibitory to the virus' life cycle. This leads us to conclude that along with the influence of specific gene knockouts there are additional factors within the cell lines that affect their permissiveness for HCV replication; we suggest that these may be epigenetically regulated, or modulated by cell line immortalization and transformation.
Asunto(s)
Línea Celular , Genoma Viral/genética , Hepacivirus/genética , Hepacivirus/fisiología , MicroARNs/genética , ARN Viral/genética , Replicación Viral , Animales , Técnicas de Cultivo de Célula , Transformación Celular Viral , Epigénesis Genética , Técnicas de Inactivación de Genes , Hígado/metabolismo , Ratones , Coactivadores de Receptor Nuclear/deficiencia , Coactivadores de Receptor Nuclear/genética , Fenotipo , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Steroid receptor coactivators (SRCs) are important transcriptional modulators that regulate nuclear receptor and transcription factor activity to adjust transcriptional output to cellular demands. Highlighting their pleiotropic effects, dysfunction of the SRCs has been found in numerous pathologies including cancer, inflammation, and metabolic disorders. The SRC family is expressed strongly in the brain including the hippocampus, cortex, and hypothalamus. Studies focusing on the effect of SRC loss using congenic SRC knockout mice (SRC(-/-)) are limited in number, yet strongly indicate that the SRCs play important roles in regulating reproductive behavior, development, and motor coordination. To better understand the unique functions of the SRCs, we performed a neurobehavioral test battery focusing on anxiety and exploratory behaviors, motor coordination, sensorimotor gating, and nociception in both male and female null mice and compared them with their wild-type (WT) littermates. Results from the test battery reveal a role for SRC1 in motor coordination. Additionally, we found that SRC1 regulates anxiety responses in SRC1(-/-) male and female mice, and nociception sensitivity in SRC1(-/-) male but not female mice. By comparison, SRC2 regulates anxiety response with SRC2(-/-) females showing decreased anxiety in novel environments, as well as increased exploratory behavior in the open field compared with WT littermates. Additionally, SRC2(-/-) males were shown to have deficits in sensorimotor gating. Loss of SRC3 also shows sex differences in anxiety and exploratory behaviors. In particular, SRC3(-/-) female mice have increased anxiety and reduced exploratory activity and impairments in prepulse inhibition, whereas SRC3(-/-) male mice show no significant behavioral differences. In both genders, ablation of SRC3 decreases nocifensive behaviors. Collectively, these resource data suggest that loss of the SRCs results in behavioral phenotypes, underscoring the importance of understanding both the general and gender-based activity of SRCs in the brain.
Asunto(s)
Ansiedad/genética , Coactivadores de Receptor Nuclear/genética , Animales , Ansiedad/metabolismo , Femenino , Estudios de Asociación Genética , Locomoción , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Destreza Motora , Nocicepción , Coactivadores de Receptor Nuclear/deficiencia , Fenotipo , Filtrado Sensorial , Caracteres SexualesRESUMEN
Uterine receptivity to embryo implantation is coordinately regulated by 17ß-estradiol (E(2)) and progesterone (P(4)). Although increased E(2) sensitivity causes infertility, the mechanisms underlying the modulation of E(2) sensitivity are unknown. We show that nuclear receptor coactivator-6 (NCOA6), a reported coactivator for estrogen receptor α (ERα), actually attenuates E(2) sensitivity to determine uterine receptivity to embryo implantation under normal physiological conditions. Specifically, conditional knockout of Ncoa6 in uterine epithelial and stromal cells does not decrease, but rather markedly increases E(2) sensitivity, which disrupts embryo implantation and inhibits P(4)-regulated genes and decidual response. NCOA6 enhances ERα ubiquitination and accelerates its degradation, while loss of NCOA6 causes ERα accumulation in stromal cells during the preimplantation period. During the same period, NCOA6 deficiency also caused a failure in downregulation of steroid receptor coactivator-3 (SRC-3), a potent ERα coactivator. Therefore, NCOA6 controls E(2) sensitivity and uterine receptivity by regulating multiple E(2)-signaling components.