RESUMEN
PKCßI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCßI in mouse oocyte meiotic maturation. PKCßI and p-PKCßI (phosphor-PKCßI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCßI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCßI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCßI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCßI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCßI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCßI is a critical regulator of meiotic cell cycle progression in oocytes. Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint.
Asunto(s)
Metafase/genética , Cuerpos Polares/metabolismo , Proteína Quinasa C beta/genética , Proteína Quinasa C beta/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Cromosomas/metabolismo , Ciclina B1/metabolismo , Citoplasma/metabolismo , Femenino , Puntos de Control de la Fase M del Ciclo Celular/genética , Ratones , Ratones Endogámicos ICR , Microinyecciones , Plásmidos/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , Huso Acromático/metabolismo , Telofase/genéticaRESUMEN
BACKGROUND: Alternative splicing is one of the post transcriptional modifications through which multiple mRNA isoforms are produced from any gene, also known as splice variants. These are expressed in tissue and developmental stage specific manner that are important during the development. Most human genes undergo alternative splicing, thus contributing to the diversity of proteins. However, many abnormal splicing processes may result in human diseases. Non-steroidal antiinflammatory drugs (NSAIDs) are medications that act as analgesics, anti-pyretics and antiinflammatory by affecting Cox genes and their products. Usually NSAIDs cause gastrotoxicity however, isozyme-specific NSAIDs exhibit a comparatively reduced gastrotoxic effect. Such NSAIDs have a broader range of application particularly as chemo-preventive drugs. It is known that changes at the active site of an enzyme may illicit a diverse range of responses. Such changes might explain the underlying reason as to why patients appear to respond differently to different NSAIDs. METHODS: An extensive literature search has been carried out using Pubmed and web of science databases considering the papers in last 10 years mainly on alternative splicing and NSAIDs. CONCLUSION: We have reviewed in detail the insight into the action of NSAIDs targeting specific isoforms of different genes. In future, the complete understanding of NSAIDs associated genes and their expression studies may be helpful in generating drugs with increased specificity.
Asunto(s)
Empalme Alternativo , Enfermedad de Alzheimer/genética , Antiinflamatorios no Esteroideos/farmacología , Neoplasias/genética , ARN Mensajero/genética , Expresión Génica/efectos de los fármacos , HumanosRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most important infectious diseases in swine industry. IL-15 is a pleiotropic cytokine and has been shown to be essential to transform NKs, CD8 T cells, and other cells of the immune systems into functional effectors. Here, we demonstrated that the broad-spectrum or conventional PKC inhibitors repressed PRRSV-induced IL-15 expression and NF-κB activation. Subsequently, we found that the PKCß specific inhibitor inhibited PRRSV-induced IL-15 production, which was also confirmed by knock-down of PKCß1, suggesting that PKCß1 is involved in the PRRSV-induced IL-15 expression. In addition, we demonstrated that PRRSV activated NF-κB through PKCß1-induced TAK1 activation. Finally, we demonstrated that PRRSV activated PKCß1 dependent on the participation of TRIF and MAVS. These data indicate that PRRSV up-regulates IL-15 through TRIF/MAVS-PKCß1-TAK1-NF-κB signaling pathway. These findings will provide new insights into the molecular mechanisms of IL-15 production induced by PRRSV.
Asunto(s)
Interleucina-15/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Proteína Quinasa C beta/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Expresión Génica , Genes Reporteros , Interleucina-15/genética , Quinasas Quinasa Quinasa PAM/genética , Modelos Biológicos , Mutación , FN-kappa B/genética , Síndrome Respiratorio y de la Reproducción Porcina/genética , Regiones Promotoras Genéticas , Unión Proteica , Proteína Quinasa C beta/genética , PorcinosRESUMEN
The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCß1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS(104) via PKCß1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21(WAF1) gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Fibroblastos/citología , Proteína Quinasa C beta/metabolismo , Proteína Quinasa C-alfa/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Animales , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Recently we identified in a wide spectrum of autoimmune diseases frequently occurring proinflammatory autoantibodies directed against progranulin, a direct inhibitor of TNFR1 & 2 and of DR3. In the present study we investigated the mechanisms for the breakdown of self-tolerance against progranulin. Isoelectric focusing identified a second, differentially electrically charged progranulin isoform exclusively present in progranulin-antibody-positive patients. Alkaline phosphatase treatment revealed this additional progranulin isoform to be hyperphosphorylated. Subsequently Ser81, which is located within the epitope region of progranulin-antibodies, was identified as hyperphosphorylated serine residue by site directed mutagenesis of candidate phosphorylation sites. Hyperphosphorylated progranulin was detected exclusively in progranulin-antibody-positive patients during the courses of their diseases. The occurrence of hyperphosphorylated progranulin preceded seroconversions of progranulin-antibodies, indicating adaptive immune response. Utilizing panels of kinase and phosphatase inhibitors, PKCß1 was identified as the relevant kinase and PP1 as the relevant phosphatase for phosphorylation and dephosphorylation of Ser81. In contrast to normal progranulin, hyperphosphorylated progranulin interacted exclusively with inactivated (pThr320) PP1, suggesting inactivated PP1 to cause the detectable occurrence of phosphorylated Ser81 PGRN. Investigation of possible functional alterations of PGRN due to Ser81 phosphorylation revealed, that hyperphosphorylation prevents the interaction and thus direct inhibition of TNFR1, TNFR2 and DR3, representing an additional direct proinflammatory effect. Finally phosphorylation of Ser81 PGRN alters the conversion pattern of PGRN. In conclusion, inactivated PP1 induces hyperphosphorylation of progranulin in a wide spectrum of autoimmune diseases. This hyperphosphorylation prevents direct inhibition of TNFR1, TNFR2 and DR3 by PGRN, alters the conversion of PGRN, and is strongly associated with the occurrence of neutralizing, proinflammatory PGRN-antibodies, indicating immunogenicity of this alternative secondary modification.
Asunto(s)
Autoanticuerpos/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Precursores de Proteínas/inmunología , Serina/inmunología , Animales , Autoanticuerpos/genética , Autoanticuerpos/metabolismo , Sitios de Unión/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Citometría de Flujo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Progranulinas , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Proteína Quinasa C beta/genética , Proteína Quinasa C beta/inmunología , Proteína Quinasa C beta/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Miembro 25 de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 25 de Receptores de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Serina/genética , Serina/metabolismoRESUMEN
NELL2 is an abundant glycoprotein containing EGF-like domain in the neural tissues where it has multiple physiological functions by interacting with protein kinase C (PKC). There are two different splicing variant forms of NELL2 identified so far. One is secreted NELL2 (sNELL2) which is a neuron-specific variant and the other is cytosolic NELL2 (cNELL2) which is non-secreted splicing variant of NELL2. Although cNELL2 structure was well characterized, the expression pattern or the cellular function of cNELL2 is not fully determined. In this study, we found that cNELL2 specifically interacts with PKCß isotypes and inhibits PKCß1 through direct binding to the N-terminal pseudosubstrate domain of PKCß1. Here, we also demonstrate that cNELL2 is predominantly expressed and has inhibitory effects on the PKC downstream signaling pathways in astrocytes thereby establishing cNELL2 as an endogenous inhibitor of PKCß1 in glia.
Asunto(s)
Astrocitos/metabolismo , Citosol/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Quinasa C beta/metabolismo , Animales , Células CHO , Células Cultivadas , Cricetulus , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/análisis , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Isoformas de Proteínas/análisis , Isoformas de Proteínas/metabolismo , Proteína Quinasa C beta/análisis , Transducción de SeñalRESUMEN
It is widely known that ionizing radiation is a physical agent broadly used to kill tumor cells during human cancer therapy. Unfortunately, adjacent normal tissues can concurrently undergo undesirable cell injury. Previous data of our laboratory demonstrated that exposure of developing rats to ionizing radiations induced a variety of behavioral differences respect to controls, including changes in associative memory and in anxiety state. However, there is a lack of data concerning modifications in different related pharmacological intermediaries. Therefore, the aim of the present study was to investigate whether the behavioral differences observed in young animals irradiated at birth might be underlain by early changes in PKCß1 levels which, in turn, could lead to changes in hippocampal GABAergic neurotransmission. Male Wistar rats were irradiated with 5Gy of X rays between 24 and 48 h after birth. Different pharmacological markers related to the affected behavioral tasks were assessed in control and irradiated hippocampus at 15 and 30 days, namely GABAA receptor, GAD65-67, ROS and PKCß1. Results showed that all measured parameters were increased in the hippocampus of 30-days-old irradiated animals. In contrast, in the hippocampus of 15-days-old irradiated animals only the levels of PKCß1 were decreased. These data suggest that PKCß1 might constitute a primary target for neonatal radiation damage on the hippocampus. Therefore, it could be hypothesized that an initial decrease in the levels of this protein can trigger a subsequent compensatory increase that, in turn, could be responsible for the plethora of biochemical changes that might underlie the previously observed behavioral alterations.