RESUMEN
Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30 > RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may have detrimental effects for RMS cells and opens a window for further research into its role in RMS pathogenesis and for potential therapeutic targeting.
Asunto(s)
Proteínas Nucleares , Complejo de la Endopetidasa Proteasomal , Proteínas Represoras , Rabdomiosarcoma , Humanos , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Rabdomiosarcoma/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Represoras/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Musculares/metabolismo , Proteínas Musculares/análisis , Línea Celular TumoralRESUMEN
Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of pro-inflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-κB (NF-κB) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases.
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Bronquios , Levilactobacillus brevis , Enfermedades Pulmonares , Sistema de Señalización de MAP Quinasas , FN-kappa B , Antiinflamatorios/farmacología , Bronquios/citología , Bronquios/metabolismo , Bronquios/microbiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Levilactobacillus brevis/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/terapia , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismoRESUMEN
Striated muscle signaling protein and transcriptional regulator ANKRD2 participates in myogenesis, myogenic differentiation, muscle adaptation and stress response. It is preferentially expressed in slow, oxidative fibers of mammalian skeletal muscle. In this study, we report on characterization of chicken ANKRD2. The chicken ANKRD2 coding region contains 1002 bp and encodes a 334-amino acid protein which shares approximately 58% identity with human and mouse orthologs, mostly in the conserved region of ankyrin repeats. Comprehensive analysis of the ANKRD2 gene and protein expression in adult chicken demonstrated its predominant expression in red muscles of thigh and drumstick, compared to white muscle. It was not detected in heart and white pectoral muscle. Uneven expression of ANKRD2 in chicken skeletal muscles, observed by immunohistochemistry, was attributed to its selective expression in slow, oxidative, type I and fast, oxidative-glycolytic, type IIA myofibers. Association of chicken ANKRD2 with phenotypic differences between red and white muscles points to its potential role in the process of myofiber-type specification. In addition to expression in slow oxidative myofibers, as demonstrated for mammalian protein, chicken ANKRD2 was also detected in fast fibers with mixed oxidative and glycolytic metabolism. This finding suggests that ANKRD2 is responsive to metabolic differences between types of avian myofibers and orientates future studies towards investigation of its role in molecular mechanisms of myofiber-type-specific gene expression.
Asunto(s)
Proteínas Musculares/genética , Animales , Pollos , Clonación Molecular , Perfilación de la Expresión Génica , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismoRESUMEN
Four human Ankrd2 transcripts, reported in the Ensembl database, code for distinct protein isoforms (360, 333, 327 and 300 aa), and so far, their existence, specific expression and localization patterns have not been studied in detail. Ankrd2 is preferentially expressed in the slow fibers of skeletal muscle. It is found in both the nuclei and the cytoplasm of skeletal muscle cells, and its localization is prone to change during differentiation and upon stress. Ankrd2 has also been detected in the heart, in ventricular cardiomyocytes and in the intercalated disks (ICDs). The main objective of this study was to distinguish between the Ankrd2 isoforms and to determine the contribution of each one to the general profile of Ankrd2 expression in striated muscles. We demonstrated that the known expression and localization pattern of Ankrd2 in striated muscle can be attributed to the isoform of 333 aa which is dominant in both tissues, while the designated cardiac and canonical isoform of 360 aa was less expressed in both tissues. The 360 aa isoform has a distinct nuclear localization in human skeletal muscle, as well as in primary myoblasts and myotubes. In contrast to the isoform of 333 aa, it was not preferentially expressed in slow fibers and not localized to the ICDs of human cardiomyocytes. Regulation of the expression of both isoforms is achieved at the transcriptional level. Our results set the stage for investigation of the specific functions and interactions of the Ankrd2 isoforms in healthy and diseased human striated muscles.
Asunto(s)
Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Humanos , Proteínas Musculares/análisis , Proteínas Musculares/química , Músculo Esquelético/patología , Miocardio/patología , Proteínas Nucleares/análisis , Proteínas Nucleares/química , Isoformas de Proteínas/análisis , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Represoras/análisis , Proteínas Represoras/química , Alineación de SecuenciaRESUMEN
INTRODUCTION: Angiogenesis is the growth of both new vascular and lymphatic blood vessels from the existing vasculature. During this process, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) express specific markers, which help their discrimination and easier identification. Since the coronary thrombi material aspirated from patients with ST-elevation myocardial infarction (STEMI) proved as good angiogenesis model, we investigated the expression of CD34 and CD31 as BECs markers, and D2-40, LYVE-1 and VEGFR3 as LEC markers in this material. MATERIALS AND METHODS: Aspirated thrombi were stained immunohistochemically for CD34, CD31, D2-40, LYVE-1 and VEGFR3. Organizational patterns of immunopositive cells were graded as single cells, clusters or microvessels. Double immunofluorescence for CD31, D2-40, LYVE-1 and VEGRF3 was done. Thrombi were also graded as fresh (<1day old), lytic (1-5days old) and organized (>5days old). RESULTS: Serial sections of aspirated thrombi showed concordant BEC and LEC markers immunopositivity. Double immunoflorescence proved co-expression of CD31 and LEC markers on the same cells. Cells expressing LEC markers organized in clusters and microvessels were mainly present in lytic and organized thrombi. CONCLUSION: Co-expression of BEC and LEC markers on the same non-tumorous cell during thrombus neovascularization indicates existing in vivo plasticity of endothelial cells under non-tumorous pathological conditions. It also points that CD34 and CD31 on one hand, and D2-40, LYVE-1 and VEGFR3 immunostaining on the other hand, cannot solely be a reliable indicators whether vessel is lymphatic or not.
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Antígenos CD34/metabolismo , Células Endoteliales/metabolismo , Infarto del Miocardio/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Biomarcadores/análisis , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/sangre , Infarto del Miocardio/patología , Neovascularización Patológica/metabolismo , Trombosis/metabolismoRESUMEN
Muscle-specific mechanosensors Ankrd2/Arpp (ankyrin repeat protein 2) and Ankrd1/CARP (cardiac ankyrin repeat protein) have an important role in transcriptional regulation, myofibrillar assembly, cardiogenesis and myogenesis. In skeletal muscle myofibrils, Ankrd2 has a structural role as a component of a titin associated stretch-sensing complex, while in the nucleus it exerts regulatory function as transcriptional co-factor. It is also involved in myogenic differentiation and coordination of myoblast proliferation. Although expressed in the heart, the role of Ankrd2 in the cardiac muscle is completely unknown. Recently, we have shown that hypertrophic and dilated cardiomyopathy pathways are altered upon Ankrd2 silencing suggesting the importance of this protein in cardiac tissue. Here we provide the underlying basis for the functional investigation of Ankrd2 in the heart. We confirmed reduced Ankrd2 expression levels in human heart in comparison with Ankrd1 using RNAseq and Western blot. For the first time we demonstrated that, apart from the sarcomere and nucleus, both proteins are localized to the intercalated disks of human cardiomyocytes. We further tested the expression and localization of endogenous Ankrd2 in rat neonatal cardiomyocytes, a well-established model for studying cardiac-specific proteins. Ankrd2 was found to be expressed in both the cytoplasm and nucleus, independently from maturation status of cardiomyocytes. In contrast to Ankrd1, it is not responsive to the cardiotoxic drug Doxorubicin, suggesting that different mechanisms govern their expression in cardiac cells.
Asunto(s)
Proteínas Musculares/análisis , Músculo Esquelético/química , Miocardio/química , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/análisis , Proteínas Represoras/análisis , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Inmunohistoquímica , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Proteínas Nucleares/metabolismo , Ratas , Ratas Wistar , Proteínas Represoras/metabolismoRESUMEN
Transcription factor Nkx2.5, essential for heart development, regulates cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted (GATA4 and dHAND) or ubiquitous (p300) transcription regulators. Here we demonstrate that Nkx2.5 and p53 synergistically activate the promoter of the striated muscle stress responsive transcriptional cofactor Ankrd2, involved in coordination of proliferation and apoptosis during myogenic differentiation. Moreover, the p53 protein is able to interact with both wild type Nkx2.5 and its mutant ΔNkx2.5 (aa 1-198) found in patients with diverse cardiac malformations. Nkx2.5 interaction site of p53 maps to the C terminal region, while p53 binding site on Nkx2.5 lies outside its C terminus. In addition, overexpression of Nkx2.5 has a modulatory, promoter dependent effect on p53 transactivation, while the mutant significantly abolished p53 activity on the Mdm2, p21(WAF1/CIP1) and Bax promoters. Their physical interaction contributes to the observed behavior in the case of the Mdm2 promoter. Our data provide a new evidence for the role of p53 in cardiac function through interaction with Nkx2.5.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Miocardio/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Sitios de Unión/genética , Células COS , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación de la Expresión Génica , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Ratones , Desarrollo de Músculos/genética , Desarrollo de Músculos/fisiología , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Mapas de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Alpha-1 antitrypsin (AAT) gene is highly polymorphic, with a large number of rare variants whose phenotypic consequences often remain inconclusive. Studies addressing functional characteristics of AAT variants are of significant biomedical importance since deficiency and dysfunctionality of AAT are associated with liver and lung diseases. We report the results of the functional analysis of two naturally occurring AAT variants, G320R and V321F, previously identified in patients with lung disease. Neither of variants has been fully functionally characterized. In order to perform their functional analysis both variants were expressed in prokaryotic and eukaryotic systems and their intracellular localization, activity, stability, and polymerization were determined. The results of this study demonstrated that variants G320R and V321F have neither impaired activity against porcine pancreatic elastase nor propensity to form polymers. However, both variants had altered electrophoretic mobility and reduced thermostability when compared to M variant of the protein, indicating a slightly impaired secondary or tertiary structure.
Asunto(s)
alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Electroforesis , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Variación Genética , Células Hep G2 , Humanos , Hepatopatías/genética , Enfermedades Pulmonares/genética , Microscopía Confocal , Fenotipo , Porcinos , TransfecciónRESUMEN
PURPOSE: Sarcomas are rare and heterogenic tumors with unclear etiology. They develop in bone and connective tissue, mainly in pediatric patients. To increase efficacy of current therapeutic options, natural products showing selective toxicity to tumor cells are extensively investigated. Here, we evaluated antitumor activity of bacterial pigment violacein in osteosarcoma (OS) and rhabdomyosarcoma (RMS) cell lines. METHODS: The toxicity of violacein was assessed in vitro and in vivo, using MTT assay and FET test. The effect of violacein on cell migration was monitored by wound healing assay, cell death by flow cytometry, uptake of violacein by fluorescence microscopy, generation of reactive oxygen species (ROS) by DCFH-DA assay and lipid peroxidation by TBARS assay. RESULTS: Violacein IC50 values for OS and RMS cells were in a range from 0.35 to 0.88 µM. Its selectivity toward malignant phenotype was confirmed on non-cancer V79-4 cells, and it was safe in vivo, for zebrafish embryos in doses up to 1 µM. Violacein induced apoptosis and affected the migratory potential of OS and RMS cells. It was found on the surfaces of tested cells. Regarding the mechanism of action, violacein acted on OS and RMS cells independently of oxidative signaling, as judged by no increase in intracellular ROS generation and no lipid peroxidation. CONCLUSION: Our study provided further evidence that reinforces the potential of violacein as an anticancer agent and candidate to consider for improvement of the effectiveness of traditional OS and RMS therapies.
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Osteosarcoma , Rabdomiosarcoma , Animales , Especies Reactivas de Oxígeno/metabolismo , Pez Cebra/metabolismo , Línea Celular , Apoptosis , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma/patología , Osteosarcoma/tratamiento farmacológico , Línea Celular Tumoral , Proliferación CelularRESUMEN
The biological activity of Cd compounds has been investigated scarce since Cd has been recognized as a human carcinogen. However, the toxicity of cadmium is comparable to the toxicity of noble metals such as Pt and Pd. The paradigm of metal toxicity has been challenged suggesting that metal toxicity is not a constant property, yet it depends on many factors like the presence of appropriate ligands. Studies on anticancer activity of cadmium complexes showed that the complexation of various ligands resulted in complexes that showed better activities than approved drugs. In the present study, cadmium complexes with biologically potent thiazolyl/selenazoyl-hydrazone ligands have been prepared, and tested for their activity against different types of tumor cell models. The complexation of ligands with Cd(II) resulted in a synergistic effect. The antiproliferative activity study revealed that all complexes are more active compared to 5-fluorouracil and cisplatin. The mechanism of tumor cell growth inhibition reveal that selenium-based compounds induce cell death in T-47D (gland carcinoma) cells through apoptosis via caspase-3/7 activation. Additionally, their pro-apoptotic effect was stronger compared to etoposide and cisplatin. Nuclease activity, detected by gel electrophoresis, may be the possible mechanism of anticancer action of investigated complexes.
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Antineoplásicos , Complejos de Coordinación , Neoplasias , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Cadmio/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Complejos de Coordinación/farmacología , Complejos de Coordinación/uso terapéutico , Humanos , Hidrazonas/farmacología , Hidrazonas/uso terapéutico , Ligandos , Neoplasias/tratamiento farmacológico , Azufre/farmacología , Azufre/uso terapéuticoRESUMEN
Remodeling is a stringently controlled process that enables adequate response of muscle cells to constant physical stresses. In this process, different kinds of stimuli have to be sensed and converted into biochemical signals that ultimately lead to alterations of muscle phenotype. Several multiprotein complexes located in the sarcomere and organized on the titin molecular spring have been identified as stress sensors and signal transducers. In this review, we focus on Ankrd1/CARP and Ankrd2/Arpp proteins,which belong to the muscle ankyrin repeat protein family (MARP) involved in a mechano-signaling pathway that links myofibrillar stress response to muscle gene expression. Apart from the mechanosensory function, they have an important role in transcriptional regulation, myofibrillar assembly, cardiogenesis and myogenesis. Their altered expression has been demonstrated in neuromuscular disorders, cardiovascular diseases, as well as in tumors, suggesting a role in pathological processes. Although analyzed in a limited number of patients, there is a considerable body of evidence that MARP proteins could be suitable candidates for prognostic and diagnostic biomarkers.
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Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Adaptación Fisiológica/fisiología , Biomarcadores/metabolismo , Enfermedades Cardiovasculares/metabolismo , Humanos , Desarrollo de Músculos/fisiología , Neoplasias/metabolismo , Enfermedades Neuromusculares/metabolismoRESUMEN
Ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic protein found in the nuclei and sarcomeres of cardiac and skeletal muscles, with a proposed role in linking myofibrilar stress and transcriptional regulation. Rapid upregulation of its expression in response to both physiological and pathological stress supports the involvement of ANKRD1 in muscle tissue adaptation and remodeling. However, the exact role of ANKRD1 remains poorly understood. To begin to investigate its function at higher resolution, we have generated and characterized a TgBAC(ankrd1a:EGFP) zebrafish line. This reporter line displays transgene expression in slow skeletal muscle fibers during development and exercise responsiveness in adult cardiac muscle. To better understand the role of Ankrd1a in pathological conditions in adult zebrafish, we assessed ankrd1a expression after cardiac ventricle cryoinjury and observed localized upregulation in cardiomyocytes in the border zone. We show that this expression in injured hearts is recapitulated by the TgBAC(ankrd1a:EGFP) reporter. Our results identify novel expression domains of ankrd1a and suggest an important role for Ankrd1a in the early stress response and regeneration of cardiac tissue. This new reporter line will help decipher the role of Ankrd1a in striated muscle stress response, including after cardiac injury.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Musculares/genética , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Estrés Fisiológico/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ventrículos Cardíacos/crecimiento & desarrollo , Ventrículos Cardíacos/lesiones , Ventrículos Cardíacos/metabolismo , Desarrollo de Músculos/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/patología , Proteínas Nucleares/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Ankrd2 is a protein known for being mainly expressed in muscle fibers, where it participates in the mechanical stress response. Since both myocytes and osteoblasts are mesenchymal-derived cells, we were interested in examining the role of Ankrd2 in the progression of osteosarcoma which features a mechano-stress component. Although having been identified in many tumor-derived cell lines and -tissues, no study has yet described nor hypothesized any involvement for this protein in osteosarcoma tumorigenesis. In this paper, we report that Ankrd2 is expressed in cell lines obtained from human osteosarcoma and demonstrate a contribution by this protein in the pathogenesis of this insidious disease. Ankrd2 involvement in osteosarcoma development was evaluated in clones of Saos2, U2OS, HOS and MG63 cells stably expressing Ankrd2, through the investigation of hallmark processes of cancer cells. Interestingly, we found that exogenous expression of Ankrd2 influenced cellular growth, migration and clonogenicity in a cell line-dependent manner, whereas it was able to improve the formation of 3D spheroids in three out of four cellular models and enhanced matrix metalloproteinase (MMP) activity in all tested cell lines. Conversely, downregulation of Ankrd2 expression remarkably reduced proliferation and clonogenic potential of parental cells. As a whole, our data present Ankrd2 as a novel player in osteosarcoma development, opening up new therapeutic perspectives.
RESUMEN
The muscle ankyrin repeat protein (MARP) family member Ankrd1/CARP is a part of the titin-mechanosensory signaling complex in the sarcomere and in response to stretch it translocates to the nucleus where it participates in the regulation of cardiac genes as a transcriptional co-repressor. Several studies have focused on its structural role in muscle, but its regulatory role is still poorly understood. To gain more insight into the regulatory function of Ankrd1/CARP we searched for transcription factors that could interact and modulate its activity. Using protein array methodology we identified the tumor suppressor protein p53 as an Ankrd1/CARP interacting partner and confirmed their interaction both in vivo and in vitro. We demonstrate a novel role for Ankrd1/CARP as a transcriptional co-activator, moderately up regulating p53 activity. Furthermore, we show that p53 operates as an upstream effector of Ankrd1/CARP, by up regulating the proximal ANKRD1 promoter. Our findings suggest that, besides acting as a transcriptional co-repressor, Ankrd1/CARP could have a stimulatory effect on gene expression in cultured skeletal muscle cells. It is probable that Ankrd1/CARP has a role in the propagation of signals initiated by myogenic regulatory factors (MRFs) during myogenesis.
Asunto(s)
Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Repetición de Anquirina , Secuencia de Bases , Células COS , Línea Celular , Chlorocebus aethiops , Cartilla de ADN/genética , Humanos , Técnicas In Vitro , Ratones , Desarrollo de Músculos/genética , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Proteína MioD/genética , Proteína MioD/metabolismo , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Análisis por Matrices de Proteínas , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Transducción de Señal , Activación Transcripcional , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Ankrd2 (ankyrin repeats containing domain 2) or Arpp (ankyrin repeat, PEST sequence, and proline-rich region) is a member of the muscle ankyrin repeat protein family. Ankrd2 is mostly expressed in skeletal muscle, where it plays an intriguing role in the transcriptional response to stress induced by mechanical stimulation as well as by cellular reactive oxygen species. Our studies in myoblasts from Emery-Dreifuss muscular dystrophy 2, a LMNA-linked disease affecting skeletal and cardiac muscles, demonstrated that Ankrd2 is a lamin A-binding protein and that mutated lamins found in Emery-Dreifuss muscular dystrophy change the dynamics of Ankrd2 nuclear import, thus affecting oxidative stress response. In this review, besides describing the latest advances related to Ankrd2 studies, including novel discoveries on Ankrd2 isoform-specific functions, we report the main findings on the relationship of Ankrd2 with A-type lamins and discuss known and potential mechanisms involving defective Ankrd2-lamin A interplay in the pathogenesis of muscular laminopathies.
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Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Emery-Dreifuss/patología , Proteínas Nucleares/metabolismo , Estrés Oxidativo , Proteínas Represoras/metabolismo , Humanos , Lamina Tipo A/metabolismo , Mecanotransducción Celular , Proteínas Musculares/química , Distrofia Muscular de Emery-Dreifuss/metabolismo , Miocardio/metabolismo , Proteínas Nucleares/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras/químicaRESUMEN
Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle.
Asunto(s)
Repetición de Anquirina , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Condicionamiento Físico Animal , Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica , Humanos , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Filogenia , Alineación de Secuencia , Estrés Fisiológico , Sintenía , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The aetiology of chronic obstructive pulmonary disease (COPD) is complex. While cigarette smoking is a well-established cause of COPD, a myriad of assessed genetic factors has given conflicting data. Since gene-environment interactions are thought to be implicated in aetiopathogenesis of COPD, we aimed to examine the matrix metalloproteinase (MMP) 9 C-1562T (rs3918242) functional variant and cigarette smoke in the pathogenesis of this disease. The distribution of the MMP9 C-1562T variant was analyzed in COPD patients and controls with normal pulmonary function from Serbia. Interaction between the C-1562T genetic variant and cigarette smoking was assessed using a case-control model. The response of the C-1562T promoter variant to cigarette smoke condensate (CSC) exposure was examined using a dual luciferase reporter assay. The frequency of T allele carriers was higher in the COPD group than in smoker controls (38.4% vs. 20%; OR = 2.7, P = 0.027). Interaction between the T allele and cigarette smoking was identified in COPD occurrence (OR = 4.38, P = 0.005) and severity (P = 0.001). A functional analysis of the C-1562T variant demonstrated a dose-dependent and allele-specific response (P < 0.01) to CSC. Significantly higher MMP9 promoter activity following CSC exposure was found for the promoter harboring the T allele compared to the promoter harboring the C allele (P < 0.05). Our study is the first to reveal an interaction between the MMP9-1562T allele and cigarette smoke in COPD, emphasising gene-environment interactions as a possible cause of lung damage in the pathogenesis of COPD. Environ. Mol. Mutagen. 57:447-454, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Interacción Gen-Ambiente , Variación Genética , Metaloproteinasa 9 de la Matriz/genética , Regiones Promotoras Genéticas , Enfermedad Pulmonar Obstructiva Crónica/etiología , Fumar/efectos adversos , Estudios de Casos y Controles , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Enfermedad Pulmonar Obstructiva Crónica/genética , Células U937RESUMEN
Ankrd2 may be a link between the sarcomere and the nucleus; a similar role has recently been proposed for CARP that has a high level of structural and functional conservation with Ankrd2. Both Ankrd2 and CARP are involved in striated muscle hypertrophy. The mechanism by which muscle stretch is sensed and signals are transduced is still unknown; however, Ankrd2 and CARP could play similar roles in pathways leading to hypertrophy, the triggering mechanisms being heart pressure overload monitored by CARP and mechanical stretch in skeletal muscle monitored by Ankrd2. Recently Ankrd2 and CARP have been proposed as members of a family of muscle ankyrin repeat proteins (MARPs) that form a complex with titin, myopalladin and calpain protease p94, involved in signaling and regulation of gene expression in response to muscle stress. Here, we show that Ankrd2 is able to interact with the Z-disc protein telethonin as well as being able to interact with three transcription factors: YB-1, PML and p53. Ankrd2 binding to the ubiquitous transcription factor YB-1 can be demonstrated both in vitro and in vivo; this is not very surprising, since a similar interaction was previously described for CARP. However, the interactions with PML and p53 are unexpected new findings, with interesting implications in the Ankrd2 signaling cascade. Ankrd2 co-localizes with the transcriptional co-activator and co-repressor PML in nuclear bodies (NBs) in human myoblasts as detected by confocal immunofluorescence. Interestingly, we show that Ankrd2 not only binds the tumor suppressor protein p53 both in vitro and in vivo but also enhances the up-regulation of the p21(WAFI/CIPI) promoter by p53. Therefore, our findings strengthen the hypothesis that Ankrd2 may be involved in sensing stress signals and linking these to muscle gene regulation.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Sarcómeros/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , Conectina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , Humanos , Microscopía Fluorescente , Factores de Transcripción NFI , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteína de la Leucemia Promielocítica , Unión Proteica , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor , Regulación hacia Arriba , Proteína 1 de Unión a la Caja YRESUMEN
Eleven Lactobacillus strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of Lactobacillus paracasei subsp. paracasei BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the aggLb gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of aggLb causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the aggLb gene of BGNJ1-64 enabled detection of the same type of aggLb gene in five of eleven selected aggregation-positive Lactobacillus strains. Heterologous expression of aggLb confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization.
Asunto(s)
Proteínas Bacterianas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Colágeno/metabolismo , Lactobacillus/metabolismo , Secuencia de Aminoácidos , Adhesión Bacteriana , Proteínas Bacterianas/genética , Moléculas de Adhesión Celular/genética , Colágeno/química , Interacciones Hidrofóbicas e Hidrofílicas , Lactobacillus/patogenicidad , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
Diminished glucocorticoid signaling is associated with an age-related decline in hippocampal functioning. In this study we demonstrate the effect of intermittent, every other day (EOD) feeding on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the hippocampus of middle-aged (18-month-old) and aged (24-month-old) Wistar rats. In aged ad libitum-fed rats, a decrease in the level of total GR and GR phosphorylated at Ser(232) (pGR) was detected. Conversely, aged rats subjected to EOD feeding, starting from 6 months of age, showed an increase in GR and pGR levels and a higher content of hippocampal corticosterone. Furthermore, prominent nuclear staining of pGR was observed in CA1 pyramidal and DG granule neurons of aged EOD-fed rats. These changes were accompanied by increased Sgk-1 and decreased GFAP transcription, pointing to upregulated transcriptional activity of GR. EOD feeding also induced an increase in the expression of the mineralocorticoid receptor. Our results reveal that intermittent feeding restores impaired GR signaling in the hippocampus of aged animals by inducing rather than by stabilizing GR signaling during aging.