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Glomerular epithelial protein-1 (Glepp1), a R3 subtype family of receptor-type protein tyrosine phosphatases, plays important role in the activation of Src family kinases and regulates cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, we firstly examined the functional evaluation of Glepp1 in tooth development and morphogenesis. The precise expression level and developmental function of Glepp1 were examined by RT-qPCR, in situ hybridization, and loss and gain of functional study using a range of in vitro organ cultivation methods. Expression of Glepp1 was detected in the developing tooth germs in cap and bell stage of tooth development. Knocking down Glepp1 at E13 for 2 days showed the altered expression levels of tooth development-related signaling molecules, including Bmps, Dspp, Fgf4, Lef1, and Shh. Moreover, transient knock down of Glepp1 revealed alterations in cellular physiology, examined by the localization patterns of Ki67 and E-cadherin. Similarly, knocking down of Glepp1 showed disrupted enamel rod and interrod formation in 3-week renal transplanted teeth. In addition, due to attrition of odontoblastic layers, the expression signals of Dspp and the localization of NESTIN were almost not detected after knock down of Glepp1; however, their expressions were increased after Glepp1 overexpression. Thus, our results suggested that Glepp1 plays modulating roles during odontogenesis by regulating the expression levels of signaling molecules and cellular events to achieve the proper structural formation of hard tissue matrices in mice molar development.
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Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Diente , Animales , Ratones , Regulación del Desarrollo de la Expresión Génica , Morfogénesis , Odontogénesis , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de Señal , Diente/metabolismoRESUMEN
Peptidoglycan recognition proteins (PGRPs) and Toll-like receptors (TLRs) are highly conserved pattern recognition receptors (PRRs). Earthworms possess genes encoding TLRs that specifically respond to Gram-positive bacteria. In addition, several PGRPs have been recently identified, which are predicted to exhibit amidase activity but lack receptor function. In lophotrochozoans, a membrane-bound PRR responsible for detecting Gram-negative bacteria remains unidentified. This study reveals several novel transmembrane peptidoglycan recognition proteins (Ean-PGRPLs) in earthworms, whose mRNA expression increases in response to Gram-negative but not Gram-positive bacteria. This indicates that Ean-PGRPLs may serve as a PRR associated with intracellular signaling for Gram-negative bacteria.
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Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.
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Oligoquetos , Animales , Filogenia , Receptor Toll-Like 1/genética , Ligandos , Receptor Toll-Like 6/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Receptores de Reconocimiento de Patrones/genética , Bacterias/metabolismo , Inmunidad Innata/genética , Mamíferos/metabolismoRESUMEN
Antistasin, which was originally discovered in the salivary glands of the Mexican leech Haementeria officinalis, was newly isolated from Helobdella austinensis. To confirm the temporal expression of antistasin during embryogenesis, we carried out semi-quantitative RT-PCR. Hau-antistasin1 was uniquely expressed at stage 4 of the cleavage and was strongly expressed in the late stages of organogenesis, as were other antistasin members. In order to confirm the spatial expression of antistasin, we performed fluorescence in situ hybridization in the late stages of organogenesis. The expression of each antistasin in the proboscis showed a similar pattern and varied in expression in the body. In addition, the spatial expression of antistasin orthologs in different leeches showed the possibility of different function across leech species. Hau-antistasin1 was expressed in the same region as hedgehog, which is a known mediator of signal transduction pathway. Hau-antistasin1 is probably a downstream target of Hedgehog signaling, involved in segment polarity signal pathway.
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Anticoagulantes/análisis , Hormonas de Invertebrados/análisis , Sanguijuelas/química , Animales , Anticoagulantes/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/análisis , Proteínas Hedgehog/metabolismo , Hormonas de Invertebrados/genética , Hormonas de Invertebrados/metabolismo , Sanguijuelas/embriología , Sanguijuelas/genética , Sanguijuelas/metabolismo , Filogenia , Transducción de SeñalRESUMEN
The Forkhead box (Fox) gene family is an evolutionarily ancient gene family named after the Drosophila melanogaster forkhead gene (fkh). Fox genes are highly conserved transcription factors critical for embryogenesis and carcinogenesis. In the current study, we report a whole-genome survey of Fox genes and their expression patterns in the leech Helobdella austienesis. Phylogenetic analysis suggests that some Fox genes of leeches are correlated with other Lophotrochozoa and vertebrate Fox genes. Here we have performed semiquantitative reverse transcription polymerase chain reaction and whole-mount in situ hybridization of Fox genes throughout the embryonic development of H. austinensis. We found that each one of the leech Fox genes (FoxA1, FoxA3, FoxC, FoxL2, FoxO1, and FoxO2) is expressed in a specific set of cells or tissue type. From Stages 9-11, Hau-FoxA1 was expressed in the foregut of the anterior region, and Hau-FoxL2 was expressed in mesodermal muscle fiber. Hau-FoxA3 was temporally expressed in the ventral neuroectoderm. At Stage 11, Hau-FoxC was expressed in the foregut. Hau-FoxO genes have a ubiquitous expression. Our results provide more insight on the evolutionary linkage and role of the Fox gene function in Bilateria.
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Factores de Transcripción Forkhead/metabolismo , Sanguijuelas/embriología , Sanguijuelas/metabolismo , Animales , Ectodermo/embriología , Ectodermo/metabolismo , Embrión no Mamífero/metabolismo , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Mesodermo/metabolismo , Filogenia , Secuenciación del ExomaRESUMEN
Genes of the twist family encode bHLH transcription factors known to be involved in the regulation and differentiation of early mesoderm. Here, we report our characterization of Hau-twist, a twist homolog from the leech Helobdella austinensis, a tractable lophotrochozoan representative. Hau-twist was expressed in segmental founder cells of the mesodermal lineage, in subsets of cells within the mesodermal lineage of the germinal plate, in circumferential muscle fibers of a provisional integument during segmentation and organogenesis stages and on the ventral side of the developing proboscis. Thus, consistent with other systems, our results suggest that twist gene of the leech Helobdella might function in mesoderm differentiation.
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Sanguijuelas/crecimiento & desarrollo , Sanguijuelas/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Embrión no Mamífero/metabolismo , Sanguijuelas/citología , Mesodermo/citología , Mesodermo/metabolismo , FilogeniaRESUMEN
snail gene family members are zinc-finger transcription factors with key roles in morphogenesis. Involvement of snail family genes in mesoderm formation has been observed in insects and mammals. The snail genes are also involved in cell motility, neural differentiation, cell fate, survival decision, and left-right identity. The functions of snail genes have been studied primarily among ecdysozoans and deuterostomes, with relatively little work carried out in lophotrochozoans. In this study, we isolated two snail homologs (Hau-snail1 and Hau-snail2) from the leech Helobdella austinensis. We characterized the temporal and spatial expression patterns of these two genes by semi-quantitative RT-PCR and in situ hybridization. The expression of Hau-snail1 and Hau-snail2 correlates with ventral nerve cord (VNC) development, segmental mesoderm, and with a ring of cells that comes to lie at the base of the leech proboscis, respectively, showing similarity to the divergent expression of duplicated snail genes in polychaetes. Our results do not support the function of lophotrochozoan snail genes in mesoderm specification.
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Sanguijuelas/crecimiento & desarrollo , Sanguijuelas/genética , Factores de Transcripción de la Familia Snail/genética , Transcriptoma , Secuencia de Aminoácidos , Animales , Clonación Molecular , Duplicación de Gen , Sanguijuelas/metabolismo , Filogenia , Análisis de Secuencia de ADN , Factores de Transcripción de la Familia Snail/química , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
BACKGROUND: The need for the adaptation of species of annelids as "Evo-Devo" model organisms of the superphylum Lophotrochozoa to refine the understanding of the phylogenetic relationships between bilaterian organisms, has promoted an increase in the studies dealing with embryonic development among related species such as leeches from the Glossiphoniidae family. The present study aims to describe the embryogenesis of Alboglossiphonia lata (Oka, 1910), a freshwater glossiphoniid leech, chiefly distributed in East Asia, and validate standard molecular biology techniques to support the use of this species as an additional model for "Evo-Devo" studies. RESULTS: A. lata undergoes direct development, and follows the highly conserved clitellate annelid mode of spiral cleavage development; the duration from the egg laying to the juvenile stage is ~7.5 days, and it is iteroparous, indicating that it feeds and deposits eggs again after the first round of brooding, as described in several other glossiphoniid leech species studied to date. The embryos hatch only after complete organ development and proboscis retraction, which has not yet been observed in other glossiphoniid genera. The phylogenetic position of A. lata within the Glossiphoniidae family has been confirmed using cytochrome c oxidase subunit 1 (CO1) sequencing. Lineage tracer injections confirmed the fates of the presumptive meso- and ectodermal precursors, and immunostaining showed the formation of the ventral nerve system during later stages of development. Further, the spatiotemporal expression of an EF-hand calcium-binding protein Calsensin ortholog was characterized, which showed a specific pattern in both the ventral and peripheral nervous systems during the later stages. CONCLUSIONS: Our description of the embryonic development of A. lata under laboratory conditions provides new data for further comparative studies with other leech and lophotrochozoa model organisms. Moreover, it offers a basis for the establishment of this species as a model for future "Evo-Devo" studies.
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BACKGROUND: The blood-brain barrier (BBB) is a specialized layer between blood vessels and tissue in the brain, which is comprised of a neuro-glia-vascular (NGV) unit, thus play a vital role in various brain diseases. NEW METHOD: We developed the in vitro NGV units by co-culturing brain microvascular endothelial cells (BMECs; bEnd.3) and primary neural stem cells extracted from subventricular zone of adult mice. This approach was designed to mimic the RNA profile conditions found in the microvessels of a mouse brain and confirmed through various comparative transcriptome analyses. RESULTS: Optimal NGV unit development was achieved by adjusting cell density-dependent co-culture ratios. Specifically, the morphogenic development and neuronal association of astrocyte endfeet were well observed in the contact region with BMECs in the NGV unit. Through transcriptome analysis, we compared co-cultured bEnd.3/NSCs with monocultured bEnd.3 or NSCs and additionally compared them with previously reported mouse brain vascular tissue to show that this NGV unit model is a suitable in vitro model for neurological disease such as Alzheimer's disease (AD). COMPARISON WITH EXISTING METHOD(S): This in vitro NGV unit was formed from neural stem cells and vascular cells in the brain of adult mice, not embryos. It is very useful for studying brain disease mechanisms by identifying proteins and genes associated with diseases progress. CONCLUSIONS: We suggest that this simple in vitro NGV model is appropriate to investigate the relationship between BBB changes and pathological factors in the fields of neurovascular biology and cerebrovascular diseases including AD.
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Células-Madre Neurales , Animales , Ratones , Enfermedad de Alzheimer/patología , Barrera Hematoencefálica/fisiología , Encéfalo , Técnicas de Cocultivo , Células Endoteliales/fisiología , Perfilación de la Expresión Génica , Neuroglía/patologíaRESUMEN
Currently used visible light catalysts either operate with high-power light sources or require prolonged periods of time for catalytic reactions. This presents a limitation regarding facile application in indoor environments and spaces frequented by the public. Furthermore, this gives rise to elevated power consumption. Here, we enhance photocatalytic performance with blue TiO2 and WO3 complexes covalently coupled through an organic molecule, 3-mercaptopropionic acid, under indoor light. Antibacterial experiments against 108 CFU/mL Escherichia coli (E. coli) suspensions were conducted under indoor light exposure conditions. They showed a sterilization effect of almost 90% within 70 min and nearly 100% after 110 min. The complex generates reactive oxygen species (ROS), such as â¢OH and O2â¢-, under natural air conditions. We also showed that h+ and â¢OH are important for sterilizing E. coli using common scavengers. This research highlights the potential of these complexes to generate ROS, effectively playing a crucial role in antibacterial effects under indoor light.
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Antibacterianos , Escherichia coli , Luz , Especies Reactivas de Oxígeno , Titanio , Tungsteno , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Titanio/química , Titanio/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Tungsteno/química , Tungsteno/farmacología , Catálisis , Especies Reactivas de Oxígeno/metabolismo , Óxidos/farmacología , Óxidos/química , Pruebas de Sensibilidad MicrobianaRESUMEN
Unlike vertebrates, the number of toothed taxa in invertebrates is very few, with leeches being the only tooth-bearing organisms in the phylum Annelida. Copious studies have been conducted regarding vertebrate teeth; however, studies regarding the structure and function of invertebrate teeth are limited. In this study, the tooth structure of leeches, specifically Hirudo nipponia and Haemadipsa rjukjuana, was revealed, which showed sharp and pointed teeth along the apex of three jaws. Understanding conserved signaling regulations among analogous organs is crucial for uncovering the underlying mechanisms during organogenesis. Therefore, to shed light on the evolutionary perspective of odontogenesis to some extent, we conducted de novo transcriptome analyses using embryonic mouse tooth germs, Hirudo teeth, and Helobdella proboscises to identify conserved signaling molecules involved in tooth development. The selection criteria were particularly based on the presence of tooth-related genes in mice, Hirudo teeth, and Helobdella proboscis, wherein 4113 genes were commonly expressed in all three specimens. Furthermore, the chemical nature of leech teeth was also examined via TEM-EDS to compare the chemical composition with vertebrate teeth. The examination of tissue-specific genetic information and chemical nature between leeches and mice revealed chemical similarities between leech and mice teeth, as well as conserved signaling molecules involved in tooth formation, including Ptpro, Prickle2, and Wnt16. Based on our findings, we propose that leech teeth express signaling molecules conserved in mice and these conserved tooth-specific signaling for dental hard tissue formation in mice would corresponds to the structural formation of the toothed jaw in leeches.
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BACKGROUND: Slit and Robo are evolutionarily conserved ligand and receptor proteins, respectively, but the number of slit and robo gene paralogs varies across recent bilaterian genomes. Previous studies indicate that this ligand-receptor complex is involved in axon guidance. Given the lack of data regarding Slit/Robo in the Lophotrochozoa compared to Ecdysozoa and Deuterostomia, the present study aims to identify and characterize the expression of Slit/Robo orthologs in leech development. RESULTS: We identified one slit (Hau-slit), and two robo genes (Hau-robo1 and Hau-robo2), and characterized their expression spatiotemporally during the development of the glossiphoniid leech Helobdella austinensis. Throughout segmentation and organogenesis, Hau-slit and Hau-robo1 are broadly expressed in complex and roughly complementary patterns in the ventral and dorsal midline, nerve ganglia, foregut, visceral mesoderm and/or endoderm of the crop, rectum and reproductive organs. Before yolk exhaustion, Hau-robo1 is also expressed where the pigmented eye spots will later develop, and Hau-slit is expressed in the area between these future eye spots. In contrast, Hau-robo2 expression is extremely limited, appearing first in the developing pigmented eye spots, and later in the three additional pairs of cryptic eye spots in head region that never develop pigment. Comparing the expression of robo orthologs between H. austinensis and another glossiphoniid leech, Alboglossiphonia lata allows to that robo1 and robo2 operate combinatorially to differentially specify pigmented and cryptic eyespots within the glossiphoniid leeches. CONCLUSIONS: Our results support a conserved role in neurogenesis, midline formation and eye spot development for Slit/Robo in the Lophotrochozoa, and provide relevant data for evo-devo studies related to nervous system evolution.
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Several pattern recognition receptors (PRRs) involved in innate immunity have been identified and characterized in earthworms. Peptidoglycan recognition proteins (PGRPs) are highly conserved PRRs that activate effector pathways such as prophenoloxidase cascade and Toll-like receptor pathway. In addition, PGRPs function as an enzyme, N-acetylmuramoyl-l-alanine amidase (NAMLAA), to directly hydrolyze peptidoglycan. We identified four full-length complementary DNA (cDNA) sequences, Ean-PGRP1/2/3/4, in Eisenia andrei, an earthworm. Sequence and phylogenetic analyses indicate that earthworm PGRP orthologs resemble short PGRP member proteins. The subcellular localizations of four Ean-PGRPs lacking the transmembrane domain are predicted to be extracellular or cytoplasmic. All Ean-PGRPs contain a highly conserved PGRP domain with a conserved Zn2+ binding site including a tyrosine residue essential for active amidase activity. Three highly conserved amino-acid residues (His, Trp, and Thr) necessary for amidase activity are also present, indicating that the Ean-PGRPs can be predicted to have amidase activity. Furthermore, we demonstrate that the Ean-PGRP genes are differentially induced by certain bacterial species, suggesting that the innate immune system of earthworms is likely to be somewhat specific rather than entirely non-specific. Tissue expression patterns indicate that Ean-PGRP mRNAs are primarily expressed in the immune-competent tissues and that their expression is tissue-specific according to Ean-PGRP types, particularly for Ean-PGRP1.
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Oligoquetos , Secuencia de Aminoácidos , Animales , Proteínas Portadoras , ADN Complementario , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Oligoquetos/genética , Peptidoglicano/metabolismo , FilogeniaRESUMEN
Cephalization refers to the evolutionary trend towards the concentration of neural tissues, sensory organs, mouth and associated structures at the front end of bilaterian animals. Comprehensive studies on gene expression related to the anterior formation in invertebrate models are currently lacking. In this study, we performed de novo transcriptional profiling on a proboscis-bearing leech (Helobdella austinensis) to identify differentially expressed genes (DEGs) in the anterior versus other parts of the body, in particular to find clues as to the development of the proboscis. Between the head and the body, 132 head-specific DEGs were identified, of which we chose 11 to investigate their developmental function during embryogenesis. Analysis of the spatial expression of these genes using in situ hybridization showed that they were characteristically expressed in the anterior region of the developing embryo, including the proboscis. Our results provide information on the genes related to head formation and insights into the function of proboscis-related genes during organogenesis with the potential roles of genes not yet characterized.
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Sanguijuelas , Animales , Perfilación de la Expresión Génica , Sanguijuelas/genética , Sanguijuelas/metabolismo , Organogénesis/genética , TranscriptomaRESUMEN
Adaptive radiation is a phenomenon in which various organs are diversified morphologically or functionally as animals adapt to environmental inputs. Leeches exhibit a variety of ingestion behaviors and morphologically diverse ingestion organs. In this study, we investigated the correlation between behavioral pattern and feeding organ structure of leech species. Among them, we found that Alboglossiphonia sp. swallows prey whole using its proboscis, whereas other leeches exhibit typical fluid-sucking behavior. To address whether the different feeding behaviors are intrinsic, we investigated the behavioral patterns and muscle arrangements in the earlier developmental stage of glossiphoniid leeches. Juvenile Glossiphoniidae including the Alboglossiphonia sp. exhibit the fluid ingestion behavior and have the proboscis with the compartmentalized muscle layers. This study provides the characteristics of leeches with specific ingestion behaviors, and a comparison of structural differences that serves as the first evidence of the proboscis diversification.
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Evolución Biológica , Conducta Alimentaria , Tracto Gastrointestinal , Sanguijuelas/anatomía & histología , Sanguijuelas/fisiología , Animales , Carnivoría , Sanguijuelas/genéticaRESUMEN
Hyaluronic acid (HA) has been implicated in cell adhesion, motility, and tumor progression in gliomas. We previously reported that HA stimulates secretion of matrix metalloproteinase-9 (MMP-9) and induces glioma invasion. However, the molecular mechanism of HA action and therapeutic strategies for blocking HA-induced MMP-9 secretion remain unknown. Here, we report that the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) blocks MMP-9 secretion and that HA-induced nuclear factor-kappaB (NF-kappaB) activation is mediated by IkappaB kinase, which phosphorylates the NF-kappaB inhibitor IkappaBalpha and promotes its degradation. In addition, using an RNA interference approach, we show that the focal adhesion kinase plays a critical role in mediating HA-induced NF-kappaB activation, which resulted in increased MMP-9 expression and secretion, cell migration, and invasion. Importantly, we show that 17-AAG acts by blocking focal adhesion kinase activation, thereby inhibiting IkappaB kinase-dependent IkappaBalpha phosphorylation/degradation, NF-kappaB activation, and MMP-9 expression. This leads to suppression of HA-induced cell migration and invasion. Based on our data, we propose that 17-AAG is a candidate drug for treatment of highly invasive gliomas resulting from HA-induced, NF-kappaB-mediated MMP-9 secretion.
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Benzoquinonas/farmacología , Glioma/metabolismo , Ácido Hialurónico/metabolismo , Lactamas Macrocíclicas/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Línea Celular Tumoral , Movimiento Celular , Núcleo Celular/metabolismo , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Matriz Extracelular/patología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Glioma/tratamiento farmacológico , Glioma/patología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/farmacología , Humanos , Ácido Hialurónico/farmacología , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , Interferencia de ARN , Transducción de SeñalRESUMEN
53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and gammaH2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.
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Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Células HeLa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Radiación Ionizante , Serina/genética , Serina/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Heat-shock protein 27 (HSP27), a member of the small heat-shock protein family, is a molecule involved in cellular protection in response to a variety of stresses such as heat shock, toxicants, and oxidative stress. HSP27 is also known to modulate cell functions via interaction with the actin cytoskeleton. To elucidate the functions of HSP27 in adhesion and invasion in more detail, we examined NIH3T3 cells overexpressing HSP27. HSP27 overexpression affected FAK phosphorylation and focal adhesion formation, depending on integrin-mediated actin cytoskeleton polymerization. In addition, the HSP27-overexpressing cells showed a retarded cell migration and invasion in wound-healing assays. Such HSP27-mediated retarded wound healing was correlated with reduced matrix metalloproteinase-2 (MMP-2) expression. The transcription factor for MMP-2 expression, signal transducer and activator or transcription 3 (STAT3), was correspondingly less phosphorylated. When a phosphomimetic form of HSP27 was transiently transfected, migration and invasion were similarly decreased via the regulation of the FAK/STAT3/MMP-2 signaling pathway, whereas a non-phosphorylatable form of HSP27 blocked HSP27-mediated phenotypes probably due to a dominant-negative effect on phosphorylation of endogenous HSP27. Altogether, our results suggest that HSP27 can enhance cell adhesion and modulate cell migration and invasion via the coordination of FAK-dependent actin organization and STAT3-dependent MMP-2 expression, and that phosphorylation of HSP27 is indispensable to regulate this signal pathway.
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Adhesión Celular , Movimiento Celular , Fibroblastos/enzimología , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas de Choque Térmico/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Regulación hacia Abajo , Adhesiones Focales/enzimología , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/genética , Humanos , Integrinas/metabolismo , Ratones , Chaperonas Moleculares , Células 3T3 NIH , Proteínas de Neoplasias/genética , Fosforilación , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Transfección , Cicatrización de HeridasRESUMEN
Although in vitro studies have been previously conducted to determine the biological effects of radio frequency (RF) radiation, it has not yet been determined whether or not RF radiation poses a potential hazard. This study was conducted to determine whether RF radiation exposure exerts detectable effects on cell cycle distribution, cellular invasion, and migration. NIH3T3 mouse fibroblasts were exposed to 849 MHz of RF radiation at average SAR values of 2 or 10 W/kg for either 1 h, or for 1 h per day for 3 days. During the exposure period, the temperature in the exposure chamber was maintained isothermally by circulating water throughout the cavity. Cell cycle distribution was analyzed at 24 and 48 h after exposure, by flow cytometry. We detected no statistically significant differences between the sham-exposed and RF radiation-exposed cells. Cellular invasion and migration were assessed by in vitro Matrigel invasion and Transwell migration assays. The RF radiation-exposed groups evidenced no significant changes in motility and invasiveness compared to the sham-exposed group. However, the ionizing radiation-exposed cells, used as a positive control group, manifested dramatic alterations in their cell cycle distribution, cellular invasiveness, and migration characteristics. Our results show that 849 MHz RF radiation exposure exerts no detectable effects on cell cycle distribution, cellular migration, or invasion at average SAR values of 2 or 10 W/kg.
Asunto(s)
Ciclo Celular/efectos de la radiación , Movimiento Celular/efectos de la radiación , Animales , Relación Dosis-Respuesta en la Radiación , Ratones , Microondas , Células 3T3 NIH , Dosis de Radiación , Ondas de RadioRESUMEN
The net balance of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) system has been known to be a key factor in tumor cell invasion. In the present study, we investigated the molecular mechanisms of anti-invasive and antimigrative activity of transforming growth factor (TGF)-beta1 on HT1080 human fibrosarcoma cells. In in vitro Matrigel invasion and Transwell migration assays, TGF-beta1 dose-dependently inhibited the invasion and migration of HT1080 cells, respectively. Gelatin zymography, Western blot, and real-time PCR analysis showed that TGF-beta1 enhanced the expression and secretion of MMP-2, TIMP-1, and, to a lesser degree, MMP-9 but not membrane type 1-MMP and TIMP-2. The addition of recombinant TIMP-1 protein reduced the Matrigel invasion and Transwell migration of HT1080 cells, similar to TGF-beta1. Because augmentation of TIMP-1 might be the major factor for the anti-invasive and antimigrative activity of TGF-beta1, we investigated possible molecular mechanisms responsible for the expression of TIMP-1 induced by TGF-beta1. Treatment of HT1080 cells with TGF-beta1 rapidly phosphorylated three mitogen-activated protein kinases [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase] and Akt. Among these kinases, the inhibition of only ERK1/2 pathway by PD98059, a specific inhibitor of MAPK/ERK kinase(MEK)-1, and transfection of dominant-negative MEK 1 effectively blocked the TIMP-1 induction by TGF-beta1. Mithramycin, a specific inhibitor of Sp1 transcription factor, but not curcumin, an inhibitor of activator protein-1, and transfection of Sp1 small interfering RNA significantly inhibited the TGF-beta1-induced expression of TIMP-1. In addition, electrophoretic mobility shift assay showed that TGF-beta1 up-regulated Sp1 DNA-binding activity, and PD98059 and mithramycin effectively inhibited these events. Finally, pretreatment of HT1080 cells with PD98059 and mithramycin, but not curcumin, restored the invasive activity of these cells. Taken together, these data suggest that TGF-beta1 modulates the net balance of the MMPs/TIMPs the systems in HT1080 cells for anti-invasion and antimigration by augmenting TIMP-1 through ERK1/2 pathway and Sp1 transcription factor.