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1.
Int J Mol Sci ; 25(13)2024 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-39000307

RESUMO

Hydronephrosis, the dilation of kidneys due to abnormal urine retention, occurs spontaneously in certain inbred mouse strains. In humans, its occurrence is often attributed to acquired urinary tract obstructions in adults, whereas in children, it can be congenital. However, the genetic factors underlying hydronephrosis pathogenesis remain unclear. We investigated the cause of hydronephrosis by analyzing tetraspanin 7 (Tspan7) gene-modified mice, which had shown a high incidence of hydronephrosis-like symptoms. We found that these mice were characterized by low liver weights relative to kidney weights and elevated blood ammonia levels, suggesting liver involvement in hydronephrosis. Gene expression analysis of the liver suggested that dysfunction of ornithine transcarbamylase (OTC), encoded by the X chromosome gene Otc and involved in the urea cycle, may contribute as a congenital factor in hydronephrosis. This OTC dysfunction may be caused by genomic mutations in X chromosome genes contiguous to Otc, such as Tspan7, or via the genomic manipulations used to generate transgenic mice, including the introduction of Cre recombinase DNA cassettes and cleavage of loxP by Cre recombinase. Therefore, caution should be exercised in interpreting the hydronephrosis phenotype observed in transgenic mice as solely a physiological function of the target gene.


Assuntos
Hidronefrose , Camundongos Transgênicos , Fenótipo , Animais , Hidronefrose/genética , Camundongos , Tetraspaninas/genética , Tetraspaninas/metabolismo , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Fígado/metabolismo , Fígado/patologia , Modelos Animais de Doenças , Rim/patologia , Rim/metabolismo , Masculino
2.
FASEB J ; 38(1): e23339, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38069905

RESUMO

Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox 3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.


Assuntos
Tecido Adiposo Marrom , Obesidade , Camundongos , Animais , Obesidade/genética , Obesidade/metabolismo , Tecido Adiposo Marrom/metabolismo , Aumento de Peso/genética , Tecido Adiposo Branco/metabolismo , Metabolismo Energético/genética , Fenótipo , Camundongos Endogâmicos C57BL , Dieta Hiperlipídica , Camundongos Knockout
3.
Cytokine ; 62(1): 115-22, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23478175

RESUMO

We previously identified the tumor suppressor INT6/eIF3e as a novel down regulator of HIF2α. Small interfering RNA targeting Int6 (siRNA-Int6) in HeLa cells led to normoxic stabilization of HIF2α, with concomitant transcription of angiogenic factors, including angiopoietin, basic fibroblast growth factor, and vascular endothelial growth factor. Here we used HIF2α normoxic up-regulation via Int6 silencing to investigate the role of HIF2α in endothelial cells. As a result Int6 silencing in human umbilical vein endothelial cells (HUVECs) and in human aortic endothelial cells (HAECs) led to robust enhanced cord formation and the medium supernatant of Int6 silenced HUVECs enhanced migration of untreated HUVECs, indicating a HIF2α triggered secretory signaling. Within the responsible genes were the cytokines interleukin-6 (IL-6) and IL-8 and not unlike in HeLa cells vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and epidermal growth factor (EGF). In addition application of IL-6 and IL-8 antibodies to the medium of Int6 silenced HUVECs could reverse the enhanced migration effect and also abrogated their tube formation. Finally, a CHIP assay analysis confirmed hypoxia-responsive elements (HREs) in the IL-6 and IL-8 promoters. Our results demonstrate that expression of both IL-6 and IL-8 is regulated by HIF2α and we suggest that IL-6 and IL-8 are HIF2α controlled cytokines for angiogenesis particularly in endothelial cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular , Fator de Iniciação 3 em Eucariotos/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Neovascularização Fisiológica , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , DNA/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-6/genética , Interleucina-8/genética , Dados de Sequência Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Oxigênio/farmacologia , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Elementos de Resposta/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção
4.
Cancer Sci ; 101(11): 2483-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20726856

RESUMO

Spliceostatin A (SSA) is a methylated derivative of an antitumor natural product FR901464, which specifically binds and inhibits the SF3b spliceosome sub-complex. To investigate the selective antitumor activity of SSA, we focused on the regulation of vascular endothelial growth factor (VEGF) mRNA, since VEGF is a key regulatory component in tumor angiogenesis and known for the intricate regulation of mRNA processing, such as alternative splicing. We found that in HeLa cells SSA reduced the amount of both mRNA and protein of VEGF. Spliceostatin A not only inhibited the splicing reaction of VEGF pre-mRNA but also reduced the total amount of VEGF's transcripts, while SSA affected GAPDH mRNA to a lesser extent. Given a significant reduction in VEGF gene expression, SSA was expected to possess anti-angiogenic activity in vivo. Indeed, SSA inhibited cancer cell-derived angiogenesis in vivo in a chicken chorioallantoic membrane (CAM) assay. The inhibition of angiogenesis with SSA was abolished by addition of exogenous VEGF. We also performed global gene expression analyses of HeLa cells and found that the expression levels of 38% of total genes including VEGF decreased to <50% of the basal levels following 16 h of SSA treatment. These results suggest that the global interference of gene expression including VEGF in tumor cells is at least one of the mechanisms by which SSA (or FR901464) exhibits its strong antitumor activity.


Assuntos
Inibidores da Angiogênese/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neovascularização Patológica/genética , Piranos/farmacologia , Compostos de Espiro/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Inibidores da Angiogênese/química , Animais , Western Blotting , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Estrutura Molecular , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Análise de Sequência com Séries de Oligonucleotídeos , Piranos/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Compostos de Espiro/química , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Circulation ; 122(9): 910-9, 2010 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-20713899

RESUMO

BACKGROUND: We previously identified INT6/eIF3e as a novel regulator of hypoxia-inducible factor 2alpha (HIF2alpha) activity. Small interfering RNA (siRNA)-Int6 adequately stabilized HIF2alpha, even under normoxic conditions, and thereby enhanced the expression of several angiogenic factors in vitro, suggesting that siRNA-Int6 may induce angiogenesis in vivo. METHODS AND RESULTS: We demonstrated a 6- to 8-fold enhanced formation of normal arteries and veins in the subcutaneous regions of adult mice 5 days after a single siRNA-Int6 application. Subcutaneous fibroblasts were identified as the major source of secreted angiogenic factors that led to the formation of functional vessels during Int6 silencing. Fibroblasts transfected ex vivo with siRNA-Int6 induced potent neoangiogenesis when transplanted into a subcutaneous region of nude mice. Application of siRNA-Int6 promoted neoangiogenesis in the area surrounding the injury in wound healing models, including genetically diabetic mice, thereby accelerating the closure of the injury. HIF2alpha accumulation caused by siRNA-Int6 was confirmed as the unequivocal cause of the angiogenesis by an in vivo angiogenesis assay. Further analysis of the Int6 silencing-induced neoangiogenesis revealed that a negative feedback regulation of HIF2alpha stability was caused by HIF2alpha-induced transcription of Int6 via hypoxia-response elements in its promoter. Thus, siRNA-Int6 temporarily facilitates an accumulation of HIF2alpha protein, leading to hypoxia-independent transcription of angiogenic factors and concomitant neoangiogenesis. CONCLUSIONS: We suggest that the pathway involving INT6/HIF2alpha acts as a hypoxia-independent master switch of functional angiogenesis; therefore, siRNA-Int6 application might be of clinical value in treating ischemic diseases such as heart and brain ischemia, skin injury, and diseases involving obstructed vessels.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fator de Iniciação 3 em Eucariotos/genética , Isquemia/fisiopatologia , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Actinas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama , Fator de Iniciação 3 em Eucariotos/metabolismo , Retroalimentação Fisiológica/fisiologia , Feminino , Fibroblastos/fisiologia , Fibroblastos/transplante , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/genética , Isquemia/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Nus , Células NIH 3T3 , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Regiões Promotoras Genéticas/fisiologia , RNA Interferente Pequeno , Transdução de Sinais/fisiologia , Tela Subcutânea/irrigação sanguínea , Tela Subcutânea/fisiologia
6.
Rinsho Byori ; 56(9): 802-10, 2008 Sep.
Artigo em Japonês | MEDLINE | ID: mdl-18975559

RESUMO

Recently, various sets of protein biomarkers have been discovered in important diseases such as cancers, brain stroke, heart attack, diabetes, and so on. Many of these biomarkers are expected to be extremely valuable as targets for clinical diagnosis and drug development; however, the clinical validation is difficult and time-consuming by individual assays or due to very low concentration in an early stage of disease. For the super-sensitive and multiplex detection of target biomarkers, we have developed MUSTag (Multiple Simultaneous Tag) assay technology with innovative modification of the immuno-PCR method. In MUSTag technology, specific antibodies against several important biomarkers were linked to 100-300bp long oligonucleotides as detection tags. Each different oligo-tag simultaneously detects multiplex protein targets with extremely high sensitivity(more than 10 fg (10(-15) g)/ml) in a dose-dependent manner by qRT-PCR-based (maximum 3 plexes) or capillary electrophoretic amplification (over 30 plexes). Here we report our recent results of multiple cytokine assay or disease-specific biomarker assay using MUSTag technology, and further, clinical results from patients with cancers, ischemic brain or heart attack, who need prompt and predictive diagnosis for adequate treatment.


Assuntos
Biomarcadores/análise , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Oligonucleotídeos , Sensibilidade e Especificidade
7.
J Biol Chem ; 282(17): 12707-16, 2007 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-17324924

RESUMO

The hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha are structurally similar as regards their DNA-binding and dimerization domains, but differ in their transactivation domains and, as is shown by experiments using hif-1 alpha(-/-) and hif-2 alpha(-/-) mice, in their functions. This implies that HIF-1 alpha and HIF-2 alpha may have unique target genes. To address this discrepancy and identify HIF-2 alpha-specific target genes, we performed yeast two-hybrid analysis and identified the tumor suppressor Int6/eIF3e/p48 as a novel target gene product involved in HIF-2 alpha regulation. The int6 gene was first identified from a screen in which the mouse mammary tumor virus was employed as an insertional mutagen to identify genes whose functions are critical for breast tumor formation. Here, by using two-hybrid analysis, immunoprecipitation in mammalian cells, and HRE-reporter assays, we report the specific interaction of HIF-2 alpha (but not HIF-1 alpha or HIF-3 alpha) with Int6. The results indicate that the direct interaction of Int6 induces proteasome inhibitor-sensitive HIF-2 alpha degradation. This degradation was clearly observed in renal cell carcinoma 786-O cells, and was found to be both hypoxia- and pVHL-independent. Furthermore, Int6 protein knockdown by int6-siRNA vectors or the dominant-negative mutant Int6-Delta C increased endogenous HIF-2 alpha expression, even under normoxia, and induced sets of critical angiogenic factors comprising vascular endoplasmic growth factor, angiopoietin, and basic fibroblast growth factor mRNA. These results indicate that Int6 is a novel and critical determinant of HIF-2 alpha-dependent angiogenesis as well as cancer formation, and that int6-siRNA transfer may be an effective therapeutic strategy in pathological conditions such as heart and brain ischemia, hepatic cirrhosis, and obstructive vessel diseases.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular , Fator de Iniciação 3 em Eucariotos/antagonistas & inibidores , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico
8.
J Neurochem ; 93(3): 641-53, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15836623

RESUMO

Huntington disease is caused by polyglutamine (polyQ) expansion in huntingtin. Selective and progressive neuronal loss is observed in the striatum and cerebral cortex in Huntington disease. We have addressed whether expanded polyQ aggregates appear in regions of the brain apart from the striatum and cortex and whether there is a correlation between expanded polyQ aggregate formation and dysregulated transcription. We generated transgenic mouse lines expressing mutant truncated N-terminal huntingtin (expanded polyQ) fused with enhanced green fluorescent protein (EGFP) and carried out a high-density oligonucleotide array analysis using mRNA extracted from the cerebrum, followed by TaqMan RT-PCR and in situ hybridization. The transgenic mice formed expanded polyQ-EGFP fluorescent aggregates and this system allowed us to directly visualize expanded polyQ aggregates in various regions of the brain without performing immunohistochemical studies. We show here that polyQ-EGFP aggregates were intense in the hypothalamus, where the expression of six hypothalamic neuropeptide mRNAs, such as oxytocin, vasopressin and cocaine-amphetamine-regulated transcript, was down-regulated in the transgenic mouse brain without observing a significant loss of hypothalamic neurons. These results indicate that the hypothalamus is susceptible to aggregate formation in these mice and this may result in the down-regulation of specific genes in this region of the brain.


Assuntos
Regulação para Baixo/genética , Proteínas de Fluorescência Verde/genética , Doença de Huntington/metabolismo , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/antagonistas & inibidores , Proteínas Nucleares/genética , Peptídeos/metabolismo , Animais , Química Encefálica/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Hipotálamo/química , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/metabolismo , Ocitocina/antagonistas & inibidores , Ocitocina/biossíntese , Ocitocina/genética , Peptídeos/genética , Regiões Promotoras Genéticas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Vasopressinas/antagonistas & inibidores , Vasopressinas/biossíntese , Vasopressinas/genética
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