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1.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 38-48, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31836883

RESUMO

Obstructive sleep apnea is characterized by chronic intermittent hypoxia (CIH), which is a risk factor for renal peritubular capillary (PTC) loss, and angiotensin II receptor blockers can alleviate PTC loss. However, the mechanism by which losartan (an angiotensin II receptor blocker) reduces CIH-induced PTC loss and attenuates kidney damage is still unknown. Thus, in this study, we examined the protective effects of losartan against CIH-induced PTC loss and explored the underlying mechanisms in rat CIH model. The immunohistochemical staining of CD34 and morphological examination showed that CIH reduced PTC density and damaged tubular epithelial cells. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR, and western blot analysis results revealed that CIH increased the expression of hypoxia inducible factor-1α (HIF-1α), angiotensin II (Ang II), angiotensin II type 1 receptor (AT1R), pro-angiogenesis factor vascular endothelial growth factor (VEGF), and anti-angiogenesis factor thrombospondin-1 (TSP-1) in the renal cortex of rats. CIH may up-regulate VEGF expression and simultaneously increase TSP-1 production. By histopathological, immunohistochemistry, ELISA, RT-qPCR, and western blot analysis, we found that the expressions of renal renin-angiotensin system (RAS), HIF-1α, VEGF, and TSP-1 were decreased, and PTC loss and tubular epithelial cell injury were attenuated with losartan treatment. Losartan ameliorated CIH-induced PTC loss by modulating renal RAS to improve the crosstalk between endothelial cells and tubular epithelial cells and subsequently regulate the balance of angiogenesis factors. Our study provided novel insights into the mechanisms of CIH-induced kidney damage and indicated that losartan could be a potential therapeutic agent for renal protection by alleviating CIH-induced PTC loss.


Assuntos
Indutores da Angiogênese/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Capilares/patologia , Hipóxia/complicações , Losartan/farmacologia , Substâncias Protetoras/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Angiotensina II/sangue , Animais , Peso Corporal/efeitos dos fármacos , Creatinina/sangue , Células Epiteliais/efeitos dos fármacos , Hipóxia/etiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Córtex Renal/irrigação sanguínea , Córtex Renal/metabolismo , Masculino , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/metabolismo , Apneia Obstrutiva do Sono/complicações , Trombospondina 1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(9): 828-831, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31750826

RESUMO

Objective To investigate the correlations between the expression of vascular endothelial growth factor (VEGF) and thrombospondin 1 (TSP-1) in breast cancer and the prognosis. Methods Immunohistochemical staining was used to detect the expression of VEGF and TSP-1 in 160 cases of breast cancer tissues and adjacent tissues, and the relationships between them were analyzed. Results The expression of TSP-1 significantly decreased and the expression of VEGF significantly increased in breast cancer tissues. Low expression of TSP-1 and high expression of VEGF were significantly associated with high clinical stage, poor differentiation, and lymph node metastasis. After 3 years of follow-up, the recurrence rate was 15.6%. Spearman rank correlation analysis showed that there was a positive correlation between the prognosis recurrence rate and the positive expression rate of VEGF (r=0.459), but negatively correlated with the positive expression rate of TSP-1 (r=-0.543). Logistic regression analysis showed that TSP-1 positive expression rate, VEGF positive expression rate, lymph node metastasis and clinical stage were the main independent risk factors for prognosis and recurrence. Conclusion The high expression of VEGF and the low expression of TSP-1 in breast cancer tissues are significantly correlated with the main clinical features. The recurrence rate of patients with high expression of VEGF and low expression of TSP is high.


Assuntos
Neoplasias da Mama/diagnóstico , Trombospondina 1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neoplasias da Mama/metabolismo , Feminino , Humanos , Recidiva Local de Neoplasia , Prognóstico
3.
Genes (Basel) ; 10(8)2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31412643

RESUMO

BACKGROUND: Pancreatic cancer is one of the malignant tumors that threaten human health. METHODS: The gene expression profiles of GSE15471, GSE19650, GSE32676 and GSE71989 were downloaded from the gene expression omnibus database including pancreatic cancer and normal samples. The differentially expressed genes between the two types of samples were identified with the Limma package using R language. The gene ontology functional and pathway enrichment analyses of differentially-expressed genes were performed by the DAVID software followed by the construction of a protein-protein interaction network. Hub gene identification was performed by the plug-in cytoHubba in cytoscape software, and the reliability and survival analysis of hub genes was carried out in The Cancer Genome Atlas gene expression data. RESULTS: The 138 differentially expressed genes were significantly enriched in biological processes including cell migration, cell adhesion and several pathways, mainly associated with extracellular matrix-receptor interaction and focal adhesion pathway in pancreatic cancer. The top hub genes, namely thrombospondin 1, DNA topoisomerase II alpha, syndecan 1, maternal embryonic leucine zipper kinase and proto-oncogene receptor tyrosine kinase Met were identified from the protein-protein interaction network. The expression levels of hub genes were consistent with data obtained in The Cancer Genome Atlas. DNA topoisomerase II alpha, syndecan 1, maternal embryonic leucine zipper kinase and proto-oncogene receptor tyrosine kinase Met were significantly linked with poor survival in pancreatic adenocarcinoma. CONCLUSIONS: These hub genes may be used as potential targets for pancreatic cancer diagnosis and treatment.


Assuntos
Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias Pancreáticas/genética , Transcriptoma , Algoritmos , Biomarcadores Tumorais/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Sindecana-1/genética , Sindecana-1/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo
4.
Int J Mol Sci ; 20(13)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261874

RESUMO

Esophageal squamous cell carcinoma (ESCC) is the most common primary esophageal malignancy. Telmisartan, an angiotensin II type 1 (AT1) receptor blocker (ARB) and a widely used antihypertensive, has been shown to inhibit proliferation of various cancer types. This study evaluated the effects of telmisartan on human ESCC cell proliferation in vitro and in vivo and sought to identify the microRNAs (miRNAs) involved in these antitumor effects. We examined the effects of telmisartan on three human ESCC cell lines (KYSE150, KYSE180, and KYSE850). Telmisartan inhibited proliferation of these three cell lines by inducing S-phase arrest, which was accompanied by decreased expression of cyclin A2, cyclin-dependent kinase 2, and other cell cycle-related proteins. Additionally, telmisartan reduced levels of phosphorylated ErbB3 and thrombospondin-1 in KYSE180 cells. Furthermore, expression of miRNAs was remarkably altered by telmisartan in vitro. Telmisartan also inhibited tumor growth in vivo in a xenograft mouse model. In conclusion, telmisartan inhibited cell proliferation and tumor growth in ESCC cells by inducing cell-cycle arrest.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Fase S/efeitos dos fármacos , Telmisartan/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Linhagem Celular Tumoral , Ciclina A2/genética , Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Telmisartan/uso terapêutico , Trombospondina 1/genética , Trombospondina 1/metabolismo
5.
DNA Cell Biol ; 38(7): 670-677, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31188027

RESUMO

Cutis laxa represents a heterogeneous group of rare, inherited, or acquired connective tissue disorders with the common feature of loose and redundant skin with decreased elasticity. The skin of affected deer showed abnormal collagen fiber morphology. To identify the differentially expressed genes of the unusual localized skin laxity in sika deer, we performed transcriptome analysis in the affected and control sika deer. The transcriptome analysis showed 700 genes with significant differential expression in the affected skin as compared with normal skin. Pathway analysis revealed an enrichment of genes involved in tumor necrosis factor signaling, the extracellular matrix-receptor interaction, platelet activation, and Huntington's disease. A gene network was constructed, and the hub nodes such as PTGS2, THBS1, COL1A1, FOS, and NOS3 were found through PPI network analysis, which may contributed to the unusual localized skin laxity in sika deer. Abnormal expression patterns of genes during the development of the affected sika deer were successfully uncovered in the present study, which provides a reference for revealing the related mechanism underlying cutis laxa in sika deer and human beings.


Assuntos
Cútis Laxa/veterinária , Cervos/genética , Transcriptoma , Animais , Colágeno/genética , Colágeno/metabolismo , Cútis Laxa/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Redes Reguladoras de Genes , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo
6.
Nat Commun ; 10(1): 2344, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138806

RESUMO

Infection by soil transmitted parasitic helminths, such as Trichuris spp, are ubiquitous in humans and animals but the mechanisms determining persistence of chronic infections are poorly understood. Here we show that p43, the single most abundant protein in T. muris excretions/secretions, is non-immunogenic during infection and has an unusual sequence and structure containing subdomain homology to thrombospondin type 1 and interleukin (IL)-13 receptor (R) α2. Binding of p43 to IL-13, the key effector cytokine responsible for T. muris expulsion, inhibits IL-13 function both in vitro and in vivo. Tethering of p43 to matrix proteoglycans presents a bound source of p43 to facilitate interaction with IL-13, which may underpin chronic intestinal infection. Our results suggest that exploiting the biology of p43 may open up new approaches to modulating IL-13 function and control of Trichuris infections.


Assuntos
Proteínas de Helminto/metabolismo , Interleucina-13/metabolismo , Enteropatias Parasitárias/metabolismo , Proteoglicanas/metabolismo , Trichuris/metabolismo , Animais , Matriz Extracelular/metabolismo , Proteínas de Helminto/imunologia , Interleucina-13/imunologia , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Enteropatias Parasitárias/imunologia , Camundongos , Homologia de Sequência de Aminoácidos , Trombospondina 1/metabolismo , Tricuríase
7.
Cell ; 177(5): 1280-1292.e20, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31031006

RESUMO

Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABAB) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders.


Assuntos
Astrócitos/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Comportamento Animal , Comunicação Celular , Neurônios/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Animais , Astrócitos/patologia , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/patologia , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Sinapses/genética , Trombospondina 1/genética , Trombospondina 1/metabolismo , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismo
9.
Cell Mol Biol (Noisy-le-grand) ; 65(3): 48-57, 2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30942155

RESUMO

The plateau zokor (Myospalaxbaileyi) is a specialized subterranean rodent that lives on the Qinghai-Tibet Plateau, and has successfully adapted to hypoxic environment. Raised expression of vascular endothelial growth factor (VEGF) and enhanced microvessel density (MVD) in tissues enable subterranean rodents to adapt to hypoxic sealed burrows. However, the expression of VEGF is inhibited by decreases in oxygen content, which is different from what obtains in Sprague Dawley (SD)rats. Thromspondin-1(TSP-1) is the first endogenous angiogenesis inhibitor identified inp53 pathway. It has several domains that bind to different proteins which regulate cell-to-cell interactions, inhibit endothelial cell proliferation and induce endothelial cell apoptosis (anti-angiogenesis). In this study, we analyzed the coding region and the expression pattern of TSP-1 gene in plateau zokor under different oxygen partial pressures using bioinformatics and qRT-PCR, respectively. Our results showed that the base and amino acid homologies between plateau zokor and Northern Israeli blind subterranean mole rat (Nannospalaxgalili) were 95.08 and 97.61%, respectively. There were eight parallel evolution sites with Nannospalaxgalili. Evaluation by 'Sorting Tolerant From Intolerant' (SIFT) algorithm showed four sites with significant effects on the function of TSP-1. Three-dimensional (3D) structures revealed that Asp185 and Thr270 were located in the NH2 terminal domain, with Glu536 in the Type I repeat domain, and Thr1092 in the COOH terminal domain. Compared to SD rats, the polarities of these four mutation sites changed. The expression levels of TSP-1 in plateau zokor tissues increased significantly from 2 260 m(16.12kPa) to 3 300 m(14.13kPa), but there was no significant difference in TSP-1 expression in SD rats. In conclusion, due to long-term adaption to the hypoxic environment of sealed burrows, plateau zokor upregulates the expression of TSP-1 to effect anti-angiogenesis. Moreover, mutations in gene structure of TSP-1 may play an important role in inhibiting angiogenesis.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica , Hipóxia/genética , Roedores/genética , Trombospondina 1/genética , Animais , DNA Mitocondrial/genética , Modelos Moleculares , Filogenia , Subunidades Proteicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Seleção Genética , Homologia de Sequência do Ácido Nucleico , Trombospondina 1/química , Trombospondina 1/metabolismo
10.
Neuroscience ; 408: 68-80, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30928339

RESUMO

Thrombospondins (TSPs) are cell adhesion molecules that play an important role in the maintenance of hearing and afferent synaptic connections. Based on their reported function in restoring synaptic connections after stroke, we tested a potential role for TSP1 and TSP2 genes in repairing cochlear synapses following noise injury. We observed a tonotopic gradient in the expression of TSP1 and TSP2 mRNA in control mouse cochleae and an upregulation of these genes following noise exposure. Examining the functional sequelae of these changes revealed that afferent synaptic counts and auditory brainstem responses (ABRs) in noise-exposed TSP1 and TSP2 knockout (-/-) mice exhibited a worst recovery when compared to controls. Consistent with their tonotopic expression, TSP1-/- mice showed greater susceptibility to noise-induced hearing loss (NIHL) at 8 kHz and 16 kHz frequencies, whereas NIHL in TSP2-/- mice occurred only at mid and high frequencies. Further analysis of the ABR waveforms indicated peripheral neuronal damage in TSP2-/- but not in TSP1-/- mice. Noise trauma affecting mid to high frequencies triggered severe seizures in the TSP2-/- mice. We found that decreased susceptibility to audiogenic seizures in TSP1-/- mice was correlated with increased TSP2 protein levels in their inner ears, suggesting that TSP2 might functionally compensate for the loss of TSP1 in these mice. Our data indicate that TSP1 and TSP2 are both involved in susceptibility to NIHL, with TSP2 playing a more prominent role.


Assuntos
Limiar Auditivo/fisiologia , Cóclea/metabolismo , Perda Auditiva Provocada por Ruído/metabolismo , Trombospondina 1/metabolismo , Trombospondinas/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Regulação da Expressão Gênica , Perda Auditiva Provocada por Ruído/fisiopatologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Gânglio Espiral da Cóclea/metabolismo , Trombospondina 1/genética , Trombospondinas/genética
11.
Cell Physiol Biochem ; 52(3): 532-552, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30897320

RESUMO

BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated. METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins. RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells. CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.


Assuntos
Sistemas CRISPR-Cas/genética , Corpo Lúteo/metabolismo , Edição de Genes , Trombospondina 1/genética , Animais , Apoptose , Búfalos/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismo , Antígeno CD47/genética , Antígeno CD47/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Sobrevivência Celular , Corpo Lúteo/citologia , Corpo Lúteo/patologia , Dinoprosta/metabolismo , Regulação para Baixo , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Trombospondina 1/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Iran J Allergy Asthma Immunol ; 18(1): 72-79, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30848575

RESUMO

Atherosclerosis is developed due to the formation of atheroma plaques in the coronary arteries. In this process, M1 macrophages and vascular smooth muscle cells (VSMCs) are the main functional cells. Inflammatory mediators such as histamine may inflame M1 macrophages. The aim of this study was to determine the effect of M1 macrophage secretion contents on the gene and protein expression levels of focal adhesion kinase (FAK), vasodilator-stimulated phosphoprotein (VASP), and thrombospondin1 (THBS1). Whole blood samples from the six healthy subjects (stenosis<5%), and six patients (stenosis>70%) were prepared and peripheral blood mononuclear cells (PBMCs) were isolated. Then monocytes were differentiated into M1 macrophages using 100 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF). The differentiated M1 macrophages were treated with histamine (10-6 M), and their secretion contents were harvested and added to the culture medium of VSMCs. The FAK, VASP, and THBS1 gene expression and protein levels were measured using RT-qPCR and western blot techniques in VSMCs, respectively. The FAK and THBS1 gene expression levels significantly increased in VSMCs after adding secretion contents obtained from histamine-treated M1 macrophages (p=0.023 and 0.05, respectively), while significant results were not observed for VASP gene (p=0.45). In converse with the phosphorylated VASP (pVASP) (p<0.34), the phosphorylated FAK (pFAK) and THBS1 protein levels increased in VSMCs (p<0.001). We concluded that in inflammatory conditions, the immune events could affect the macrophages by histamine. The activated macrophages could locally activate signaling pathways via FAK and THBS1 genes that are effective in the proliferation and migration of VSMCs.


Assuntos
Quinase 1 de Adesão Focal/metabolismo , Macrófagos/metabolismo , Miócitos de Músculo Liso/fisiologia , Trombospondina 1/metabolismo , Idoso , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Células Cultivadas , Estenose Coronária/metabolismo , Feminino , Quinase 1 de Adesão Focal/genética , Histamina/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Transdução de Sinais , Trombospondina 1/genética
13.
Nat Commun ; 10(1): 1146, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850588

RESUMO

We undertook a systematic study focused on the matricellular protein Thrombospondin-1 (THBS1) to uncover molecular mechanisms underlying the role of THBS1 in glioblastoma (GBM) development. THBS1 was found to be increased with glioma grades. Mechanistically, we show that the TGFß canonical pathway transcriptionally regulates THBS1, through SMAD3 binding to the THBS1 gene promoter. THBS1 silencing inhibits tumour cell invasion and growth, alone and in combination with anti-angiogenic therapy. Specific inhibition of the THBS1/CD47 interaction using an antagonist peptide decreases cell invasion. This is confirmed by CD47 knock-down experiments. RNA sequencing of patient-derived xenograft tissue from laser capture micro-dissected peripheral and central tumour areas demonstrates that THBS1 is one of the gene with the highest connectivity at the tumour borders. All in all, these data show that TGFß1 induces THBS1 expression via Smad3 which contributes to the invasive behaviour during GBM expansion. Furthermore, tumour cell-bound CD47 is implicated in this process.


Assuntos
Neoplasias Encefálicas/genética , Antígeno CD47/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Proteína Smad3/genética , Trombospondina 1/genética , Fator de Crescimento Transformador beta1/genética , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Córtex Cerebral , Glioblastoma/irrigação sanguínea , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Microdissecção e Captura a Laser , Masculino , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Peptídeos/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise de Sobrevida , Trombospondina 1/antagonistas & inibidores , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Mol Cell Proteomics ; 18(5): 818-836, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30705123

RESUMO

Kallikrein-related peptidase 7 (KLK7) is a serine peptidase that is over expressed in ovarian cancer. In vitro functional analyses have suggested KLK7 to play a cancer progressive role, although monitoring of KLK7 expression has suggested a contradictory protective role for KLK7 in ovarian cancer patients. In order to help delineate its mechanism of action and thereby the functional roles, information on its substrate repertoire is crucial. Therefore, in this study a quantitative proteomics approach-PROtein TOpography and Migration Analysis Platform (PROTOMAP)-coupled with SILAC was used for in-depth analysis of putative KLK7 substrates from a representative ovarian cancer cell line, SKOV-3, secreted proteins. The Terminal Amine Isotopic Labeling of Substrates (TAILS) approach was used to determine the exact cleavage sites and to validate qPROTOMAP-identified putative substrates. By employing these two technically divergent approaches, exact cleavage sites on 16 novel putative substrates and two established substrates, matrix metalloprotease (MMP) 2 and insulin growth factor binding protein 3 (IGFBP3), were identified in the SKOV-3 secretome. Eight of these substrates were also identified on TAILS analysis of another ovarian cancer cell (OVMZ-6) secretome, with a further seven OVMZ-6 substrates common to the SKOV-3 qPROTOMAP profile. Identified substrates were significantly associated with the common processes of cell adhesion, extracellular matrix remodeling and cell migration according to the gene ontology (GO) biological process analysis. Biochemical validation supports a role for KLK7 in directly activating pro-MMP10, hydrolysis of IGFBP6 and cleavage of thrombospondin 1 with generation of a potentially bioactive N-terminal fragment. Overall, this study constitutes the most comprehensive analysis of the putative KLK7 degradome in any cancer to date, thereby opening new avenues for KLK7 research.


Assuntos
Calicreínas/metabolismo , Neoplasias Ovarianas/metabolismo , Proteólise , Proteoma/metabolismo , Proteômica , Sequência de Aminoácidos , Linhagem Celular Tumoral , Quimotripsina/metabolismo , Meios de Cultivo Condicionados/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Ontologia Genética , Humanos , Hidrólise , Metaloproteinase 10 da Matriz/metabolismo , Neoplasias Ovarianas/patologia , Peptídeos/química , Peptídeos/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Trombospondina 1/química , Trombospondina 1/metabolismo
15.
Front Immunol ; 10: 27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30733718

RESUMO

Approximately 5% of individuals chronically infected with Schistosoma mansoni develop pulmonary hypertension (PH). The disease is progressive and often fatal, and treatment options are palliative, not curative. Recent studies have unraveled major players of the Th2 inflammation axis in the Schistosoma-induced PH pathology using murine models and studying human samples. TGF-ß signaling is a link between the Type 2 inflammation and vascular remodeling, and specifically Thrombospondin-1 (TSP-1) is upregulated by the inflammation and activates TGF-ß. Overall, the current model for the pathogenesis of Schistosoma-induced PH is that deposition of Schistosoma mansoni eggs in the pulmonary vasculature results in localized Th2 inflammation, leading to TGF-ß activation by TSP-1, and the active TGF-ß then results in vascular remodeling and PH.


Assuntos
Suscetibilidade a Doenças/imunologia , Hipertensão Pulmonar/etiologia , Schistosoma/imunologia , Esquistossomose/complicações , Esquistossomose/imunologia , Células Th2/imunologia , Animais , Biomarcadores , Diagnóstico por Imagem , Testes de Função Cardíaca , Humanos , Hipertensão Pulmonar/diagnóstico , Esquistossomose/parasitologia , Células Th2/metabolismo , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Remodelação Vascular
16.
J Biomol Struct Dyn ; 37(3): 811-822, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29380675

RESUMO

Cell surface calreticulin (CRT) binding to thrombospondin-1 (TSP1), regulates cell adhesion, migration, anoikis resistance, and collagen production. Due to the essential role of membrane microdomains in CRT-mediated focal adhesion disassembly, we previously studied the effect of raft-like bilayers on TSP1-CRT interactions with all-atom molecular dynamics (AAMD) simulations. However, the simulated systems of protein on the surface of the bilayer(s) in the explicit solvent are too large for long timescale AAMD simulations due to computational expense. In this study, we adopted a multiscale modeling approach of combining AAMD, coarse-grained molecule dynamics (CGMD), and reversed AAMD (REV AAMD) simulations to investigate the interactions of single CRT or of the TSP1-CRT complex with a membrane microdomain at microsecond timescale. Results showed that CRT conformational stabilization by binding of TSP1 in AAMD simulation was undetectable in CGMD simulation, but it was recovered in REV AAMD simulation. Similarly, interactions of the CRT N-domain and TSP1 with the membrane microdomain were lost in CGMD simulations but they were re-gained in the REV AAMD simulations. There was the higher coordination of the CRT P-domain in the TSP1-CRT complex with the lipid components of membrane microdomain compared to that of single CRT, which could directly affect the conformation of CRT and further mediate CRT recruitment of LDL receptor-related protein for signaling events. This study provides structural and molecular insights into TSP1-CRT interactions in a membrane microdomain environment and demonstrates the feasibility of using multiscale simulations to investigate the interactions between protein and membrane microdomains at a long timescale.


Assuntos
Calreticulina/química , Calreticulina/metabolismo , Microdomínios da Membrana/química , Simulação de Dinâmica Molecular , Trombospondina 1/química , Trombospondina 1/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Termodinâmica
17.
Nephrology (Carlton) ; 24(4): 405-413, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30129267

RESUMO

AIM: Long non-coding RNA (lncRNAs) have been shown to play a critical role in a variety of pathophysiological processes, such as cell proliferation, apoptosis and migration. However, there were few studies addressing the function of lncRNAs in renal ischaemia/reperfusion (I/R) injury. Apoptosis is an important pathogenesis during I/R injury. Here, we identified the effect of hypoxia-responsive lncRNA growth arrest-specific 5 (GAS5) on apoptosis in renal I/R injury. METHODS: Ischaemia/reperfusion injury in mice or hypoxia/re-oxygenation (H/R) in human proximal renal tubular epithelial cells (HK-2) was practiced to induce apoptosis. The kidneys and blood were collected at 24 h after reperfusion. The GAS5 messenger RNA (mRNA) expression and apoptosis-related gene mRNA and protein levels, including p53, cellular inhibitor of apoptosis protein 2 (cIAP2) and thrombospondin-1 (TSP-1), were analysed. GAS5 small-interfering RNA was transfected with H/R induced cells. Over-expression of GAS5 was performed by plasmid transfection. RESULTS: Apoptotic cells significantly increased in I/R-injured kidneys. GAS5 could be up-regulated in kidneys at 24 h after reperfusion and 3 h after re-oxygenation, combined with increased expression of its downstream apoptosis-related proteins p53 and cIAP2. GAS5 small-interfering RNA treatment down-regulated the mRNA and protein levels of p53 and TSP-1, and attenuated apoptosis induced by H/R in HK-2 cells. Conversely, over-expression of GAS5 up-regulated the mRNA and protein levels of p53 and TSP-1, and promoted apoptosis in HK-2 cells. CONCLUSION: Long non-coding RNA GAS5 induced by I/R injury could promote apoptosis in kidney. TSP-1 might be one of the downstream effectors of GAS5, which will be explored in the future.


Assuntos
Lesão Renal Aguda/metabolismo , Apoptose , Túbulos Renais Proximais/metabolismo , RNA Longo não Codificante/metabolismo , Traumatismo por Reperfusão/metabolismo , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Trombospondina 1/genética , Trombospondina 1/metabolismo
18.
Mol Cell Proteomics ; 18(1): 51-64, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30257878

RESUMO

Transmissible gastroenteritis virus (TGEV) is a member of Coronaviridae family. Our previous research showed that TGEV infection could induce mitochondrial dysfunction and upregulate miR-222 level. Therefore, we presumed that miR-222 might be implicated in regulating mitochondrial dysfunction induced by TGEV infection. To verify the hypothesis, the effect of miR-222 on mitochondrial dysfunction was tested and we showed that miR-222 attenuated TGEV-induced mitochondrial dysfunction. To investigate the underlying molecular mechanism of miR-222 in TGEV-induced mitochondrial dysfunction, a quantitative proteomic analysis of PK-15 cells that were transfected with miR-222 mimics and infected with TGEV was performed. In total, 4151 proteins were quantified and 100 differentially expressed proteins were obtained (57 upregulated, 43 downregulated), among which thrombospondin-1 (THBS1) and cluster of differentiation 47 (CD47) were downregulated. THBS1 was identified as the target of miR-222. Knockdown of THBS1 and CD47 decreased mitochondrial Ca2+ level and increased mitochondrial membrane potential (MMP) level. Reversely, overexpression of THBS1 and CD47 elevated mitochondrial Ca2+ level and reduced mitochondrial membrane potential (MMP) level. Together, our data establish a significant role of miR-222 in regulating mitochondrial dysfunction in response to TGEV infection.


Assuntos
Antígeno CD47/metabolismo , Gastroenterite Suína Transmissível/metabolismo , MicroRNAs/genética , Mitocôndrias/metabolismo , Trombospondina 1/metabolismo , Vírus da Gastroenterite Transmissível/patogenicidade , Animais , Antígeno CD47/genética , Cálcio/metabolismo , Linhagem Celular , Gastroenterite Suína Transmissível/genética , Regulação da Expressão Gênica , Potencial da Membrana Mitocondrial , Mapas de Interação de Proteínas , Proteômica/métodos , Suínos , Trombospondina 1/genética , Transfecção
19.
Diab Vasc Dis Res ; 16(1): 28-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30360646

RESUMO

Type 1 diabetes can have deleterious effects on skeletal muscle and its microvasculature. Our laboratory has recently identified murine double minute-2 as a master regulator of muscle microvasculature by controlling expression levels of two key molecular actors of the angio-adaptive process: the pro-angiogenic vascular endothelial growth factor-A and the anti-angiogenic thrombospondin-1. Here, we show for the first time that in the soleus and plantaris muscles of the diabetes-prone BioBreeding rats, a rodent model of autoimmune type 1 diabetes, murine double minute-2 protein levels are significantly decreased, coinciding with elevated protein levels of thrombospondin-1 and its transcription factor forkhead box O1. Significant capillary regression was observed to similar extent in soleus and plantaris muscles of type 1 diabetic rats. Elevated blood glucose levels were correlated with the loss of capillaries, the reduction in murine double minute-2 expression and with the elevations in thrombospondin-1. Vascular endothelial growth factor-A protein levels were unaltered or even increased in diabetic animals, yet type 1 diabetic animals had less vascular endothelial growth factor receptor-2 abundance. The vascular endothelial growth factor-A/thrombospondin-1 ratio, a good indicator of skeletal muscle angio-adaptive environment, was decreased in type 1 diabetic muscle. Our results suggest that the murine double minute-2-forkhead box O1-thrombospondin-1 pathway plays an important role in angio-regulation of the skeletal muscle in the pathophysiological context of type 1 diabetes.


Assuntos
Glicemia/metabolismo , Capilares/metabolismo , Diabetes Mellitus Tipo 1/sangue , Angiopatias Diabéticas/sangue , Músculo Esquelético/irrigação sanguínea , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Trombospondina 1/metabolismo , Animais , Biomarcadores/sangue , Capilares/patologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/patologia , Modelos Animais de Doenças , Ratos Endogâmicos BB , Ratos Endogâmicos , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
20.
Cancer Lett ; 442: 287-298, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30439539

RESUMO

Exosomes are implicated in cancer cell development, migration and invasion. Pigment epithelium-derived factor (PEDF) is a secreted anticancer protein that can regulate lung cancer progression; however, the role of PEDF in non-small cell lung cancer (NSCLC), including metastasis and cancer cell-derived exosome secretion, is unclear. In this study, we analyzed the effects of PEDF on exosome-mediated migration, invasion, and tumorigenicity of cultured NSCLC cells. The results showed that PEDF overexpression significantly reduced NSCLC invasion and migration, while inducing cell aggregation, whereas PEDF knockdown had the opposite effects. Exosomes from NSCLC cells treated with recombinant PEDF had a significantly reduced ability to promote cancer cell motility, migration, and invasion compared to exosomes from untreated cells. Exosomes from PEDF-treated cells contained thrombospondin 1 (THBS1), which inhibited cytoskeletal remodeling and exosome-induced lung cancer cell motility, migration, and invasion. Furthermore, PEDF-overexpressing NSCLC cells formed smaller xenograft tumors with higher THBS1 expression compared to control tumors. Our findings indicate that PEDF decreases the metastatic potential of NSCLC cells through regulation of THBS1 release in cancer cell-derived exosomes, thus uncovering a new mechanism of lung cancer progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Movimento Celular , Exossomos/metabolismo , Proteínas do Olho/metabolismo , Neoplasias Pulmonares/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Trombospondina 1/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Exossomos/genética , Exossomos/patologia , Proteínas do Olho/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos SCID , Invasividade Neoplásica , Fatores de Crescimento Neural/genética , Serpinas/genética , Transdução de Sinais , Trombospondina 1/genética , Carga Tumoral , Regulação para Cima
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