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1.
Chem Biol Interact ; 316: 108920, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31857088

RESUMO

Cedrelone is a limonoid isolated from the plant Trichilia catigua (Meliaceae). Previous studies have demonstrated that cedrelone (1) has several damaging effects on triple negative breast tumor (TNBC) cell line MDA-MB-231. In this work we investigated two new derivatives of cedrelone, the acetate (1a) and the mesylate (1b), to examine whether their effects are improved in comparison to the lead molecule. Cedrelone acetate (1a) was the most cytotoxic compound on TNBC cells and was chosen for additional analyses in traditional two-dimensional (2D) monolayer cultures and three-dimensional (3D) assays. In 2D, 1a induced cell cycle arrest, apoptosis and inhibited essential steps of the metastasis process of the MDA-MB-231 cells, in vitro. Moreover, 1a was able to revert the malignant phenotype of the T4-2 cells in 3D. These effects were concomitant with the downregulation of EGFR, ß1-integrin and phospho-Akt, which could have resulted in a decrease of NFκB levels and MMP9 activity. These results suggest that 1a could be used as an important model for the design of a new drug to be applied in cancer treatment and be further studied in vivo for its antitumor and antimetastatic effects.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Limoninas/química , Acetilação , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Limoninas/farmacologia , Meliaceae/química , Meliaceae/metabolismo , Fenótipo
2.
Vet Microbiol ; 237: 108420, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31585649

RESUMO

Mastitis is a disease that affects dairy cattle and causes a decline in milk quality as well as economic loss worldwide. TGF-ß1 levels are usually increased during mastitis; however, it is unknown whether TGF-ß1 is involved in bovine mastitis. Therefore, this study evaluated the effects of TGF-ß1 on the susceptibility of bovine mammary epithelial cells (BMECs) to Staphylococcus aureus (S. aureus). The results revealed that S. aureus adhesion to and invasion of BMECs was significantly increased after cells were treated with TGF-ß1. Adhesion of S. aureus to BMECs was increased dramatically by upregulation of fibronectin (Fn) and integrin ß1 (ITGB1), while the increase in the susceptibility of BMECs to S. aureus was blocked by specific antibodies against either Fn or ITGB1. These results indicated that adhesion and invasion were increased by TGF-ß1-induced upregulation of both Fn and ITGB1. Furthermore, TGF-ß1 treatment prior to S. aureus infection significantly increased S. aureus colonization as well as Fn and ITGB1 expression in the mammary glands of mice. These results suggest that TGF-ß1 promoted the expression of Fn and ITGB1 on the surface of BMECs and contributed to mammary gland infection in vitro and in vivo. The results of this study imply that Fn and ITGB1 may be useful therapeutic targets for the treatment of mastitis in dairy cows.


Assuntos
Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Integrina beta1/metabolismo , Glândulas Mamárias Animais/citologia , Staphylococcus aureus/fisiologia , Fator de Crescimento Transformador beta1/farmacologia , Animais , Aderência Bacteriana , Bovinos , Relação Dose-Resposta a Droga , Feminino , Fibronectinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Integrina beta1/genética , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta1/administração & dosagem
3.
Life Sci ; 238: 116963, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639396

RESUMO

AIMS: Triple-negative breast cancer (TNBC) is a special subtype of breast cancer that lacks receptor expression and is difficult to cure. Epigenetic regulators have been suggested as targets for cancer therapy in recent years. Our previous study indicated that the chromodomain-helicase-DNA-binding protein 4 (CHD4) is a prognostic biomarker of TNBC and therapeutic target in patients with TNBC. However, the exact mechanisms regulated by CHD4 are still unclear. METHODS: In this study, we compared differences in gene expression in parental and CHD4-deficient cells by next-generation sequencing and Ingenuity Pathway Analysis. KEY FINDINGS: We found that ß1 integrin is a downstream target gene of CHD4, which could be transcriptionally regulated by CHD4 in TNBC cells. Consistent with in vitro data, immunohistochemistry revealed that co-expression of ß1 integrin and CHD4 was significantly associated with metastatic state, recurrence, and survival status in TNBC patients. It also showed a positive correlation between ß1 integrin and CHD4 in vivo. SIGNIFICANCE: This is the first study to suggest that CHD4 regulates ß1 integrin in TNBC. Overall, CHD4-ß1 integrin axis could potentially be a predictive marker in patients with TNBC and the use of ß1 integrin inhibitors may be a therapeutic option for TNBC patients with high CHD4 expression.


Assuntos
Biomarcadores Tumorais/metabolismo , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Integrina beta1/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias de Mama Triplo Negativas/patologia , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina beta1/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Transdução de Sinais , Taxa de Sobrevida , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Células Tumorais Cultivadas
4.
Anticancer Res ; 39(9): 4853-4864, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519588

RESUMO

BACKGROUND/AIM: Colorectal cancer (CRC) is the leading cause of cancer mortality worldwide. Its poor prognosis can be ascribed primarily to high recurrence rates. Accordingly, the aim of this study was to identify novel prognostic biomarkers and therapeutic targets for management of CRC. MATERIALS AND METHODS: To develop prognostic biomarkers, we performed RNA-seq analysis and real-time RT-PCR in primary cancer tissues with or without systemic recurrence. To characterize the molecular functions of the encoded proteins, CRC cells underexpressing or overexpressing the candidate genes were established and appropriate cell-based assays were applied. RESULTS: ITGB1 and RHOC mRNA levels were up-regulated in the recurrence group of CRC patients. Overexpression of ITGB1 or RHOC stimulated CRC cell proliferation, invasion and migration, whereas the opposite effects were observed in cells underexpressing either protein. Five-year recurrence-free survival rates were significantly higher in the ITGB1- and RHOC-underexpression groups than those in the overexpression. CONCLUSION: ITGB1 and RHOC are potential predictors of recurrence and therapeutic targets for CRC, possibly predicting a high-risk group of stage II patients.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Integrina beta1/metabolismo , Proteína de Ligação a GTP rhoC/metabolismo , Idoso , Biomarcadores Tumorais , Proliferação de Células , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Interferente Pequeno/genética , Recidiva , Análise de Sobrevida
5.
Nat Commun ; 10(1): 3708, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420553

RESUMO

Neuropilin-1 (NRP1) is an essential transmembrane receptor with a variety of cellular functions. Here, we identify two human NRP1 splice variants resulting from the skipping of exon 4 and 5, respectively, in colorectal cancer (CRC). Both NRP1 variants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes. This increased endocytic trafficking of the two NRP1 variants, upon HGF stimulation, is due to loss of N-glycosylation at the Asn150 or Asn261 site, respectively. Moreover, these NRP1 variants enhance interactions with the Met and ß1-integrin receptors, resulting in Met/ß1-integrin co-internalization and co-accumulation on endosomes. This provides persistent signals to activate the FAK/p130Cas pathway, thereby promoting CRC cell migration, invasion and metastasis. Blocking endocytosis or endosomal Met/ß1-integrin/FAK signaling profoundly inhibits the oncogenic effects of both NRP1 variants. These findings reveal an important role for these NRP1 splice variants in the regulation of endocytic trafficking for cancer cell dissemination.


Assuntos
Neoplasias Colorretais/genética , Endossomos/metabolismo , Neuropilina-1/genética , Processamento Alternativo/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Colorretais/patologia , Proteína Substrato Associada a Crk/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Glicosilação , Células HCT116 , Células HT29 , Humanos , Integrina beta1/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Neuropilina-1/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais
6.
Int J Oncol ; 55(3): 721-732, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31364728

RESUMO

Metastasis is responsible for the majority of deaths among patients with malignant melanoma. Despite recent advances, the majority of current and modern therapies are ineffective and/or financially unfeasible. Thus, in this study, we investigated two low­cost highly­diluted natural complexes (HDNCs) that have been shown to be effective against malignant melanoma in a murine model in vivo. The aim of this study was to determine the mechanisms through which these HDNCs directly affect melanoma cells, either alone or in an artificial tumor microenvironment, suppressing the metastatic phenotype, thus explaining previous in vivo effects. For this purpose, HDNC in vitro treatments of B16­F10 melanoma cells, alone or in co­culture with Balb/3T3 fibroblasts, were carried out. Molecular biology techniques and standard functional assays were used to assess the changes in molecule expression and in cell behaviors related to the metastatic phenotype. Melanoma progression features were found to be regulated by HDNCs. Molecules related to cell adhesion (N­cadherin, ß1­integrin and CD44), and migration, extracellular matrix remodeling and angiogenesis were modulated. The cell migratory, invasive and clonogenic capacities were reduced by the HDNCs. No loss of cell proliferation or viability were observed. On the whole, the findings of this study indicate that HDNCs directly reprogram, molecularly and functionally, melanoma cells in vitro, modulating their metastatic phenotype. Such findings are likely to be responsible for the attenuation of tumor growth and lung colonization previously observed in vivo.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Receptores de Hialuronatos/metabolismo , Integrina beta1/metabolismo , Melanoma/metabolismo , Extratos Vegetais/farmacologia , Neoplasias Cutâneas/metabolismo , Animais , Células 3T3 BALB , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Camundongos , Metástase Neoplásica , Extratos Vegetais/química , Plantas/química , Neoplasias Cutâneas/tratamento farmacológico
7.
PLoS Negl Trop Dis ; 13(6): e0007339, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31233498

RESUMO

Although Mycobacterium leprae (M.leprae) is usually found in macrophages and nerves of the dermis of patients with multibacillary leprosy, it is also present in all layers of the epidermis, basal, suprabasal, prickle cells, and keratin layers. However, the mechanism by which M.leprae invades the dermis remains unknown, whereas the underlying mechanism by which M.leprae invades peripheral nerves, especially Schwann cells, is well defined. M. leprae binds to the α-dystroglycan (DG) of Schwann cells via the interaction of α-DG and laminin (LN) -α2 in the basal lamina, thus permitting it to become attached to and invade peripheral nerves. In the current study, we investigated the issue of how M.leprae is phagocytosed by human epidermal keratinocytes, neonatal (HEKn). LN-5 is the predominant form of laminin in the epidermis and allows the epidermis to be stably attached to the dermis via its interaction with α/ß-DG as well as integrins that are produced by keratinocytes. We therefore focused on the role of LN-5 when M. leprae is internalized by HEKn cells. Our results show that M.leprae preferentially binds to LN-5-coated slides and this binding to LN-5 enhances its binding to HEKn cells. The findings also show that pre-treatment with an antibody against α-DG, integrin-ß1, or -ß4 inhibited the binding of LN-5-coated M.leprae to HEKn cells. These results suggest that M. leprae binds to keratinocytes by taking advantage of the interaction of LN-5 in the basal lamina of the epidermis and a surface receptor of keratinocytes, such as α-DG, integrin-ß1, or -ß4.


Assuntos
Aderência Bacteriana , Moléculas de Adesão Celular/metabolismo , Distroglicanas/metabolismo , Integrina beta1/metabolismo , Integrina beta4/metabolismo , Queratinócitos/microbiologia , Mycobacterium leprae/fisiologia , Células Cultivadas , Humanos , Fagocitose , Ligação Proteica
8.
Cells ; 8(6)2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226820

RESUMO

Metastasis is the leading cause of cancer death worldwide. Circulating tumor cells (CTCs) are a critical step in the metastatic cascade and a good tool to study this process. We isolated CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) and a human xenograft mouse model of castration-resistant prostate cancer (CRPC). From these models, novel primary tumor and CTC cell lines were established. CTCs exhibited greater migration than primary tumor-derived cells, as well as epithelial-to-mesenchymal transition (EMT), as observed from decreased E-cadherin and increased SLUG and fibronectin expression. Additionally, when fibronectin was knocked down in CTCs, integrin B1 and SLUG were decreased, indicating regulation of these molecules by fibronectin. Investigation of cell surface molecules and secreted cytokines conferring immunomodulatory advantage to CTCs revealed decreased major histocompatibility complex class I (MHCI) expression and decreased endostatin, C-X-C motif chemokine 5 (CXCL5), and proliferin secretion by CTCs. Taken together, these findings indicate that CTCs exhibit distinct characteristics from primary tumor-derived cells. Furthermore, CTCs demonstrate enhanced migration in part through fibronectin regulation of integrin B1 and SLUG. Further study of CTC biology will likely uncover additional important mechanisms of cancer metastasis.


Assuntos
Fibronectinas/metabolismo , Integrina beta1/metabolismo , Células Neoplásicas Circulantes/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Animais , Biomarcadores Tumorais/sangue , Caderinas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Endostatinas , Transição Epitelial-Mesenquimal , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunomodulação , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Células Neoplásicas Circulantes/patologia
9.
Int J Mol Sci ; 20(11)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31195598

RESUMO

Inflammatory bowel diseases increase the risk of colorectal cancer and colitis-associated colorectal cancer (CAC). Tenascin-C, a matricellular protein, is highly expressed in inflammatory bowel diseases, especially colorectal cancer. However, the role of tenascin-C in the development of CAC is not yet fully understood. We previously showed that a peptide derived from tenascin-C, peptide TNIIIA2, induces potent and sustained activation of ß1-integrin. Moreover, we recently reported that peptide TNIIIA2 promotes invasion and metastasis in colon cancer cells. Here, we show the pathological relevance of TNIIIA2-related functional site for the development of CAC. First, expression of the TNIIIA2-containing TNC peptides/fragments was detected in dysplastic lesions of an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model. In vitro experiments demonstrated that conditioned medium from peptide TNIIIA2-stimulated human WI-38 fibroblasts induced malignant transformation in preneoplastic epithelial HaCaT cells. Indeed, these pro-proliferative effects stimulated by peptide TNIIIA2 were abrogated by peptide FNIII14, which has the ability to inactivate ß1-integrin. Importantly, peptide FNIII14 was capable of suppressing polyp formation in the AOM/DSS model. Therefore, tenascin-C-derived peptide TNIIIA2 may contribute to the formation of CAC via activation of stromal fibroblasts based on ß1-integrin activation. Peptide FNIII14 could represent a potential prophylactic treatment for CAC.


Assuntos
Colite/complicações , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Progressão da Doença , Fibroblastos/metabolismo , Integrina beta1/metabolismo , Peptídeos/metabolismo , Tenascina/metabolismo , Animais , Azoximetano , Células CACO-2 , Proliferação de Células , Pólipos do Colo/patologia , Meios de Cultivo Condicionados/farmacologia , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/patologia , Humanos , Masculino , Camundongos Endogâmicos ICR , Comunicação Parácrina
10.
ACS Appl Mater Interfaces ; 11(25): 22218-22227, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31199127

RESUMO

The surface electric potential of biomaterials has been extensively proven to play a critical role in stem cells' fate. However, there are ambiguous reports on the relation of stem cells' osteogenic capacity to surface potential characteristics (potential polarity and intensity). To address this, we adopted a surface with a wide potential range and both positive/negative polarity in a comprehensive view to get insight into surface potential-regulating cellular osteogenic differentiation. Tb xDy1- xFe2 alloy/poly(vinylidene fluoride-trifluoroethylene) magnetoelectric films were prepared, and the film could provide controllable surface potential characteristics with positive or negative polarity and potential (ϕME) intensity variation from 0 to ±120 mV as well as keep the surface chemical composition and microstructure unchanged. Cell culture results showed that osteogenic differentiation of mesenchymal stem cells on both positive and negative potential films was obviously upregulated when the /ϕME/ intensities were set from 0-55 mV. Differently, the highest upregulated osteogenic differentiation on the positive potential films corresponded to the /ϕME/ intensity from 35-55 mV and was better than that on the negative potential films whereas the highest on the negative potential films corresponded to the /ϕME/ intensity from 0-35 mV and was better than that on the positive potential films. This fact could illustrate why previous reports appeared ambiguously; i.e., the comparative result in osteogenic differentiation between the positive and negative potential films strongly depends on the selection of surface potential intensity. On the basis of assaying of the exposed functional sites (RGD and PHSRN) of the adsorbed fibronectin (FN) and the expression of cellular integrin α5 and ß1 subunits, the difference in the behavior between the positive and negative potential films was attributed to the distinct conformation of adsorbed fibronectin (FN) and the opposite changing trend with /ϕME/ for the two films, which triggers the osteogenesis-related FAK/ERK signaling pathway to a different extent. This study could provide new cognition for the in-depth understanding of the regulation mechanism underlying surface potential characteristics in cell behaviors.


Assuntos
Fibronectinas/metabolismo , Adesão Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/fisiologia , Espectroscopia Fotoeletrônica , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
11.
PLoS One ; 14(6): e0217920, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31166991

RESUMO

We previously reported that overexpression of PODXL, BCL7B, and ARHGEF4 in pancreatic cancer tissue is correlated with pancreatic cancer-related survival. The aim of this study was to investigate the use of PODXL, BCL7B, ARHGEF4, and the integrin family member ITGB1 as useful markers for the prognosis of postoperative pancreatic cancer patients in comparison with tumor size and the tumor node metastasis (TNM) staging system. Immunohistochemistry was performed using an anti-ITGB1 antibody on 102 samples of pancreatic cancer tissue surgically resected at the University of Kochi Medical School Hospital and the Matsuyama Shimin Hospital. Univariate Cox proportional hazards regression analysis showed that TNM stage and overexpression of PODXL, BCL7B, and ITGB1 were correlated with postoperative survival. However, tumor size was not significantly associated with postoperative prognosis of pancreatic cancer compared to these features. Multivariate Cox proportional hazards regression analysis showed that the overexpression of both PODXL and ITGB1 and overexpression of both BCL7B and ITGB1 increased the hazard ratio (6.27, 95% confidence interval [CI] 2.58-15.21; and 3.93, 95% CI 1.74-8.91, respectively) compared to that of TNM stage (IIA and IIB vs. III and IV; 3.05, 95% CI 1.25-7.42). These results imply that the combination of PODXL with ITGB1 and the combination of BCL7B with ITGB1 accurately predicted the postoperative outcomes of pancreatic cancer patients, and they were superior compared to the TNM staging system. The combination of PODXL with ITGB1 would be particularly useful, as it was the most highly correlated with postoperative outcomes. Importantly, the present results are useful to determine which adjuvant therapy should be selected.


Assuntos
Integrina beta1/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas/metabolismo , Sialoglicoproteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Período Pós-Operatório , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Resultado do Tratamento , Carga Tumoral
12.
Mol Cell Biol ; 39(16)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31160493

RESUMO

Cytoskeleton-associated protein 4 (CKAP4) is an endoplasmic reticulum protein that is also present in the cell surface membrane, where it acts as a receptor for Dickkopf1 (DKK1). In this study, we found that CKAP4 interacts with ß1 integrin and controls the recycling of α5ß1 integrin independently of DKK1. In S2-CP8 cells, knockdown of CKAP4 but not DKK1 enlarged the size of cell adhesion sites and enhanced cell adhesion to fibronectin, resulting in decreased cell migration. When CKAP4 was depleted, the levels of α5 but not ß1 integrin were increased in the cell surface membrane. A similar phenotype was observed in other cells expressing low levels of DKK1. In S2-CP8 cells, α5 integrin was trafficked with ß1 integrin and CKAP4 to the lysosome or recycled with ß1 integrin. In CKAP4-depleted cells, the internalization of α5ß1 integrin was unchanged, but its recycling was upregulated. Knockdown of sorting nexin 17 (SNX17), a mediator of integrin recycling, abrogated the increased α5 integrin levels caused by CKAP4 knockdown. CKAP4 bound to SNX17, and its knockdown enhanced the recruitment of α5ß1 integrin to SNX17. These results suggest that CKAP4 suppresses the recycling of α5ß1 integrin and coordinates cell adhesion sites and migration independently of DKK1.


Assuntos
Integrina alfa5beta1/metabolismo , Integrina beta1/metabolismo , Proteínas de Membrana/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Regulação da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo
13.
Int J Mol Sci ; 20(11)2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181623

RESUMO

The prognostic and therapeutic values of fibronectin have been reported in patients with renal cell carcinoma (RCC). However, the underlying mechanisms of malignancy in RCC are not completely understood. We found that silencing of fibronectin expression attenuated human RCC 786-O and Caki-1 cell growth and migration. Silencing of potential fibronectin receptor integrin α5 and integrin ß1 decreased 786-O cell ability in movement and chemotactic migration. Biochemical examination revealed a reduction of cyclin D1 and vimentin expression, transforming growth factor-ß1 (TGF-ß1) production, as well as Src and Smad phosphorylation in fibronectin-silenced 786-O and Caki-1 cells. Pharmacological inhibition of Src decreased 786-O cell growth and migration accompanied by a reduction of cyclin D1, fibronectin, vimentin, and TGF-ß1 expression, as well as Src and Smad phosphorylation. In 786-O cells, higher activities in cell growth and migration than in Caki-1 cells were noted, along with elevated fibronectin and TGF-ß1 expression. The additions of exogenous fibronectin and TGF-ß1 promoted Caki-1 cell growth and migration, and increased cyclin D1, fibronectin, vimentin, and TGF-ß1 expression, as well as Src and Smad phosphorylation. These findings highlight the role of fibronectin in RCC cell growth and migration involving Src and TGF-ß1 signaling.


Assuntos
Carcinoma de Células Renais/metabolismo , Movimento Celular , Proliferação de Células , Fibronectinas/metabolismo , Neoplasias Renais/metabolismo , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Fibronectinas/genética , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Vimentina/genética , Vimentina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
14.
Elife ; 82019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31204997

RESUMO

Functional tissue architecture originates by self-assembly of distinct cell types, following tissue-specific rules of cell-cell interactions. In the liver, a structural model of the lobule was pioneered by Elias in 1949. This model, however, is in contrast with the apparent random 3D arrangement of hepatocytes. Since then, no significant progress has been made to derive the organizing principles of liver tissue. To solve this outstanding problem, we computationally reconstructed 3D tissue geometry from microscopy images of mouse liver tissue and analyzed it applying soft-condensed-matter-physics concepts. Surprisingly, analysis of the spatial organization of cell polarity revealed that hepatocytes are not randomly oriented but follow a long-range liquid-crystal order. This does not depend exclusively on hepatocytes receiving instructive signals by endothelial cells, since silencing Integrin-ß1 disrupted both liquid-crystal order and organization of the sinusoidal network. Our results suggest that bi-directional communication between hepatocytes and sinusoids underlies the self-organization of liver tissue.


Assuntos
Polaridade Celular , Hepatócitos/citologia , Cristais Líquidos/química , Fígado/citologia , Algoritmos , Animais , Capilares/química , Capilares/citologia , Capilares/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Hepatócitos/química , Hepatócitos/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Fígado/irrigação sanguínea , Fígado/química , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , Interferência de RNA
15.
Inflammation ; 42(5): 1575-1584, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31111299

RESUMO

Integrins can function synergistically with syndecan-4 (SYND4) and bind to the fibronectin (FN) matrix, resulting in the regulation of tissue regeneration. This study aimed to explore the effects of TNF-α on the formation of FN/ITGß1/SYND4 complex and the relative mechanism in NP cells. The expression of FN-ITG-SYND4 at the cellular level under TNF-α stimulation was detected by immunofluorescent staining, western blotting, and RT-PCR. ITGß1 is a crucial component of ITG FN-induced FAK signaling, which was detected using dual mode. And, the involved signaling down stream pathways were also detected. FN is a preferred adhesion substrate for NP cells and that integrin ß1 (ITGß1) and SYND4 work synergistically during ECM engagement in a focal adhesion kinase (FAK)-dependent fashion. The PI3k/Akt pathway is obviously down-regulated, resulting in decreased adherence capacity and increased anoikis. TNF-α induction could weaken FAK activity and downstream levels of phospho-PI3K and Akt, resulting in decreased adherence capacity and increased apoptosis. Thus, TNF-α is essential for the formation of FN/ITGß1/SYND4 complex in NP cells and further elucidates the inflammatory mechanism of NP cells degeneration.


Assuntos
Integrina beta1/metabolismo , Núcleo Pulposo/metabolismo , Sindecana-4/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adesão Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina beta1/efeitos dos fármacos , Núcleo Pulposo/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
J Biomed Sci ; 26(1): 37, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096970

RESUMO

BACKGROUND: The blood-spinal cord barrier (BSCB) is composed of a monolayer of endothelium linked with tight junctions and extracellular matrix (ECM)-rich basement membranes and is surrounded by astrocyte foot processes. Endothelial permeability is regulated by interaction between endothelial cells and ECM proteins. Fibronectin (FN) is a principal ECM component of microvessels. Excessive FN deposition disrupts cell-cell adhesion in fibroblasts through ß1 integrin ligation. To determine whether excessive FN deposition contributes to the disruption of endothelial integrity, we used an in vitro model of the endothelial monolayer to investigate whether the FN inhibitor pUR4 prevents FN deposition into the subendothelial matrix and attenuates endothelial leakage. METHODS: To correlate the effects of excessive FN accumulation in microvessels on BSCB disruption, spinal nerve ligation-which induces BSCB leakage-was applied, and FN expression in the spinal cord was evaluated through immunohistochemistry and immunoblotting. To elucidate the effects by which pUR4 modulates endothelial permeability, brain-derived endothelial (bEND.3) cells treated with tumor necrosis factor (TNF)-α were used to mimic a leaky BSCB. A bEND.3 monolayer was preincubated with pUR4 before TNF-α treatment. The transendothelial electrical resistance (TEER) measurement and transendothelial permeability assay were applied to assess the endothelial integrity of the bEND.3 monolayer. Immunofluorescence analysis and immunoblotting were performed to evaluate the inhibitory effects of pUR4 on TNF-α-induced FN deposition. To determine the mechanisms underlying pUR4-mediated endothelial permeability, cell morphology, stress fiber formation, myosin light chain (MLC) phosphorylation, and ß1 integrin-mediated signaling were evaluated through immunofluorescence analysis and immunoblotting. RESULTS: Excessive FN was accumulated in the microvessels of the spinal cord after spinal nerve ligation; moreover, pUR4 inhibited TNF-α-induced FN deposition in the bEND.3 monolayer and maintained intact TEER and endothelial permeability. Furthermore, pUR4 reduced cell morphology alteration, actin stress fiber formation, and MLC phosphorylation, thereby attenuating paracellular gap formation. Moreover, pUR4 reduced ß1 integrin activation and downstream signaling. CONCLUSIONS: pUR4 reduces TNF-α-induced ß1 integrin activation by depleting ECM FN, leading to a decrease in endothelial hyperpermeability and maintenance of monolayer integrity. These findings suggest therapeutic benefits of pUR4 in pathological vascular leakage treatment.


Assuntos
Fibronectinas/antagonistas & inibidores , Integrina beta1/metabolismo , Fragmentos de Peptídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
17.
J Am Soc Nephrol ; 30(6): 1006-1019, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31097607

RESUMO

BACKGROUND: Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. METHODS: To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model. RESULTS: Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo, nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. We also found that activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin ß1 in cultured podocytes. CONCLUSIONS: Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin ß1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.


Assuntos
Regulação da Expressão Gênica/genética , Integrina beta1/metabolismo , Proteínas de Membrana/genética , Fosforilação/genética , Podócitos/metabolismo , Insuficiência Renal Crônica/genética , Animais , Células Cultivadas , Drosophila/citologia , Citometria de Fluxo , Humanos , Microscopia Eletrônica de Transmissão , Insuficiência Renal Crônica/patologia , Transdução de Sinais/genética , Estatísticas não Paramétricas
18.
Biomed Res ; 40(2): 79-85, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30982803

RESUMO

The vitreous of perinatal mice temporarily develops a unique vascular system, called the vasa hyaloidea propria (VHP). Observations showed the vessels possessed an extracellular matrix including the basement membrane in their entire length. Immunostaining of whole mount preparations of VHP with integrin ß1 antibody displayed a bush-like network consisting of long and straight fibers which were associated with the VHP but extended apart from the blood vessels. Electron microscopically, each fiber was composed of a bundle of thin filaments different from collagen fibrils. Macrophages associated with the VHP appeared to be arrested by the integrin bushes. The integrin bushes fragmented and disappeared by postnatal day 10, just before the regression of the VHP. Macrophages were involved in the digestion and clearance of integrin bushes. The vitreous integrin bushes appear to provide a scaffold for architectural maintenance of the hyaloid vessels and macrophages.


Assuntos
Membrana Basal/ultraestrutura , Vasos Sanguíneos/ultraestrutura , Citoesqueleto/ultraestrutura , Matriz Extracelular/ultraestrutura , Integrina beta1/ultraestrutura , Corpo Vítreo/ultraestrutura , Animais , Animais Recém-Nascidos , Membrana Basal/metabolismo , Vasos Sanguíneos/anatomia & histologia , Citoesqueleto/metabolismo , Embrião de Mamíferos , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Imuno-Histoquímica , Integrina beta1/genética , Integrina beta1/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica , Neovascularização Fisiológica , Gravidez , Corpo Vítreo/anatomia & histologia , Corpo Vítreo/irrigação sanguínea
19.
Mediators Inflamm ; 2019: 1208086, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30944544

RESUMO

In acute pulmonary inflammation, polymorphonuclear cells (PMNs) pass a transendothelial barrier from the circulation into the lung interstitium followed by a transepithelial migration into the alveolar space. These migration steps are regulated differentially by a concept of adhesion molecules and remain-despite decades of research-incompletely understood. Current knowledge of changes in the expression pattern of adhesion molecules mainly derives from in vitro studies or from studies in extrapulmonary organ systems, where regulation of adhesion molecules differs significantly. In a murine model of lung inflammation, we determined the expression pattern of nine relevant neutrophilic adhesion molecules on their way through the different compartments of the lung. We used a flow cytometry-based technique that allowed describing spatial distribution of the adhesion molecules expressed on PMNs during their migration through the lung in detail. For example, the highest expression of CD29 was found in the intravascular compartment, highlighting its impact on the initial adhesion to the endothelium. CD47 showed its peak of expression on the later phase of transendothelial migration, whereas CD11b and CD54 expression peaked interstitial. A pivotal role for transepithelial migration was found for the adhesion molecule CD172a. Thereby, expression may correlate with functional impact for specific migration steps. In vitro studies further confirmed our in vivo findings. In conclusion, we are the first to determine the changes in expression patterns of relevant adhesion molecules on their migration through the different compartments of the lung. These findings may help to further understand the regulation of neutrophil trafficking in the lung.


Assuntos
Moléculas de Adesão Celular/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Neutrófilos/metabolismo , Pneumonia/metabolismo , Lesão Pulmonar Aguda/metabolismo , Animais , Antígeno CD11b/metabolismo , Antígeno CD47/metabolismo , Adesão Celular/efeitos dos fármacos , Citometria de Fluxo , Inflamação/imunologia , Inflamação/metabolismo , Integrina beta1/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Receptores Imunológicos/metabolismo
20.
Dev Cell ; 49(5): 681-696.e6, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31006651

RESUMO

Hematopoietic stem cells (HSCs) emerge from hemogenic endothelium (HE) within the ventral portion of the dorsal aorta during vertebrate development. In zebrafish, Notch signaling induces HE specification from posterior lateral plate mesoderm (PLPM) cells as they migrate over the ventral surface of the somite. During migration, PLPM cells make close contact with Notch-ligand-expressing somitic cells to acquire HE identity. Herein, we show in zebrafish that the small GTPase Rap1b regulates HSC development by potentiating Notch-mediated HE specification. PLPM cells migrate toward the midline along the somite boundary where fibronectin accumulates. Rap1b stimulates integrin ß1 to enhance PLPM cell adhesion to fibronectin localized at the somite boundary. Rap1b-induced integrin-ß1-mediated adhesion to fibronectin leads to the spreading of PLPM cells to facilitate their physical contact with the Notch-ligand-expressing somitic cells, thereby promoting Notch-mediated HE specification. Thus, we have revealed an unexpected role of Rap1-induced integrin-mediated cell adhesion in HSC development.


Assuntos
Adesão Celular , Fibronectinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Integrina beta1/metabolismo , Receptores Notch/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Fibronectinas/genética , Células-Tronco Hematopoéticas/metabolismo , Integrina beta1/genética , Receptores Notch/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas rap de Ligação ao GTP/genética
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