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1.
Zhonghua Yi Xue Za Zhi ; 101(34): 2692-2697, 2021 Sep 14.
Artigo em Chinês | MEDLINE | ID: mdl-34510875

RESUMO

Objective: To elucidate the biological role and potential mechanism of integrin α5 (ITGA5) in gastric cancer (GC). Methods: From January 2019 to December 2020, 35 pairs of GC tissue [21 males and 14 females, aged (53.8±5.4) years] and matched adjacent tissue samples were collected from GC patients who underwent surgical resection in Zhejiang Provincial People's Hospital. GC and normal gastric mucosa cells were purchased from Beijing Biobw Biotech Company. Quantitative real-time PCR (qRT-PCR), immunohistochemistry, Western blotting were performed to detect the mRNA and protein expression levels of ITGA5, cell adhesion-related genes (pFAK, pSrc, aRac1) in GC cells. Cell Counting Kit-8 (CCK-8), Transwell invasion, wound healing and cell adhesion assays were conducted for GC cell phenotype detection. Results: ITGA5 was highly expressed in GC compared with normal gastric mucosa cells (relative expression increased from 1.00±0.26 to 1.23±0.27,P<0.05). In addition, ITGA5 overexpression promoted the cell proliferation [from (1.14±0.14) OD to (1.61±0.14) OD], migration ability [from (20.3±2.3)% to (56.4±6.1)%], invasion ability (from 144.0±4.6 to 216.7±6.6), and adhesion ability of matrix protein (from 99.0±8.5 to 152.0±12.3) through FAK/Src/Rac1 signaling pathway in GC.(all P<0.05) Conclusions: ITGA5 acts as a cancer-promoting factor in GC. The current study provides theoretical evidence for probing the novel molecular targets for the treatment of GC.


Assuntos
Integrina alfa5 , Integrinas/metabolismo , Neoplasias Gástricas , Movimento Celular , Proliferação de Células , Feminino , Humanos , Integrina alfa5/metabolismo , Masculino , Invasividade Neoplásica , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo
2.
PLoS One ; 16(9): e0257576, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34551004

RESUMO

Exaggerated inflammatory response results in pathogenesis of various inflammatory diseases. Tumor Necrosis Factor-alpha (TNF) is a multi-functional pro-inflammatory cytokine regulating a wide spectrum of physiological, biological, and cellular processes. TNF induces Focal Adhesion Kinase (FAK) for various activities including induction of pro-inflammatory response. The mechanism of FAK activation by TNF is unknown and the involvement of cell surface integrins in modulating TNF response has not been determined. In the current study, we have identified an oxysterol 25-hydroxycholesterol (25HC) as a soluble extracellular lipid amplifying TNF mediated innate immune pro-inflammatory response. Our results demonstrated that 25HC-integrin-FAK pathway amplifies and optimizes TNF-mediated pro-inflammatory response. 25HC generating enzyme cholesterol 25-hydroxylase (C25H) was induced by TNF via NFκB and MAPK pathways. Specifically, chromatin immunoprecipitation assay identified binding of AP-1 (Activator Protein-1) transcription factor ATF2 (Activating Transcription Factor 2) to the C25H promoter following TNF stimulation. Furthermore, loss of C25H, FAK and α5 integrin expression and inhibition of FAK and α5ß1 integrin with inhibitor and blocking antibody, respectively, led to diminished TNF-mediated pro-inflammatory response. Thus, our studies show extracellular 25HC linking TNF pathway with integrin-FAK signaling for optimal pro-inflammatory activity and MAPK/NFκB-C25H-25HC-integrin-FAK signaling network playing an essential role to amplify TNF dependent pro-inflammatory response. Thus, we have identified 25HC as the key factor involved in FAK activation during TNF mediated response and further demonstrated a role of cell surface integrins in positively regulating TNF dependent pro-inflammatory response.


Assuntos
Transdução de Sinais/efeitos dos fármacos , Esteroide Hidroxilases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator 2 Ativador da Transcrição/metabolismo , Animais , Células Cultivadas , Quimiocina CCL3/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Hidroxicolesteróis/metabolismo , Integrina alfa5/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Ligação Proteica , Esteroide Hidroxilases/deficiência , Esteroide Hidroxilases/genética , Regulação para Cima/efeitos dos fármacos
3.
Exp Cell Res ; 406(2): 112765, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34358523

RESUMO

Nasopharyngeal carcinoma (NPC) originates in the nasopharynx epithelium. Although concurrent chemoradiation therapy followed by chemotherapy is considered as an effective treatment, there is substantial drug resistance in locally advanced NPC patients. One major contributor to the chemoresistance includes aberrant expression of cell adhesion molecules, such as integrin α and ß subunits, giving rise to cell adhesion-mediated drug resistance. Thus, the aim of this study was to investigate the effect of integrin α5 on the development of intrinsic cisplatin resistance in NPC and the associated underlying mechanisms using in vitro three-dimensional (3D) spheroid models, as well as induced cisplatin-resistant NPC (NPCcisR). We demonstrated that established 3D highly- (5-8F) and lowly- (6-10B) metastatic NPC spheroids overexpressed integrin α5 and aggravated their resistance to cisplatin. Besides, enhanced integrin α5 resulted in substantially reduced growth, corresponding to G0/G1 and G2/M cell cycle arrest. In addition, 5-8FcisR and 6-10BcisR cells in 3D forms synergistically strengthened endurance of their spheroids to cisplatin treatment as observed by increased resistance index (RI) and decreased apoptosis. Mechanistically, the aberrantly expressed integrin α5 decreased drug susceptibility in NPC spheroids by inactivating ERK and inhibition of caspase-3 inducing apoptosis. Furthermore, the effect of integrin α5 inducing intrinsic resistance was verified via treatment with ATN-161, a peptide inhibitor for integrin α5ß1. The results showed dramatic reduction in integrin α5 expression, reversal of ERK phosphorylation and caspase-3 cleavage, together with elevated cisplatin sensitivity, indicating regulation of innate drug resistance via integrin α5. Taken together, our findings suggest that integrin α5 could act as a promising target to enhance the chemotherapeutic sensitivity in NPC.


Assuntos
Apoptose , Caspase 3/química , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Integrina alfa5/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/química , Carcinoma Nasofaríngeo/patologia , Esferoides Celulares/patologia , Antineoplásicos/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular , Humanos , Integrina alfa5/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/secundário , Fosforilação , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo
4.
FASEB J ; 35(8): e21679, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314542

RESUMO

The ability to form a variety of cell-matrix connections is crucial for angiogenesis to take place. Without stable anchorage to the extracellular matrix (ECM), endothelial cells (ECs) are unable to sense, integrate and disseminate growth factor stimulated responses that drive growth of a vascular bed. Neuropilin-2 (NRP2) is a widely expressed membrane-bound multifunctional non-tyrosine kinase receptor, which has previously been implicated in influencing cell adhesion and migration by interacting with α5-integrin and regulating adhesion turnover. α5-integrin, and its ECM ligand fibronectin (FN) are both known to be upregulated during the formation of neo-vasculature. Despite being descriptively annotated as a candidate biomarker for aggressive cancer phenotypes, the EC-specific roles for NRP2 during developmental and pathological angiogenesis remain unexplored. The data reported here support a model whereby NRP2 actively promotes EC adhesion and migration by regulating dynamic cytoskeletal remodeling and by stimulating Rab11-dependent recycling of α5-integrin-p-FAK complexes to newly assembling adhesion sites. Furthermore, temporal depletion of EC-NRP2 in vivo impairs primary tumor growth by disrupting vessel formation. We also demonstrate that EC-NRP2 is required for normal postnatal retinal vascular development, specifically by regulating cell-matrix adhesion. Upon loss of endothelial NRP2, vascular outgrowth from the optic nerve during superficial plexus formation is disrupted, likely due to reduced FAK phosphorylation within sprouting tip cells.


Assuntos
Actinas/metabolismo , Células Endoteliais , Integrina alfa5/metabolismo , Pulmão/irrigação sanguínea , Neovascularização Patológica/metabolismo , Neuropilina-2/fisiologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Matriz Extracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
5.
Anticancer Res ; 41(8): 3843-3849, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281844

RESUMO

BACKGROUND/AIM: Integrin-targeting compounds have shown clinically significant benefits in many patients. Here, we examined the activity of millettocalyxin B, extracted from the stem bark of Millettia erythrocalyx, in lung cancer cells. MATERIALS AND METHODS: The viability of human lung cancer cells was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazoliumbromide (MTT) assay. Migration and invasion assays were performed. Phalloidin-rhodamine staining was used to determine the formation of filopodia. Western blot analysis and immunofluorescence staining were used to identify the signaling proteins involved in migration regulation. RESULTS: Non-toxic concentrations (0-25 µM) of millettocalyxin B reduced migration and invasion of lung cancer A549 cells. Filopodia were significantly reduced in millettocalyxin B-treated cells. The migration regulatory proteins including integrin α5, active FAK, active Akt, and Cdc42 were significantly decreased in Millettocalyxin B-treated cells. CONCLUSION: Our findings revealed a novel anti-migration and anti-invasion effects and the underlying mechanism of millettocalyxin B, which may be exploited for cancer treatment.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Flavonoides/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Células A549 , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Humanos , Integrina alfa5/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pseudópodes/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/metabolismo
6.
Dev Cell ; 56(8): 1164-1181.e12, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33761321

RESUMO

Cells probe their surrounding matrix for attachment sites via integrins that are internalized by endocytosis. We find that SH3BP4 regulates integrin surface expression in a signaling-dependent manner via clathrin-coated pits (CCPs). Dephosphorylated SH3BP4 at S246 is efficiently recruited to CCPs, while upon Akt phosphorylation, SH3BP4 is sequestered by 14-3-3 adaptors and excluded from CCPs. In the absence of Akt activity, SH3BP4 binds GIPC1 and targets neuropilin-1 and α5/ß1-integrin for endocytosis, leading to inhibition of cell spreading. Similarly, chemorepellent semaphorin-3a binds neuropilin-1 to activate PTEN, which antagonizes Akt and thus recruits SH3BP4 to CCPs to internalize both receptors and induce cell contraction. In PTEN mutant non-small cell lung cancer cells with high Akt activity, expression of non-phosphorylatable active SH3BP4-S246A restores semaphorin-3a induced cell contraction. Thus, SH3BP4 links Akt signaling to endocytosis of NRP1 and α5/ß1-integrins to modulate cell-matrix interactions in response to intrinsic and extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endocitose , Integrina alfa5/metabolismo , Neuropilina-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas 14-3-3/metabolismo , Linhagem Celular Tumoral , Invaginações Revestidas da Membrana Celular/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Mutantes/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Ligação Proteica , Semaforina-3A/metabolismo , Transdução de Sinais
7.
J Immunol ; 206(7): 1549-1560, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33637617

RESUMO

Outside-in integrin signaling regulates cell fate decisions in a variety of cell types, including hematopoietic stem cells (HSCs). Our earlier published studies showed that interruption of periostin (POSTN) and integrin-αv (ITGAV) interaction induces faster proliferation in HSCs with developmental stage-dependent functional effects. In this study, we examined the role of POSTN-ITGAV axis in lymphohematopoietic activity in spleen that hosts a rare population of HSCs, the functional regulation of which is not clearly known. Vav-iCre-mediated deletion of Itgav in the hematopoietic system led to higher proliferation rates, resulting in increased frequency of primitive HSCs in the adult spleen. However, in vitro CFU-C assays demonstrated a poorer differentiation potential following Itgav deletion. This also led to a decrease in the white pulp area with a significant decline in the B cell numbers. Systemic deletion of its ligand, POSTN, phenocopied the effects noted in Vav-Itgav-/- mice. Histological examination of Postn-deficient spleen also showed an increase in the spleen trabecular areas. Importantly, these are the myofibroblasts of the trabecular and capsular areas that expressed high levels of POSTN within the spleen tissue. In addition, vascular smooth muscle cells also expressed POSTN. Through CFU-S12 assays, we showed that hematopoietic support potential of stroma in Postn-deficient splenic hematopoietic niche was defective. Overall, we demonstrate that POSTN-ITGAV interaction plays an important role in spleen lymphohematopoiesis.


Assuntos
Moléculas de Adesão Celular/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Integrina alfa5/metabolismo , Linfócitos/fisiologia , Miócitos de Músculo Liso/fisiologia , Miofibroblastos/fisiologia , Baço/imunologia , Animais , Moléculas de Adesão Celular/genética , Proliferação de Células , Técnicas de Silenciamento de Genes , Hematopoese , Integrina alfa5/genética , Camundongos , Camundongos Knockout , Transdução de Sinais , Nicho de Células-Tronco
8.
Sci Rep ; 11(1): 2368, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504916

RESUMO

In vertebrates, new bone formation via intramembranous osteogenesis is a critical biological event for development, remodeling, and fracture healing of bones. Chemotaxis of osteoblast lineage cells is an essential cellular process in new bone formation. Connective tissue growth factor (CTGF) is known to exert chemotactic properties on various cells; however, details of CTGF function in the chemotaxis of osteoblast lineage cells and underlying molecular biological mechanisms have not been clarified. The aim of the present study was to evaluate the chemotactic properties of CTGF and its underlying mechanisms during active bone formation through intramembranous osteogenesis. In our mouse tensile force-induced bone formation model, preosteoblasts were aggregated at the osteogenic front of calvarial bones. CTGF was expressed at the osteogenic front, and functional inhibition of CTGF using a neutralizing antibody suppressed the aggregation of preosteoblasts. In vitro experiments using µ-slide chemotaxis chambers showed that a gradient of CTGF induced chemotaxis of preosteoblastic MC3T3-E1 cells, while a neutralizing integrin α5 antibody and a Ras inhibitor inhibited the CTGF-induced chemotaxis of MC3T3-E1 cells. These findings suggest that the CTGF-integrin α5-Ras axis is an essential molecular mechanism to promote chemotaxis of preosteoblasts during new bone formation through intramembranous osteogenesis.


Assuntos
Quimiotaxia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Integrina alfa5/genética , Osteoblastos/metabolismo , Osteogênese/fisiologia , Resistência à Tração , Proteínas ras/genética , Células 3T3 , Animais , Biomarcadores , Osso e Ossos , Diferenciação Celular , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/genética , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Imunofluorescência , Imuno-Histoquímica , Integrina alfa5/metabolismo , Camundongos , Osteoblastos/citologia , Transdução de Sinais , Proteínas ras/metabolismo
9.
J Physiol Pharmacol ; 71(4)2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33214335

RESUMO

Burkitt lymphoma (BL) is a highly aggressive form of non-Hodgkin's B-cell lymphoma. Currently, multi-agent chemotherapy regimens are being used to significantly improve cure rates and achieve complete remissions in BL patients. However, drug resistance can often occur within 6 months in BL patients, contributing to poor prognosis. Mounting evidence suggests that cell adhesion-mediated drug resistance (CAM-DR), caused by the interaction between the bone marrow microenvironment and tumour cells may play an important role in drug resistance to chemotherapy. However, the molecular mechanism underlying CAM-DR in BL has not been identified yet. In this study, we investigated the molecular mechanism responsible for CAM-DR in BL cells. We also examined the therapeutic targets of CAM-DR in BL cells and found CD49d and CD49e to be the important adhesion molecules involved. However, CD49a, CD49b, CD11a, CD29, CD18, and CD61 were not found to be associated with CAM-DR in BL cells. Furthermore, we clarified that CD49d- and CD49e-mediated CAM-DR could be attributed to an increase in the expression of B cell leukemia-xL (Bcl-xL) and survivin proteins, and a decrease in the expression of Bcl-2 associated X (Bax), Bcl-2 interacting mediator (Bim) and p53 upregulated modulator of apoptosis (PUMA) proteins via nuclear factor kappaB (NF-κB) activation. In addition, bortezomib was found to overcome CAM-DR in BL cells by inhibiting NF-κB. Thus, bortezomib may have potential clinical applications in the treatment of CD49d- and CD49e-mediated CAM-DR in BL patients.


Assuntos
Antineoplásicos/farmacologia , Linfoma de Burkitt/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Integrina alfa4/metabolismo , Integrina alfa5/metabolismo , NF-kappa B/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Bortezomib/farmacologia , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Proteassoma/farmacologia , Transdução de Sinais , Microambiente Tumoral
10.
Med Oncol ; 37(11): 103, 2020 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-33068194

RESUMO

Multiple myeloma (MM) is a hematological malignancy characterized by the proliferation of abnormal plasma cells in bone marrow. Flow cytometry distinguishes between normal and abnormal plasma cells by evaluating cluster of differentiation (CD) 56 and CD19 expression patterns. Moreover, immunophenotyping of mature plasma cell 1 (MPC-1) and very late antigen-5 (CD49e) identifies the maturity of MM as mature (MPC-1+, CD49e+), intermediate (MPC-1+, CD49e-), or immature (MPC-1-, CD49e-). We retrospectively examined the effects of surface marker expression and maturity subtype on overall survival (OS) and time to next treatment (TNT) among 55 patients (25 males, 30 females) with symptomatic MM. All patients were treated with regimens containing bortezomib (BOR) (n = 39) or lenalidomide (LEN) (n = 16) as the initial treatment. Median age at diagnosis was 72 years (range: 36-88). The lack of CD56, an aberrant marker, was associated with significantly worse prognosis compared with CD56+ MM (median OS: 24 vs. 60 months, respectively; p = 0.0050). In CD49e+ MM, defined as mature type, no significant difference was seen in TNT of the initial treatment, regardless of whether it was a BOR-based regimen or LEN + dexamethasone (Ld) therapy. On the other hand, in CD49e- MM, defined as immature/intermediate type, TNT of Ld therapy was significantly longer than that of BOR-based regimens (median TNT: undefined vs. 12 months, respectively; p = 0.0043). These results suggest that Ld therapy is more effective than BOR-based therapy for CD49e- MM and thus may aid regimen-related decisions in the novel agents era.


Assuntos
Antineoplásicos/uso terapêutico , Antígeno CD56/metabolismo , Integrina alfa5/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Bortezomib/uso terapêutico , Feminino , Humanos , Imunofenotipagem , Lenalidomida/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/metabolismo , Plasmócitos/metabolismo , Prognóstico , Estudos Retrospectivos
11.
Microbiol Immunol ; 64(12): 825-834, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33034909

RESUMO

It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/ß1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/ß1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/ß1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/ß1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/ß1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases.


Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Proteína HMGN2/metabolismo , Integrina alfa5beta1/metabolismo , Klebsiella pneumoniae/metabolismo , Fatores de Transcrição NFI/metabolismo , Células A549 , Regulação da Expressão Gênica , Proteína HMGN2/genética , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina alfa5beta1/genética , Integrina beta1/genética , Integrina beta1/metabolismo , Infecções por Klebsiella/metabolismo , Klebsiella pneumoniae/genética , Pulmão , RNA Interferente Pequeno/metabolismo , Transcriptoma
12.
J Exp Clin Cancer Res ; 39(1): 221, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-33081836

RESUMO

BACKGROUND: Peritoneal metastasis (PM) is an important pathological process in the progression of gastric cancer (GC). The metastatic potential of tumor and stromal cells is governed by hypoxia, which is a key molecular feature of the tumor microenvironment. Mesothelial cells also participate in this complex and dynamic process. However, the molecular mechanisms underlying the hypoxia-driven mesothelial-tumor interactions that promote peritoneal metastasis of GC remain unclear. METHODS: We determined the hypoxic microenvironment in PM of nude mice by immunohistochemical analysis and screened VEGFA by human growth factor array kit. The crosstalk mediated by VEGFA between peritoneal mesothelial cells (PMCs) and GC cells was determined in GC cells incubated with conditioned medium prepared from hypoxia-treated PMCs. The association between VEGFR1 and integrin α5 and fibronectin in GC cells was enriched using Gene Set Enrichment Analysis and KEGG pathway enrichment analysis. In vitro and xenograft mouse models were used to evaluate the impact of VEGFA/VEGFR1 on gastric cancer peritoneal metastasis. Confocal microscopy and immunoprecipitation were performed to determine the effect of hypoxia-induced autophagy. RESULTS: Here we report that in the PMCs of the hypoxic microenvironment, SIRT1 is degraded via the autophagic lysosomal pathway, leading to increased acetylation of HIF-1α and secretion of VEGFA. Under hypoxic conditions, VEGFA derived from PMCs acts on VEGFR1 of GC cells, resulting in p-ERK/p-JNK pathway activation, increased integrin α5 and fibronectin expression, and promotion of PM. CONCLUSIONS: Our findings have elucidated the mechanisms by which PMCs promote PM in GC in hypoxic environments. This study also provides a theoretical basis for considering autophagic pathways or VEGFA as potential therapeutic targets to treat PM in GC.


Assuntos
Autofagia , Fibronectinas/metabolismo , Hipóxia/fisiopatologia , Integrina alfa5/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Biomarcadores Tumorais , Movimento Celular , Proliferação de Células , Epitélio/metabolismo , Epitélio/patologia , Feminino , Fibronectinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa5/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Eur Rev Med Pharmacol Sci ; 24(17): 8675-8684, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32964955

RESUMO

OBJECTIVE: To clarify the interaction between TGF-ß1 and WISP1, and the effect of Integrin α5/V subunits on the WISP1 caused chondrocyte (CH) dedifferentiated phenotype. PATIENTS AND METHODS: The knee joint cartilage from the trauma and osteoarthritis (OA) patients were collected. The patients of trauma group were confirmed to have no OA history. The protein level of WISP1, Integrin-α5/V, and type II/I collagen were analyzed by Western blotting. Besides, we isolated the CHs from the cartilage without OA and treated CHs with exogenic TGF-ß1 and WISP1 protein. In addition to this, to regulate the α5 and αV subunits expression of CHs, we silenced two genes by siRNA transfection and upregulated them by exogenic protein supplement. Then, the CHs with different α5 and αV expression were treated with WISP1. To value the chondrogenic gene expression, we determined the type II collagen and SOX9 gene expression by immunofluorescence (IF) and RT-PCR, respectively. Meanwhile, the dedifferentiation markers of CH, type I collagen, and Runx2 expression was also analyzed. Cell proliferation was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. RESULTS: The OA cartilage contains a higher level of type I collagen, WISP1, Integrin α5, and Integrin αV, but low type II collagen. The upregulation of TGF-ß1 caused the increase of WISP1, as well as the high level of Integrin α5/V, and dedifferentiated gene. Besides, the upregulation of WISP1 also contributed to the TGF-ß1 expression and CHs dedifferentiation. Apart from this, the silencing of the α5 subunit of Integrin aggravated the WISP1 induced CHs dedifferentiation, which was reversed by α5 upregulation. However, the αV subunit played an opposite role that mediated the WISP1-induced CHs dedifferentiation. Additionally, the interaction between TGF-ß1 and WISP1 promoted the CHs proliferation, which was not affected by the Integrin-α5/V expression. CONCLUSIONS: TGF-ß1 and WISP1 interact to induce CHs dedifferentiation, which was mainly by the mediation of the Integrin-αV subunit. On the contrary, Integrin-α5 shows a protective effect during the WISP1 caused CHs dedifferentiation.


Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Desdiferenciação Celular , Condrócitos/metabolismo , Integrina alfa5/metabolismo , Integrina alfaV/metabolismo , Osteoartrite/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Cartilagem Articular/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Humanos , Articulação do Joelho/metabolismo , Ferimentos e Lesões/metabolismo
14.
Medicine (Baltimore) ; 99(34): e21821, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32846824

RESUMO

BACKGROUND: Traditional Chinese medicine Tongxinluo (TXL) has been widely used to treat coronary artery disease in China, since it could reduce myocardial infarct size and ischemia/reperfusion injury in both non-diabetic and diabetic conditions. It has been shown that TXL could regulate peroxisome proliferator activated receptor-α (PPAR-α), a positive modulator of angiopoietin-like 4 (Angptl4), in diabetic rats. Endothelial junction substructure components, such as VE-cadherin, are involved in the protection of reperfusion injury. Thus, we hypothesized cell-intrinsic and endothelial-specific Angptl4 mediated the protection of TXL on endothelial barrier under high glucose condition against ischemia/reperfusion-injury via PPAR-α pathway. METHODS: Incubated with high glucose medium, the human cardiac microvascular endothelial cells (HCMECs) were then exposed to oxygen-glucose-serum deprivation (2 hours) and restoration (2 hours) stimulation, with or without TXL, insulin, or rhAngptl4 pretreatment. RESULTS: TXL, insulin, and rhAngptl4 had similar protective effects on the endothelial barrier. TXL treatment reversed the endothelial barrier breakdown in HCMECs significantly as identified by decreasing endothelial permeability, upregulating the expression of JAM-A, VE-cadherin, and integrin-α5 and increasing the membrane location of VE-cadherin and integrin-α5, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with small interfering RNA (siRNA) interference and PPAR-α inhibitor MK886 partially abrogated these beneficial effects of TXL. Western blotting also revealed that similar with insulin, TXL upregulated the expression of Angptl4 in HCMECs, which could be inhibited by Angptl4 siRNA or MK886 exposure. TXL treatment increased PPAR-α activity, which could be diminished by MK886 but not by Angptl4 siRNA. CONCLUSION: These data suggest cell-intrinsic and endothelial-specific Angptl4 mediates the protection of TXL against endothelial barrier breakdown during oxygen-glucose-serum deprivation and restoration under high glucose condition partly via the PPAR-α/Angptl4 pathway.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Endotélio/fisiopatologia , PPAR alfa/metabolismo , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/farmacologia , Caderinas/metabolismo , Permeabilidade Capilar , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Vasos Coronários/citologia , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Glucose/farmacologia , Humanos , Indóis/farmacologia , Insulina/farmacologia , Integrina alfa5/metabolismo , Inibidores de Lipoxigenase/farmacologia , Microvasos/citologia , Oxigênio/metabolismo , Oxigênio/farmacologia , Receptores de Superfície Celular/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais
15.
Dev Biol ; 465(1): 46-57, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32628938

RESUMO

Endocardium is critically important for proper function of the cardiovascular system. Not only does endocardium connect the heart to blood vasculature, it also plays an important role in heart morphogenesis, valve formation, and ventricular trabeculation. The extracellular protein Fibronectin (Fn1) promotes endocardial differentiation, but the signaling pathways downstream of Fn1 that regulate endocardial development are not understood. Here, we analyzed the role of the Fibronectin receptors Integrin alpha5 (Itga5) and Integrin alpha4 (Itga4) in zebrafish heart development. We show that itga5 mRNA is expressed in both endocardium and myocardium during early stages of heart development. Through analysis of both itga5 single mutants and itga4;itga5 double mutants, we show that loss of both itga5 and itga4 results in enhanced defects in endocardial differentiation and morphogenesis compared to loss of itga5 alone. Loss of both itga5 and itga4 results in cardia bifida and severe myocardial morphology defects. Finally, we find that loss of itga5 and itga4 results in abnormally narrow anterior endodermal sheet morphology. Together, our results support a model in which Itga5 and Itga4 cooperate to promote endocardial differentiation, medial migration of endocardial and myocardial cells, and morphogenesis of anterior endoderm.


Assuntos
Diferenciação Celular , Endocárdio/embriologia , Integrina alfa4/metabolismo , Integrina alfa5/metabolismo , Modelos Biológicos , Organogênese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Integrina alfa4/genética , Integrina alfa5/genética , Mutação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
16.
Circ Res ; 127(8): 1074-1090, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32673515

RESUMO

RATIONALE: Atherosclerosis preferentially occurs at specific sites of the vasculature where endothelial cells (ECs) are exposed to disturbed blood flow. Translocation of integrin α5 to lipid rafts promotes integrin activation and ligation, which is critical for oscillatory shear stress (OSS)-induced EC activation. However, the underlying mechanism of OSS promoted integrin α5 lipid raft translocation has remained largely unknown. OBJECTIVE: The objective of this study was to specify the mechanotransduction mechanism of OSS-induced integrin α5 translocation and subsequent EC activation. METHODS AND RESULTS: Mass spectrometry studies identified endothelial ANXA2 (annexin A2) as a potential carrier allowing integrin α5ß1 to traffic in response to OSS. Interference by siRNA of AnxA2 in ECs greatly decreased OSS-induced integrin α5ß1 translocation to lipid rafts, EC activation, and monocyte adhesion. Pharmacological and genetic inhibition of PTP1B (protein tyrosine phosphatase 1B) blunted OSS-induced integrin α5ß1 activation, which is dependent on Piezo1-mediated calcium influx in ECs. Furthermore, ANXA2 was identified as a direct substrate of activated PTP1B by mass spectrometry. Using bioluminescence resonance energy transfer assay, PTP1B-dephosphorylated ANXA2 at Y24 was found to lead to conformational freedom of the C-terminal core domain from the N-terminal domain of ANXA2. Immunoprecipitation assays showed that this unmasked ANXA2-C-terminal core domain specifically binds to an integrin α5 nonconserved cytoplasmic domain but not ß1. Importantly, ectopic lentiviral overexpression of an ANXA2Y24F mutant increased and shRNA against Ptp1B decreased integrin α5ß1 ligation, inflammatory signaling, and progression of plaques at atheroprone sites in apolipoprotein E (ApoE)-/- mice. However, the antiatherosclerotic effect of Ptp1B shRNA was abolished in AnxA2-/-ApoE-/- mice. CONCLUSIONS: Our data elucidate a novel endothelial mechanotransduction molecular mechanism linking atheroprone flow and activation of integrin α5ß1, thereby identifying a class of potential therapeutic targets for atherosclerosis. Graphic Abstract: An graphic abstract is available for this article.


Assuntos
Anexina A2/metabolismo , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Integrina alfa5/metabolismo , Integrina alfa5beta1/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Anexina A2/genética , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Integrina alfa5/genética , Integrina alfa5beta1/genética , Integrinas , Canais Iônicos/metabolismo , Masculino , Mecanotransdução Celular , Microdomínios da Membrana/patologia , Camundongos Knockout para ApoE , Placa Aterosclerótica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Fluxo Sanguíneo Regional , Estresse Mecânico , Células THP-1
17.
Development ; 147(12)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32540847

RESUMO

In the developing neocortex, radially migrating neurons stop migration and form layers beneath the marginal zone (MZ). Reelin plays essential roles in these processes via its receptors, apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR). Although we recently reported that reelin causes neuronal aggregation via ApoER2, which is thought to be important for the subsequent layer formation, it remains unknown what effect reelin exerts via the VLDLR. Here, we found that ectopic reelin overexpression in the Vldlr-mutant mouse cortex causes neuronal aggregation, but without an MZ-like cell-sparse central region that is formed when reelin is overexpressed in the normal cortex. We also found that both the early-born and late-born Vldlr-deficient neurons invade the MZ and exhibit impaired dendrite outgrowth from before birth. Rescue experiments indicate that VLDLR suppresses neuronal invasion into the MZ via a cell-autonomous mechanism, possibly mediated by Rap1, integrin and Akt. These results suggest that VLDLR is not a prerequisite for reelin-induced neuronal aggregation and that the major role of VLDLR is to suppress neuronal invasion into the MZ during neocortical development.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Córtex Cerebral/metabolismo , Dendritos/metabolismo , Embrião de Mamíferos/metabolismo , Proteínas da Matriz Extracelular/genética , Integrina alfa5/metabolismo , Proteínas Relacionadas a Receptor de LDL/deficiência , Proteínas Relacionadas a Receptor de LDL/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Piramidais/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Serina Endopeptidases/genética , Proteínas rap1 de Ligação ao GTP/metabolismo
18.
Biosci Biotechnol Biochem ; 84(8): 1614-1620, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32351169

RESUMO

Previous studies suggest an association of cardiac microvascular endothelial cells (CMECs) hyperpermeability with sepsis-related cardiac injury. Our results showed that CMECs permeability was dependent upon concentration and time of lipopolysaccharides (LPS) stimulation. Integrin ανß3 expression decreased after LPS stimulation. Pretreatment with anti-integrin ανß3 antibody enhanced LPS-induced hyperpermeability. Upregulation of integrin ανß3 decreased LPS-induced hyperpermeability. F-actin remodeling was enhanced after LPS stimulation and was inhibited by up-regulation of integrin ανß3. Inhibition of Src or Rac1 reduced CMECs permeability after LPS stimulation, but there were no differences in the phosphorylation of Src and Rac1 when over-expressing or blocking integrin ß3. After pretreatment with Src or Rac1 inhibitor, no significant difference was found in the expression of integrin ανß3 in LPS-induced CMECs. These finding suggested that integrin ανß3 overexpression decreased LPS-stimulated CMECS permeability by inhibition of cytoskeletal remodeling, but the mechanism might not be mediated via Src/Rac1 signaling.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Integrina alfa5/genética , Integrina beta3/genética , Lipopolissacarídeos/farmacologia , Actinas/genética , Actinas/metabolismo , Aminoquinolinas/farmacologia , Animais , Anticorpos Neutralizantes/farmacologia , Permeabilidade Capilar/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Integrina alfa5/metabolismo , Integrina beta3/metabolismo , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Cultura Primária de Células , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
19.
Cell Rep ; 31(1): 107472, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32268102

RESUMO

Chronic allergic itch is a common symptom affecting millions of people and animals, but its pathogenesis is not fully explained. Herein, we show that periostin, abundantly expressed in the skin of patients with atopic dermatitis (AD), induces itch in mice, dogs, and monkeys. We identify the integrin αVß3 expressed on a subset of sensory neurons as the periostin receptor. Using pharmacological and genetic approaches, we inhibited the function of neuronal integrin αVß3, which significantly reduces periostin-induced itch in mice. Furthermore, we show that the cytokine TSLP, the application of AD-causing MC903 (calcipotriol), and house dust mites all induce periostin secretion. Finally, we establish that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Altogether, our results identify a TSLP-periostin reciprocal activation loop that links the skin to the spinal cord via peripheral sensory neurons, and we characterize the non-canonical functional role of an integrin in itch.


Assuntos
Moléculas de Adesão Celular/metabolismo , Integrinas/metabolismo , Prurido/metabolismo , Animais , Moléculas de Adesão Celular/fisiologia , Dermatite Atópica/etiologia , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Cães , Feminino , Hipersensibilidade/fisiopatologia , Integrina alfa5/metabolismo , Integrina beta3/metabolismo , Queratinócitos/metabolismo , Masculino , Camundongos , Primatas , Prurido/patologia , Células Receptoras Sensoriais/metabolismo , Pele/metabolismo
20.
Commun Biol ; 3(1): 120, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170208

RESUMO

Helicobacter pylori, the most common etiologic agent of gastric diseases including gastric cancer, is auxotrophic for cholesterol and has to hijack it from gastric epithelia. Upon uptake, the bacteria convert cholesterol to cholesteryl 6'-O-acyl-α-D-glucopyranoside (CAG) to promote lipid raft clustering in the host cell membranes. However, how CAG appears in the host to exert the pathogenesis still remains ambiguous. Herein we identified hp0499 to be the gene of cholesteryl α-D-glucopyranoside acyltransferase (CGAT). Together with cholesteryl glucosyltransferase (catalyzing the prior step), CGAT is secreted via outer membrane vesicles to the host cells for direct synthesis of CAG. This significantly enhances lipid rafts clustering, gathers adhesion molecules (including Lewis antigens and integrins α5, ß1), and promotes more bacterial adhesion. Furthermore, the clinically used drug amiodarone was shown as a potent inhibitor of CGAT to effectively reduce the bacterial adhesion, indicating that CGAT is a potential target of therapeutic intervention.


Assuntos
Aciltransferases/metabolismo , Aderência Bacteriana/genética , Proteínas de Bactérias/metabolismo , Colesterol/análogos & derivados , Mucosa Gástrica/microbiologia , Helicobacter pylori/enzimologia , Aciltransferases/antagonistas & inibidores , Aciltransferases/genética , Amiodarona/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Colesterol/metabolismo , Epitélio/microbiologia , Técnicas de Inativação de Genes , Genes Bacterianos , Glucosiltransferases/metabolismo , Humanos , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Microdomínios da Membrana/metabolismo
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