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1.
PLoS One ; 15(7): e0232307, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667911

RESUMO

In the mammalian gut CD103+ve myeloid DCs are known to suppress inflammation threatened by luminal bacteria, but stimuli driving DC precursor maturation towards this beneficial phenotype are incompletely understood. We isolated CD11+ve DCs from mesenteric lymph nodes (MLNs) of healthy mice; CD103+ve DCs were 8-24 fold more likely than CD103-ve DCs to exhibit extensive of prior phagocytosis of apoptotic intestinal epithelial cells. However, CD103+ve and CD103-ve MLN DCs exhibited similar ex vivo capacity to ingest apoptotic cells, indicating that apoptotic cells might drive immature DC maturation towards the CD103+ve phenotype. When cultured with apoptotic cells, myeloid DC precursors isolated from murine bone marrow and characterised as lineage-ve CD103-ve, displayed enhanced expression of CD103 and ß8 integrin and acquired increased capacity to induce T regulatory lymphocytes (Tregs) after 7d in vitro. However, DC precursors isolated from αv-tie2 mice lacking αv integrins in the myeloid line exhibited reduced binding of apoptotic cells and complete deficiency in the capacity of apoptotic cells and/or latent TGF-ß1 to enhance CD103 expression in culture, whereas active TGF-ß1 increased DC precursor CD103 expression irrespective of αv expression. Fluorescence microscopy revealed clustering of αv integrin chains and latent TGF-ß1 at points of contact between DC precursors and apoptotic cells. We conclude that myeloid DC precursors can deploy αv integrin to orchestrate binding of apoptotic cells, activation of latent TGF-ß1 and acquisition of the immunoregulatory CD103+ve ß8+ve DC phenotype. This implies that a hitherto unrecognised consequence of apoptotic cell interaction with myeloid phagocytes is programming that prevents inflammation.


Assuntos
Antígenos CD/metabolismo , Apoptose , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Imunomodulação , Cadeias alfa de Integrinas/metabolismo , Integrina alfaV/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Feminino , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Fagocitose , Linfócitos T Reguladores/imunologia
4.
Environ Sci Pollut Res Int ; 27(23): 29530-29538, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32440878

RESUMO

Chlorpyrifos (CPF), as a worldwide pesticide, can effect on the integrins αv and ß3 which play a main role in the implantation window. Therefore, the aim of this study was to consider CPF effects on integrin alpha v and beta 3 in implantation window phase. Thirty female NMRI mice were separated into groups of CPF, sham, and control. After 6 weeks, each group was mated, and on the 5th day of gestation, all mice were euthanized. Estradiol and progesterone levels were detected by the enzyme-linked immunosorbent assay (ELISA) test; two subunits of integrins (αv and ß3) genes and proteins of endometrium were analyzed by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry method, respectively. Fibrosis of the liver which evaluated by Masson's trichrome stain was increased in the CPF group compared with the others. But estradiol and progesterone levels were significantly decreased in CPF groups. Based on the findings, the proportion of genes' expressions of integrin subunits declined by the effect of CPF, while there was not any notable consequence on mice in the sham group. Alpha v and beta 3 integrin proteins expressed in all groups, but the concentration of these proteins in CPF groups was lower than in other groups. This study has shown that the decline of estradiol and progesterone downregulates the expression of αv and ß3 integrins which were influenced by CPF exposure. Changing these patterns of proteins could have numerous influences on unsuccessful implantation. Therefore, this experimental study recommends that inclusive consideration of the effects of insecticides may be crucial to women's unrecognized cause of infertility.


Assuntos
Clorpirifos , Inseticidas , Integrina alfaV , Integrina beta3 , Animais , Clorpirifos/toxicidade , Implantação do Embrião , Endométrio , Feminino , Humanos , Camundongos , Progesterona
5.
Am J Pathol ; 190(6): 1224-1235, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32201264

RESUMO

Intratracheal instillation of apoptotic cells enhances resolution of experimental lung inflammation by incompletely understood mechanisms. We report that this intervention induces functional regulatory T lymphocytes (Tregs) in mouse lung experimentally inflamed by intratracheal administration of lipopolysaccharide. Selective depletion demonstrated that Tregs were necessary for maximal apoptotic cell-directed enhancement of resolution, and adoptive transfer of additional Tregs was sufficient to promote resolution without administering apoptotic cells. After intratracheal instillation, labeled apoptotic cells were observed in most CD11c+CD103+ myeloid dendritic cells migrating to mediastinal draining lymph nodes and bearing migratory and immunoregulatory markers, including increased CCR7 and ß8 integrin (ITGB8) expression. In mice deleted for αv integrin in the myeloid line to reduce phagocytosis of dying cells by CD103+ dendritic cells, exogenous apoptotic cells failed to induce transforming growth factor-ß1 expression or Treg accumulation and failed to enhance resolution of lipopolysaccharide-induced lung inflammation. We conclude that in murine lung, myeloid phagocytes encountering apoptotic cells can deploy αv integrin-mediated mechanisms to induce Tregs and enhance resolution of acute inflammation.


Assuntos
Apoptose/fisiologia , Integrina alfaV/metabolismo , Pneumonia/metabolismo , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Fatores de Transcrição Forkhead/metabolismo , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Fagocitose/fisiologia , Pneumonia/patologia
6.
Biochem Biophys Res Commun ; 523(2): 398-404, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31870546

RESUMO

The role of protein kinase N1 (PKN1) in cell aggregation and spheroid formation was investigated using mouse embryonic fibroblasts (MEFs) deficient in kinase activity caused by a point mutation (T778A) in the activation loop. Wild type (WT) MEFs formed cell aggregates within a few hours in suspension cultures placed in poly-2-hydroxyethylmethacrylate (poly-HEMA) coated flat-bottom dishes. By contrast, PKN1[T778A] (PKN1 T778A/T778A homozygous knock-in) MEFs showed significantly delayed aggregate formation and higher susceptibility to cell death. Video analysis of suspension cultures revealed decreased cell motility and lesser frequency of cell-cell contact in PKN1[T778A] MEFs compared to that in WT MEFs. Aggregate formation of PKN1[T778A] MEFs was compensated by shaking the cell suspension. When cultured in U-shaped ultra-low attachment well plates, initially larger-sized and loosely packed aggregates of WT MEFs underwent compaction resulting in a single round spheroid. On the other hand, image-based quantitative analysis of PKN1[T778A] MEFs revealed irregular compaction with decreased roundness, solidity, and sphericity within 24 h. Flow cytometry of PKN1[T778A] MEFs revealed decreased surface-expression of N-cadherin and integrins α5 and αV. These results suggest that kinase activity of PKN1 controls cell aggregation and spheroid compaction in MEF suspension culture, possibly by regulating the cell migration and cell-surface expression of N-cadherin and integrins.


Assuntos
Proteína Quinase C/metabolismo , Animais , Caderinas/metabolismo , Agregação Celular/fisiologia , Membrana Celular/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/enzimologia , Técnicas de Introdução de Genes , Integrina alfa5/metabolismo , Integrina alfaV/metabolismo , Camundongos , Camundongos Mutantes , Mutação Puntual , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Esferoides Celulares/citologia , Esferoides Celulares/enzimologia
7.
Elife ; 82019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31793433

RESUMO

During human immunodeficiency virus-1 (HIV-1) infection lymphoid organ follicular dendritic cells (FDCs) serve as a reservoir for infectious virus and an obstacle to curative therapies. Here, we identify a subset of lymphoid organ sinus lining macrophage (SMs) that provide a cell-cell contact portal, which facilitates the uptake of HIV-1 viral-like particles (VLPs) by FDCs and B cells in mouse lymph node. Central for portal function is the bridging glycoprotein MFG-E8. Using a phosphatidylserine binding domain and an RGD motif, MFG-E8 helps target HIV-1 VLPs to αv integrin bearing SMs. Lack of MFG-E8 or integrin blockade severely limits HIV-1 VLP spread onto FDC networks. Direct SM-FDC virion transfer also depends upon short-lived FDC network abutment, likely triggered by SCSM antigen uptake. This provides a mechanism for rapid FDC loading broadening the opportunity for rare, antigen reactive follicular B cells to acquire antigen, and a means for HIV virions to accumulate on the FDC network.


Assuntos
Antígenos de Superfície/genética , Células Dendríticas Foliculares/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Proteínas do Leite/genética , Animais , Antígenos de Superfície/imunologia , Linfócitos B/imunologia , Linhagem Celular , Células Dendríticas Foliculares/metabolismo , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Integrina alfaV/genética , Linfonodos/imunologia , Linfonodos/virologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Proteínas do Leite/imunologia
8.
Molecules ; 24(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627430

RESUMO

Twenty new 12N-substituted matrinol derivatives were synthesized and evaluated for their inhibitory effects on collagen α1 (I) (COL1A1) promotor in human hepatic stellate LX-2 cells. The structure-activity relationship (SAR) revealed that introducing a 12N-benzeneaminoacylmethyl substitution might significantly enhance the activity. Compound 8a exhibited the highest inhibitory potency against COL1A1, and its inhibition activity against COL1A1 was further confirmed on both the mRNA and protein levels. It also effectively inhibited the expression of α smooth muscle actin (α-SMA), fibronectin and transforming growth factor ß1 (TGFß1), indicating an extensive inhibitory effect on the expression of fibrogenic genes. The primary mechanism study indicated that it might take action via the Integrin/FAK/PI3K/Akt signaling pathway. The results provided powerful information for further structure optimization, and compound 8a was selected as a novel anti-fibrogenic lead for further investigation.


Assuntos
Colágeno Tipo I/genética , Células Estreladas do Fígado/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Linhagem Celular , Colágeno Tipo I/antagonistas & inibidores , Colágeno Tipo I/metabolismo , Fibrose/prevenção & controle , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Humanos , Integrina alfaV/genética , Integrina alfaV/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Relação Estrutura-Atividade , Tiazóis/síntese química
9.
Science ; 366(6462)2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31601741

RESUMO

Epithelial resident memory T (eTRM) cells serve as sentinels in barrier tissues to guard against previously encountered pathogens. How eTRM cells are generated has important implications for efforts to elicit their formation through vaccination or prevent it in autoimmune disease. Here, we show that during immune homeostasis, the cytokine transforming growth factor ß (TGF-ß) epigenetically conditions resting naïve CD8+ T cells and prepares them for the formation of eTRM cells in a mouse model of skin vaccination. Naïve T cell conditioning occurs in lymph nodes (LNs), but not in the spleen, through major histocompatibility complex class I-dependent interactions with peripheral tissue-derived migratory dendritic cells (DCs) and depends on DC expression of TGF-ß-activating αV integrins. Thus, the preimmune T cell repertoire is actively conditioned for a specialized memory differentiation fate through signals restricted to LNs.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Memória Imunológica , Fator de Crescimento Transformador beta/metabolismo , Animais , Movimento Celular , Epiderme/imunologia , Integrina alfaV/genética , Integrina alfaV/metabolismo , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/imunologia
10.
Cells Tissues Organs ; 207(1): 46-57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31261153

RESUMO

In the field of wound healing, stem cell-based strategies are gaining importance for their regenerative potential. Adipose-derived stem cells (ADSCs) are a particular subset of mesenchymal stem cells present in the stromal-vascular fraction of the adipose tissue, today considered very attractive for their relative abundance and accessibility in the human body. However, ADSCs are still not routinely used in normal clinical practice. Several studies have also reported ADSC transplantation in association with biomaterials in an attempt to enhance the local retention and growth rate of the cells. The aim of our study was to evaluate the ability of ADSCs to build a dermal scaffold to be potentially used as a dermal substitute in the field of wound healing, with optimal biocompatibility and mechanical properties. ADSCs were defined as CD90-, CD73-, and CD105-positive cells. ADSCs turned out to be capable of secreting all the main components of the extracellular matrix (ECM) upon stimulation, thus efficiently producing a collagen and fibronectin-containing dermal matrix. We also checked whether the ADSC-produced dermal scaffold could be seeded with keratinocytes. The scaffolding material directly produced by ADSCs has several advantages when compared to the commercially available ones: it is easily obtained from the patients and it is 100% biocompatible and supports cell-ECM interaction. Moreover, it represents a possible powerful therapeutic tool for patients with chronic ulcers since it appears to be potentially grafted with keratinocytes layers, thus bypassing the classical two-step grafting procedure.


Assuntos
Tecido Adiposo/citologia , Pele Artificial , Células-Tronco/citologia , Engenharia Tecidual/métodos , Tecido Adiposo/metabolismo , Colágeno Tipo IV/metabolismo , Epiderme/metabolismo , Matriz Extracelular/metabolismo , Humanos , Integrina alfaV/metabolismo , Queratinócitos/citologia , Cicatrização
11.
Pathol Res Pract ; 215(9): 152531, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31320250

RESUMO

Integrin subunit alpha V (ITGAV), a member of integrin family of extracellular matrix receptors, is involved in many types of cancer. In this study, the expression levels, clinical features and prognosis of ITGAV in gastric cancer (GC) patients were investigated, and the functional roles of ITGAV were also investigated. Cell Counting Kit-8 (CCK-8) assay was performed to examine the proliferation of GC cells. Transwell assays and wound-healing assays were conducted to explore the effect of ITGAV expression on GC cell migration and invasion. We found that ITGAV was overexpressed in both GC tissues and GC cells. ITGAV expression was positively correlated with lymph node metastasis and TNM stage of GC. High expression of ITGAV was associated with shorter overall survival (OS) and disease-free survival (DFS). Interestingly, the downregulation of ITGAV resulted in suppression of proliferation, migration, and invasion in GC cells. In conclusion, ITGAV is overexpressed in gastric cancer and is associated with poorer prognostic outcomes. ITGAV may serve as an important prognostic marker for GC staging and progression.


Assuntos
Biomarcadores Tumorais/análise , Integrina alfaV/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Adulto , Idoso , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Prognóstico , Neoplasias Gástricas/mortalidade
12.
J Exp Clin Cancer Res ; 38(1): 317, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319863

RESUMO

BACKGROUND: Molecular markers for prostate cancer (PCa) are required to improve the early definition of patient outcomes. Atypically large extracellular vesicles (EVs), referred as "Large Oncosomes" (LO), have been identified in highly migratory and invasive PCa cells. We recently developed and characterized the DU145R80 subline, selected from parental DU145 cells as resistant to inhibitors of mevalonate pathway. DU145R80 showed different proteomic profile compared to parental DU145 cells, along with altered cytoskeleton dynamics and a more aggressive phenotype. METHODS: Immunofluorescence staining and western blotting were used to identify blebbing and EVs protein cargo. EVs, purified by gradient ultra-centrifugations, were analyzed by tunable resistive pulse sensing and multi-parametric flow cytometry approach coupled with high-resolution imaging technologies. LO functional effects were tested in vitro by adhesion and invasion assays and in vivo xenograft model in nude mice. Xenograft and patient tumor tissues were analyzed by immunohistochemistry. RESULTS: We found spontaneous blebbing and increased shedding of LO from DU145R80 compared to DU145 cells. LO from DU145R80, compared to those from DU145, carried increased amounts of key-molecules involved in PCa progression including integrin alpha V (αV-integrin). By incubating DU145 cells with DU145R80-derived LO we demonstrated that αV-integrin on LO surface was functionally involved in the increased adhesion and invasion of recipient cells, via AKT. Indeed either the pre-incubation of LO with an αV-integrin blocking antibody, or a specific AKT inhibition in recipient cells are able to revert the LO-induced functional effects. Moreover, DU145R80-derived LO also increased DU145 tumor engraftment in a mice model. Finally, we identified αV-integrin positive LO-like structures in tumor xenografts as well as in PCa patient tissues. Increased αV-integrin tumor expression correlated with high Gleason score and lymph node status. CONCLUSIONS: Overall, this study is the first to demonstrate the critical role of αV-integrin positive LO in PCa aggressive features, adding new insights in biological function of these large EVs and suggesting their potential use as PCa prognostic markers.


Assuntos
Vesículas Extracelulares/patologia , Integrina alfaV/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Vesículas Extracelulares/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Gradação de Tumores , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , Proteômica/métodos , Regulação para Cima
13.
N Engl J Med ; 380(15): 1480-1482, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30970196
14.
Brain Inj ; 33(7): 836-845, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31033358

RESUMO

Background: Α few genetic variants are associated with the outcome after traumatic brain injury (TBI). Integrins are glycoprotein receptors that play an important role in the integrity of microvasculature of the brain. Objective: To examine the role of integrin-AV (ITGAV) and integrin-B8 (ITGB8) tag single nucleotide polymorphisms (SNPs) on the outcome of patients with TBI. Methods: 363 participants were included and genotyped for 11 SNPs for ITGAV and 11 for ITGB8 gene. SNPs were tested for associations with the 6-month outcome after TBI, the presence of a hemorrhagic event after TBI, and the initial TBI severity after adjustment for TBI's main predictors. Results: The ITGAV rs3911239 CC and rs7596996 GG genotypes were associated with an unfavorable outcome after TBI, compared to the TT and AA genotypes, respectively. The ITGB8 rs10239099 CC and rs3757727 CC genotypes were associated with increased risk of any cerebral hemorrhagic event after TBI compared to GG and TT respectively. The ITGAV rs7589470 and rs7565633 were associated with the TBI's initial severity. Conclusions: ITGAV gene SNPs may be implicated in the outcome after TBI, as well as in the initial TBI severity, and also of ITGB8 gene SNPs in the risk of hemorrhagic event after a TBI.


Assuntos
Lesões Encefálicas Traumáticas/genética , Hemorragia Cerebral/genética , Genótipo , Integrina alfaV/genética , Cadeias beta de Integrinas/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Lesões Encefálicas Traumáticas/complicações , Hemorragia Cerebral/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
15.
Proc Natl Acad Sci U S A ; 116(10): 4462-4470, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30770452

RESUMO

CD4+ follicular helper T cells (Tfh) are essential for germinal center (GC) reactions in the lymph node that generate high-affinity, long-lived plasma cells (LLPCs). Temporal GC analysis suggests B memory cells (Bmem) are generated early, while LLPCs are generated late in the GC reaction. Distinct roles for Tfh at these temporally different stages are not yet clear. Tfh entry into the GC is highly dynamic and the signals that maintain Tfh within the GC for support of late LLPC production are poorly understood. The GC is marked by inflammation-induced presentation of specific ECM components. To determine if T cell recognition of these ECM components played a role in Tfh support of the GC, we immunized mice with a T cell-restricted deletion of the ECM-binding integrin αV (αV-CD4 cKO). T cell integrin αV deletion led to a striking defect in the number and size of the GCs following immunization with OVA protein in complete Freund's adjuvant. The GC defect was not due to integrin αV deficiency impeding Tfh generation or follicle entry or the ability of αV-CD4 cKO Tfh to contact and support B cell activation. Instead, integrin αV was essential for T cell-intrinsic accumulation within the GC. Altered Tfh positioning resulted in lower-affinity antibodies and a dramatic loss of LLPCs. Influenza A infection revealed that αV integrin was not required for Tfh support of Bmem but was essential for Tfh support of LLPCs. We highlight an αV integrin-ECM-guided mechanism of Tfh GC accumulation that selectively impacts GC output of LLPCs but not Bmem.


Assuntos
Centro Germinativo/imunologia , Integrina alfaV/fisiologia , Plasmócitos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Matriz Extracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
17.
Cell Death Dis ; 10(2): 68, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683841

RESUMO

KDM4/JMJD2 Jumonji C-containing histone lysine demethylases (KDM4A-D) constitute an important class of epigenetic modulators in the transcriptional activation of cellular processes and genome stability. Interleukin-8 (IL-8) is overexpressed in gastric cancer, but the mechanisms and particularly the role of the epigenetic regulation of IL-8, are unclear. Here, we report that KDM4B, but not KDM4A/4C, upregulated IL-8 production in the absence or presence of Helicobacter pylori. Moreover, KDM4B physically interacts with c-Jun on IL-8, MMP1, and ITGAV promoters via its demethylation activity. The depletion of KDM4B leads to the decreased expression of integrin αV, which is exploited by H. pylori carrying the type IV secretion system, reducing IL-8 production and cell migration. Elevated KDM4B expression is significantly associated with the abundance of p-c-Jun in gastric cancer and is linked to a poor clinical outcome. Together, our results suggest that KDM4B is a key regulator of JNK/c-Jun-induced processes and is a valuable therapeutic target.


Assuntos
Carcinogênese/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Helicobacter pylori/metabolismo , Humanos , Integrina alfaV/metabolismo , Interleucina-8/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Metaloproteinase 1 da Matriz/metabolismo , Prognóstico , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Ativação Transcricional , Transfecção
18.
Arthritis Rheumatol ; 71(2): 302-314, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30175895

RESUMO

OBJECTIVE: Several studies have demonstrated that the secreted glycoprotein and integrin ligand milk fat globule-associated protein with epidermal growth factor- and factor VIII-like domains (MFG-E8) negatively regulates fibrosis in the liver, lungs, and respiratory tract. However, the mechanisms and roles of MFG-E8 in skin fibrosis in systemic sclerosis (SSc) have not been characterized. We undertook this study to elucidate the role of MFG-E8 in skin fibrosis in SSc. METHODS: We assessed expression of MFG-E8 in the skin and serum in SSc patients. We examined the effect of recombinant MFG-E8 (rMFG-E8) on latent transforming growth factor ß (TGFß)-induced gene/protein expression in SSc fibroblasts. We examined the effects of deficiency or administration of MFG-E8 on fibrosis mouse models. RESULTS: We demonstrated that MFG-E8 expression around dermal blood vessels and the serum MFG-E8 level in SSc patients (n = 7 and n = 44, respectively) were lower than those in healthy individuals (n = 6 and n = 28, respectively). Treatment with rMFG-E8 significantly inhibited latent TGFß-induced expression of type I collagen, α-smooth muscle actin, and CCN2 in SSc fibroblasts (n = 3-8), which suggested that MFG-E8 inhibited activation of latent TGFß as well as TGFß signaling via binding to αv integrin. In a mouse model of bleomycin-induced fibrosis (n = 5-8) and in a TSK mouse model (a genetic model of SSc) (n = 5-10), deficient expression of MFG-E8 significantly enhanced both pulmonary and skin fibrosis, and administration of rMFG-E8 significantly inhibited bleomycin-induced dermal fibrosis. CONCLUSION: These results suggest that vasculopathy-induced dysfunction of pericytes and endothelial cells, the main cells secreting MFG-E8, may be associated with the decreased expression of MFG-E8 in SSc and that the deficient inhibitory regulation of latent TGFß-induced skin fibrosis by MFG-E8 may be involved in the pathogenesis of SSc and may be a therapeutic target for fibrosis in SSc patients.


Assuntos
Antígenos de Superfície/metabolismo , Fibroblastos/metabolismo , Integrina alfaV/metabolismo , Proteínas do Leite/metabolismo , Escleroderma Sistêmico/metabolismo , Pele/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Antígenos de Superfície/genética , Antígenos de Superfície/farmacologia , Bleomicina/toxicidade , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibrose , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Leite/genética , Proteínas do Leite/farmacologia , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/patologia , Pele/efeitos dos fármacos , Pele/patologia
19.
Exp Cell Res ; 374(1): 128-139, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30472097

RESUMO

Vang-like 2 (VANGL2) is a four-pass transmembrane protein required for a variety of polarized cell behaviors underlying embryonic development. Recent data show human VANGL2 interacts with integrin αv to control cell adhesion to extracellular matrix proteins. The goal of this study was to further define the functional relationship between integrin αv and VANGL2. We demonstrate integrin αv regulates VANGL2 protein levels both in vitro and in the zebrafish embryo. While integrin αv knockdown reduces VANGL2 expression at membrane compartments, it does not affect VANGL2 transcription. Knockdown of integrin ß5, but not ß1 or ß3, also decreases VANGL2 protein levels. Inhibition of protein translation using cycloheximide demonstrates that integrin αv knockdown cells have increased VANGL2 degradation while interference with either proteasome or lysosome function restores VANGL2. We further show integrin activation and stimulation of cell-matrix adhesion using MnCl2 fails to influence VANGL2. However, MnCl2 treatment stabilizes VANGL2 protein expression levels in the presence of cycloheximide. In the converse experiment, blockage of integrin-mediated cell-matrix adhesion using a cyclic RGD peptide causes a reduction in VANGL2 protein levels. Together, our findings support a model where integrin αv and cellular interactions with the extracellular matrix are required to maintain VANGL2 protein levels and thus function at the plasma membrane.


Assuntos
Matriz Extracelular/metabolismo , Integrina alfaV/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Junções Célula-Matriz , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Cadeias beta de Integrinas/metabolismo , Integrina beta3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Fenótipo , Estabilidade Proteica , Proteólise , Transcrição Genética , Peixe-Zebra
20.
J Cell Biochem ; 120(1): 283-289, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30145815

RESUMO

Human osteosarcoma 143B cells were previously stably transfected with an αv integrin green flourescent protein (GFP) vector. 143B cells expressing αv integrin-GFP were transplanted orthotopically in the tibia of transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in noncolored nude mice, which maintained the RFP stroma. The interaction of αv integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) which persisted even 3 weeks after passage to nontransgenic nude mice. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing αv integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing αv integrin-GFP could be identified. TAMs expressing RFP accompanied metastasized osteosarcoma cells expressing αv integrin-GFP in the lung. The current study demonstrates the importance of αv integrin interaction with stromal elements in osteosarcoma.


Assuntos
Neoplasias Ósseas/metabolismo , Colágeno/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Integrina alfaV/metabolismo , Proteínas Luminescentes/metabolismo , Neoplasias Pulmonares/metabolismo , Osteossarcoma/metabolismo , Células Estromais/metabolismo , Microambiente Tumoral , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Integrina alfaV/genética , Neoplasias Pulmonares/secundário , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Microscopia Confocal , Osteossarcoma/patologia , Transfecção , Transplante Heterólogo
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