RESUMO
In zebrafish, brain lymphatic endothelial cells (BLECs) are essential for meningeal angiogenesis and cerebrovascular regeneration. Although epidermal growth factor-like domain 7 (Egfl7) has been reported to act as a pro-angiogenic factor, its roles in lymphangiogenesis remain unclear. Here, we show that Egfl7 is expressed in both blood and lymphatic endothelial cells. We generate an egfl7 cq180 mutant with a 13-bp-deletion in exon 3 leading to reduced expression of Egfl7. The egfl7 cq180 mutant zebrafish exhibit defective formation of BLEC bilateral loop-like structures, although trunk and facial lymphatic development remains unaffected. Moreover, while the egfl7 cq180 mutant displays normal BLEC lineage specification, the migration and proliferation of these cells are impaired. Additionally, we identify integrin αvß3 as the receptor for Egfl7. αvß3 is expressed in the CVP and sprouting BLECs, and blocking this integrin inhibits the formation of BLEC bilateral loop-like structures. Thus, this study identifies a role for Egfl7 in BLEC development that is mediated through the integrin αvß3.
Assuntos
Encéfalo , Células Endoteliais , Integrina alfaVbeta3 , Linfangiogênese , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Humanos , Animais Geneticamente Modificados , Encéfalo/metabolismo , Movimento Celular/genética , Proliferação de Células , Família de Proteínas EGF/metabolismo , Família de Proteínas EGF/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/genética , Linfangiogênese/genética , Mutação , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Colorectal cancer (CRC) commonly exhibits tolerance to cisplatin treatment, but the underlying mechanisms remain unclear. Within the tumor microenvironment, macrophages play a role in resisting the cytotoxic effects of chemotherapy by engaging in efferocytosis to clear apoptotic cells induced by chemotherapeutic agents. The involvement of extracellular vesicles (EVs), an intercellular communicator within the tumor microenvironment, in regulating the efferocytosis for the promotion of drug resistance has not been thoroughly investigated. METHODS: We constructed GFP fluorescent-expressing CRC cell lines (including GFP-CT26 and GFP-MC38) to detect macrophage efferocytosis through flow cytometric analysis. We isolated and purified CRC-secreted EVs using a multi-step ultracentrifugation method and identified them through electron microscopy and nanoflow cytometry. Proteomic analysis was conducted to identify the protein molecules carried by CRC-EVs. MFGE8 knockout CRC cell lines were constructed using CRISPR-Cas9, and their effects were validated through in vitro and in vivo experiments using Western blotting, immunofluorescence, and flow cytometric analysis, confirming that these EVs activate the macrophage αvß3-Src-FAK-STAT3 signaling pathway, thereby promoting efferocytosis. RESULTS: In this study, we found that CRC-derived EVs (CRC-EVs) enhanced macrophage efferocytosis of cisplatin-induced apoptotic CRC cells. Analysis of The Cancer Genome Atlas (TCGA) database revealed a high expression of the efferocytosis-associated gene MFGE8 in CRC patients, suggesting a poorer prognosis. Additionally, mass spectrometry-based proteomic analysis identified a high abundance of MFGE8 protein in CRC-EVs. Utilizing CRISPR-Cas9 gene edition system, we generated MFGE8-knockout CRC cells, demonstrating that their EVs fail to upregulate macrophage efferocytosis in vitro and in vivo. Furthermore, we demonstrated that MFGE8 in CRC-EVs stimulated macrophage efferocytosis by increasing the expression of αvß3 on the cell surface, thereby activating the intracellular Src-FAK-STAT3 signaling pathway. CONCLUSIONS: Therefore, this study highlighted a mechanism in CRC-EVs carrying MFGE8 activated the macrophage efferocytosis. This activation promoted the clearance of cisplatin-induced apoptotic CRC cells, contributing to CRC resistance against cisplatin. These findings provide novel insights into the potential synergistic application of chemotherapy drugs, EVs inhibitors, and efferocytosis antagonists for CRC treatment.
Assuntos
Neoplasias Colorretais , Vesículas Extracelulares , Macrófagos , Fagocitose , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Macrófagos/metabolismo , Humanos , Animais , Linhagem Celular Tumoral , Camundongos , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Transdução de Sinais , Cisplatino/farmacologia , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/genética , EferocitoseRESUMO
Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVß3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVß3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVß3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVß3 ligands. Moreover, we describe for the first time co-fractionation of αVß3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Antagonistas de Androgênios , Transdução de Sinais , Anticorpos Monoclonais , Integrinas , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Linhagem Celular TumoralRESUMO
Medulloblastoma is one of the most prevalent solid tumors found in children, occurring in the brain's posterior fossa. The standard treatment protocol involves maximal resection surgery followed by craniospinal irradiation and chemotherapy. Despite a long-term survival rate of 70%, wide disparities among patients have been observed. The identification of pertinent targets for both initial and recurrent medulloblastoma cases is imperative. Both primary and recurrent medulloblastoma are marked by their aggressive infiltration into surrounding brain tissue, robust angiogenesis, and resistance to radiotherapy. While the significant role of integrin-αvß3 in driving these characteristics has been extensively documented in glioblastoma, its impact in the context of medulloblastoma remains largely unexplored. Integrin-αvß3 was found to be expressed in a subset of patients with medulloblastoma. We investigated the role of integrin-αvß3 using medulloblastoma-derived cell lines with ß3-subunit depletion or overexpression both in vitro and in vivo settings. By generating radioresistant medulloblastoma cell lines, we uncovered an increased integrin-αvß3 expression, which correlated with increased susceptibility to pharmacologic integrin-αvß3 inhibition with cilengitide, a competitive ligand mimetic. Finally, we conducted single-photon emission computed tomography (SPECT)/MRI studies on orthotopic models using a radiolabeled integrin-αvß3 ligand (99mTc-RAFT-RGD). This innovative approach presents the potential for a novel predictive imaging technique in the realm of medulloblastoma. Altogether, our findings lay the foundation for employing SPECT/MRI to identify a specific subset of patients with medulloblastoma eligible for integrin-αvß3-directed therapies. This breakthrough offers a pathway toward more targeted and effective interventions in the treatment of medulloblastoma. SIGNIFICANCE: This study demonstrates integrin-αvß3's fundamental role in medulloblastoma tumorigenicity and radioresistance and the effect of its expression on cilengitide functional activity.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Criança , Humanos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Cerebelares/tratamento farmacológico , Integrina alfaVbeta3/genética , Ligantes , Meduloblastoma/tratamento farmacológico , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
Interleukin-32 (IL-32), first reported in 2005, and its isoforms have been the subject of numerous studies investigating their functions in virus infection, cancer, and inflammation. IL-32θ, one of the IL-32 isoforms, has been shown to modulate cancer development and inflammatory responses. A recent study identified an IL-32θ mutant with a cytosine to thymine replacement at position 281 in breast cancer tissues. It means that alanine was also replaced to valine at position 94 in amino acid sequence (A94V). In this study, we investigated the cell surface receptors of IL-32θA94V and evaluated their effect on human umbilical vein endothelial cells (HUVECs). Recombinant human IL-32θA94V was expressed, isolated, and purified using Ni-NTA and IL-32 mAb (KU32-52)-coupled agarose columns. We observed that IL-32θA94V could bind to the integrins αVß3 and αVß6, suggesting that integrins act as cell surface receptors for IL-32θA94V. IL-32θA94V significantly attenuated monocyte-endothelial adhesion by inhibiting the expression of Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor (TNF)-α-stimulated HUVECs. IL-32θA94V also reduced the TNF-α-induced phosphorylation of protein kinase B (AKT) and c-jun N-terminal kinases (JNK) by inhibiting phosphorylation of focal adhesion kinase (FAK). Additionally, IL-32θA94V regulated the nuclear translocation of nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1), which are involved in ICAM-1 and VCAM-1 expression. Monocyte-endothelial adhesion mediated by ICAM-1 and VCAM-1 is an important early step in atherosclerosis, which is a major cause of cardiovascular disease. Our findings suggest that IL-32θA94V binds to the cell surface receptors, integrins αVß3 and αVß6, and attenuates monocyte-endothelial adhesion by suppressing the expression of ICAM-1 and VCAM-1 in TNF-α-stimulated HUVECs. These results demonstrate that IL-32θA94V can act as an anti-inflammatory cytokine in a chronic inflammatory disease such as atherosclerosis.
Assuntos
Aterosclerose , Molécula 1 de Adesão de Célula Vascular , Humanos , Aterosclerose/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
TRAIL (TNF-related apoptosis-inducing ligand) and its derivatives are potentials for anticancer therapy due to the selective induction of apoptosis in tumor cells upon binding to death receptors DR4 or DR5. Previously, we generated a DR5-selective TRAIL mutant variant DR5-B overcoming receptor-dependent resistance of tumor cells to TRAIL. In the current study, we improved the antitumor activity of DR5-B by fusion with a tumor-homing iRGD peptide, which is known to enhance the drug penetration into tumor tissues. The obtained bispecific fusion protein DR5-B-iRGD exhibited dual affinity for DR5 and integrin αvß3 receptors. DR5-B-iRGD penetrated into U-87 tumor spheroids faster than DR5-B and demonstrated an enhanced antitumor effect in human glioblastoma cell lines T98G and U-87, as well as in primary patient-derived glioblastoma neurospheres in vitro. Additionally, DR5-B-iRGD was highly effective in a xenograft mouse model of the U-87 human glioblastoma cell line in vivo. We suggest that DR5-B-iRGD may become a promising candidate for targeted therapy for glioblastoma.
Assuntos
Glioblastoma , Ligante Indutor de Apoptose Relacionado a TNF , Humanos , Camundongos , Animais , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Integrina alfaVbeta3/genética , Linhagem Celular Tumoral , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , ApoptoseRESUMO
Placentas from pregnancies complicated by severe early-onset fetal growth restriction (FGR) exhibit diminished vascular development mediated by impaired angiogenesis, but underlying mechanisms remain unknown. In this study, we show that FGR endothelial cells demonstrate inherently reduced migratory capacity despite the presence of fibronectin, a matrix protein abundant in placental stroma that displays abnormal organization in FGR placentas. Thus, we hypothesized that aberrant endothelial-fibronectin interactions in FGR are a key mechanism underlying impaired FGR endothelial migration. Using human fetoplacental endothelial cells isolated from uncomplicated term control and FGR pregnancies, we assessed integrin α5ß1 and αvß3 regulation during cell migration. We show that endothelial integrin α5ß1 and αvß3 interactions with fibronectin are required for migration and that FGR endothelial cells responded differentially to integrin inhibition, indicating integrin dysregulation in FGR. Whole-cell expression was not different between groups. However, there were significantly more integrins in focal adhesions and reduced intracellular trafficking in FGR. These newly identified changes in FGR endothelial cellular processes represent previously unidentified mechanisms contributing to persistent angiogenic deficiencies in FGR.
Assuntos
Retardo do Crescimento Fetal , Integrina alfaVbeta3 , Células Endoteliais/metabolismo , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Placenta/metabolismo , GravidezRESUMO
Integrins are α-ß heterodimeric cell receptors that can bind the protein components of pathogens, and play crucial roles in mammalian immune responses, but the immune functions mediated by integrins remains largely unknown in teleost fish. In this study, an integrin αvß3 (GCαvß3) originally assembled by αv (GCαv) and ß3 (GCß3) subunits, was identified from a teleost fish grass carp Ctenopharyngodon idella. The pairwise alignment analyses showed that the amino acid sequences of GCαv and GCß3 shared high similarity (75.2-95.1%) and identity (58.6-90.7%) with their homologs from other vertebrates. Both GCαv and GCß3 harbored the conserved protein domains and motifs, and were clustered in fish branch of the phylogenetic tree containing the counterparts from various vertebrates. Co-immunoprecipitation displayed that GCß3 could interact with the grass carp reovirus (GCRV) outer capsid protein VP5. Two incubation experiments revealed that the interaction of GCRV or VP5 proteins with GCß3 could induce the expressions of type I interferons (IFNs) including IFN2 and IFN3 in grass carp ovary cell line. The functional analysis demonstrated that GCαvß3 served as a receptor of viral protein components to be involved in antiviral immunity as human integrin αvß3 did. In addition, both GCαv and GCß3 were significantly upregulated in various tissues of grass carp after GCRV infection. This study might provide fundamental basis for understanding the molecular characteristics and immune functions of GCαvß3, and offer a new insight into the antiviral immune mechanism specific to the integrins in grass carp.
Assuntos
Carpas , Doenças dos Peixes , Interferon Tipo I , Infecções por Reoviridae , Reoviridae , Animais , Antivirais , Proteínas do Capsídeo , Carpas/genética , Carpas/metabolismo , Proteínas de Peixes/química , Humanos , Integrina alfaVbeta3/genética , Mamíferos/metabolismo , Filogenia , Reoviridae/fisiologiaRESUMO
OBJECTIVE: The present study investigates the effects and mechanisms of Yikang decoction on embryo implantation in mice. METHODS: Totally, mature female mice were randomly divided into four groups: normal, implantation failure (mifepristone treatment), Yikang decoction treatment (mifepristone and Yikang decoction treatment), and control (mifepristone and physiological saline treatment) groups. The efficacy of Yikang decoction was evaluated by the adhesion of uterine endometrial cells. The expression of leukemia inhibitory factor (LIF) and integrin αvß3 in the endometrium were detected by real-time quantitative PCR and western blot. In addition, mouse endometrial cells were transfected with LIF specific siRNA-1, and the expression of LIF and αvß3 were detected. RESULTS: The number of embryos markedly decreased after mifepristone treatment. The adhesion of endometrial cells in the Yikang decoction treatment group significantly increased, when compared to the control group. The expression of LIF and integrin αvß3 was significantly reduced by mifepristone, but the attenuated expression of LIF and αvß3 was markedly reversed by treatment with Yikang decoction. In addition, after LIF siRNA-1 transfection, the expression of integrin αvß3 significantly decreased (P < 0.01). The correlation analysis revealed a significant positive correlation between LIF and αvß3 protein expression. CONCLUSION: Yikang decoction can regulate the expression of αvß3 and increase cell adhesion by upregulating the expression of LIF, thereby improving embryo implantation in mice. These data suggest that Yikang decoction may have therapeutic effect in treating infertility.
Assuntos
Integrina alfaVbeta3 , Mifepristona , Feminino , Camundongos , Animais , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Regulação para Cima , Mifepristona/farmacologia , RNA Interferente Pequeno/genética , Implantação do Embrião/fisiologiaRESUMO
Tumor metastasis is the leading cause of cancer-associated mortality. Unfortunately, the underlying mechanism of metastasis is poorly understood. Expression of legumain (LGMN), an endo-lysosomal cysteine protease, positively correlates with breast cancer metastatic progression and poor prognosis. Here, we report that LGMN is secreted in the zymogen form by motile breast cancer cells. Through binding to cell surface integrin αvß3 via an RGD motif, the autocrine pro-LGMN activates FAK-Src-RhoA signaling in cancer cells and promotes cancer cell migration and invasion independent of LGMN protease activity. Either silencing LGMN expression or mutationally abolishing pro-LGMNâαvß3 interaction significantly inhibits cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Finally, we developed a monoclonal antibody against LGMN RGD motif, which blocks pro-LGMNâαvß3 binding, and effectively suppresses cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Thus, disruption of pro-LGMNâintegrin αvß3 interaction may be a potentially promising strategy for treating breast cancer metastasis.
Assuntos
Neoplasias da Mama , Integrina alfaVbeta3 , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Cisteína Endopeptidases , Feminino , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Metástase Neoplásica , OligopeptídeosRESUMO
The arginine-glycine-aspartic acid (RGD) motif is a cell adhesion sequence that binds to integrins. Some RGD-containing peptides promote adhesion of both embryonic stem cells and induced pluripotent stem cells (iPSCs); however, not all such RGD-containing peptides are active. In this study, we elucidated the role of RGD-neighboring sequences on iPSC adhesion using diverse synthetic peptides and recombinant proteins. Our results indicate that iPSC adhesion requires RGDX1 X2 sequences, such as RGDVF and RGDNY, and that the X1 X2 residues are essential for the adhesion via integrin αvß5 but not αvß3. iPSCs express integrin αvß5 but not αvß3; therefore, iPSC adhesion requires the RGDX1 X2 -containing sequences. The importance of the X1 X2 residues was confirmed with both HeLa and A549 cells, which express integrin αvß5 but not αvß3. Analysis of RGD-neighboring sequences provides important insights into ligand-binding specificity of integrins. Identification of integrin αvß5-binding motifs is potentially useful in drug development, drug delivery, cell culture, and tissue engineering.
Assuntos
Células-Tronco Pluripotentes , Receptores de Vitronectina , Adesão Celular/fisiologia , Integrina alfaVbeta3/genética , Oligopeptídeos/química , Peptídeos/química , Células-Tronco Pluripotentes/metabolismo , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismoRESUMO
In the context of breast cancer metastasis study, we have shown in an in vitro model of cell migration that IGDQ-exposing (IsoLeu-Gly-Asp-Glutamine type I Fibronectin motif) monolayers (SAMs) on gold sustain the adhesion of breast cancer MDA-MB-231 cells by triggering Focal Adhesion Kinase and integrin activation. Such tunable scaffolds are used to mimic the tumor extracellular environment, inducing and controlling cell migration. The observed migratory behavior induced by the IGDQ-bearing peptide gradient along the surface allows to separate cell subpopulations with a "stationary" or "migratory" phenotype. In this work, we knocked down the integrins α5(ß1) and (αv)ß since they are already known to be implicated in cell migration. To this aim, a whole proteomic analysis was performed in beta 3 integrin (ITGB3) or alpha 5 integrin (ITGA5) knock-down MDA-MB-231 cells, in order to highlight the pathways implied in the integrin-dependent cell migration. Our results showed that i) ITGB3 depletion influenced ITGA5 mRNA expression, ii) ITGB3 and ITGA5 were both necessary for IGDQ-mediated directional single cell migration and iii) integrin (αv)ß3 was activated by IGDQ fibronectin type I motif. Finally, the proteomic analysis suggested that co-regulation of recycling transport of ITGB3 by ITGA5 is potentially necessary for directional IGDQ-mediated cell migration.
Assuntos
Integrina alfaVbeta3 , Neoplasias , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Fibronectinas/genética , Humanos , Integrina alfaVbeta3/genética , Peptídeos , ProteômicaRESUMO
Based on in vitro and in silico assays as well as proteome analysis, this study explored the nongenomic mechanism for butyl benzyl phthalate (BBP)-induced thyroid disruption. Molecular docking simulations showed that BBP could dock into the Arg-Gly-Asp (RGD) domain of integrin αvß3 and form hydrogen bonds with a docking energy of -35.80 kcal/mol. This chemical enhanced rat pituitary tumor cell (GH3) proliferation and exhibited thyroid hormone-disrupting effects at 5-10 µmol/L. Meanwhile, BBP upregulated ß3 gene expression and activated the downstream mitogen-activated protein kinase (MAPK) pathway in GH3 cells. Interestingly, GH3 cell proliferation was attenuated by integrin αvß3 inhibitor (RGD peptide) or ERK1/2 inhibitor (PD98059), suggesting that the disruptions might be partly attributed to its interaction with integrin αvß3 and activation of MAPK. Furthermore, quantitative proteomic analysis of zebrafish embryos exposed to BBP at an environmentally relevant concentration of 0.3 µmol/L revealed that BBP perturbed proteins and pathways related to cell communication (e.g., integrin binding) and signal transduction (e.g., MAPK signaling pathway). Taken together, our results supported that the biological effects of BBP-activated integrin αvß3 mediated by the nongenomic pathway play an important role in its thyroid disruption. CAPSULE: The nongenomic pathway plays a vital role in the thyroid disruption-inducing actions of BBP.
Assuntos
Ácidos Ftálicos , Glândula Tireoide , Animais , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/toxicidade , Proteoma/metabolismo , Proteômica , Ratos , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Integrin αvß3 has been proposed as crucial determinant for tumor sustained progression and a molecular marker for the estimation of tumor angiogenesis. Our study suggested that integrin αvß3 could efficiently promote lung cancer cell proliferation and stem-like phenotypes in a tribbles homolog 3 (TRIB3) dependent manner. RESULT: Integrin αvß3 could mediate the activation of FAK/AKT pro-survival signaling pathway. Meanwhile, activated TRIB3 interacted with AKT to upregulated FOXO1 and SOX2 expression, resulting in sustained tumor progression in lung cancer. Our further analysis revealed that TRIB3 was significantly upregulated in lung tumor tissues and correlated with the poor outcome in clinical patients, indicating the potential role of TRIB3 in diagnostic and prognostic estimation for patients with lung cancer. CONCLUSION: Our study showed here for the first time that integrin αvß3 promote lung cancer development by activating the FAK/AKT/SOX2 axis in a TRIB3 dependent signaling pathway, and interrupting TRIB3/AKT interaction significantly improved the outcome of chemotherapy in tumor-bearing mice, representing a promising therapeutic strategy in lung cancer.
Assuntos
Proteínas de Ciclo Celular , Integrina alfaVbeta3 , Neoplasias Pulmonares , Proteínas Serina-Treonina Quinases , Proteínas Repressoras , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/genética , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: The development of thoracic aortic dissection (TAD) is closely related to extracellular matrix degradation and vascular smooth muscle cell (VSMC) transformation from contractile to synthetic type. LGMN (legumain) degrades extracellular matrix components directly or by activating downstream signals. The role of LGMN in VSMC differentiation and the occurrence of TAD remains elusive. METHODS: Microarray datasets concerning vascular dissection or aneurysm were downloaded from the Gene Expression Omnibus database to screen differentially expressed genes. Four-week-old male Lgmn knockout mice (Lgmn-/-), macrophage-specific Lgmn knockout mice (LgmnF/F;LysMCre), and RR-11a-treated C57BL/6 mice were given BAPN (ß-aminopropionitrile monofumarate; 1 g/kg/d) in drinking water for 4 weeks for TAD modeling. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Cell interaction was examined in macrophage and VSMC coculture system. The reciprocity of macrophage-derived LGMN with integrin αvß3 in VSMCs was tested by coimmunoprecipitation assay and colocalization analyses. RESULTS: Microarray datasets from the Gene Expression Omnibus database indicated upregulated LGMN in aorta from patients with TAD and mice with angiotensin II-induced AAA. Elevated LGMN was evidenced in aorta and sera from patients with TAD and mice with BAPN-induced TAD. BAPN-induced TAD progression was significantly ameliorated in Lgmn-deficient or inhibited mice. Macrophage-specific deletion of Lgmn alleviated BAPN-induced extracellular matrix degradation. Unbiased profiler polymerase chain reaction array and Gene Ontology analysis displayed that LGMN regulated VSMC phenotype transformation. Macrophage-specific deletion of Lgmn ameliorated VSMC phenotypic switch in BAPN-treated mice. Macrophage-derived LGMN inhibited VSMC differentiation in vitro as assessed by macrophages and the VSMC coculture system. Macrophage-derived LGMN bound to integrin αvß3 in VSMCs and blocked integrin αvß3, thereby attenuating Rho GTPase activation, downregulating VSMC differentiation markers and eventually exacerbating TAD development. ROCK (Rho kinase) inhibitor Y-27632 reversed the protective role of LGMN depletion in vascular dissection. CONCLUSIONS: LGMN signaling may be a novel target for the prevention and treatment of TAD.
Assuntos
Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/metabolismo , Dissecção Aórtica/metabolismo , Cisteína Endopeptidases/metabolismo , Integrina alfaVbeta3/metabolismo , Amidas/farmacologia , Dissecção Aórtica/tratamento farmacológico , Dissecção Aórtica/genética , Animais , Aneurisma da Aorta Torácica/tratamento farmacológico , Aneurisma da Aorta Torácica/genética , Cisteína Endopeptidases/genética , Feminino , Humanos , Integrina alfaVbeta3/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Actin rings are unique structures that facilitate the attachment of osteoclasts to the bone matrix during bone resorption. Previous studies have shown that tetraspanin7 (TSPAN7) plays an important role in the reorganization of the cytoskeleton necessary for the bone-resorbing activity of osteoclasts. However, questions remain as to the mechanisms by which TSPAN7 regulates this cytoskeletal rearrangement. In this study, we investigated the roles of TSPAN7 in osteoclasts by deleting the Tm4sf2 gene in mice, which encodes TSPAN7. The Tm4sf2 global knockout model showed protective effects on pathological bone loss, but no discernible changes in bone phenotypes under physiological conditions. In vitro study revealed that ablation of Tm4sf2 caused significant defects in integrin-mediated actin ring formation, thereby leading to significantly decreased bone resorption. Additionally, we demonstrated an association between TSPAN7 and the receptor activator of nuclear factor-кB/αvß3 integrin. Overall, our findings suggest that TSPAN7 acts as a novel modulator regulating the bone-resorbing function of osteoclasts.
Assuntos
Reabsorção Óssea , Osteoclastos , Actinas , Animais , Reabsorção Óssea/patologia , Diferenciação Celular , Integrina alfaVbeta3/genética , Integrinas/genética , Proteínas de Membrana , Camundongos , Proteínas do Tecido Nervoso , Osteoclastos/patologia , Ligante RANK/genética , Tetraspaninas/genéticaRESUMO
AIMS: Von Willebrand factor (VWF) is a plasma glycoprotein involved in primary haemostasis, while also having additional roles beyond haemostasis namely in cancer, inflammation, angiogenesis, and potentially in vascular smooth muscle cell (VSMC) proliferation. Here, we addressed how VWF modulates VSMC proliferation and investigated the underlying molecular pathways and the in vivo pathophysiological relevance. METHODS AND RESULTS: VWF induced proliferation of human aortic VSMCs and also promoted VSMC migration. Treatment of cells with a siRNA against αv integrin or the RGT-peptide blocking αvß3 signalling abolished proliferation. However, VWF did not bind to αvß3 on VSMCs through its RGD-motif. Rather, we identified the VWF A2 domain as the region mediating binding to the cells. We hypothesized the involvement of a member of the LDL-related receptor protein (LRP) family due to their known ability to act as co-receptors. Using the universal LRP-inhibitor receptor-associated protein, we confirmed LRP-mediated VSMC proliferation. siRNA experiments and confocal fluorescence microscopy identified LRP4 as the VWF-counterreceptor on VSMCs. Also co-localization between αvß3 and LRP4 was observed via proximity ligation analysis and immuno-precipitation experiments. The pathophysiological relevance of our data was supported by VWF-deficient mice having significantly reduced hyperplasia in carotid artery ligation and artery femoral denudation models. In wild-type mice, infiltration of VWF in intimal regions enriched in proliferating VSMCs was found. Interestingly, also analysis of human atherosclerotic lesions showed abundant VWF accumulation in VSMC-proliferating rich intimal areas. CONCLUSION: VWF mediates VSMC proliferation through a mechanism involving A2 domain binding to the LRP4 receptor and integrin αvß3 signalling. Our findings provide new insights into the mechanisms that drive physiological repair and pathological hyperplasia of the arterial vessel wall. In addition, the VWF/LRP4-axis may represent a novel therapeutic target to modulate VSMC proliferation.
Assuntos
Aterosclerose/metabolismo , Proliferação de Células , Integrina alfaVbeta3/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de von Willebrand/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Movimento Celular , Células Cultivadas , Hiperplasia , Integrina alfaVbeta3/genética , Proteínas Relacionadas a Receptor de LDL/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima , Placa Aterosclerótica , Transdução de Sinais , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologia , Fator de von Willebrand/genéticaRESUMO
Osteosarcoma is the most common type of primary malignant bone cancer, and it is associated with high rates of pulmonary metastasis. Integrin αvß3 is critical for osteosarcoma cell migratory and invasive abilities. Chemokine (C-C motif) ligand 4 (CCL4) has diverse effects on different cancer cells through its interaction with its specific receptor, C-C chemokine receptor type 5 (CCR5). Analysis of mRNA expression in human osteosarcoma tissue identified upregulated levels of CCL4, integrin αv and ß3 expression. Similarly, an analysis of records from the Gene Expression Omnibus (GEO) dataset showed that CCL4 was upregulated in human osteosarcoma tissue. Importantly, the expression of both CCL4 and integrin αvß3 correlated positively with osteosarcoma clinical stages and lung metastasis. Analysis of osteosarcoma cell lines identified that CCL4 promotes integrin αvß3 expression and cell migration by activating the focal adhesion kinase (FAK), protein kinase B (AKT), and hypoxia inducible factor 1 subunit alpha (HIF-1α) signaling pathways, which can downregulate microRNA-3927-3p expression. Pharmacological inhibition of CCR5 by maraviroc (MVC) prevented increases in integrin αvß3 expression and cell migration. This study is the first to implicate CCL4 as a potential target in the treatment of metastatic osteosarcoma.
Assuntos
Biomarcadores Tumorais/metabolismo , Movimento Celular , Quimiocina CCL4/metabolismo , Regulação Neoplásica da Expressão Gênica , Integrina alfaVbeta3/metabolismo , MicroRNAs/genética , Osteossarcoma/patologia , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Quimiocina CCL4/genética , Humanos , Integrina alfaVbeta3/genética , Osteossarcoma/genética , Osteossarcoma/metabolismo , Células Tumorais CultivadasRESUMO
Blood-brain barrier (BBB) disruption is a common and critical pathology following subarachnoid hemorrhage (SAH). We investigated the BBB disruption property of secreted protein acidic and rich in cysteine (SPARC) after SAH. A total of 197 rats underwent endovascular perforation to induce SAH or sham operation. Small interfering ribonucleic acid (siRNA) for SPARC or scrambled siRNA was administered intracerebroventricularly to rats 48 h before SAH. Anti-SPARC monoclonal antibody (mAb) 236 for functional blocking or normal mouse immunoglobulin G (IgG) was administered intracerebroventricularly 1 h after SAH. Selective integrin αVß3 inhibitor cyclo(-RGDfK) or phosphate-buffered saline was administered intranasally 1 h before SAH, along with recombinant SPARC treatment. Neurobehavior, SAH severity, brain edema, immunohistochemical staining, and Western blot were evaluated. The expression of SPARC and integrin αVß3 was upregulated after SAH in the endothelial cells. SPARC siRNA and anti-SPARC mAb 236 prevented neuroimpairments and brain edema through protection of BBB as measured by IgG extravasation 24 and 72 h after SAH. Recombinant SPARC aggravated neuroimpairments and cyclo(-RGDfK) suppressed the harmful neurological effects via inhibition of activated c-Jun N-terminal kinase, p38, and matrix metalloproteinase-9 followed by retention of endothelial junction proteins. SPARC may induce post-SAH BBB disruption via integrin αVß3 signaling pathway.
Assuntos
Barreira Hematoencefálica/patologia , Regulação da Expressão Gênica , Integrina alfaVbeta3/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/metabolismo , Osteonectina/metabolismo , Hemorragia Subaracnóidea/fisiopatologia , Animais , Barreira Hematoencefálica/metabolismo , Integrina alfaVbeta3/genética , Masculino , Metaloproteinase 9 da Matriz/genética , Osteonectina/genética , Ratos , Ratos Sprague-DawleyRESUMO
Integrins are a family of heterodimeric plasma membrane glycoproteins, which regulate tumor growth, angiogenesis, migration, and metastasis. Integrin αvß3 has been recognized as a putative target for the treatment of several cancers. Thus, the characterization of αvß3 distribution in different human tumors is of substantial interest in tumor targeting and its suppression. In this study we evaluated the expression of integrin αvß3 in different cancer types to define the expression pattern in cancer model. Furthermore, we investigated the effect of novel αvß3 antagonist Diaminopropane Tetraiodothyroacetic acid conjugated to poly (lactic-co-glycolic acid) polymer and its nanoformulated form (NDAT), on different cancer cell lines both in vitro and in xenografts. In vitro, NDAT downregulated αv and ß3 monomer expression. In vivo in tumor xenografts, similarly, NDAT downregulated αv and ß3. Distinct reduction in tumor weight and viability was observed in glioblastoma xenografts treated with NDAT. Furthermore, NDAT was safe and tolerable in mice treated with high doses. In conclusion, NDAT is an effective and safe inhibitor of integrin αvß3 expression in various cancer types, which indicates its impact on the targetability and suppression of αvß3-associated tumor functions.