RESUMO
PRELP is thought to be an inhibitor of the development and progression of a variety of malignancies. Metastasis is a major cause of death in patients with colorectal cancer, but the mechanism underlying the role of PRELP in colorectal cancer metastasis remains poorly understood. In this study, we found that PRELP was significantly higher in metastatic tissues than in non-metastatic tissues of colorectal cancer and was closely associated with poor prognosis of colorectal cancer patients. PRELP promotes growth and metastasis of colorectal cancer cells. PRELP reduces cell stiffness and adhesion. PRELP promoted EMT in colorectal cancer cells and that PRELP bind to integrin α5 to activate the integrin α5/FAK/AKT signaling pathway. In conclusion, we demonstrate that PRELP is upregulated in metastatic colorectal cancer, providing a potential prognostic marker and therapeutic target for the clinical management of metastatic colorectal cancer from a biomechanical perspective.
Assuntos
Adesão Celular , Proliferação de Células , Neoplasias Colorretais , Proteínas da Matriz Extracelular , Glicoproteínas , Integrina alfa5 , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Integrina alfa5/metabolismo , Integrina alfa5/genética , Integrinas , Camundongos Nus , Metástase Neoplásica , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismoRESUMO
Metastatic melanoma, a deadly form of skin cancer, often develops resistance to the BRAF inhibitor drug vemurafenib, highlighting the need for understanding the underlying mechanisms of resistance and exploring potential therapeutic strategies targeting integrins and TGF-ß signalling. In this study, the role of integrins and TGF-ß signalling in vemurafenib resistance in melanoma was investigated, and the potential of combining vemurafenib with cilengitide as a therapeutic strategy was investigated. In this study, it was found that the transcription of PAI1 and p21 was induced by acquired vemurafenib resistance, and ITGA5 levels were increased as a result of this resistance. The transcription of ITGA5 was mediated by the TGF-ß pathway in the development of vemurafenib resistance. A synergistic effect on the proliferation of vemurafenib-resistant melanoma cells was observed with the combination therapy of vemurafenib and cilengitide. Additionally, this combination therapy significantly decreased invasion and colony formation in these resistant cells. In conclusion, it is suggested that targeting integrins and TGF-ß signalling, specifically ITGA5, ITGB3, PAI1, and p21, may offer promising approaches to overcoming vemurafenib resistance, thereby improving outcomes for metastatic melanoma patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma , Venenos de Serpentes , Vemurafenib , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Melanoma/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Venenos de Serpentes/farmacologia , Integrina beta3/metabolismo , Integrina beta3/genética , Fator de Crescimento Transformador beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Integrinas/metabolismo , Integrinas/antagonistas & inibidores , Integrina alfa5/metabolismo , Integrina alfa5/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Indóis/farmacologia , Indóis/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Background: Parkinson's disease (PD) is marked by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to motor and cognitive dysfunctions. The molecular mechanisms underlying synaptic alterations in PD remain elusive, with a focus on the role of Itga5 in synaptic integrity and motor coordination and TAT-Itga5 was designed to suppress PTEN activity in this investigation. Methods: This study utilized MPTP-induced PD animal models to investigate the expression and role of Itga5 in the striatum. Techniques included quantitative PCR, Western blotting, immunostaining, CRISPR-CasRx-mediated knockdown, electrophysiological assays, behavioral tests, and mass spectrometry. Results: Itga5 expression was significantly reduced in MPTP-induced PD models. In these models, a marked decrease in dendritic spine density and a shift towards thinner spines in striatal GABA neurons were observed, suggesting impaired synaptic integration. Knockdown of Itga5 resulted in reduced dendritic branching, decreased mushroom spines, and increased thin spines, altering synaptic architecture. Electrophysiological analyses revealed changes in action potential and spontaneous excitatory postsynaptic currents, indicating altered synaptic transmission. Motor behavior assessments showed that Itga5 deficiency led to impairments in fine motor control and coordination. Furthermore, Itga5 was found to interact with PTEN, affecting AKT signaling crucial for synaptic development and motor coordination. Conclusion: The study demonstrates that Itga5 plays a critical role in maintaining synaptic integrity and motor coordination in PD. The Itga5-PTEN-AKT pathway represents a potential therapeutic target for addressing synaptic and motor dysfunctions in PD.
Assuntos
PTEN Fosfo-Hidrolase , Doença de Parkinson , Transdução de Sinais , Animais , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Masculino , Camundongos , Corpo Estriado/metabolismo , Camundongos Endogâmicos C57BL , Integrina alfa5/metabolismo , Integrina alfa5/genética , Sinapses/metabolismo , Modelos Animais de DoençasRESUMO
INTRODUCTION: Hypertension can accelerate and aggravate the process of arterial ageing and calcification. However, the mechanism behind has yet to be well elucidated. METHODS: Here, we monitored the dynamic changes of fibronectin (FN)/α5 integrin, bone morphogenetic protein 2/matrix Gla protein (BMP2/MGP), and Runx2 in the aorta of spontaneously hypertensive rats (SHRs) and thoracic aortic vascular smooth muscle cells (VSMCs), also the phenotypic transformation of VSMCs during the process of arterial ageing and calcification. Further, study on arterial ageing and calcification through antagonist experiments at the molecular level was explored. RESULTS: We found extracellular FN and its α5 integrin receptor expressions were positively associated with arterial ageing and calcification in SHR during ageing, as well in VSMCs from SHR in vitro. Integrin receptor inhibitor of GRGDSP would delay this arterial ageing and calcification process. Moreover, the elevated FN and α5 integrin receptor expression evoked the disequilibrium of BMP2/MGP, where the expression of BMP2, a potent osteogenic inducer, increased while MGP, a calcification inhibitor, decreased. Furthermore, it was followed by the upregulation of Runx2 and the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Notably, BMP2 antagonist of rmNoggin was sufficient to ameliorate the ageing and calcification process of VSMCs and exogenous BMP2-adding accelerate and aggregate the process. CONCLUSION: Our study revealed that hypertension-associated arterial ageing and calcification might be a consequence that hypertension up-regulated FN and its high binding affinity integrin α5 receptor in the aortic wall, which in turn aggravated the imbalance of BMP2/MGP, promoted the transcription of Runx2, and induced the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Our study would provide insights into hypertension-associated arterial ageing and calcification and shed new light on the control of arterial calcification, especially for those with hypertension.
Assuntos
Envelhecimento , Proteína Morfogenética Óssea 2 , Subunidade alfa 1 de Fator de Ligação ao Core , Fibronectinas , Hipertensão , Proteína de Matriz Gla , Músculo Liso Vascular , Fenótipo , Ratos Endogâmicos SHR , Calcificação Vascular , Proteína Morfogenética Óssea 2/metabolismo , Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Ratos , Fibronectinas/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Calcificação Vascular/etiologia , Masculino , Proteínas da Matriz Extracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Integrina alfa5/metabolismo , Integrina alfa5/genética , Células CultivadasRESUMO
In an attempt to repair injured central nervous system (CNS) nerves/tracts, immune cells are recruited into the injury site, but endogenous response in adult mammals is insufficient for promoting regeneration of severed axons. Here, we found that a portion of retinal ganglion cell (RGC) CNS projection neurons that survive after optic nerve crush (ONC) injury are enriched for and upregulate fibronectin (Fn)-interacting integrins Itga5 and ItgaV, and that Fn promotes long-term survival and long-distance axon regeneration of a portion of axotomized adult RGCs in culture. We then show that, Fn is developmentally downregulated in the axonal tracts of optic nerve and spinal cord, but injury-activated macrophages/microglia upregulate Fn while axon regeneration-promoting zymosan augments their recruitment (and thereby increases Fn levels) in the injured optic nerve. Finally, we found that Fn's RGD motif, established to interact with Itga5 and ItgaV, promotes long-term survival and long-distance axon regeneration of adult RGCs after ONC in vivo, with some axons reaching the optic chiasm when co-treated with Rpl7a gene therapy. Thus, experimentally augmenting Fn levels in the injured CNS is a promising approach for therapeutic neuroprotection and axon regeneration of at least a portion of neurons.
Assuntos
Axônios , Fibronectinas , Regeneração Nervosa , Traumatismos do Nervo Óptico , Células Ganglionares da Retina , Animais , Regeneração Nervosa/fisiologia , Fibronectinas/metabolismo , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Axônios/patologia , Axônios/fisiologia , Camundongos , Células Ganglionares da Retina/metabolismo , Camundongos Endogâmicos C57BL , Células Cultivadas , Integrina alfa5/metabolismo , Integrina alfa5/genética , Compressão Nervosa , FemininoRESUMO
Studies have shown that the abnormal expression of circular RNA (circRNA) is inextricably linked to hepatocellular carcinoma (HCC). Recently, hsa_circ_0000518 (circ_0000518) was discovered in many cancer progressions. However, its function in HCC is still unclear. Through GEO database analysis combined with gene expression detection of HCC related clinical samples and cell lines, we identified that circ_0000518 was abnormally overexpressed in HCC. Cell and animal model experiments jointly indicated that circ_0000518 can stimulate HCC cell proliferation, migration, invasion and suppress apoptosis. Furthermore, we also found that knocking down the circ_0000518 could inhibit the Warburg effect in HCC cells. Mechanistically, circ_0000518 was found to be primarily localized in the cytoplasm, and sponge hsa-miR-326 (miR-326) promoted integrin alpha 5 (ITGA5) expression. In addition, circ_0000518 could enhance the stability of HuR-mediated ITGA5 mRNA, thereby activating the Warburg effect. In conclusion, this study elucidated that circ_0000518 was a cancer-promoting circRNA, which could enhance ITGA5 expression through competing endogenous RNAs (ceRNA) and RNA Binding Protein (RBP) mechanisms, thus facilitating the development of HCC. It provides a meaningful diagnostic and therapeutic target for HCC.
Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Neoplasias Hepáticas , MicroRNAs , RNA Circular , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Efeito Warburg em Oncologia , Integrina alfa5/metabolismo , Integrina alfa5/genética , Movimento Celular , Camundongos Nus , Camundongos , Apoptose , Progressão da Doença , Camundongos Endogâmicos BALB C , Masculino , IntegrinasRESUMO
Podocyte apoptosis or loss is the pivotal pathological characteristic of diabetic kidney disease (DKD). Insulin-like growth factor-binding protein 2 (IGFBP2) have a proinflammatory and proapoptotic effect on diseases. Previous studies have shown that serum IGFBP2 level significantly increased in DKD patients, but the precise mechanisms remain unclear. Here, we found that IGFBP2 levels obviously increased under a diabetic state and high glucose stimuli. Deficiency of IGFBP2 attenuated the urine protein, renal pathological injury and glomeruli hypertrophy of DKD mice induced by STZ, and knockdown or deletion of IGFBP2 alleviated podocytes apoptosis induced by high concentration of glucose or in DKD mouse. Furthermore, IGFBP2 facilitated apoptosis, which was characterized by increase in inflammation and oxidative stress, by binding with integrin α5 (ITGA5) of podocytes, and then activating the phosphorylation of focal adhesion kinase (FAK)-mediated mitochondrial injury, including membrane potential decreasing, ROS production increasing. Moreover, ITGA5 knockdown or FAK inhibition attenuated the podocyte apoptosis caused by high glucose or IGFBP2 overexpression. Taken together, these findings unveiled the insight mechanism that IGFBP2 increased podocyte apoptosis by mitochondrial injury via ITGA5/FAK phosphorylation pathway in DKD progression, and provided the potential therapeutic strategies for diabetic kidney disease.
Assuntos
Apoptose , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina , Mitocôndrias , Podócitos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/genética , Podócitos/metabolismo , Podócitos/patologia , Animais , Camundongos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/genética , Masculino , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Estresse Oxidativo , Integrina alfa5/metabolismo , Integrina alfa5/genética , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fosforilação , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Camundongos Knockout , IntegrinasRESUMO
Integrin trafficking to and from membrane adhesions is a crucial mechanism that dictates many aspects of a cell's behaviour, including motility, polarisation, and invasion. In endothelial cells (ECs), the intracellular traffic of α5 integrin is regulated by both neuropilin 1 (NRP1) and neuropilin 2 (NRP2), yet the redundancies in function between these co-receptors remain unclear. Moreover, the endocytic complexes that participate in NRP-directed traffic remain poorly annotated. Here we identify an important role for the GTPase-activating protein p120RasGAP in ECs, promoting the recycling of α5 integrin from early endosomes. Mechanistically, p120RasGAP enables transit of endocytosed α5 integrin-NRP1-NRP2 complexes to Rab11+ recycling endosomes, promoting cell polarisation and fibronectin (FN) fibrillogenesis. Silencing of both NRP receptors, or p120RasGAP, resulted in the accumulation of α5 integrin in early endosomes, a loss of α5 integrin from surface adhesions, and attenuated EC polarisation. Endothelial-specific deletion of both NRP1 and NRP2 in the postnatal retina recapitulated our in vitro findings, severely impairing FN fibrillogenesis and polarised sprouting. Our data assign an essential role for p120RasGAP during integrin traffic in ECs and support a hypothesis that NRP receptors co-traffic internalised cargoes. Importantly, we utilise comparative proteomics analyses to isolate a comprehensive map of NRP1-dependent and NRP2-dependent α5 integrin interactions in ECs.
Assuntos
Endossomos , Células Endoteliais , Fibronectinas , Integrina alfa5 , Neuropilina-1 , Neuropilina-2 , Proteômica , Proteína p120 Ativadora de GTPase , Animais , Camundongos , Endossomos/metabolismo , Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Integrina alfa5/metabolismo , Integrina alfa5/genética , Integrinas , Neuropilina-1/metabolismo , Neuropilina-1/genética , Neuropilina-2/metabolismo , Neuropilina-2/genética , Proteína p120 Ativadora de GTPase/metabolismo , Proteína p120 Ativadora de GTPase/genética , Transporte Proteico , Proteômica/métodosRESUMO
BACKGROUND: Dental pulp regeneration therapy is a challenge to achieve early vascularization during treatment. Studying the regulatory mechanisms of vascular formation during human dental pulp development may provide insights for related therapies. In this study, we utilized single-cell sequencing analysis to compare the gene expression of dental pulp stem cells (DPSCs) and vascular endothelial cells (ECs) from developing and mature dental pulps. METHOD: Immunohistochemistry, Western blot, and real-time polymerase chain reaction (RT-PCR) were used to detect fibronectin 1 (FN1) expression and molecules, such as PI3K/AKT. Cell proliferation assay, scratch assay, tube formation assay and were used to investigate the effects of DPSCs on the vasculogenetic capability of ECs. Additionally, animal experiments involving mice were conducted. RESULT: The results revealed that DPSCs exist around dental pulp vasculature. FN1 expression was significantly higher in DPSCs from young permanent pulps than mature pulps, promoting HUVEC proliferation, migration, and tube formation via ITGA5 and the downstream PI3K/AKT signaling pathway. CONCLUSION: Our data indicate that intercellular communication between DPSCs and ECs mediated by FN1-ITGA5 signaling is crucial for vascularizationduring dental pulp development, laying an experimental foundation for future clinical studies.
Assuntos
Proliferação de Células , Polpa Dentária , Fibronectinas , Integrina alfa5 , Neovascularização Fisiológica , Transdução de Sinais , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Humanos , Fibronectinas/metabolismo , Fibronectinas/genética , Animais , Camundongos , Integrina alfa5/metabolismo , Integrina alfa5/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Comunicação Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Movimento Celular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Diferenciação Celular , Fosfatidilinositol 3-Quinases/metabolismo , IntegrinasRESUMO
BACKGROUND: The role of microRNA (miRNA) in modulating the function of cancer stem cells through diverse signaling pathway has been evidenced. We here identified a role of microRNA (miRNA) family, specifically miR-148/152, in gastric cancer and delineated its functional effects on gastric cancer stem cells. METHODS: Bioinformatics analysis was conducted to analyze expression of integrin α5 (ITGA5) which was verified through expression determination in clinical tissue samples. Next, the upstream regulatory factors of ITGA5 were determined. CD44+EpCAM (high) cells sorted from AGS cells subjected to gain-of-function experiments, followed by evaluation of their capacity of colony formation, generation of tumorosphere, cell migration and viability in vitro and xenograft tumor formation in vivo. RESULTS: ITGA5 was elevated in gastric cancer tissues and confirmed as a target gene of the miR-148/152 family members. The miR-148/152 family members were downregulated in gastric cancer tissues and cells. Decreased expression of miR-148/152 family members was also detected in gastric cancer stem cells. However, the raised expression led to reduced colony formation, tumorosphere, cell migration, cell viability, and drug resistance of CD44+EpCAM (high) AGS cells in vitro, and tumorigenesis in vitro. ITGA5 overexpression reversed the effect of the miR-148/152 family members. CONCLUSIONS: This study demonstrates that the miR-148/152 family members may prevent gastric cancer stem cell-like properties by targeting ITGA5, which can serve as an appealing target for gastric cancer treatment.
Assuntos
MicroRNAs , Neoplasias Gástricas , Humanos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Regulação Neoplásica da Expressão Gênica , Integrina alfa5/genética , Integrina alfa5/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
OBJECTIVE: Haematogenous dissemination is a prevalent route of colorectal cancer (CRC) metastasis. However, as the gatekeeper of vessels, the role of tumour pericytes (TPCs) in haematogenous metastasis remains largely unknown. Here, we aimed to investigate the heterogeneity of TPCs and their effects on CRC metastasis. DESIGN: TPCs were isolated from patients with CRC with or without liver metastases and analysed by single-cell RNA sequencing (scRNA-seq). Clinical CRC specimens were collected to analyse the association between the molecular profiling of TPCs and CRC metastasis. RNA-sequencing, chromatin immunoprecipitation-sequencing and bisulfite-sequencing were performed to investigate the TCF21-regulated genes and mechanisms underlying integrin α5 on TCF21 DNA hypermethylation. Pericyte-conditional Tcf21-knockout mice were constructed to investigate the effects of TCF21 in TPCs on CRC metastasis. Masson staining, atomic force microscopy, second-harmonic generation and two-photon fluorescence microscopy were employed to observe perivascular extracellular matrix (ECM) remodelling. RESULTS: Thirteen TPC subpopulations were identified by scRNA-seq. A novel subset of TCF21high TPCs, termed 'matrix-pericytes', was associated with liver metastasis in patients with CRC. TCF21 in TPCs increased perivascular ECM stiffness, collagen rearrangement and basement membrane degradation, establishing a perivascular metastatic microenvironment to instigate colorectal cancer liver metastasis (CRCLM). Tcf21 depletion in TPCs mitigated perivascular ECM remodelling and CRCLM, whereas the coinjection of TCF21high TPCs and CRC cells markedly promoted CRCLM. Mechanistically, loss of integrin α5 inhibited the FAK/PI3K/AKT/DNMT1 axis to impair TCF21 DNA hypermethylation in TCF21high TPCs. CONCLUSION: This study uncovers a previously unidentified role of TPCs in haematogenous metastasis and provides a potential diagnostic marker and therapeutic target for CRC metastasis.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Animais , Camundongos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , DNA , Regulação Neoplásica da Expressão Gênica , Integrina alfa5/genética , Integrina alfa5/metabolismo , Neoplasias Hepáticas/patologia , Metástase Neoplásica , Pericitos/metabolismo , Pericitos/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Microambiente TumoralRESUMO
Craniofacial skeletal elements are derived from cranial neural crest cells (CNCCs), which migrate along discrete paths and populate distinct pharyngeal arches, structures that are separated by the neighboring endodermal pouches (EPs). Interactions between the CNCCs and the endoderm are critical for proper craniofacial development. In zebrafish, integrin α5 (Itga5) functions in the endoderm to regulate formation of specifically the first EP (EP1) and the development of the hyoid cartilage. Here we show that fibronectin (Fn), a major component of the extracellular matrix (ECM), is also required for these developmental processes, and that the penetrance of defects in mutants is temperature-dependent. fn1a-/- embryos exhibited defects that are similar to, but much more severe than, those of itga5-/- embryos, and a loss of integrin av (itgav) function enhanced both endoderm and cartilage defects in itga5-/- embryos, suggesting that Itga5 and Itgav cooperate to transmit signals from Fn to regulate the development of endoderm and cartilage. Whereas the endodermal defects in itga5; itga5v-/- double mutant embryos were comparable to those of fn1a-/- mutants, the cartilage defects were much milder. Furthermore, Fn assembly was detected in migrating CNCCs, and the epithelial organization and differentiation of CNCC-derived arches were impaired in fn1a-/- embryos, indicating that Fn1 exerts functions in arch development that are independent of Itga5 and Itgav. Additionally, reduction of itga5 function in fn1a-/- embryos led to profound defects in body axis elongation, as well as in endoderm and cartilage formation, suggesting that other ECM proteins signal through Itga5 to regulate development of the endoderm and cartilage. Thus, our studies reveal that Fn1a and Itga5 have both overlapping and independent functions in regulating development of the pharyngeal endoderm and cartilage.
Assuntos
Endoderma , Integrina alfa5 , Animais , Região Branquial/metabolismo , Cartilagem/metabolismo , Endoderma/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Integrina alfa5/genética , Integrina alfa5/metabolismo , Crista Neural , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
This study aims to investigate the expression levels of fibrinogen α chain (FGA) in human gastric cancer (GC) tissues and cell lines, clarify its role in gastric cancer progression, and explore its underlying mechanism. Bioinformatics analysis, Immunoblot, Immunohistochemical (IHC), and quantitative PCR assays were performed to assess the expression of FGA in human gastric cancer tissues and cell lines. CCK-8 and colony formation assays were performed to detect its role in the proliferation of gastric cancer cells. Wound healing, transwell, and Immunofluorescence were performed to detect its effects on gastric cancer cell motility and epithelial-mesenchymal transition (EMT) processes. Luciferase and CHIP assays were performed to confirm the transcriptional regulation of FGA on ITGA5. Immunoblot assays and double-label RFP-GFP-LC3 immunofluorescence analysis were conducted to detect its effects on gastric cancer cell autophagy and FAK/ERK pathway, and in vivo tumor growth assays were further performed. We found the low expression of FGA in human gastric cancer tissues and cell lines. FGA suppressed gastric cancer cell proliferation, motility, and EMT process, and stimulated cell autophagy. We further found that FGA suppressed the expression of Integrin-α5 (ITGA5) and inhibited the FAK/ERK pathway, therefore suppressing the progression of gastric cancer. The in vivo assays further confirmed that FGA suppressed tumor growth of gastric cancer cells in the BALB/c nude mice (18-22 g, female, 8-week-old) through suppressing ITGA5-mediated FAK/ERK pathway in mice. We demonstrated the mechanism underlying FGA suppressing gastric cancer progression, and therefore we thought FGA could serve as a tumor suppressor protein in gastric cancer.
Assuntos
Morte Celular Autofágica , Fibrinogênio , Integrina alfa5 , Sistema de Sinalização das MAP Quinases , Neoplasias Gástricas , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrinogênio/genética , Fibrinogênio/metabolismo , Integrina alfa5/genética , Integrina alfa5/metabolismo , Camundongos , Camundongos Nus , Metástase NeoplásicaRESUMO
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/metabolismoRESUMO
MAP30 (Momordica antiviral protein 30kD) is a single-chain â -type ribosome inactivating protein with a variety of biological activities, including anti-tumor ability. It was reported that MAP30 would serve as a novel and relatively safe agent for prophylaxis and treatment of liver cancer. To determine whether adding two tumor targeting peptides could improve the antitumor activities of MAP30, we genetically modified MAP30 with an RGD motif and a EGFRi motif, which is a ligand with high affinity for αvß3 integrins and with high affinity for EGFR. The recombinant protein ELRL-MAP30 (rELRL-MAP30) containing a GST-tag was expressed in E. coli. The rELRL-MAP30 was highly expressed in the soluble fraction after induction with 0.15 mM IPTG for 20 h at 16 °C. The purified rELRL-MAP30 appeared as a band on SDS-PAGE. It was identified by western blotting. Cytotoxicity of recombinant protein to HepG2, MDA-MB-231, HUVEC and MCF-7 cells was detected by MTT analysis. Half maximal inhibitory concentration (IC50) values were 54.64 µg/mL, 70.13 µg/mL, 146 µg/mL, 466.4 µg/mL, respectively. Proliferation inhibition assays indicated that rELRL-MAP30 could inhibit the growth of Human liver cancer cell HepG2 effectively. We found that rELRL-MAP30 significantly induced apoptosis in liver cancer cells, as evidenced by nuclear staining of DAPI. In addition, rELRL-MAP30 induced apoptosis in human liver cancer HepG2 cells by up-regulation of Bax as well as down-regulation of Bcl-2. Migration of cell line were markedly inhibited by rELRL-MAP30 in a dose-dependent manner compared to the recombinant MAP30 (rMAP30). In summary, the fusion protein displaying extremely potent cytotoxicity might be highly effective for tumor therapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Momordica charantia/química , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Inativadoras de Ribossomos Tipo 2/genética , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Células MCF-7 , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 2/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2/farmacologia , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
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-TroncoRESUMO
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/metabolismoRESUMO
Disturbed flow leads to increased inflammatory responses of endothelial cells (ECs) prone to atherogenic state. Currently, little is known about the physiological mechanisms protecting vasculature against disturbed flow-activated ECs leading to atherosclerosis. Understanding the protective mediators involved in EC activation could provide novel therapeutic strategies for atherosclerosis. The extracellular matrix microenvironment profoundly regulates cellular homeostasis. A non-EC resident ECM protein, cartilage oligomeric matrix protein (COMP), has diverse protective roles in the cardiovascular system. To determine whether COMP could protect against disturbed flow-activated EC and atherosclerosis, we compared oscillatory shear stress (OSS) induced EC activation coated with various ECM proteins. Purified COMP inhibited EC activation caused by OSS. EC activation was upregulated in the aortic arch where the flow is disturbed in COMP-/- mice as compared with wild-type mice under physiological conditions or pathologically in partially ligated mouse carotid arteries. Mechanistically, co-immunoprecipitation, mammalian two-hybrid and FRET assay results suggest that COMP bound directly to integrin α5 via its C-terminus. We next synthesized a COMP-derived peptidomimetics (CCPep24) mimicking a specific COMP-integrin α5 interaction and found that CCPep24 protected against EC activation and atherogenesis in vivo. This study extends our current understanding of how ECM and flow coordinately fine-tune EC homeostasis and reveals the potential therapeutic effect of COMP or COMP-derived peptidomimetics on blocking aberrant integrin α5 activation, inflammatory EC activation and atherosclerosis pathogenesis.
Assuntos
Aterosclerose/genética , Proteína de Matriz Oligomérica de Cartilagem/genética , Integrina alfa5/genética , Animais , Aterosclerose/patologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Microambiente Celular/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Matriz Extracelular/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Peptidomiméticos/farmacologia , Mapas de Interação de Proteínas/genéticaRESUMO
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 XenoenxertoRESUMO
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.