RESUMEN
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.
Asunto(s)
Resistencia a Antineoplásicos , Melanoma , Venenos de Serpiente , Vemurafenib , Vemurafenib/farmacología , Vemurafenib/uso terapéutico , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Melanoma/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Venenos de Serpiente/farmacología , Integrina beta3/metabolismo , Integrina beta3/genética , Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Integrinas/metabolismo , Integrinas/antagonistas & inhibidores , Integrina alfa5/metabolismo , Integrina alfa5/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Indoles/farmacología , Indoles/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
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.
Asunto(s)
Fosfohidrolasa PTEN , Enfermedad de Parkinson , Transducción de Señal , Animales , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Masculino , Ratones , Cuerpo Estriado/metabolismo , Ratones Endogámicos C57BL , Integrina alfa5/metabolismo , Integrina alfa5/genética , Sinapsis/metabolismo , Modelos Animales de EnfermedadRESUMEN
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.
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Adhesión Celular , Proliferación Celular , Neoplasias Colorrectales , Proteínas de la Matriz Extracelular , Glicoproteínas , Integrina alfa5 , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Movimiento Celular , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Integrina alfa5/metabolismo , Integrina alfa5/genética , Integrinas , Ratones Desnudos , Metástasis de la Neoplasia , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismoRESUMEN
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.
Asunto(s)
Carcinoma Hepatocelular , Proliferación Celular , Neoplasias Hepáticas , MicroARNs , ARN Circular , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Efecto Warburg en Oncología , Integrina alfa5/metabolismo , Integrina alfa5/genética , Movimiento Celular , Ratones Desnudos , Ratones , Apoptosis , Progresión de la Enfermedad , Ratones Endogámicos BALB C , Masculino , IntegrinasRESUMEN
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.
Asunto(s)
Envejecimiento , Proteína Morfogenética Ósea 2 , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Fibronectinas , Hipertensión , Proteína Gla de la Matriz , Músculo Liso Vascular , Fenotipo , Ratas Endogámicas SHR , Calcificación Vascular , Proteína Morfogenética Ósea 2/metabolismo , Animales , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Hipertensión/metabolismo , Hipertensión/fisiopatología , Ratas , Fibronectinas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Envejecimiento/metabolismo , Envejecimiento/fisiología , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/etiología , Masculino , Proteínas de la Matriz Extracelular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas de Unión al Calcio/metabolismo , Integrina alfa5/metabolismo , Integrina alfa5/genética , Células CultivadasRESUMEN
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.
Asunto(s)
Axones , Fibronectinas , Regeneración Nerviosa , Traumatismos del Nervio Óptico , Células Ganglionares de la Retina , Animales , Regeneración Nerviosa/fisiología , Fibronectinas/metabolismo , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Axones/patología , Axones/fisiología , Ratones , Células Ganglionares de la Retina/metabolismo , Ratones Endogámicos C57BL , Células Cultivadas , Integrina alfa5/metabolismo , Integrina alfa5/genética , Compresión Nerviosa , FemeninoRESUMEN
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.
Asunto(s)
Apoptosis , Diabetes Mellitus Experimental , Nefropatías Diabéticas , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Mitocondrias , Podocitos , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/genética , Podocitos/metabolismo , Podocitos/patología , Animales , Ratones , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/genética , Masculino , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Estrés Oxidativo , Integrina alfa5/metabolismo , Integrina alfa5/genética , Ratones Endogámicos C57BL , Transducción de Señal , Fosforilación , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Ratones Noqueados , IntegrinasRESUMEN
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.
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Endosomas , Células Endoteliales , Fibronectinas , Integrina alfa5 , Neuropilina-1 , Neuropilina-2 , Proteómica , Proteína Activadora de GTPasa p120 , Animales , Ratones , Endosomas/metabolismo , Células Endoteliales/metabolismo , Fibronectinas/metabolismo , Integrina alfa5/metabolismo , Integrina alfa5/genética , Integrinas , Neuropilina-1/metabolismo , Neuropilina-1/genética , Neuropilina-2/metabolismo , Neuropilina-2/genética , Proteína Activadora de GTPasa p120/metabolismo , Proteína Activadora de GTPasa p120/genética , Transporte de Proteínas , Proteómica/métodosRESUMEN
Langerhans cells (LCs) are mainly present in the epidermis and mucosa, and have important roles during skin infection. Migration of LCs to lymph nodes is essential for antigen presentation. However, due to the difficulties in isolating and culturing human LCs, it is not fully understood how LCs move and interact with the extracellular matrix (ECM) through their adhesion molecules such as integrin, during the immune responses. In this study, we aimed to investigate LC motility, cell shape and the role of integrin under inflammatory conditions using monocyte-derived Langerhans cells (moLCs) as a model. As a result, lipopolysaccharide (LPS) stimulation increased adhesion on fibronectin coated substrate and integrin α5 expression in moLCs. Time-lapse imaging of moLCs revealed that stimulation with LPS elongated cell shape, whilst decreasing their motility. Additionally, this decrease in motility was not observed when pre-treated with a neutralising antibody targeting integrin α5. Together, our data suggested that activation of LCs decreases their motility by promoting integrin α5 expression to enhance their affinity to the fibronectin, which may contribute to their migration during inflammation.
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Integrina alfa5 , Células de Langerhans , Humanos , Fibronectinas/metabolismo , Inmunidad , Integrina alfa5/metabolismo , Integrinas/metabolismo , Lipopolisacáridos/farmacología , MonocitosRESUMEN
Bone marrow macrophages (Mφ) are essential components of the bone marrow niche that regulate the function of hematopoietic stem cells. Poor graft function and inhibition of hematopoietic production can result from abnormal macrophage function; however, the underlying mechanism is unclear. Clodronate liposomes (Clo-Lip) have been used widely to deplete macrophages and study their functions. Our previous results showed that Clod-Lip-mediated clearance of macrophages plays a vital role in regulating hematopoietic reconstruction after allogeneic hematopoietic cell transplantation (HCT). In this study, using an isogenic hematopoietic stem cell transplantation model, we found that Clod-Lip-mediated clearance of macrophages suppressed hematopoietic reconstruction by inhibiting the homing process of hematopoietic cells. We also demonstrated that macrophage depletion inhibited the direct supportive effect of macrophages on hematopoietic stem and progenitor cells and erythroid differentiation but promoted the production of megakaryocytic progenitors ex vivo. We showed that macrophages increase CD49e expression on hematopoietic stem and progenitor cells (HSPCs). However, CD49e inhibitors did not support the proliferative effect of macrophages on hematopoietic cells. In contrast, macrophage E-selectin/ intercellular cell adhesion molecule-1 (ICAM-1) may be involved in directly regulating HSPCs. In conclusion, macrophage depletion with Clo-Lip partially disrupts bone marrow hematopoiesis after HCT by impeding donor cell homing and macrophage-HSPCs interactions.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Integrina alfa5 , Integrina alfa5/metabolismo , Células Madre Hematopoyéticas , Trasplante de Células Madre Hematopoyéticas/métodos , Hematopoyesis , Macrófagos/metabolismoRESUMEN
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.
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Proliferación Celular , Pulpa Dental , Fibronectinas , Integrina alfa5 , Neovascularización Fisiológica , Transducción de Señal , Pulpa Dental/citología , Pulpa Dental/metabolismo , Humanos , Fibronectinas/metabolismo , Fibronectinas/genética , Animales , Ratones , Integrina alfa5/metabolismo , Integrina alfa5/genética , Células Madre/metabolismo , Células Madre/citología , Células Endoteliales/metabolismo , Células Endoteliales/citología , Comunicación Celular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Movimiento Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , Diferenciación Celular , Fosfatidilinositol 3-Quinasas/metabolismo , IntegrinasRESUMEN
The bacteria that invade the periapical tissue of teeth can directly damage tissue cells such as periapical fibroblasts, leading to an inflammatory response in the periapical tissue and ultimately resulting in bone destruction. We investigated the role of fibroblast activation protein α (FAPα) and integrin α5 (ITGA5) in periapical bone destruction. This study found that FAPα and ITGA5 were highly expressed in human tissues from patients with chronic apical periodontitis. Osteoclast differentiation decreased when FAPα or ITGA5 was silenced and inhibited. The results of protein molecular docking showed that FAPα had good binding affinity to ITGA5, and its free energy was -14.5â¯kcal/mol. Immunofluorescence staining and co-immunoprecipitation showed that FAPα and ITGA5 formed protein complexes in the inflammatory microenvironment. In conclusion, this study proved that FAPα and ITGA5 participate in the regulation of osteoclast differentiation by forming protein complexes in the inflammatory microenvironment, which then regulates the occurrence and development of chronic apical periodontitis.
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Proteínas de la Membrana , Periodontitis Periapical , Periodontitis , Humanos , Integrina alfa5/metabolismo , Simulación del Acoplamiento Molecular , EndopeptidasasRESUMEN
Macrophages sense changes in the extracellular matrix environment through the integrins and play a central role in regulation of the reparative response after myocardial infarction. Here we show that macrophage integrin α5 protects the infarcted heart from adverse remodeling and that the protective actions are associated with acquisition of an angiogenic macrophage phenotype. We demonstrate that myeloid cell- and macrophage-specific integrin α5 knockout mice have accentuated adverse post-infarction remodeling, accompanied by reduced angiogenesis in the infarct and border zone. Single cell RNA-sequencing identifies an angiogenic infarct macrophage population with high Itga5 expression. The angiogenic effects of integrin α5 in macrophages involve upregulation of Vascular Endothelial Growth Factor A. RNA-sequencing of the macrophage transcriptome in vivo and in vitro followed by bioinformatic analysis identifies several intracellular kinases as potential downstream targets of integrin α5. Neutralization assays demonstrate that the angiogenic actions of integrin α5-stimulated macrophages involve activation of Focal Adhesion Kinase and Phosphoinositide 3 Kinase cascades.
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Integrina alfa5 , Infarto del Miocardio , Ratones , Animales , Integrina alfa5/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Macrófagos/metabolismo , Ratones Noqueados , ARN/metabolismoRESUMEN
Staphylococcus aureus, a Gram-positive bacterial pathogen, is an urgent health threat causing a wide range of clinical infections. Originally viewed as a strict extracellular pathogen, accumulating evidence has revealed S. aureus to be a facultative intracellular pathogen subverting host cell signalling to support invasion. The majority of clinical isolates produce fibronectin-binding proteins A and B (FnBPA and FnBPB) to interact with host integrin α5ß1, a key component of focal adhesions. S. aureus binding of integrin α5ß1 promotes its clustering on the host cell surface, triggering activation of focal adhesion kinase (FAK) and cytoskeleton rearrangements to promote bacterial invasion into non-phagocytic cells. Here, we discover that septins, a component of the cytoskeleton that assembles on membranes, are recruited as collar-like structures with actin to S. aureus invasion sites engaging integrin α5ß1. To investigate septin recruitment to the plasma membrane in a bacteria-free system, we used FnBPA-coated latex beads and showed that septins are recruited upon activation of integrin α5ß1. SEPT2 depletion reduced S. aureus invasion, but increased surface expression of integrin α5 and adhesion of S. aureus to host cells. Consistent with this, SEPT2 depletion increased cellular protein levels of integrin α5 and ß1 subunits, as well as FAK. Collectively, these results provide insights into regulation of integrin α5ß1 and invasion of S. aureus by the septin cytoskeleton.
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Integrina alfa5beta1 , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Integrina alfa5beta1/metabolismo , Septinas/metabolismo , Integrina alfa5/metabolismo , Fibronectinas , Citoesqueleto/metabolismoRESUMEN
BACKGROUND: TP53, encoding the tumor suppressor p53, is frequently mutated in various cancers, producing mutant p53 proteins (mutp53) which can exhibit neomorphic, gain-of-function properties. The latter transform p53 into an oncoprotein that promotes metastatic tumor progression via downstream effectors such as ENTPD5, an endoplasmic reticulum UDPase involved in the calnexin/calreticulin cycle of N-glycoprotein biosynthesis. Elucidating the mechanisms underlying the pro-metastatic functions of the mutp53-ENTPD5 axis is crucial for developing targeted therapies for aggressive metastatic cancer. METHODS: We analyzed pancreatic, lung, and breast adenocarcinoma cells with p53 missense mutations to study the impact of mutp53 and ENTPD5 on the N-glycoproteins integrin-α5 (ITGA5) and integrin-ß1 (ITGB1), which heterodimerize to form the key fibronectin receptor. We assessed the role of the mutp53-ENTPD5 axis in integrin-dependent tumor-stroma interactions and tumor cell motility using adhesion, migration, and invasion assays, identifying and validating therapeutic intervention targets. We employed an orthotopic xenograft model of pancreatic ductal adenocarcinoma to examine in vivo targeting of mutp53-ENTPD5-mediated ITGA5 regulation for cancer therapy. RESULTS: Mutp53 depletion diminished ITGA5 and ITGB1 expression and impaired tumor cell adhesion, migration, and invasion, rescued by ENTPD5. The mutp53-ENTPD5 axis maintained ITGA5 expression and function via the calnexin/calreticulin cycle. Targeting this axis using ITGA5-blocking antibodies, α-glucosidase inhibitors, or pharmacological degradation of mutp53 by HSP90 inhibitors, such as Ganetespib, effectively inhibited ITGA5-mediated cancer cell motility in vitro. In the orthotopic xenograft model, Ganetespib reduced ITGA5 expression and metastasis in an ENTPD5-dependent manner. CONCLUSIONS: The mutp53-ENTPD5 axis fosters ITGA5 and ITGB1 expression and tumor cell motility through the calnexin/calreticulin cycle, contributing to cancer metastasis. ITGA5-blocking antibodies or α-glucosidase inhibitors target this axis and represent potential therapeutic options worth exploring in preclinical models. The pharmacologic degradation of mutp53 by HSP90 inhibitors effectively blocks ENTPD5-ITGA5-mediated cancer cell motility and metastasis in vivo, warranting further clinical evaluation in p53-mutant cancers. This research underscores the significance of understanding the complex interplay between mutp53, ENTPD5, and the calnexin/calreticulin cycle in integrin-mediated metastatic tumor progression, offering valuable insights for the development of potential therapeutic strategies.
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Adenocarcinoma , Antineoplásicos , Animales , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Calnexina/genética , Calnexina/metabolismo , Integrina alfa5/metabolismo , Calreticulina/metabolismo , Anticuerpos Bloqueadores/metabolismo , Inhibidores de Glicósido Hidrolasas , Línea Celular Tumoral , Chaperonas Moleculares/metabolismo , Modelos Animales de Enfermedad , Pirofosfatasas/metabolismo , Proteínas Oncogénicas/metabolismoRESUMEN
BACKGROUND: The effect of platelet factor 4 (PF4) on bone marrow mesenchymal stem cells (BMMSCs) and osteoporosis is poorly understood. Therefore, this study aimed to evaluate the effects of PF4-triggered bone destruction in mice and determine the underlying mechanism. METHODS: First, in vitro cell proliferation and cell cycle of BMMSCs were assessed using a CCK8 assay and flow cytometry, respectively. Osteogenic differentiation was confirmed using staining and quantification of alkaline phosphatase and Alizarin Red S. Next, an osteoporotic mouse model was established by performing bilateral ovariectomy (OVX). Furthermore, the PF4 concentrations were obtained using enzyme-linked immunosorbent assay. The bone microarchitecture of the femur was evaluated using microCT and histological analyses. Finally, the key regulators of osteogenesis and pathways were investigated using quantitative real-time polymerase chain reaction and Western blotting. RESULTS: Human PF4 widely and moderately decreased the cell proliferation and osteogenic differentiation ability of BMMSCs. Furthermore, the levels of PF4 in the serum and bone marrow were generally increased, whereas bone microarchitecture deteriorated due to OVX. Moreover, in vivo mouse PF4 supplementation triggered bone deterioration of the femur. In addition, several key regulators of osteogenesis were downregulated, and the integrin α5-focal adhesion kinase-extracellular signal-regulated kinase (ITGA5-FAK-ERK) pathway was inhibited due to PF4 supplementation. CONCLUSIONS: PF4 may be attributed to OVX-induced bone loss triggered by the suppression of bone formation in vivo and alleviate BMMSC osteogenic differentiation by inhibiting the ITGA5-FAK-ERK pathway.
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Integrina alfa5 , Osteogénesis , Animales , Femenino , Humanos , Ratones , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/farmacología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrina alfa5/metabolismo , Integrina alfa5/farmacología , Sistema de Señalización de MAP Quinasas , Factor Plaquetario 4/metabolismo , Factor Plaquetario 4/farmacología , Quinasa 1 de Adhesión Focal/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal disease affecting upper and lower motor neurons. Microglia directly interact with motor neurons and participate in the progression of ALS. Single-cell mass cytometry (CyTOF) analysis revealed prominent expression of α5 integrin in microglia and macrophages in a superoxide dismutase-1 G93A mouse model of ALS (SOD1G93A). In postmortem tissues from ALS patients with various clinical ALS phenotypes and disease duration, α5 integrin is prominent in motor pathways of the central and peripheral nervous system and in perivascular zones associated with the blood-brain barrier. In SOD1G93A mice, administration of a monoclonal antibody against α5 integrin increased survival compared to an isotype control and improved motor function on behavioral testing. Together, these findings in mice and in humans suggest that α5 integrin is a potential therapeutic target in ALS.
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Esclerosis Amiotrófica Lateral , Corteza Motora , Ratones , Humanos , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Integrina alfa5/metabolismo , Ratones Transgénicos , Superóxido Dismutasa/metabolismo , Macrófagos/metabolismo , Modelos Animales de EnfermedadRESUMEN
γ-bungarotoxin (γ-BGT) is an RGD motif-containing protein, derived from the venom of Bungarus multicinctus, leading to acute death in mice. These RGD motif-containing proteins from snake venom belonging to the disintegrin family can interfere with vascular endothelial homeostasis by directly binding cell surface integrins. Targeting integrins that generate vascular endothelial dysfunction may contribute to γ-BGT poisoning, however, the underlying mechanisms have not been investigated in detail. In this study, the results showed that γ-BGT played a role in -promoting the permeability of the vascular endothelial barrier. Depending on its selective binding to integrin α5 in vascular endothelium (VE), γ-BGT initiated downstream events, including focal adhesion kinase dephosphorylation and cytoskeleton remodeling, resulting in the intercellular junction interruption. Those alternations facilitated paracellular permeability of VE and barrier dysfunction. Proteomics profiling identified that as a downstream effector of the integrin α5 / FAK signaling pathway cyclin D1 partially mediated the cellular structural changes and barrier dysfunction. Furthermore, VE-released plasminogen activator urokinase and platelet-derived growth factor D could serve as potential diagnostic biomarkers for γ-BGT-induced vascular endothelial dysfunction. Our results indicate the mechanisms through which γ-BGT as a novel disintegrin directly interacts with the VE, with consequences for barrier dysfunction.
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Bungarotoxinas , Endotelio Vascular , Integrina alfa5 , Venenos de Serpiente , Animales , Ratones , Bungarotoxinas/toxicidad , Desintegrinas/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Integrina alfa5/metabolismo , Integrinas/metabolismo , Oligopéptidos , Venenos de Serpiente/toxicidadRESUMEN
Integrins act as cellmatrix adhesion molecules involved in cell attachment to the extracellular matrix and generate signals that respond to cancer metastasis. Integrin α5ß1 is a heterodimer with α5 and ß1 subunits and mediates cell adhesion and migration of cancer cells. Integrins are transcriptionally regulated by the Janus kinase (JAK)/STAT signaling pathways. Our previous study revealed that Helicobacter pylori increased the levels of reactive oxygen species (ROS), which activate JAK1/STAT3 in gastric cancer AGS cells in vitro. Astaxanthin (ASX) has been reported to be an effective antioxidant and anticancer nutrient. The present study investigated whether ASX suppresses H. pyloriinduced integrin α5 expression, cell adhesion and migration and whether ASX reduces ROS levels and suppresses phosphorylation of JAK1/STAT3 in gastric cancer AGS cells stimulated with H. pylori. The effect of ASX was determined using a dichlorofluorescein fluorescence assay, western blot analysis, adhesion assay and woundhealing assay in AGS cells stimulated with H. pylori. The results demonstrated that H. pylori increased the expression levels of integrin α5, without affecting integrin ß1, and increased cell adhesion and migration of AGS cells. ASX reduced ROS levels and suppressed JAK1/STAT3 activation, integrin α5 expression, cell adhesion and migration of H. pyloristimulated AGS cells. In addition, both a JAK/STAT inhibitor, AG490, and an integrin α5ß1 antagonist, K34C, suppressed cell adhesion and migration in H. pyloristimulated AGS cells. AG490 inhibited integrin α5 expression in AGS cells stimulated with H. pylori. In conclusion, ASX inhibited H. pyloriinduced integrin α5mediated cell adhesion and migration by decreasing the levels of ROS and suppressing JAK1/STAT3 activation in gastric epithelial cells.
Asunto(s)
Helicobacter pylori , Neoplasias Gástricas , Humanos , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Helicobacter pylori/fisiología , Integrina alfa5/metabolismo , Integrina alfa5beta1/metabolismo , Janus Quinasa 1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
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.