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Purpose: Castration Resistant Prostate Cancer (CRPC) is characterized by poor prognosis and limited therapeutic options. AgNPs functionalized with glucose (G-AgNPs) were observed cytotoxic to CRPC cell lines (PC-3 and Du-145) and not LNCaP. This study aims to evaluate AgNPs and G-AgNPs' uptake mechanisms in these cells and understand their role in the selective effect against CRPC cells. Methods: Uptake of AgNPs and G-AgNPs was assessed through transmission electron microscopy (TEM). A microRNA (miRNAs) analysis approach was used to uncover the main molecular differences responsible for the endocytic mechanisms' regulation. Caveolin (Cav) 1 and 2 mRNA and protein levels were assessed in the three cell lines. Caveolae-dependent endocytosis was inhibited with genistein or siCav1- and siCav2- in PC-3 and Du-145 and resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration. Caveolae-dependent endocytosis was induced with Cav1+ and Cav2+ plasmids in LNCaP, resazurin assay was used to evaluate viability after AgNPs and G-AgNPs administration and TEM to assess their location. Results: AgNPs and G-AgNPs were not uptaked by LNCaP. miRNA analysis revealed 37 upregulated and 90 downregulated miRNAs. Functional enrichment analysis of miRNAs' targets resulted in enrichment of terms related to endocytosis and caveolae. We observed that Cav1 and Cav2 are not expressed in LNCaP. Inhibiting caveolae-dependent endocytosis in Du-145 and PC-3 led to a significative reduction of cytotoxic capacity of AgNPs and G-AgNPs and induction of caveolae-dependent endocytosis in LNCaP lead to a significative increase as well as their uptake by cells. Conclusion: This study shows the potential of these AgNPs as a new therapeutic approach directed to CRPC patients, uncovers caveolae-dependent endocytosis as the uptake mechanism of these AgNPs and highlights deregulation of Cav1 and Cav2 expression as a key difference in hormone sensitive and resistant PCa cells which may be responsible for drug resistance.
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Caveolas , Caveolina 1 , Endocitosis , Nanopartículas del Metal , MicroARNs , Neoplasias de la Próstata Resistentes a la Castración , Plata , Masculino , Humanos , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Caveolas/metabolismo , Caveolas/efectos de los fármacos , Plata/química , Plata/farmacología , Plata/farmacocinética , Caveolina 1/metabolismo , Caveolina 1/genética , Nanopartículas del Metal/química , Línea Celular Tumoral , MicroARNs/metabolismo , MicroARNs/genética , Supervivencia Celular/efectos de los fármacos , Caveolina 2/metabolismo , Caveolina 2/genética , Antineoplásicos/farmacología , Células PC-3RESUMEN
Peritoneal metastasis is one of the most common risk factors contributing to the poor prognosis of gastric cancer. We previously reported that extracellular vesicles from gastric cancer cells could facilitate peritoneal metastasis. However, their impact on gastric cancer-induced peritoneal metastasis under hypoxic conditions remains unclear. This study aims to elucidate how hypoxia-resistant gastric cancer cell-derived extracellular vesicles affect the peritoneal metastasis of normoxic gastric cancer cells. Proteomic analysis revealed elevated levels of Caveolin1 and Laminin ß2 in hypoxia-resistant gastric cancer cells and their corresponding extracellular vesicles. Importantly, Caveolin1 was found to play a central role in mediating Laminin ß2 sorting into extracellular vesicles derived from hypoxia-resistant gastric cancer cells, and subsequently, extracellular vesicle-associated Laminin ß2 promoted peritoneal metastasis in normoxic gastric cancer cells by activating the AKT pathway. Further investigation confirmed that Caveolin1 activation by Rho-related Coiled-coil kinase 1-mediated phosphorylation of Y14 residue is a key factor facilitating Laminin ß2 sorting into extracellular vesicles. Moreover, Y14 phosphorylated- Caveolin1 enhanced Laminin ß2 sorting by activating Rab11. Finally, our study demonstrated that a combined assessment of plasma extracellular vesicle-associated Caveolin1 and extracellular vesicle-associated Laminin ß2 could provide an accurate predictive tool for peritoneal metastasis occurrence in gastric cancer.
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Caveolina 1 , Vesículas Extracelulares , Neoplasias Peritoneales , Neoplasias Gástricas , Proteínas de Unión al GTP rab , Quinasas Asociadas a rho , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/metabolismo , Animales , Quinasas Asociadas a rho/metabolismo , Vesículas Extracelulares/metabolismo , Ratones , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Línea Celular Tumoral , Transducción de Señal , Masculino , FemeninoRESUMEN
PURPOSE: Cholesterol metabolism reprograming has been acknowledged as a novel feature of cancers. Pancreatic ductal adenocarcinoma (PDAC) is a cancer with a high demand of cholesterol for rapid growth. The underlying mechanism of how cholesterol metabolism homestasis are disturbed in PDAC is explored. EXPERIMENTAL DESIGN: The relevance between PDAC and cholesterol was confirmed in TCGA database. The expression and clinical association were discovered in TCGA and GEO datasets. Knockdown and overexpression of AGFG1 was adopted to perform function studies. RNA sequencing, cholesterol detection, transmission electron microscope, co-immunoprecipitation, and immunofluorescence et al. were utilized to reveal the underlying mechanism. RESULTS: AGFG1 was identified as one gene positively correlated with cholesterol metabolism in PDAC as revealed by bioinformatics analysis. AGFG1 expression was then found associated with poor prognosis in PDAC. AGFG1 knockdown led to decreased proliferation of tumor cells both in vitro and in vivo. By RNA sequencing, we found AGFG1 upregulated expression leads to enhanced intracellular cholesterol biosynthesis. AGFG1 knockdown suppressed cholesterol biosynthesis and an accumulation of cholesterol in the ER. Mechanistically, we confirmed that AGFG1 interacted with CAV1 to relocate cholesterol for the proceeding of cholesterol biosynthesis, therefore causing disorders in intracellular cholesterol metabolism. CONCLUSIONS: Our study demonstrates the tumor-promoting role of AGFG1 by disturbing cholesterol metabolism homestasis in PDAC. Our study has present a new perspective on cancer therapeutic approach based on cholerstrol metabolism in PDAC.
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Carcinoma Ductal Pancreático , Proliferación Celular , Colesterol , Homeostasis , Neoplasias Pancreáticas , Humanos , Colesterol/metabolismo , Colesterol/biosíntesis , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Animales , Línea Celular Tumoral , Ratones , Regulación Neoplásica de la Expresión Génica , Progresión de la Enfermedad , Pronóstico , Caveolina 1/genética , Caveolina 1/metabolismo , Ratones Desnudos , MasculinoRESUMEN
BACKGROUND: The roles of Caveolin-1 (Cav-1) and the Wnt/ß-catenin signaling pathways in cerebral ischemia-reperfusion (I/R) injury are well established. The translocation of ß-catenin into the nucleus is critical for regulating neuronal apoptosis, repair, and neurogenesis within the ischemic brain. It has been reported that the scaffold domain of Caveolin-1 (Cav-1) (residues 95-98) interacts with ß-catenin (residues 330-337). However, the specific contribution of the Cav-1/ß-catenin complex to I/R injury remains unknown. METHODS AND RESULTS: To investigate the mechanism underlying the involvement of the Cav-1/ß-catenin complex in the subcellular translocation of ß-catenin and its subsequent effects on cerebral I/R injury, we treated ischemic brains with ASON (Cav-1 antisense oligodeoxynucleotides) or FTVT (a competitive peptide antagonist of the Cav-1 and ß-catenin interaction). Our study demonstrated that the binding of Cav-1 to ß-catenin following I/R injury prevented the nuclear accumulation of ß-catenin. Treatment with ASON or FTVT after I/R injury significantly increased the levels of nuclear ß-catenin. Furthermore, ASON reduced the phosphorylation of ß-catenin at Ser33, Ser37, and Thr41, which contributes to its proteasomal degradation, while FTVT increased phosphorylation at Tyr333, which is associated with its nuclear translocation. CONCLUSIONS: The above results indicate that the formation of the Cav-1/ß-catenin complex anchors ß-catenin in the cytoplasm following I/R injury. Additionally, both ASON and FTVT treatments attenuated neuronal death in ischemic brains. Our study suggests that targeting the interaction between Cav-1 and ß-catenin serve as a novel therapeutic strategy to protect against neuronal damage during cerebral injury.
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Caveolina 1 , Núcleo Celular , Neuronas , Daño por Reperfusión , beta Catenina , beta Catenina/metabolismo , Animales , Daño por Reperfusión/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Neuronas/metabolismo , Neuronas/patología , Núcleo Celular/metabolismo , Masculino , Ratas , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Apoptosis , Vía de Señalización Wnt , Ratas Sprague-Dawley , Unión Proteica , Transporte de Proteínas , Muerte CelularRESUMEN
BACKGROUND: In early atherosclerosis, circulating LDLs (low-density lipoproteins) traverse individual endothelial cells by an active process termed transcytosis. The CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) treated advanced atherosclerosis using a blocking antibody for IL-1ß (interleukin-1ß); this significantly reduced cardiovascular events. However, whether IL-1ß regulates early disease, particularly LDL transcytosis, remains unknown. METHODS: We used total internal reflection fluorescence microscopy to quantify transcytosis by human coronary artery endothelial cells exposed to IL-1ß. To investigate transcytosis in vivo, we injected wild-type and knockout mice with IL-1ß and LDL to visualize acute LDL deposition in the aortic arch. RESULTS: Exposure to picomolar concentrations of IL-1ß induced transcytosis of LDL but not of albumin by human coronary artery endothelial cells. Surprisingly, expression of the 2 known receptors for LDL transcytosis, ALK-1 (activin receptor-like kinase-1) and SR-BI (scavenger receptor BI), was unchanged or decreased. Instead, IL-1ß increased the expression of the LDLR (LDL receptor); this was unexpected because LDLR is not required for LDL transcytosis. Overexpression of LDLR had no effect on basal LDL transcytosis. However, knockdown of LDLR abrogated the effect of IL-1ß on transcytosis rates while the depletion of Cav-1 (caveolin-1) did not. Since LDLR was necessary but overexpression had no effect, we reasoned that another player must be involved. Using public RNA sequencing data to curate a list of Rab (Ras-associated binding) GTPases affected by IL-1ß, we identified Rab27a. Overexpression of Rab27a alone had no effect on basal transcytosis, but its knockdown prevented induction by IL-1ß. This was phenocopied by depletion of the Rab27a effector JFC1 (synaptotagmin-like protein 1). In vivo, IL-1ß increased LDL transcytosis in the aortic arch of wild-type but not Ldlr-/- or Rab27a-deficient mice. The JFC1 inhibitor nexinhib20 also blocked IL-1ß-induced LDL accumulation in the aorta. CONCLUSIONS: IL-1ß induces LDL transcytosis by a distinct pathway requiring LDLR and Rab27a; this route differs from basal transcytosis. We speculate that induction of transcytosis by IL-1ß may contribute to the acceleration of early disease.
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Vasos Coronarios , Células Endoteliales , Interleucina-1beta , Lipoproteínas LDL , Ratones Noqueados , Receptores de LDL , Transducción de Señal , Transcitosis , Proteínas de Unión al GTP rab , Interleucina-1beta/metabolismo , Animales , Humanos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Lipoproteínas LDL/metabolismo , Vasos Coronarios/metabolismo , Vasos Coronarios/efectos de los fármacos , Células Cultivadas , Ratones Endogámicos C57BL , Caveolina 1/metabolismo , Caveolina 1/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Modelos Animales de Enfermedad , Aorta Torácica/metabolismo , Aorta Torácica/efectos de los fármacos , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Masculino , RatonesRESUMEN
BACKGROUND: Currently, there are few treatment-predictive and prognostic biomarkers in triple-negative breast cancer (TNBC). Caveolin-1 (CAV1) is linked to chemoresistance and several important processes involved in tumor progression and metastasis, such as epithelial-mesenchymal transition (EMT). Herein, we report that high CAV1 gene expression is an independent factor of poor prognosis in TNBC. METHODS: CAV1 gene expression was compared across different molecular features (e.g., PAM50 subtypes). CAV1 expression was assessed in relation to clinical outcomes using Cox regression adjusted for clinicopathological predictors. Differential gene expression and gene set enrichment analyses were applied to compare high- and low-expressing CAV1 tumors. Tumor microenvironment composition of high- and low-expressing CAV1 tumors was estimated using ECOTYPER. Tumor tissue microarrays were used to evaluate CAV1 protein levels in stromal and malignant cells. RESULTS: In the SCAN-B (n = 525) and GSE31519 (n = 327) cohorts, patients with CAV1-high tumors had an increased incidence of early recurrence adjusted HR 1.78 (95% CI 1.12-2.81) and 2.20 (95% CI 1.39-3.47), respectively. In further analysis, high CAV1 gene expression was associated with a molecular profile indicating altered metabolism, neovascularization, chemoresistance, EMT, suppressed immune response, and active tumor microenvironment. Protein levels of CAV1 in malignant and stromal cells were not correlated with CAV1 gene expression. CONCLUSION: CAV1 gene expression in TNBC is a biomarker that merits further investigation in clinical trials and as a therapeutic target.
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Caveolina 1 , Resistencia a Antineoplásicos , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Humanos , Caveolina 1/genética , Caveolina 1/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Microambiente Tumoral/genética , Femenino , Resistencia a Antineoplásicos/genética , Persona de Mediana Edad , Regulación Neoplásica de la Expresión Génica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pronóstico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Transición Epitelial-Mesenquimal/genética , AncianoRESUMEN
Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.
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Caveolina 1 , Movimiento Celular , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Macrófagos/metabolismo , Fosforilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/genética , Transporte Biológico , Cicatrización de Heridas/fisiología , Orgánulos/metabolismoRESUMEN
The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.
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Vectores Genéticos , Glutatión , Polietileneimina , Transfección , Polietileneimina/química , Transfección/métodos , Glutatión/metabolismo , Glutatión/química , Animales , Humanos , Vectores Genéticos/química , Vectores Genéticos/genética , Manitol/química , Ratones , Caveolina 1/metabolismo , Caveolina 1/genética , Terapia Genética/métodos , Técnicas de Transferencia de Gen , Disulfuros/químicaRESUMEN
Caveolin-1 is critical for interacting with the TGF-ß receptor (TGFßR) and EGF receptor (EGFR) signaling, often observed in advanced cancers and tissue fibrosis. However, the mechanism underlying caveolin-1-mediated transactivation of TGFßR and EGFR signaling remains unclear. Therefore, we sought to determine whether caveolin-1 is involved in canonical and non-canonical TGFßR and EGFR signaling transactivation in this study. Methyl-ß-cyclodextrin (MßCD) was used to disrupt the cholesterol-containing membranes domains, and the caveolin-1 scaffolding domain (CSD) peptide was used to mimic the CSD of caveolin-1. Additionally, we transfected the Madin-Darby canine kidney cells with wild-type or phosphorylation-defective caveolin-1. We discovered that tyrosine 14 of caveolin-1 was critical for the negative regulation of TGFßR and EGFR canonical signaling. On the contrary, caveolin-1 inhibited TGF-ß1-induced ERK2 activation independent of tyrosine 14 phosphorylation. Although EGF failed to induce Smad3 phosphorylation in caveolin-1 knockdown cells, it activated Smad3 upon MßCD co-treatment, indicating that caveolin-1 indirectly regulated the non-canonical pathway of EGF. In conclusion, caveolin-1 differentially modulates TGFßR and EGFR signaling. Thus, targeting caveolin-1 is a potential strategy for treating diseases involving TGF-ß1 and EGF signaling.
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Caveolina 1 , Receptores ErbB , Transducción de Señal , Animales , Perros , Caveolina 1/metabolismo , Caveolina 1/genética , Células de Riñón Canino Madin Darby , Receptores ErbB/metabolismo , Fosforilación , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Humanos , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: Radiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury. METHODS: Paired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing. RESULTS: Comparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0-4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses. CONCLUSION: In response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.
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Caveolina 1 , Fibroblastos , Análisis de la Célula Individual , Piel , Humanos , Femenino , Fibroblastos/efectos de la radiación , Fibroblastos/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Caveolina 1/biosíntesis , Piel/efectos de la radiación , Piel/patología , Piel/metabolismo , Neoplasias de la Mama/radioterapia , Neoplasias de la Mama/patología , Caveolina 2/metabolismo , Caveolina 2/genética , Traumatismos por Radiación/patología , Traumatismos por Radiación/metabolismo , Fibrosis , Persona de Mediana EdadRESUMEN
Airway remodelling in lung diseases can be treated by inhibiting excessive smooth muscle cell proliferation. Zedoarondiol (Zed) is a natural compound isolated from the Chinese herb Curcuma longa. The caveolin-1 (CAV-1) is widely expressed in lung cells and plays a key role in platelet-derived growth factor (PDGF) signalling and cell proliferation. This study aims to investigate the effect of Zed on human bronchial smooth muscle cell (HBSMC) proliferation and explore its potential molecular mechanisms. We assessed the effect of Zed on the proliferation of PDGF-stimulated HBSMCs and performed proteomic analysis to identify potential molecular targets and pathways. CAV1 siRNA was used to validate our findings in vitro. In PDGF-stimulated HBSMCs, Zed significantly inhibited excessive proliferation of HBSMCs. Proteomic analysis of zedoarondiol-treated HBSMCs revealed significant enrichment of differentially expressed proteins in cell proliferation-related pathways and biological processes. Zed inhibition of HBSMC proliferation was associated with upregulation of CAV1, regulation of the CAV-1/PDGF pathway and inhibition of MAPK and PI3K/AKT signalling pathway activation. Treatment of HBSMCs with CAV1 siRNA partly reversed the inhibitory effect of Zed on HBSMC proliferation. Thus, this study reveals that zedoarondiol potently inhibits HBSMC proliferation by upregulating CAV-1 expression, highlighting its potential value in airway remodelling and related diseases.
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Bronquios , Caveolina 1 , Proliferación Celular , Miocitos del Músculo Liso , Factor de Crecimiento Derivado de Plaquetas , Transducción de Señal , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Bronquios/metabolismo , Bronquios/citología , Bronquios/patología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteómica/métodos , Fosfatidilinositol 3-Quinasas/metabolismo , Células CultivadasRESUMEN
Deficiencies in mice and in humans have brought to the fore the importance of the caveolar network in key aspects of adipocyte biology. The conserved N-terminal caveolin-binding motif (CBM) of the ubiquitous Na/K-ATPase (NKA) α1 isoform, which allows NKA/caveolin-1 (Cav1) interaction, influences NKA signaling and caveolar distribution. It has been shown to be critical for animal development and ontogenesis, as well as lineage-specific differentiation of human induced pluripotent stem cells (hiPSCs). However, its role in postnatal adipogenesis has not been fully examined. Using a genetic approach to alter CBM in hiPSC-derived adipocytes (iAdi-mCBM) and in mice (mCBM), we investigated the regulatory function of NKA CBM signaling in adipogenesis. Seahorse XF cell metabolism analyses revealed impaired glycolysis and decreased ATP synthesis-coupled respiration in iAdi-mCBM. These metabolic dysfunctions were accompanied by evidence of extensive remodeling of the extracellular matrix (ECM), including increased collagen staining, overexpression of ECM marker genes, and heightened TGF-ß signaling uncovered by RNAseq analysis. Rescue of mCBM by lentiviral delivery of WT NKA α1 or treatment of mCBM hiPSCs with the TGF-ß inhibitor SB431542 normalized ECM, suggesting that NKA CBM signaling integrity is required for adequate control of TGF-ß signaling and ECM stiffness during adipogenesis. The physiological impact was revealed in mCBM male mice with reduced fat mass accompanied by histological and transcriptional evidence of elevated adipose fibrosis and decreased adipocyte size. Based on these findings, we propose that the genetic alteration of the NKA/Cav1 regulatory path uncovered in human iAdi leads to lipodystrophy in mice.NEW & NOTEWORTHY A Na/K-ATPase α1 caveolin-binding motif regulates adipogenesis. Mutation of this binding motif in the mouse leads to reduced fat with increased extracellular matrix production and inflammation. RNA-seq analysis and pharmacological interventions in human iPSC-derived adipocytes revealed that TGF-ß signal, rather than Na/K-ATPase-mediated ion transport, is a key mediator of NKA regulation of adipogenesis.
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Adipocitos , Adipogénesis , Caveolina 1 , Células Madre Pluripotentes Inducidas , ATPasa Intercambiadora de Sodio-Potasio , Adipogénesis/genética , Animales , Caveolina 1/metabolismo , Caveolina 1/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Humanos , Ratones , Adipocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal , Diferenciación Celular , Masculino , Matriz Extracelular/metabolismo , Secuencias de Aminoácidos , Ratones Endogámicos C57BLRESUMEN
Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, and this supports an antiinflammatory phenotype. High laminar shear stress also induces flow-aligned cell elongation and front-rear polarity, but whether these are required for the antiinflammatory phenotype is unclear. Here, we showed that caveolin-1-rich microdomains polarize to the downstream end of ECs that are exposed to continuous high laminar flow. These microdomains were characterized by high membrane rigidity, filamentous actin (F-actin), and raft-associated lipids. Transient receptor potential vanilloid (TRPV4) ion channels were ubiquitously expressed on the plasma membrane but mediated localized Ca2+ entry only at these microdomains where they physically interacted with clustered caveolin-1. These focal Ca2+ bursts activated endothelial nitric oxide synthase within the confines of these domains. Importantly, we found that signaling at these domains required both cell body elongation and sustained flow. Finally, TRPV4 signaling at these domains was necessary and sufficient to suppress inflammatory gene expression and exogenous activation of TRPV4 channels ameliorated the inflammatory response to stimuli both in vitro and in vivo. Our work revealed a polarized mechanosensitive signaling hub in arterial ECs that dampened inflammatory gene expression and promoted cell resilience.
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Calcio , Células Endoteliales , Inflamación , Mecanotransducción Celular , Canales Catiónicos TRPV , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Animales , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Calcio/metabolismo , Ratones , Humanos , Microdominios de Membrana/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Señalización del Calcio , Estrés Mecánico , Aorta Torácica/metabolismo , Aorta Torácica/patologíaRESUMEN
Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.
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Neuronas Dopaminérgicas , Proteínas Hedgehog , Células Madre Pluripotentes Inducidas , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad de Parkinson , Receptor Patched-1 , Proteína con Dedos de Zinc GLI1 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Neuronas Dopaminérgicas/metabolismo , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , AMP Cíclico/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Mutación/genética , Calcio/metabolismo , Diferenciación Celular/genética , Transducción de Señal/genéticaRESUMEN
AIMS: CD4+ T cells are activated during inflammatory dilated cardiomyopathy (iDCM) development to induce immunogenic responses that damage the myocardium. Low-intensity pulsed ultrasound (LIPUS), a novel physiotherapy for cardiovascular diseases, has recently been shown to modulate inflammatory responses. However, its efficacy in iDCM remains unknown. Here, we investigated whether LIPUS could improve the severity of iDCM by orchestrating immune responses and explored its therapeutic mechanisms. METHODS AND RESULTS: In iDCM mice, LIPUS treatment reduced cardiac remodelling and dysfunction. Additionally, CD4+ T-cell inflammatory responses were suppressed. LIPUS increased Treg cells while decreasing Th17 cells. LIPUS mechanically stimulates endothelial cells, resulting in increased secretion of extracellular vesicles (EVs), which are taken up by CD4+ T cells and alter their differentiation and metabolic patterns. Moreover, EVs selectively loaded with microRNA (miR)-99a are responsible for the therapeutic effects of LIPUS. The hnRNPA2B1 translocation from the nucleus to the cytoplasm and binding to caveolin-1 and miR-99a confirmed the upstream mechanism of miR-99a transport. This complex is loaded into EVs and taken up by CD4+ T cells, which further suppress mTOR and TRIB2 expression to modulate cellular differentiation. CONCLUSION: Our findings revealed that LIPUS uses an EVs-dependent molecular mechanism to protect against iDCM progression. Therefore, LIPUS is a promising new treatment option for iDCM.
Asunto(s)
Cardiomiopatía Dilatada , Modelos Animales de Enfermedad , Vesículas Extracelulares , Ratones Endogámicos C57BL , MicroARNs , Transducción de Señal , Terapia por Ultrasonido , Función Ventricular Izquierda , Animales , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/trasplante , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/terapia , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/inmunología , Cardiomiopatía Dilatada/fisiopatología , MicroARNs/metabolismo , MicroARNs/genética , Ondas Ultrasónicas , Remodelación Ventricular , Masculino , Células Th17/inmunología , Células Th17/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Serina-Treonina Quinasas TOR/metabolismo , Células Cultivadas , Humanos , RatonesRESUMEN
ZFYVE21 is an ancient, endosome-associated protein that is highly expressed in endothelial cells (ECs) but whose function(s) in vivo are undefined. Here, we identified ZFYVE21 as an essential regulator of vascular barrier function in the aging kidney. ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we generated EC-specific Zfyve21-/- reporter mice. These knockout mice developed accelerated aging phenotypes including reduced endothelial nitric oxide (ENOS) activity, failure to thrive, and kidney insufficiency. Kidneys from Zfyve21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that in a GTPase Rab5- and protein kinase Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Thus, our work defines a ZFYVE21- mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging.
Asunto(s)
Envejecimiento , Caveolina 1 , Células Endoteliales , Riñón , Óxido Nítrico Sintasa de Tipo III , Óxido Nítrico , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Envejecimiento/metabolismo , Envejecimiento/fisiología , Caveolina 1/metabolismo , Caveolina 1/genética , Células Endoteliales/metabolismo , Aparato de Golgi/metabolismo , Riñón/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión al GTP rab5/genética , Insuficiencia Renal/metabolismo , Insuficiencia Renal/fisiopatología , Insuficiencia Renal/genéticaRESUMEN
The YTH-domain family (YTHDF) of RNA binding proteins can control gene expression at the post-transcriptional level by regulating mRNAs with N6-methyladenosine (m6A) modifications. Despite the established importance of m6A in the heart, the cardiac role of specific m6A-binding proteins remains unclear. Here, we characterized the function of YTHDF1 in cardiomyocytes using a newly generated cardiac-restricted mouse model. Deletion of YTHDF1 in adult cardiomyocytes led to hypertrophy, fibrosis, and dysfunction. Using mass spectrometry, we identified the necessity of YTHDF1 for the expression of cardiomyocyte membrane raft proteins. Specifically, YTHDF1 bound to m6A-modified Caveolin 1 (Cav1) mRNA and favored its translation. We further demonstrated that YTHDF1 regulates downstream ERK signaling. Altogether, our findings highlight a novel role for YTHDF1 as a post-transcriptional regulator of caveolar proteins which is necessary for the maintenance of cardiac function.
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Homeostasis , Miocitos Cardíacos , Proteínas de Unión al ARN , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Miocitos Cardíacos/metabolismo , Ratones , Caveolina 1/metabolismo , Caveolina 1/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Miocardio/metabolismo , Regulación de la Expresión Génica , Adenosina/análogos & derivados , Adenosina/metabolismo , Ratones Noqueados , Biosíntesis de ProteínasRESUMEN
Alteration in the normal mechanical forces of breathing can contribute to changes in contractility and remodeling characteristic of airway diseases, but the mechanisms that mediate these effects in airway cells are still under investigation. Airway smooth muscle (ASM) cells contribute to both contractility and extracellular matrix (ECM) remodeling. In this study, we explored ASM mechanisms activated by mechanical stretch, focusing on mechanosensitive piezo channels and the key Ca2+ regulatory protein stromal interaction molecule 1 (STIM1). Expression of Ca2+ regulatory proteins, including STIM1, Orai1, and caveolin-1, mechanosensitive ion channels Piezo-1 and Piezo-2, and NLRP3 inflammasomes were upregulated by 10% static stretch superimposed on 5% cyclic stretch. These effects were blunted by STIM1 siRNA. Histamine-induced [Ca2+]i responses and inflammasome activation were similarly blunted by STIM1 knockdown. These data show that the effects of mechanical stretch in human ASM cells are mediated through STIM1, which activates multiple pathways, including Piezo channels and the inflammasome, leading to potential downstream changes in contractility and ECM remodeling.NEW & NOTEWORTHY Mechanical forces on the airway can contribute to altered contractility and remodeling in airway diseases, but the mechanisms are not clearly understood. Using human airway smooth muscle cells exposed to cyclic forces with static stretch to mimic breathing and static pressure, we found that the effects of stretch are mediated through STIM1, resulting in the activation of multiple pathways, including Piezo channels and the inflammasome, with potential downstream influences on contractility and remodeling.
Asunto(s)
Miocitos del Músculo Liso , Molécula de Interacción Estromal 1 , Humanos , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 1/genética , Miocitos del Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Inflamasomas/metabolismo , Estrés Mecánico , Mecanotransducción Celular , Músculo Liso/metabolismo , Canales Iónicos/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Transducción de Señal , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Calcio/metabolismo , Células Cultivadas , Contracción Muscular/fisiología , Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Proteína ORAI1/metabolismo , Proteína ORAI1/genéticaRESUMEN
In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients' surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.
Asunto(s)
Caveolina 1 , Glaucoma de Ángulo Abierto , Malla Trabecular , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Negro o Afroamericano/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolina 2/genética , Caveolina 2/metabolismo , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/metabolismo , Glaucoma de Ángulo Abierto/patología , Glaucoma de Ángulo Abierto/etnología , Malla Trabecular/metabolismo , Malla Trabecular/patología , Blanco , Población Blanca/genéticaRESUMEN
Zika virus (ZIKV) has gained notoriety in recent years because there are no targeted therapies or vaccines available so far. Caveolin-1 (Cav-1) in host cells plays crucial functions in the invasion of many viruses. However, its specific involvement in ZIKV infection has remained unclear. Here, we reveal that depleting Cav-1 leads to a substantial reduction in ZIKV RNA levels, protein expression and viral particle production, indicating that ZIKV exploits Cav-1 for its infection. By dissecting each stage of the viral life cycle, we unveil that, unlike its invasion role in many other viruses, Cav-1 depletion selectively impairs ZIKV replication, resulting in altered replication dynamics and reduced strand-specific RNA levels, but does not affect viral entry, maturation and release. These results reveal an unforeseen function of Cav-1 in facilitating ZIKV replication, which provides new insights into the intricate interaction between Cav-1 and ZIKV and underscores Cav-1 as a potential candidate for anti-ZIKV approaches.