Project description:EMILIN1 promotes ?4?1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-? processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to ?4?1 and ?9?1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-?4/?9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte ?9?1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.
Project description:ObjectiveOsteoarthritis (OA) is closely related to aging, and autophagy is implicated in the retardation of aging. Activated synoviocytes play important roles in OA; the synoviocytes could produce osteopontin (OPN) and its main receptors CD44 and integrin, which are all involved in OA. The purpose of this study is to investigate whether OPN has an effect on autophagy in osteoarthritic synoviocytes.MethodsWe cultured human OA fibroblast-like synoviocytes (FLS) and treated them with rhOPN and antibodies against CD44 and CD51/61 (αvβ3 integrin) or isotype IgG to block the interaction between receptors and ligands. Infection with lentivirus mRFP-GFP-LC3, laser confocal imaging and Western blotting were used to determine changes in the expression of autophagy markers, and cell proliferation of FLS was assessed with a CCK-8 assay.ResultsOur results showed the expression level of autophagy marker protein LC3 II and the mRFP-GFP-LC3 puncta were significantly decreased after treatment with rhOPN when compared with the control group, when the FLS were incubated with antibodies against CD44 or CD51/61 (αvβ3 integrin) or with control isotype IgG for 1 h, followed by rhOPN treatment for 48 h, rhOPN could suppress the relative expression of LC3 II and Beclin1 via integrin and CD44 in the FLS, CCK-8 assay also showed that rhOPN significantly increased the cell proliferation and viability of FLS.ConclusionsOPN could inhibit autophagy via CD44 and αvβ3 integrin and promote the proliferation of FLS, playing an important role in OA synovitis.
Project description:Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here, we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI-P878A) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.
Project description:The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF) is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes) and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs.
Project description:Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix-OptoIntegrin system may serve as a blueprint for rendering matrix-receptor interactions amendable to precise control with light.
Project description:Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting β2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced β2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and β2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.
Project description:We have previously shown that glioblastoma stem cells (GSCs) are enriched in the hypoxic tumor microenvironment, and that monocarboxylate transporter-4 (MCT4) is critical for mediating GSC signaling in hypoxia. Basigin is involved in many physiological functions during early stages of development and in cancer and is required for functional plasma membrane expression of MCT4. We sought to determine if disruption of the MCT-Basigin interaction may be achieved with a small molecule. Using a cell-based drug-screening assay, we identified Acriflavine (ACF), a small molecule that inhibits the binding between Basigin and MCT4. Surface plasmon resonance and cellular thermal-shift-assays confirmed ACF binding to basigin in vitro and in live glioblastoma cells, respectively. ACF significantly inhibited growth and self-renewal potential of several glioblastoma neurosphere lines in vitro, and this activity was further augmented by hypoxia. Finally, treatment of mice bearing GSC-derived xenografts resulted in significant inhibition of tumor progression in early and late-stage disease. ACF treatment inhibited intratumoral expression of VEGF and tumor vascularization. Our work serves as a proof-of-concept as it shows, for the first time, that disruption of MCT binding to their chaperon, Basigin, may be an effective approach to target GSC and to inhibit angiogenesis and tumor progression.
Project description:Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.
Project description:N-myc downstream-regulated gene 1 (NDRG1) is a multifunctional protein associated with carcinogenesis and tumor progression. The function of NDRG1 in hepatocellular carcinoma (HCC) cells remains controversial. The present study investigated the role of NDRG1 in HCC as well as its molecular mechanism using a range of techniques, including western blot analysis, cellular proliferation test, wound healing assay and Transwell assay. In HCC, the levels of NDRG1 expression were highest in the cytoplasm, followed by the membrane, and were lowest in the nucleus. NDRG1 was revealed to inhibit the proliferation and invasion of BEL7402 cells, which facilitated the hypothesis that NDRG1 expression levels may be lower in cell line with a high metastatic potential compared with those in cell lines with a low metastatic potential. However, the present study identified that NDRG1 expression was higher in detached BEL7402 cells and MHCC-97H cells compared with that in attached BEL7402 cells and MHCC-97L cells. Thus, this finding was contrary to what was expected, suggesting that NDRG1 overexpression in the HCC with a high metastatic potential may be the compensatory mechanism. The human HCC BEL7402 cell line demonstrated a significant increase in the capability of motility, invasion and cellular proliferation following NDRG1-short hairpin RNA transfection. Integrin β3 (ITGB3) protein expression was increased in NDRG1-downregulated BEL7402 cells and SMMC7721 cells compared with that in the control cells. The present study suggested that NDRG1 may be a potential anti-tumor target for the treatment of patients with HCC. A potential mechanism for these roles of NDRG1 is by regulating ITGB3 expression; however, this requires additional investigation.
Project description:Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against β1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. β1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial β1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1β decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via β1- and α5-integrins and ANGPT2. Additionally, β1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5β1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, β1-integrin promotes endothelial barrier disruption during inflammation, and targeting β1-integrin signaling could serve as a novel means of blocking pathological vascular leak.