Redox-active endosomes mediate α5ß1 integrin signaling and promote chondrocyte matrix metalloproteinase production in osteoarthritis.

Mechanical cues sensed by integrins induce cells to produce proteases to remodel the extracellular matrix. Excessive protease production occurs in many degenerative diseases, including osteoarthritis, in which articular cartilage degradation is associated with the genesis of matrix protein fragments that can activate integrins. We investigated the mechanisms by which integrin signals may promote protease production in response to matrix changes in osteoarthritis. Using a fragment of the matrix protein fibronectin (FN) to activate the α5ß1 integrin in primary human chondrocytes, we found that endocytosis of the integrin and FN fragment complex drove the production of the matrix metalloproteinase MMP-13. Activation of α5ß1 by the FN fragment, but not by intact FN, was accompanied by reactive oxygen species (ROS) production initially at the cell surface, then in early endosomes. These ROS-producing endosomes (called redoxosomes) contained the integrin-FN fragment complex, the ROS-producing enzyme NADPH oxidase 2 (NOX2), and SRC, a redox-regulated kinase that promotes MMP-13 production. In contrast, intact FN was endocytosed and trafficked to recycling endosomes without inducing ROS production. Articular cartilage from patients with osteoarthritis showed increased amounts of SRC and the NOX2 complex component p67phox. Furthermore, we observed enhanced localization of SRC and p67phox at early endosomes, suggesting that redoxosomes could transmit and sustain integrin signaling in response to matrix damage. This signaling mechanism not only amplifies the production of matrix-degrading proteases but also establishes a self-perpetuating cycle that contributes to the ongoing degradation of cartilage matrix in osteoarthritis.

SEMA7a primes integrin α5ß1 engagement instructing fibroblast mechanotransduction, phenotype and transcriptional programming.

Integrins are cellular receptors that bind the extracellular matrix (ECM) and facilitate the transduction of biochemical and biophysical microenvironment cues into cellular responses. Upon engaging the ECM, integrin heterodimers must rapidly strengthen their binding with the ECM, resulting in the assembly of force-resistant and force-sensitive integrin associated complexes (IACs). The IACs constitute an essential apparatus for downstream signaling and fibroblast phenotypes. During wound healing, integrin signaling is essential for fibroblast motility, proliferation, ECM reorganization and, ultimately, restoration of tissue homeostasis. Semaphorin 7A (SEMA7a) has been previously implicated in post-injury inflammation and tissue fibrosis, yet little is known about SEMA7a's role in directing stromal cell, particularly fibroblast, behaviors. We demonstrate that SEMA7a regulates integrin signaling through cis-coupling with active integrin α5ß1 on the plasma membrane, enabling rapid integrin adhesion strengthening to fibronectin (Fn) and normal downstream mechanotransduction. This molecular function of SEMA7a potently regulates fibroblast adhesive, cytoskeletal, and migratory phenotype with strong evidence of downstream alterations in chromatin structure resulting in global transcriptomic reprogramming such that loss of SEMA7a expression is sufficient to impair the normal migratory and ECM assembly phenotype of fibroblasts resulting in significantly delayed tissue repair in vivo.

Implication of integrin α5ß1 in senescence of SK-Mel-147 human melanoma cells.

Downregulation of α5ß1 integrin in the SK-Mel-147 human melanoma culture model sharply inhibits the phenotypic manifestations of tumor progression: cell proliferation and clonal activity. This was accompanied by a 2-3-fold increase in the content of SA-ß-Gal positive cells thus indicating an increase in the cellular senescence phenotype. These changes were accompanied by a significant increase in the activity of p53 and p21 tumor suppressors and components of the PI3K/Akt/mTOR/p70 signaling pathway. Pharmacological inhibition of mTORC1 reduced the content of SA-ß-Gal positive cells in the population of α5ß1-deficient SK-Mel-147 cells. A similar effect was observed with pharmacological and genetic inhibition of the activity of Akt1, one of the three Akt protein kinase isoenzymes; suppression of other Akt isozymes did not affect melanoma cell senescence. The results presented in this work and previously obtained indicate that α5ß1 shares with other integrins of the ß1 family the function of cell protection from senescence. This function is realized via regulation of the PI3K/Akt1/mTOR signaling pathway, in which Akt1 exhibits a non-canonical activity.

Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5ß1.

Reticular adhesions (RAs) consist of integrin αvß5 and harbor flat clathrin lattices (FCLs), long-lasting structures with similar molecular composition as clathrin-mediated endocytosis (CME) carriers. Why FCLs and RAs colocalize is not known. Here, we show that RAs are assembled at FCLs in a process controlled by fibronectin (FN) and its receptor, integrin α5ß1. We observed that cells on FN-rich matrices displayed fewer FCLs and RAs. CME machinery inhibition abolished RAs and live-cell imaging showed that RA establishment requires FCL coassembly. The inhibitory activity of FN was mediated by the activation of integrin α5ß1 at Tensin1-positive fibrillar adhesions. Conventionally, endocytosis disassembles cellular adhesions by internalizing their components. Our results present a novel paradigm in the relationship between these two processes by showing that endocytic proteins can actively function in the assembly of cell adhesions. Furthermore, we show this novel adhesion assembly mechanism is coupled to cell migration via unique crosstalk between cell-matrix adhesions.

Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist.

Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against αv and ß3 but not α5 or ß1 integrins. Similarly, we observed direct binding of S1-RBD to recombinant human αvß3 and αvß6 integrins, but not α5ß1 integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an αv-selective integrin agonist. This study provides evidence that cell surface αv-containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.

Dysregulation of integrin αvß3 and α5ß1 impedes migration of placental endothelial cells in fetal growth restriction.

Placentas from pregnancies complicated by severe early-onset fetal growth restriction (FGR) exhibit diminished vascular development mediated by impaired angiogenesis, but underlying mechanisms remain unknown. In this study, we show that FGR endothelial cells demonstrate inherently reduced migratory capacity despite the presence of fibronectin, a matrix protein abundant in placental stroma that displays abnormal organization in FGR placentas. Thus, we hypothesized that aberrant endothelial-fibronectin interactions in FGR are a key mechanism underlying impaired FGR endothelial migration. Using human fetoplacental endothelial cells isolated from uncomplicated term control and FGR pregnancies, we assessed integrin α5ß1 and αvß3 regulation during cell migration. We show that endothelial integrin α5ß1 and αvß3 interactions with fibronectin are required for migration and that FGR endothelial cells responded differentially to integrin inhibition, indicating integrin dysregulation in FGR. Whole-cell expression was not different between groups. However, there were significantly more integrins in focal adhesions and reduced intracellular trafficking in FGR. These newly identified changes in FGR endothelial cellular processes represent previously unidentified mechanisms contributing to persistent angiogenic deficiencies in FGR.

The interactions between integrin α5ß1 of liver cancer cells and fibronectin of fibroblasts promote tumor growth and angiogenesis.

Hepatocellular carcinoma (HCC) progression is closely related to pathological fibrosis, which involves heterotypic intercellular interactions (HIIs) between liver cancer cells and fibroblasts. Here, we studied them in a direct coculture model, and identified fibronectin from fibroblasts and integrin-α5ß1 from liver cancer cells as the primary responsible molecules utilizing CRISPR/Cas9 gene-editing technology. Coculture led to the formation of 3D multilayer microstructures, and obvious fibronectin remodeling was caused by upregulated integrin-α5ß1, which greatly promoted cell growth in 3D microstructures. Integrin-α5 was more sensitive and specific than integrin-ß1 in this process. Subsequent mechanistic exploration revealed the activation of integrin-Src-FAK, AKT and ERK signaling pathways. Importantly, the growth-promoting effect of HIIs was verified in a xenograft tumor model, in which more blood vessels were observed in bigger tumors derived from the coculture group than that derived from monocultured groups. Hence, we conducted triculture by introducing human umbilical vein endothelial cells, which aligned to and differentiated along multilayer microstructures in an integrin-α5ß1 dependent manner. Furthermore, fibronectin, integrin-α5, and integrin-ß1 were upregulated in 52 HCC tumors, and fibronectin was related to microvascular invasion. Our findings identify fibronectin, integrin-α5, and integrin-ß1 as tumor microenvironment-related targets and provide a basis for combination targeted therapeutic strategies for future HCC treatment.

Characterization of the Role of Integrin α5ß1 in Platelet Function, Hemostasis, and Experimental Thrombosis.

OBJECTIVE: Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5ß1, a receptor for fibronectin. The aim of this study was to characterize the role of α5ß1 in megakaryopoiesis, platelet function, and to determine its importance in hemostasis and arterial thrombosis. APPROACH AND RESULTS: We generated a mouse strain deficient for integrin α5ß1 on megakaryocytes and platelets (PF4Cre-α5-/-). PF4Cre-α5-/- mice were viable, fertile, and presented no apparent signs of abnormality. Megakaryopoiesis appears unaltered as evidence by a normal megakaryocyte morphology and development, which is in agreement with a normal platelet count. Expression of the main platelet receptors and the response of PF4Cre-α5-/- platelets to a series of agonists were all completely normal. Adhesion and aggregation of PF4Cre-α5-/- platelets under shear flow on fibrinogen, laminin, or von Willebrand factor were unimpaired. In contrast, PF4Cre-α5-/- platelets displayed a marked decrease in adhesion, activation, and aggregation on fibrillar cellular fibronectin and collagen. PF4Cre-α5-/- mice presented no defect in a tail-bleeding time assay and no increase in inflammatory bleeding in a reverse passive Arthus model and a lipopolysaccharide pulmonary inflammation model. Finally, no defects were observed in three distinct experimental models of arterial thrombosis based on ferric chloride-induced injury of the carotid artery, mechanical injury of the abdominal aorta, or laser-induced injury of mesenteric vessels. CONCLUSION: In summary, this study shows that platelet integrin α5ß1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis.

More Is Not Always Better-the Double-Headed Role of Fibronectin in Staphylococcus aureus Host Cell Invasion.

While Staphylococcus aureus has classically been considered an extracellular pathogen, these bacteria are also capable of being taken up by host cells, including nonprofessional phagocytes such as endothelial cells, epithelial cells, or osteoblasts. The intracellular S. aureus lifestyle contributes to infection development. The predominant recognition and internalization pathway appears to be the binding of the bacteria via a fibronectin bridge to the α5ß1-integrin on the host cell membrane, followed by phagocytosis. Although osteoblasts showed high expression of α5ß1-integrin and fibronectin, and bacteria adhered to osteoblasts to a high proportion, here we demonstrate by internalization assays and immunofluorescence microscopy that S. aureus was less engulfed in osteoblasts than in epithelial cells. The addition of exogenous fibronectin during the infection of cells with S. aureus resulted in an increased uptake by epithelial cells but not by osteoblasts. This contrasts with the previous conception of the uptake mechanism, where high expression of integrin and fibronectin would promote the bacterial uptake into host cells. Extracellular fibronectin surrounding osteoblasts, but not epithelial cells, is organized in a fibrillary network. The inhibition of fibril formation, the short interfering RNA-mediated reduction of fibronectin expression, and the disruption of the fibronectin-fibril meshwork all resulted in a significant increase in S. aureus uptake by osteoblasts. Thus, the network of fibronectin fibrils appears to strongly reduce the uptake of S. aureus into a given host cell, indicating that the supramolecular structure of fibronectin determines the capacity of particular host cells to internalize the pathogen. IMPORTANCE Traditionally, Staphylococcus aureus has been considered an extracellular pathogen. However, among other factors, the frequent failure of antimicrobial therapy and the ability of the pathogen to cause recurrent disease have established the concept of eukaryotic invasion of the pathogen, thereby evading the host's immune system. In the current model of host cell invasion, bacteria initially bind to α5ß1 integrin on the host cell side via a fibronectin bridge, which eventually leads to phagocytosis of S. aureus by host cells. However, in this study, we demonstrate that not the crude amount but the supramolecular structure of fibronectin molecules deposited on the eukaryotic cell surface plays an essential role in bacterial uptake by host cells. Our findings explain the large differences of S. aureus uptake efficacy in different host cell types as well as in vivo differences between courses of bacterial infections and the localization of bacteria in different clinical settings.

Receptor and Molecular Mechanism of AGGF1 Signaling in Endothelial Cell Functions and Angiogenesis.

Objective: Angiogenic factor AGGF1 (angiogenic factor with G-patch and FHA [Forkhead-associated] domain 1) promotes angiogenesis as potently as VEGFA (vascular endothelial growth factor A) and regulates endothelial cell (EC) proliferation, migration, specification of multipotent hemangioblasts and venous ECs, hematopoiesis, and vascular development and causes vascular disease Klippel-Trenaunay syndrome when mutated. However, the receptor for AGGF1 and the underlying molecular mechanisms remain to be defined. Approach and Results: Using functional blocking studies with neutralizing antibodies, we identified [alpha]5[beta]1 as the receptor for AGGF1 on ECs. AGGF1 interacts with [alpha]5[beta]1 and activates FAK (focal adhesion kinase), Src (proto-oncogene tyrosine-protein kinase), and AKT (protein kinase B). Functional analysis of 12 serial N-terminal deletions and 13 C-terminal deletions by every 50 amino acids mapped the angiogenic domain of AGGF1 to a domain between amino acids 604-613 (FQRDDAPAS). The angiogenic domain is required for EC adhesion and migration, capillary tube formation, and AKT activation. The deletion of the angiogenic domain eliminated the effects of AGGF1 on therapeutic angiogenesis and increased blood flow in a mouse model for peripheral artery disease. A 40-mer or 15-mer peptide containing the angiogenic domain blocks AGGF1 function, however, a 15-mer peptide containing a single amino acid mutation from -RDD- to -RGD- (a classical RGD integrin-binding motif) failed to block AGGF1 function. Conclusions: We have identified integrin [alpha]5[beta]1 as an EC receptor for AGGF1 and a novel AGGF1-mediated signaling pathway of [alpha]5[beta]1-FAK-Src-AKT for angiogenesis. Our results identify an FQRDDAPAS angiogenic domain of AGGF1 crucial for its interaction with [alpha]5[beta]1 and signaling.

CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation.

The Scar/WAVE complex drives actin nucleation during cell migration. Interestingly, the same complex is important in forming membrane ruffles during macropinocytosis, a process mediating nutrient uptake and membrane receptor trafficking. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A, has not been characterized. Here, we implicate CYRI-A as a key regulator of macropinosome formation and integrin internalization. We find that CYRI-A is transiently recruited to nascent macropinosomes, dependent on PI3K and RAC1 activity. CYRI-A recruitment precedes RAB5A recruitment but follows sharply after RAC1 and actin signaling, consistent with it being a local inhibitor of actin polymerization. Depletion of both CYRI-A and -B results in enhanced surface expression of the α5ß1 integrin via reduced internalization. CYRI depletion enhanced migration, invasion, and anchorage-independent growth in 3D. Thus, CYRI-A is a dynamic regulator of macropinocytosis, functioning together with CYRI-B to regulate integrin trafficking.

Integrin α5ß1 Mediated Cellular Reorganization in Human Mesenchymal Stem Cells During Neuronal Differentiation.

BACKGROUND/AIM: Mesenchymal stem cells (MSCs) have been widely used for yielding neurons in culture to study nervous system pathologies and develop regenerative approaches. In this study, cellular rearrangements of human MSCs related to the expression of the fibronectin common receptor integrin α5ß1 and its cell surface localization during neuronal differentiation, were examined. MATERIALS AND METHODS: Proliferation kinetics of neuronal induced hMSCs (hMd-Neurons) were quantified by BrdU assay, and hMd-Neurons were immunostained for neuronal marker expression. Additionally, cDNA and protein samples were collected at different time points for integrin α5ß1 expression analysis. RESULTS: Endogenous integrin α5ß1 expression was significantly upregulated by day 6 and maintained until day 12. Cell surface localization of α5ß1 integrin was increased by day 6; the integrin was internalized into the cytosol by day 12. CONCLUSION: Integrin dynamics around day 6 of differentiation might be involved in neuronal differentiation and maturation or specification of hMd-Neurons.

Integrin αVß1 regulates procollagen I production through a non-canonical transforming growth factor ß signaling pathway in human hepatic stellate cells.

Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor ß (TGF-ß), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVß1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-ß1-activated primary human HSCs. Unexpectedly, either a selective αVß1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-ß1-activated procollagen I production in primary human HSCs, in which the role of ß1 integrin was confirmed by ITGB1 siRNA. In contrast with an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-ß1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-ß-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVß1 integrin is involved in non-canonical TGF-ß signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-ß1 induced phosphorylation of ERK1/2 and decreased TGF-ß1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-ß1 induced procollagen I production. Taken together, current data indicate that αVß1 integrin can regulate TGF-ß signaling independent of its reported role in activating latent TGF-ß. Our data further support that αVß1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.

IL-1ß mediated nanoscale surface clustering of integrin α5ß1 regulates the adhesion of mesenchymal stem cells.

Clinical use of human mesenchymal stem cells (hMSCs) is limited due to their rapid clearance, reducing their therapeutic efficacy. The inflammatory cytokine IL-1ß activates hMSCs and is known to enhance their engraftment. Consequently, understanding the molecular mechanism of this inflammation-triggered adhesion is of great clinical interest to improving hMSC retention at sites of tissue damage. Integrins are cell-matrix adhesion receptors, and clustering of integrins at the nanoscale underlies cell adhesion. Here, we found that IL-1ß enhances adhesion of hMSCs via increased focal adhesion contacts in an α5ß1 integrin-specific manner. Further, through quantitative super-resolution imaging we elucidated that IL-1ß specifically increases nanoscale integrin α5ß1 availability and clustering at the plasma membrane, whilst conserving cluster area. Taken together, these results demonstrate that hMSC adhesion via IL-1ß stimulation is partly regulated through integrin α5ß1 spatial organization at the cell surface. These results provide new insight into integrin clustering in inflammation and provide a rational basis for design of therapies directed at improving hMSC engraftment.

Interrupting TGF-ß1/CCN2/integrin-α5ß1 signaling alleviates high mechanical-stress caused chondrocyte fibrosis.

OBJECTIVE: Mechanical-stress has been reported to trigger cartilage fibrosis, in which transforming growth factor (TGF)-ß and connective tissue growth factor (CCN2) are involved. However, the function of integrin-α5ß1, a cytomembrane receptor, on mechanical-stress related fibrosis has not yet been elucidated. This study aims to reveal the interaction of TGF-ß1/CCN2/integrin-α5ß1 in the mechanical-stress induced chondrocyte (CH) fibrosis. PATIENTS AND METHODS: We used different levels (5% and 10%) of cyclic tension simulation (CTS) to stretch CHs and observed the gene expression of TGF-ß1/CCN2/integrin-α5ß1 as well as the fibrous related genes containing collagen I/II/III, Runx2, MMP13, and ADAMTS-5 by real-time polymerase chain reaction (RT-PCR) or immunofluorescence. We used the siRNA or the corresponding antagonist of TGF-ß1, CCN2, integrin-α5ß1 during the CTS to clear the effect of them in the fibrosis progress. In addition, to verify the crosstalk between TGF-ß1, CCN2, and integrin-α5ß1, we used the recombinant human (rh)-TGF-ß1 and CCN2 to culture CHs without CST. RESULTS: 24 hours-10% CTS was sufficient to induce a decrease of collagen II and increase the collagen I/III, Runx2, MMP13, and ADAMTS-5 gene expression. Under CTS, TGF-ß1 silencing resulted in a decline of CCN2, integrin-α5ß1, and alleviated the CHs fibrosis. Apart from this, blocking CCN2 or integrin-α5ß1 expression also contributed to the suppression of 10% CTS induced CHs fibrosis. Meanwhile, the exogenic protein supplement raised the cellular TGF-ß1 or CCN2 expression and increased the integrin-α5ß1 mRNA level. However, the downregulation of TGF-ß1 or CCN2 did not affect integrin-α5ß1 expression, whether the CTS exited or not. CONCLUSIONS: High mechanical-stress induces CHs fibrosis via the activation of TGF-ß1/CCN2/integrin-α5ß1 signaling, and interrupting the TGF-ß1, CCN2, or integrin-α5ß1 expression can alleviate the fibrous process.

Cell-Extracellular Matrix Interactions Play Multiple Essential Roles in Aortic Arch Development.

RATIONALE: Defects in the morphogenesis of the fourth pharyngeal arch arteries (PAAs) give rise to lethal birth defects. Understanding genes and mechanisms regulating PAA formation will provide important insights into the etiology and treatments for congenital heart disease. OBJECTIVE: Cell-ECM (extracellular matrix) interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin α5ß1 and Fn1 (fibronectin) expressed in the Isl1 lineages regulate PAA formation. The objective of the current studies was to investigate cellular mechanisms by which integrin α5ß1 and Fn1 regulate aortic arch artery morphogenesis. METHODS AND RESULTS: Using temporal lineage tracing, whole-mount confocal imaging, and quantitative analysis of the second heart field (SHF) and endothelial cell (EC) dynamics, we show that the majority of PAA EC progenitors arise by E7.5 in the SHF and contribute to pharyngeal arch endothelium between E7.5 and E9.5. Consequently, SHF-derived ECs in the pharyngeal arches form a plexus of small blood vessels, which remodels into the PAAs by 35 somites. The remodeling of the vascular plexus is orchestrated by signals dependent on the pharyngeal ECM microenvironment, extrinsic to the endothelium. Conditional ablation of integrin α5ß1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates aortic arch artery morphogenesis at multiple steps: (1) accumulation of SHF-derived ECs in the pharyngeal arches, (2) remodeling of the EC plexus in the fourth arches into the PAAs, and (3) differentiation of neural crest-derived cells adjacent to the PAA endothelium into vascular smooth muscle cells. CONCLUSIONS: PAA formation is a multistep process entailing dynamic contribution of SHF-derived ECs to pharyngeal arches, the remodeling of endothelial plexus into the PAAs, and the remodeling of the PAAs into the aortic arch artery and its major branches. Cell-ECM interactions regulated by integrin α5ß1 and Fn1 play essential roles at each of these developmental stages.

Quantification of Integrin Activation and Ligation in Adherent Cells.

Integrin activation is a crucial event for multiple biological functions. Therefore, methods to detect integrin activation are vital. Since the main cellular function of integrins is adhesion, we and others utilize this feature to measure integrin activation. Here, we describe how to detect the activity of the fibronectin-binding integrin α5ß1 using a fusion of glutathione S-transferase (GST) to the 9th, 10th, and 11th type III repeats on fibronectin (GST-FNIII9-11). Moreover, we detail how to measure αvß3 integrin activity using the ligand-mimetic WOW-1 antibody that selectively binds unoccupied αvß3 integrins. Finally, we describe methods of testing ligation of fibronectin-binding integrins utilizing monoclonal antibodies against ligand-induced binding sites (LIBS).

Quantifying Polarized Extracellular Matrix Secretion in Cultured Endothelial Cells.

In endothelial cells (ECs), the onset of apicobasal polarity is primarily regulated by the interaction of integrins with the surrounding extracellular matrix (ECM). ECs secrete and polymerize fibronectin (FN), a unique, permissive substrate that allows for vascular morphogenesis and lumen formation. We previously identified a signaling pathway that, under the control of the adhesion site adaptor protein PPFIA1, integrates the polarized secretion of freshly synthesized FN with the recycling of conformationally active α5ß1 integrin, the main FN receptor in ECs. To characterize the functional role of PPFIA1-dependent signaling in ECs, we set up a Transwell-based assay to quantify the polarized secretion of ECM proteins. To this aim, we allowed ECs to form a confluent monolayer on the Transwell membrane and checked its integrity by measuring transendothelial electric resistance and controlling the stability of tight junctions over time by fluorescent confocal microscope analysis. Finally, we quantified apical and basolateral FN secretion in control and PPFIA1-silenced EC culture medium by western blot analysis coupled to spike-in normalization.

Gefitinib induces EGFR and α5ß1 integrin co-endocytosis in glioblastoma cells.

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.

Implication of integrins α3ß1 and α5ß1 in invasion and anoikis of SK-Mel-147 human melanoma cells: non-canonical functions of protein kinase Akt.

Downregulation of integrins α3ß1 and α5ß1 strongly decreased cell colony formation and in vitro invasion and markedly enhanced anoikis in SK-Mel-147 human melanoma cells. These modifications were accompanied by a marked increase in the levels of active Akt protein kinase, which indicated it played a non-canonical function in the melanoma cells. Pharmacological inhibition of Akt1, an Akt isozyme, in cells depleted of α3ß1 or α5ß1 restored their invasive activity, while inhibition of the Akt 2 isoform did not cause a visible effect. Similar to our previous results with the α2ß1 integrin, this finding suggested that in signaling pathways initiated by α3ß1 and α5ß1, the Akt1 isoform performs a non-canonical function in regulating invasive phenotype of melanoma cells. In contrast, when the effects of Akt inhibitors on anoikis of the melanoma cells were compared, the Akt2 isoform demonstrated a non-canonical activity in which Akt2 suppression led to a significant attenuation of apoptosis in cells with downregulated α3ß1 or α5ß1. Our results were the first evidence that, in the same tumor cells, different integrins can control various manifestations of tumor progression through distinct signaling pathways that are both common to various integrins and specific to a particular receptor.