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Cells intricately sense mechanical forces from their surroundings, driving biophysical and biochemical activities. This mechanosensing phenomenon occurs at the cell-matrix interface, where mechanical forces resulting from cellular motion, such as migration or matrix stretching, are exchanged through surface receptors, primarily integrins, and their corresponding matrix ligands. A pivotal player in this interaction is the α 5 β 1 integrin and fibronectin (FN) bond, known for its role in establishing cell adhesion sites for migration. However, upregulation of the α 5 β 1 -FN bond is associated with uncontrolled cell metastasis. This bond operates through catch bond dynamics, wherein the bond lifetime paradoxically increases with greater force. The mechanism sustaining the characteristic catch bond dynamics of α 5 β 1 -FN remains unclear. Leveraging molecular dynamics simulations, our approach unveils a pivot-clip mechanism. Two key binding sites on FN, namely the synergy site and the RGD (Arg-Gly-Asp) motif, act as active points for structural changes in α 5 β 1 integrin. Conformational adaptations at these sites are induced by a series of hydrogen bond formations and breaks at the synergy site. We disrupt these adaptations through a double mutation on FN, known to reduce cell adhesion. A whole-cell finite-element model is employed to elucidate how the synergy site may promote dynamic α 5 β 1 -FN binding, resisting cell contraction. In summary, our study integrates molecular- and cellular-level modeling to propose that FN's synergy site reinforces cell adhesion through enhanced binding dynamics and a mechanosensitive pivot-clip mechanism. This work sheds light on the interplay between mechanical forces and cell-matrix interactions, contributing to our understanding of cellular behaviors in physiological and pathological contexts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5β1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 CY Cergy Paris University. Published by Elsevier Inc. All rights reserved.)

The physical properties of nanoscale cell-extracellular matrix (ECM) ligands profoundly impact biological processes, such as adhesion, motility, and differentiation. While the mechanoresponse of cells to static ligands is well-studied, the effect of dynamic ligand presentation with "adaptive" properties on cell mechanotransduction remains less understood. Utilizing a controllable diffusible ligand interface, we demonstrated that cells on surfaces with rapid ligand mobility could recruit ligands through activating integrin α5β1, leading to faster focal adhesion growth and spreading at the early adhesion stage. By leveraging UV-light-sensitive anchor molecules to trigger a "dynamic to static" transformation of ligands, we sequentially activated α5β1 and αvβ3 integrins, significantly promoting osteogenic differentiation of mesenchymal stem cells. This study illustrates how manipulating molecular dynamics can directly influence stem cell fate, suggesting the potential of "sequentially" controlled mobile surfaces as adaptable platforms for engineering smart biomaterial coatings.

Background and Purpose: Icariin, an 8-prenylated flavonoid glycoside, is an anabolic agent that could exert rapid estrogenic actions via ligand-independent activation of estrogen receptor alpha (ERα) in osteoblastic cells to promote osteogenesis. However, relatively little is known about its direct cellular target, its protective effects, and cell adhesion activities in bone marrow stromal cells (BMSCs) against microgravity. In the present study, the effects of icariin on osteogenesis and cell adhesion under microgravity were examined with the involvement of integrin receptor α5β1, connexin 43, and CAMs., Study Design and Methods: Icariin was orally administered to 6-month-old ovariectomized (OVX) Sprague-Dawley (SD) rats for 3 months through daily intake of phytoestrogen-free rodent diets containing icariin at 2 different dosages (50 and 500 ppm). BMSCs were harvested for experiments and RNA-sequencing analysis to examine the mechanism of action of icariin and its direct cellular target in stimulating osteogenesis., Results: The results revealed that icariin induced the expression of cell adhesion molecules (CAMs) and protected against microgravity-induced disruption of actin cytoskeleton and the loss of osteogenic activities in BMSCs through the activation of connexin-43 (Cx43) and Ras homolog family member A (RhoA) and Rac family small GTPase 1 (Rac1)-mediated signaling pathways. Computerized molecular docking techniques and the competitive solid-phase binding ELISA assay confirmed that icariin could be a direct ligand of integrin alpha 5 beta 1 (α5β1), and it could also increase the protein expression of integrin α5β1 for mechanosensing., Conclusion: Our findings suggest that icariin could directly activate cell adhesion signaling by binding to integrin α5β1, which opens up new avenues for the development of integrin α5β1 ligand as an agent to protect against unloading-induced bone loss., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier GmbH.)

Age-related macular degeneration (AMD) is a progressive, degenerative disease of the macula. The formation of macular neovascularization (MNV) and subretinal fibrosis of AMD is the most classic cause of the loss of vision in older adults worldwide. While the underlying causes of MNV and subretinal fibrosis remain elusive, the common feature of many common retinal diseases is changes the proportions of protein deposition in extracellular matrix (ECM) when compared to normal tissue. In ECM, fibronectin (FN) is a crucial component and plays a pivotal part not only in fibrotic diseases but also in the process of angiogenesis. The study aims to understand the role of ligand FN and its common integrin receptor α5β1 on MNV, and to understand the molecular mechanism involved. To study this, the laser-induced MNV mouse model and the rhesus macaque choroid-retinal endothelial cell line (RF/6A) chemical hypoxia mode were established, and the FN-α5β1 expression levels were detected by immunohistochemistry (IHC) and quantitative real-time PCR analysis (qRT-PCR). Fibronectin expression was silenced using small interfering RNA (siRNA) targeting FN. The tube formation and vitro scratch assays were used to assess the ability to form blood vessels and cell migration. To measure the formation of MNV, immunofluorescence, and Western blot assays were used. These results revealed that the expressions of FN and integrin α5β1 were distinctly increased in the laser-induced MNV mouse model and in the RF/6A cytochemically induced hypoxia model, and the expression tendency was identical. After the use of FN siRNA, the tube formation and migration abilities of the RF/6A cells were lower, the ability of endothelial cells to proliferate was confined and the scope of damage caused by the laser in animal models was significantly cut down. In addition, FN gene knockdown dramatically inhibited the expression of Wnt/β-catenin signal. The interaction of FN with the integrin receptor α5β1 in the constructed model, which may act through the Wnt/β-catenin signaling pathway, was confirmed in this study. In conclusion, FN may be a potential new molecular target for the prevention and treatment of subretinal fibrosis and MNV., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Acute type A aortic dissection (AAAD) is a devastating disease. Human aortic smooth muscle cells (HASMCs) exhibit decreased proliferation and increased apoptosis, and integrin α5β1 and FAK are important proangiogenic factors involved in regulating angiogenesis. The aim of this study was to investigate the role of integrin α5β1 and FAK in patients with AAAD and the potential underlying mechanisms., Methods: Aortic tissue samples were obtained from 8 patients with AAAD and 4 organ donors at Zhongshan Hospital of Fudan University. The level of apoptosis in the aortic tissues was assessed by immunohistochemical (IHC) staining and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assays. The expression of integrin α5β1 and FAK was determined. Integrin α5β1 was found to be significantly expressed in HASMCs, and its interaction with FAK was assessed via coimmunoprecipitation (Co-IP) analysis. Proliferation and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assays and flow cytometry after integrin α5β1 deficiency., Results: The levels of integrin α5β1 and FAK were both significantly decreased in patients with AAAD. Downregulating the expression of integrin α5β1-FAK strongly increased apoptosis and decreased proliferation in HASMCs, indicating that integrin α5β1-FAK might play an important role in the development of AAAD., Conclusions: Downregulation of integrin α5β1-FAK is associated with increased apoptosis and decreased proliferation in aortic smooth muscle cells and may be a potential therapeutic strategy for AAAD., (© 2024. The Author(s).)

Primary tumor growth and metastasis in triple-negative breast cancer (TNBC) require supporting vasculature, which develop through a combination of endothelial angiogenesis and vasculogenic mimicry (VM), a process associated with aggressive metastatic behavior in which vascular-like structures are lined by tumor cells. We developed αEGFR-E-P125A, an antibody-endostatin fusion protein that delivers a dimeric, mutant endostatin (E-P125A) payload that inhibits TNBC angiogenesis and VM in vitro and in vivo. To characterize the mechanisms associated with induction and inhibition of VM, RNA sequencing (RNA-seq) of MDA-MB-231-4175 TNBC cells grown in a monolayer (two-dimensional) was compared with cells plated on Matrigel undergoing VM [three-dimensional (3D)]. We then compared RNA-seq between TNBC cells in 3D and cells in 3D with VM inhibited by αEGFR-E-P125A (EGFR-E-P125A). Gene set enrichment analysis demonstrated that VM induction activated the IL6-JAK-STAT3 and angiogenesis pathways, which were downregulated by αEGFR-E-P125A treatment.Correlative analysis of the phosphoproteome demonstrated decreased EGFR phosphorylation at Y1069, along with decreased phosphorylation of focal adhesion kinase Y397 and STAT3 Y705 sites downstream of α5β1 integrin. Suppression of phosphorylation events downstream of EGFR and α5β1 integrin demonstrated that αEGFR-E-P125A interferes with ligand-receptor activation, inhibits VM, and overcomes oncogenic signaling associated with EGFR and α5β1 integrin cross-talk. In vivo, αEGFR-E-P125A treatment decreased primary tumor growth and VM, reduced lung metastasis, and confirmed the inhibition of signaling events observed in vitro. Simultaneous inhibition of EGFR and α5β1 integrin signaling by αEGFR-E-P125A is a promising strategy for the inhibition of VM, tumor growth, motility, and metastasis in TNBC and other EGFR-overexpressing tumors., Significance: αEGFR-E-P125A reduces VM, angiogenesis, tumor growth, and metastasis by inhibiting EGFR and α5β1 integrin signaling, and is a promising therapeutic agent for TNBC treatment, used alone or in combination with chemotherapy., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Endothelial dysfunction plays a pivotal role in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Dipeptidyl peptidase IV (DPP-4), a cell surface glycoprotein, has been implicated in endothelial inflammation and barrier dysfunction. In this study, the role of DPP-4 on lipopolysaccharide (LPS)-induced pulmonary microvascular endothelial cells (HPMECs) dysfunction and the underlying mechanism were investigated by siRNA-mediated knockdown of DPP-4. Our results indicated that LPS (1 μg/ml) challenge resulted in either the production and releasing of DPP-4, as well as the secretion of IL-6 and IL-8 in HPMECs. DPP-4 knockdown inhibited chemokine releasing and monolayer hyper-permeability in LPS challenged HPMECs. When cocultured with human polymorphonuclear neutrophils (PMNs), DPP4 knockdown suppressed LPS-induced neutrophil-endothelial adhesion, PMN chemotaxis and trans-endothelial migration. Western blotting showed that DPP-4 knockdown attenuated LPS-induced activation of TLR4/NF-κB pathway. Immunoprecipitation and liquid chromatography-tandem mass spectrometry revealed that DPP-4 mediated LPS-induced endothelial inflammation by interacting with integrin-α5β1. Moreover, exogenous soluble DPP-4 treatment sufficiently activated integrin-α5β1 downstream FAK/AKT/NF-κB signaling, thereafter inducing ICAM-1 upregulation in HPMECs. Collectively, our results suggest that endothelia synthesis and release DPP-4 under the stress of endotoxin, which interact with integrin-α5β1 complex in an autocrine or paracrine manner to exacerbate endothelial inflammation and enhance endothelial cell permeability. Therefore, blocking DDP-4 could be a potential therapeutic strategy to prevent endothelial dysfunction in ALI/ARDS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Integrins are cell surface nanosized receptors crucial for cell motility and mechanosensing of the extracellular environment, which are often targeted for the development of biomaterials and nanomedicines. As a key feature of integrins, their activity, structure and behavior are highly mechanosensitive, which are regulated by mechanical forces down to pico-Newton scale. Using single-molecule biomechanical approaches, we compared the force-modulated ectodomain bending/unbending conformational changes of two integrin species, α 5 β 1 and α V β 3 . It was found that the conformation of integrin α 5 β 1 is determined by a threshold head-to-tail tension. By comparison, integrin α V β 3 exhibits bistability even without force and can spontaneously transition between the bent and extended conformations with an apparent transition time under a wide range of forces. Molecular dynamics simulations observed almost concurrent disruption of ∼2 hydrogen bonds during integrin α 5 β 1 unbending, but consecutive disruption of ∼7 hydrogen bonds during integrin α V β 3 unbending. Accordingly, we constructed a canonical energy landscape for integrin α 5 β 1 with a single energy well that traps the integrin in the bent state until sufficient force tilts the energy landscape to allow the conformational transition. In contrast, the energy landscape of integrin α V β 3 conformational changes was constructed with hexa-stable intermediate states and intermediate energy barriers that segregate the conformational change process into multiple small steps. Our study elucidates the different biomechanical inner workings of integrins α 5 β 1 and α V β 3 at the submolecular level, helps understand their mechanosignaling processes and how their respective functions are facilitated by their distinctive mechanosensitivities, and provides useful design principles for the engineering of protein-based biomechanical nanomachines.

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects host cells by engaging its spike (S) protein with human ACE2 receptor. Recent studies suggest the involvement of integrins in SARS-CoV-2 infection through interaction with the S protein, but the underlying mechanism is not well understood. This study investigated the role of integrin α 5 β 1 , which recognizes the Arg-Gly-Asp (RGD) motif in its physiological ligands, in S-mediated virus entry and cell-cell fusion. Our results showed that α 5 β 1 does not directly contribute to S-mediated cell entry, but it enhances S-mediated cell-cell fusion in collaboration with ACE2. This effect cannot be inhibited by the putative α 5 β 1 inhibitor ATN-161 or the high-affinity RGD-mimetic inhibitor MK-0429 but requires the participation of α 5 cytoplasmic tail (CT). We detected a direct interaction between α 5 β 1 and the S protein, but this interaction does not rely on the RGD-containing receptor binding domain of the S1 subunit of the S protein. Instead, it involves the S2 subunit of the S protein and α 5 β 1 homo-oligomerization. Furthermore, we found that the S protein induces inflammatory responses in human endothelial cells, characterized by NF-κB activation, gasdermin D cleavage, and increased secretion of proinflammatory cytokines IL-6 and IL-1β. These effects can be attenuated by the loss of α 5 expression or inhibition of the α 5 CT binding protein phosphodiesterase-4D (PDE4D), suggesting the involvement of α 5 CT and PDE4D pathway. These findings provide molecular insights into the pathogenesis of SARS-CoV-2 mediated by a nonclassical RGD-independent ligand-binding and signaling function of integrin α 5 β 1 and suggest potential targets for antiviral treatment., Competing Interests: Competing interests statement:The authors declare no competing interest.

Staphylococcus aureus, a Gram-positive bacterial pathogen, is an urgent health threat causing a wide range of clinical infections. Originally viewed as a strict extracellular pathogen, accumulating evidence has revealed S. aureus to be a facultative intracellular pathogen subverting host cell signalling to support invasion. The majority of clinical isolates produce fibronectin-binding proteins A and B (FnBPA and FnBPB) to interact with host integrin α5β1, a key component of focal adhesions. S. aureus binding of integrin α5β1 promotes its clustering on the host cell surface, triggering activation of focal adhesion kinase (FAK) and cytoskeleton rearrangements to promote bacterial invasion into non-phagocytic cells. Here, we discover that septins, a component of the cytoskeleton that assembles on membranes, are recruited as collar-like structures with actin to S. aureus invasion sites engaging integrin α5β1. To investigate septin recruitment to the plasma membrane in a bacteria-free system, we used FnBPA-coated latex beads and showed that septins are recruited upon activation of integrin α5β1. SEPT2 depletion reduced S. aureus invasion, but increased surface expression of integrin α5 and adhesion of S. aureus to host cells. Consistent with this, SEPT2 depletion increased cellular protein levels of integrin α5 and β1 subunits, as well as FAK. Collectively, these results provide insights into regulation of integrin α5β1 and invasion of S. aureus by the septin cytoskeleton., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Interests The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Metastasis leads to a high mortality rate in colorectal cancer (CRC). Increased neutrophil extracellular traps (NETs) formation is one of the main causes of metastasis. However, the mechanism of NETs-mediated metastasis remains unclear and effective treatments are lacking. In this study, we found neutrophils from CRC patients have enhanced NETs formation capacity and increased NETs positively correlate with CRC progression. By quantitative proteomic analysis of clinical samples and cell lines, we found that decreased secreted protein acidic and rich in cysteine (SPARC) results in massive NETs formation and integrin α5β1 is the hub protein of NETs-tumor cell interaction. Mechanistically, SPARC regulates the activation of the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) pathway by interacting with the receptor for activated C kinase 1 (RACK1). Over-activated NADPH oxidase generates more reactive oxygen species (ROS), leading to the release of NETs. Then, NETs upregulate the expression of integrin α5β1 in tumor cells, which enhances adhesion and activates the downstream signaling pathways to promote proliferation and migration. The combination of NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and integrin α5β1 inhibitor ATN-161 (Ac-PHSCN-NH2) effectively suppresses tumor progression in vivo. Our work reveals the mechanistic link between NETs and tumor progression and suggests a combination therapy against NETs-mediated metastasis for CRC.

Fibronectin (FN) fragments stimulate catabolic signaling, and, by binding to integrins, they induce chondrocytes to increase the production of matrix metalloproteinases, including MMP-13. In this issue of Science Signaling , Miao et al. reveal that internalization of a FN fragment, but not intact FN, by α5β1 integrin results in the formation of ROS-producing endosomes (redoxosomes) through which chondrocytes detect and respond to damaged matrix by producing more MMP-13.

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 α5β1 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 α5β1 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 p67 phox . Furthermore, we observed enhanced localization of SRC and p67 phox 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.

During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro , little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. Using the HSAEC1-KT human small airway epithelial (HSAE) cell line, we developed an in vitro model to study the interaction of two strains of A. fumigatus with these cells. We then compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAE cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5β1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5β1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro . Importance During the initiation of invasive aspergillosis, Aspergillus fumigatus interacts with the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus -epithelial cell interactions in vitro used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line; the interactions of fungi with terminal bronchiolar epithelial cells were not investigated. Using the TERT-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line, we developed an in vitro model of the interactions of A. fumigatus with bronchiolar epithelial cells. We discovered that A. fumigatus invades and damages A549 and HSAE cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells., Competing Interests: The authors declare no conflict of interest.

Tumor initiation at either primary or metastatic sites is an inefficient process in which tumor cells must fulfill a series of conditions. One critical condition involves the ability of individual tumor-initiating cells to overcome 'isolation stress', enabling them to survive within harsh isolating microenvironments that can feature nutrient stress, hypoxia, oxidative stress and the absence of a proper extracellular matrix (ECM). In response to isolation stress, tumor cells can exploit various adaptive strategies to develop stress tolerance and gain stemness features. In this Opinion, we discuss how strategies such as the induction of certain cell surface receptors and deposition of ECM proteins enable tumor cells to endure isolation stress, thereby gaining tumor-initiating potential. As examples, we highlight recent findings from our group demonstrating how exposure of tumor cells to isolation stress upregulates the G-protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), its downstream target fibronectin and two fibronectin-binding integrins, α5β1 and αvβ3. These responses create a fibronectin-rich niche for tumor cells, ultimately driving stress tolerance, cancer stemness and tumor initiation. We suggest that approaches to prevent cancer cells from adapting to stress by suppressing LPAR4 induction, blocking its downstream signaling or disrupting fibronectin-integrin interactions hold promise as potential strategies for cancer treatment., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Objective: Clostridium perfringens causes food poisoning and gas gangrene, a serious wound-associated infection. C. perfringens cells adhere to collagen via fibronectin (Fn). We investigated whether the peptidoglycan hydrolase of C. perfringens, i.e., autolysin (Acp), is implicated in Fn binding to C. perfringens cells., Methods: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA., Results: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells., Conclusions: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5β1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5β1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Reticular adhesions (RAs) consist of integrin αvβ5 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 α5β1. 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 α5β1 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., (© 2023 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.)

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 α5β1 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., Competing Interests: Declaration of Competing Interest The authors declare that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Cells regulate adhesion to the fibrillar extracellular matrix (ECM) of which fibronectin is an essential component. However, most studies characterize cell adhesion to globular fibronectin substrates at time scales long after cells polarize and migrate. To overcome this limitation, a simple and scalable method to engineer biomimetic 3D fibrillar fibronectin matrices is introduced and how they are sensed by fibroblasts from the onset of attachment is characterized. Compared to globular fibronectin substrates, fibroblasts accelerate adhesion initiation and strengthening within seconds to fibrillar fibronectin matrices via α5β1 integrin and syndecan-4. This regulation, which additionally accelerates on stiffened fibrillar matrices, involves actin polymerization, actomyosin contraction, and the cytoplasmic proteins paxillin, focal adhesion kinase, and phosphoinositide 3-kinase. Furthermore, this immediate sensing and adhesion of fibroblast to fibrillar fibronectin guides migration speed, persistency, and proliferation range from hours to weeks. The findings highlight that fibrillar fibronectin matrices, compared to widely-used globular fibronectin, trigger short- and long-term cell decisions very differently and urge the use of such matrices to better understand in vivo interactions of cells and ECMs. The engineered fibronectin matrices, which can be printed onto non-biological surfaces without loss of function, open avenues for various cell biological, tissue engineering and medical applications., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

CD40L is expressed in activated T cells, and it plays a major role in immune response and is a major therapeutic target for inflammation. High IgM syndrome type 1 (HIGM1) is a congenital functional defect in CD40L/CD40 signaling due to defective CD40L. CD40L is also stored in platelet granules and transported to the surface upon platelet activation. Platelet integrin αIIbβ3 is known to bind to fibrinogen and activation of αIIbβ3 is a key event that triggers platelet aggregation. Also, the KGD motif is critical for αIIbβ3 binding and the interaction stabilizes thrombus. Previous studies showed that CD40L binds to and activates integrins αvβ3 and α5β1 and that HIGM1 mutations are clustered in the integrin-binding sites. However, the specifics of CD40L binding to αIIbβ3 were unclear. Here, we show that CD40L binds to αIIbβ3 in a KGD-independent manner using CD40L that lacks the KGD motif. Two HIGM1 mutants, S128E/E129G and L155P, reduced the binding of CD40L to the classical ligand-binding site (site 1) of αIIbβ3, indicating that αIIbβ3 binds to the outer surface of CD40L trimer. Also, CD40L bound to the allosteric site (site 2) of αIIbβ3 and allosterically activated αIIbβ3 without inside-out signaling. Two HIMG1 mutants, K143T and G144E, on the surface of trimeric CD40L suppressed CD40L-induced αIIbβ3 activation. These findings suggest that CD40L binds to αIIbβ3 in a manner different from that of αvβ3 and α5β1 and induces αIIbβ3 activation. HIGM1 mutations are clustered in αIIbβ3 binding sites in CD40L and are predicted to suppress thrombus formation and immune responses through αIIbβ3.

Integrins act as cell‑matrix adhesion molecules involved in cell attachment to the extracellular matrix and generate signals that respond to cancer metastasis. Integrin α5β1 is a heterodimer with α5 and β1 subunits and mediates cell adhesion and migration of cancer cells. Integrins are transcriptionally regulated by the Janus kinase (JAK)/STAT signaling pathways. Our previous study revealed that Helicobacter pylori increased the levels of reactive oxygen species (ROS), which activate JAK1/STAT3 in gastric cancer AGS cells in vitro . Astaxanthin (ASX) has been reported to be an effective antioxidant and anticancer nutrient. The present study investigated whether ASX suppresses H. pylori ‑induced integrin α5 expression, cell adhesion and migration and whether ASX reduces ROS levels and suppresses phosphorylation of JAK1/STAT3 in gastric cancer AGS cells stimulated with H. pylori . The effect of ASX was determined using a dichlorofluorescein fluorescence assay, western blot analysis, adhesion assay and wound‑healing assay in AGS cells stimulated with H. pylori. The results demonstrated that H. pylori increased the expression levels of integrin α5, without affecting integrin β1, and increased cell adhesion and migration of AGS cells. ASX reduced ROS levels and suppressed JAK1/STAT3 activation, integrin α5 expression, cell adhesion and migration of H. pylori ‑stimulated AGS cells. In addition, both a JAK/STAT inhibitor, AG490, and an integrin α5β1 antagonist, K34C, suppressed cell adhesion and migration in H. pylori‑stimulated AGS cells. AG490 inhibited integrin α5 expression in AGS cells stimulated with H. pylori . In conclusion, ASX inhibited H. pylori ‑induced integrin α5‑mediated cell adhesion and migration by decreasing the levels of ROS and suppressing JAK1/STAT3 activation in gastric epithelial cells.

Fibronectin (FN)-binding integrins control a variety of cellular responses through Rho GTPases. The FN-binding integrins, αvβ3 and α5β1, are known to induce different effects on cell morphology and motility. Here, we report that FN-bound αvβ3 integrin, but not FN-bound α5β1 integrin, triggers the dissociation of the RhoA GEF Lfc (also known as GEF-H1 and ARHGEF2 in humans) from microtubules (MTs), leading to the activation of RhoA, formation of stress fibres and maturation of focal adhesions (FAs). Conversely, loss of Lfc expression decreases RhoA activity, stress fibre formation and FA size, suggesting that Lfc is the major GEF downstream of FN-bound αvβ3 that controls RhoA activity. Mechanistically, FN-engaged αvβ3 integrin activates a kinase cascade involving MARK2 and MARK3, which in turn leads to phosphorylation of several phospho-sites on Lfc. In particular, S151 was identified as the main site involved in the regulation of Lfc localization and activity. Our findings indicate that activation of Lfc and RhoA is orchestrated in FN-adherent cells in an integrin-specific manner., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: Antiangiogenic tyrosine kinase inhibitors (TKIs) provide one of the few therapeutic options for effective treatment of hepatocellular carcinoma (HCC). However, patients with HCC often develop resistance toward antiangiogenic TKIs, and the underlying mechanisms are not understood. The aim of this study was to determine the mechanisms underlying antiangiogenic TKI resistance in HCC., Methods: We used an unbiased proteomic approach to define proteins that were responsible for the resistance to antiangiogenic TKIs in HCC patients. We evaluated the prognosis, therapeutic response, and serum insulin-like growth factor-binding protein-1 (IGFBP-1) levels of 31 lenvatinib-treated HCC patients. Based on the array of results, a retrospective clinical study and preclinical experiments using mouse and human hepatoma cells were conducted. Additionally, in vivo genetic and pharmacological gain- and loss-of-function experiments were performed., Results: In the patient cohort, IGFBP-1 was identified as the signaling molecule with the highest expression that was inversely associated with overall survival. Mechanistically, antiangiogenic TKI treatment markedly elevated tumor IGFBP-1 levels via the hypoxia-hypoxia inducible factor signaling. IGFBP-1 stimulated angiogenesis through activation of the integrin α5β1-focal adhesion kinase pathway. Consequently, loss of IGFBP-1 and integrin α5β1 by genetic and pharmacological approaches re-sensitized HCC to lenvatinib treatment., Conclusions: Together, our data shed light on mechanisms underlying acquired resistance of HCC to antiangiogenic TKIs. Antiangiogenic TKIs induced an increase of tumor IGFBP-1, which promoted angiogenesis through activating the IGFBP-1-integrin α5β1 pathway. These data bolster the application of a new therapeutic concept by combining antiangiogenic TKIs with IGFBP-1 inhibitors., (© 2023 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.)

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 α v and β 3 but not α 5 or β 1 integrins. Similarly, we observed direct binding of S1-RBD to recombinant human α v β 3 and α v β 6 integrins, but not α 5 β 1 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 α v -selective integrin agonist. This study provides evidence that cell surface α v -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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5β1 integrin (Intα5β1) activity. Although the binding of Intα5β1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5β1 activation and accelerates tumor cells toward invasive disease, involving the protein β-arrestin1 (β-arr1) and the activation of the endothelin A receptor (ET A R) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intβ1 and downstream FAK/paxillin activation. Mechanistically, β-arr1 directly interacts with talin1 and Intβ1, promoting talin1 phosphorylation and its recruitment to Intβ1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ET A R/β-arr1-driven Intα5β1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ET A R, Ambrisentan (AMB), and of Intα5β1, ATN161, inhibits ET-1-driven Intα5β1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intβ1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ET A R/β-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ET A R/β-arr1 regulates Intα5β1 functional pathway., (© 2023. The Author(s).)

Background: Tendon injury is associated with oxidative stress, leading to reactive oxygen species (ROS) production and inflammation. N-acetyl-L-cysteine (NAC) is a potent antioxidant. However, how NAC affects the biological functions of tendon stem/progenitor cells (TSPCs) and tendon repair has not been clarified.  METHOD: The impacts of NAC on the viability, ROS production, and differentiation of TSPCs were determined with the cell counting kit-8, fluorescence staining, Western blotting, and immunofluorescence. The effect of NAC on gene transcription in TSPCs was analyzed by transcriptomes and bioinformatics and validated by Western blotting. The potential therapeutic effect of NAC on tendon repair was tested in a rat model of Achilles tendon injury., Results: Compared with the untreated control, treatment with 500 µM NAC greatly promoted the proliferation of TSPCs and significantly mitigated hydrogen peroxide-induced ROS production and cytotoxicity in vitro. NAC treatment significantly increased the relative protein expression of collagen type 1 alpha 1 (COL1A1), tenascin C (TNC), scleraxis (SCX), and tenomodulin (TNMD) in TPSCs. Bioinformatics analyses revealed that NAC modulated transcriptomes, particularly in the integrin-related phosphoinositide 3-kinase (PI3K)/AKT signaling, and Western blotting revealed that NAC enhanced integrin α5β1 expression and PI3K/AKT activation in TSPCs. Finally, NAC treatment mitigated the tendon injury, but enhanced the protein expression of SCX, TNC, TNMD, and COLIA1 in the injured tissue regions of the rats., Conclusion: NAC treatment promoted the survival and differentiation of TSPCs to facilitate tendon repair after tendon injury in rats. Thus, NAC may be valuable for the treatment of tendon injury., (© 2023. The Author(s).)

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 α5β1 and αvβ3 regulation during cell migration. We show that endothelial integrin α5β1 and αvβ3 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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Hyperlipidemia with high blood levels of free fatty acids (FFA) is the leading cause of non-alcoholic steatohepatitis. CCN1 is a secreted matricellular protein that drives various cellular functions, including proliferation, migration, and differentiation. However, its role in mediating FFA-induced pro-inflammatory cell death and its underlying molecular mechanisms have not been characterized. In this study, we demonstrated that CCN1 was upregulated in the livers of obese mice. The increase in FFA-induced CCN1 was evaluated in vitro by treating hepatocytes with a combination of oleic acid and palmitic acid (2:1). Gene silencing using specific small interfering RNAs (siRNA) revealed that CCN1 participated in FFA-induced intracellular lipid accumulation, caspase-1 activation, and hepatocyte pyroptosis. Next, we identified integrin α 5 β 1 as a potential receptor of CCN1. Co-immunoprecipitation demonstrated that the binding between CCN1 and integrin α 5 β 1 increased in hepatocytes upon FFA stimulation in the livers of obese mice. Similarly, the protein levels of integrin α 5 and β 1 were increased in vitro and in vivo. Experiments with specific siRNAs confirmed that integrin α 5 β 1 played a part in FFA-induced intracellular lipid accumulation, NLRP3 inflammasome activation, and pyroptosis in hepatocytes. In conclusion, these results provide novel evidence that the CCN1/integrin α 5 β 1 is a novel mediator that drives hepatic lipotoxicity via NLRP3-dependent pyroptosis.

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEVs). The tumor-supportive CAFs possess a distinct phenotypic profile, compared to normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α 5 β 1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1 + CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Further, we show that NetG1 expression in CAFs is required for the pro-survival properties of sEVs. Additionally, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α 5 β 1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α 5 β 1 were detected in sEVs collected from plasma of PDAC patients, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor-stroma interactions and pathogenic stroma detection., Competing Interests: Conflicts: C.A. Lyssiotis has received consulting fees from Astellas Pharmaceuticals, Odyssey Therapeutics, and T-Knife Therapeutics, and is an inventor on patents pertaining to Kras regulated metabolic pathways, redox control pathways in pancreatic cancer, and targeting the GOT1-pathway as a therapeutic approach (US Patent No: 2015126580-A1, 05/07/2015; US Patent No: 20190136238, 05/09/2019; International Patent No: WO2013177426-A2, 04/23/2015). E. Cukierman has two patents pending that relate to this study and wishes to disclose she is currently a consultant and SAB member for Phenomic AI.

Background: The migration, proliferation and apoptosis of vascular smooth muscle cells (VSMCs) are critical for plaque stability. WNT-inducible signalling pathway protein-1 (WISP1), a member of the CCN family of extracellular matrix proteins, can expedite the migration and proliferation of VSMCs. However, its underlying mechanism and relationship with atherosclerosis remain elusive. The relationship between WISP1 and apoptosis of VSMCs has not been determined previously., Method: In the study, we aimed to investigate the relationship between WISP1 and plaque stability and its related mechanism.ApoE-/- mice were divided following groups: the null lentivirus (NC), lentivirus WISP1 (IvWISP1) and WISP1-shRNA (shWISP1) groups. Immunofluorescence, Oil Red O and Masson's staining of the carotid arteries were performed. Transwell wound healing assay, CCK8 assay, and TdT-mediated dUTP nick-end labeling (TUNEL) staining were performed using VSMCs. The levels of WISP1, P38, C-Jun N-terminal kinase, extracellular signal-regulated kinase (ERK), mitogen-activated extracellular signal-regulated kinase (MEK), focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt (also known as PKB, protein kinase B), mammalian target of rapamycin (mTOR), cleaved caspase3, Bcl2 and Bax were detected by western blotting., Results: The relative area of lipids and monocytes/macrophages in the shWISP1 group increased compared with that of the NC group. However, the relative area of smooth muscle cell and collagen in the IvWISP1 group increased compared with that in the NC group. Therefore, WISP1 could stabilize atherosclerotic plaques. Besides, WISP1 accelerate the migration and proliferation of VSMCs via integrin α5β1 and FAK/MEK/ERK signalling pathways. In addition, WISP1 can inhibit the apoptosis of VSMCs via the PI3K/Akt/mTOR pathway., Conclusion: WISP1 not only inhibits the apoptosis of VSMCs via the PI3K/Akt/mTOR pathway but also enhances the migration and proliferation of VSMCs via the integrin α5β1 and FAK/MEK/ERK pathways. Therefore, WISP1 could enhance the stability of atherosclerotic plaques., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α 5 β 1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.

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-α 5 β 1 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-α 5 β 1 , which greatly promoted cell growth in 3D microstructures. Integrin-α 5 was more sensitive and specific than integrin-β 1 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-α 5 β 1 dependent manner. Furthermore, fibronectin, integrin-α 5 , and integrin-β 1 were upregulated in 52 HCC tumors, and fibronectin was related to microvascular invasion. Our findings identify fibronectin, integrin-α 5 , and integrin-β 1 as tumor microenvironment-related targets and provide a basis for combination targeted therapeutic strategies for future HCC treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100-1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL- g -PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at ∼7 nm localization precision and with a surface density of 38-46 μm -2 , revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.

Skin is constantly exposed to injuries that are repaired with different outcomes, either regeneration or scarring. Scars result from fibrotic processes modulated by cellular physical forces transmitted by integrins. Fibronectin (FN) is a major component in the provisional matrix assembled to repair skin wounds. FN enables cell adhesion binding of α5β1/αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5/αIIb is the synergy site located in close proximity to the RGD motif. The mutation to impair the FN synergy region ( Fn1 syn/syn ) demonstrated that its absence permits complete development. However, only with the additional engagement to the FN synergy site do cells efficiently resist physical forces. To test how the synergy site-mediated adhesion affects the course of wound healing fibrosis, we used a mouse model of skin injury and in-vitro migration studies with keratinocytes and fibroblasts on FN syn . The loss of FN synergy site led to normal re-epithelialization caused by two opposing migratory defects of activated keratinocytes and, in the dermis, induced reduced fibrotic responses, with lower contents of myofibroblasts and FN deposition and diminished TGF-β1-mediated cell signalling. We demonstrate that weakened α5β1-mediated traction forces on FN syn cause reduced TGF-β1 release from its latent complex.

Objective: Due to the increasing prevalence of obesity and insulin resistance, there is an urgent need for better treatment of obesity and its related metabolic disorders. This study aimed to elucidate the role of SERPINA3C, an adipocyte secreted protein, in obesity and related metabolic disorders., Methods: Male wild type (WT) and knockout (KO) mice were fed with high-fat diet (HFD) for 16 weeks, adiposity, insulin resistance, and inflammation were assessed. AAV-mediated overexpression of SERPINA3C was injected locally in inguinal white adipose tissue (iWAT) to examine the effect of SERPINA3C. In vitro analyses were conducted in 3T3-L1 adipocytes to explore the molecular pathways underlying the function of SERPINA3C., Results: Functional exploration of the SERPINA3C knockout mice revealed that SERPINA3C deficiency led to an impaired metabolic phenotype (more severe obesity, lower metabolic rates, worse glucose intolerance and insulin insensitivity), which was associated with anabatic inflammation and apoptosis of white adipose tissues. Consistent with these results, overexpression of SERPINA3C in inguinal adipose tissue protected mice against diet-induced obesity and metabolic disorders with less inflammation and apoptosis in adipose tissue. Mechanistically, SERPINA3C inhibited Cathepsin G activity, acting as a serine protease inhibitor, which blocked Cathepsin G-mediated turnover of α5/β1 Integrin protein. Then, the preserved integrity (increase) of α5/β1 Integrin signaling activated AKT to decrease JNK phosphorylation, thereby inhibiting inflammation and promoting insulin sensitivity in adipocytes., Conclusions/interpretation: These findings demonstrate a previously unknown SERPINA3C/Cathepsin G/Integrin/AKT pathway in regulating adipose tissue inflammation, and suggest the therapeutic potential of targeting SERPINA3C/Cathepsin G axis in adipose tissue for the treatment of obesity and metabolic diseases., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Lung cancer is the leading cause of cancer-associated death worldwide and is classified into non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC accounts for approximately 80%-85% of all lung cancer cases. Chenodeoxycholic acid (CDCA), a primary bile acid, has been reported to inhibit carcinoma cell proliferation. Here, we aimed to determine the effects and mechanism of action of CDCA against lung adenocarcinoma (LUAD)., Methods: Western blotting and quantitative real-time polymerase chain reaction were used to evaluate the protein and mRNA expression levels in LUAD cell lines, respectively. Cell Counting Kit-8 and clone formation assays were performed to evaluate the proliferation ability of different cell types in vitro. Tumor cell motility was evaluated using Transwell assays. The transcriptional profile of A549 cells treated with CDCA was determined through RNA sequencing analysis. A xenograft model was established to evaluate the effects of CDCA on LUAD progression in vivo., Results: CDCA inhibited LUAD cell proliferation, migration, and invasion. Furthermore, it promoted apoptosis in LUAD cells. Mechanistically, CDCA inhibited the integrin α5β1 signaling pathway in LUAD cells by inhibiting the expression of the α5 and β1 subunits of integrin and phosphorylated FAK. Moreover, CDCA induced an increase in the levels of p53, a downstream gene of the integrin α5β1/FAK pathway. In addition, CDCA significantly decreased tumor volume in mice without inducing significant toxicity., Conclusions: Our findings indicate that CDCA attenuates LUAD pathogenesis in vitro and in vivo via the integrin α5β1/FAK/p53 axis., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Activation of integrins by Mn 2+ is a benchmark in the integrin field, but how Mn 2+ works and whether it reproduces physiological activation is unknown. We show that Mn 2+ and high Mg 2+ concentrations compete with Ca 2+ at the ADMIDAS and shift the conformational equilibrium toward the open state, but the shift is far from complete. Additionally, replacement of Mg 2+ by Mn 2+ at the MIDAS increases the intrinsic affinities of both the high-affinity open and low-affinity closed states of integrins, in agreement with stronger binding of Mn 2+ than Mg 2+ to oxygen atoms. Mutation of the ADMIDAS increases the affinity of closed states and decreases the affinity of the open state and thus reduces the difference in affinity between the open and closed states. An important biological function of the ADMIDAS may be to stabilize integrins in highly discrete states, so that when integrins support cell adhesion and migration, their high and low affinity correspond to discrete on and off states, respectively.

Objective:  Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5β1, a receptor for fibronectin. The aim of this study was to characterize the role of α5β1 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 α5β1 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 α5β1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

The value of plasma fibronectin (pFN) in the diagnosis and prognosis of sepsis has not been fully established. Previous studies finding that pFN is significantly reduced in sepsis, however, whether reduced pFn affects the prognosis of sepsis has not been clarified. Here, we detected and analyzed pFN and other conventional inflammatory markers in advanced sepsis patients and performed correlation analysis with SOFA score. We also used Fn gene conditional knockout mice which were performed by cecum ligation and puncture (CLP) to investigate the effect of FN deficiency on sepsis prognosis. We found, compared with procalcitonin, c-reactive protein, and interleukin-6, pFN was more correlated with SOFA score in advanced sepsis patients (r -.720, p < .001). In animal experiments, Fn gene knockout mice showed significantly greater mortality after CLP compared with the control group because of inhibited phagocytosis and bacterial clearance ability of macrophages, with double cytokine storm. Furthermore, FN can regulate macrophages through the integrin α5β1/Fak/Src signaling pathway. Overall, we found pFN can more accurately reflect the severity and prognosis of advanced sepsis. The absence of FN altered the cytokine storm and phagocytic function of macrophages, suggesting that FN could be a potential therapeutic target in sepsis., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The most promising therapy for leukemia is hematopoietic stem cell transplantation. Engraftment of HPSCs mainly depends on some factors such as adhesion molecules, including VLAs. This study tried to delineate the relationship between HPSCs engraftment and expression level of PSGL1 and VLA4, 5, and 6 genes in candidate MM patients for autologous bone marrow transplantation. Firstly, the CD 34+ HPSCs were collected from multiple myeloma (MM) patients after five days of G-CSF therapy through apheresis processes. Then, the patients were categorized into two groups of good and bad prognosis depending on engraftment time (Less or more than 18 days). Followingly, the expression of PSGL1 and VLA4, VLA5, and VLA6 genes were assessed by the qRT-PCR technique in each patient. Finally, the correlation between the genes and engraftment time was investigated to determine the prognostic role of each gene on HPSCs transplantation. Our findings demonstrated that there is a significant correlation between VLA4 (P=< 0.0001) and 5 (P = 0.005) levels and HPSCs engraftment time. As the higher levels of VLA4 and 5, the shorter time HPSCs engraftment occurs. In contrast, there was no significant correlation between VLA6 (P = 0.2) and PSGL1 (P = 0.3) genes levels and engraftment time. So that, the patients with a good prognosis had a higher level of VLA4 and VLA5, but no relation was found between VLA6 and PSGL1. It is concluded that VLA4 and VLA5 expression could be considered a significant prognostic factor for HPSC transplantation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We previously demonstrated that F4 peptide (CNPEDCLYPVSHAHQR) from collagen XIX was able to inhibit melanoma cell migration in vitro  and cancer progression in a mouse melanoma model. The aim of the present work was to study the anti-angiogenic properties of F4 peptide. We demonstrated that F4 peptide inhibited VEGF-induced pseudo-tube formation on Matrigel by endothelial cells and endothelial sprouting in a rat aortic ring assay. By affinity chromatography, we identified αvβ3 and α5β1 integrins as potential receptors for F4 peptide on endothelial cell surface. Using solid phase assays, we proved the direct interaction between F4 and both integrins. Taken together, our results demonstrate that F4 peptide is a potent antitumor agent inhibiting both angiogenesis and tumor cell migration.

Background: Angiopoietin-like protein 2 (ANGPTL2) is a secreted molecule with numerous physiologic and pathologic functions, for example, in angiogenesis, hematopoiesis, and tumorigenesis. Although recent studies implicated ANGPTL2 in chronic inflammation in mouse peritoneal macrophages, human ligamentum flavum fibroblasts, and human retinal microvascular endothelial cells, the mechanism underlying ANGPTL2-associated inflammation in chondrocytes remains unclear. Therefore, it was investigated whether ANGPTL2 is expressed in or functions in chondrocytes., Methods: Expression of ANGPTL2 and its receptor, integrin α5β1 were examined over time in ATDC5 cells using real-time RT-PCR (reverse transcription-polymerase chain reaction) analysis. ATDC5 cells were then incubated with or without ANGPTL2 for 3 hours, and expression of the IL-1β, TNF-α, COX-2, aggrecanase (ADAMTS)-5, matrix metalloproteinase (MMP)-3, and MMP-13 genes were examined using real-time RT-PCR. Additionally, phosphorylation of ERK, JNK, p38, Akt, and NF-κB was examined by western blotting. Furthermore, it was also investigated for the effect of anti-integrin α 5 β 1 antibody on the expression of inflammatory markers and intracellular signaling pathways., Results: ANGPTL2 induced the phosphorylation of all 3 MAPKs, Akt, and NF-κB and dramatically upregulated the expression of inflammation-related factor genes. Inhibiting the activation of integrin α5β1 suppressed these reactions., Conclusion: ANGPTL2 may induce inflammatory factors by stimulating the integrin α5β1/MAPKs, Akt, and NF-κB signaling pathway.

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.

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 α5β1-integrin on the host cell membrane, followed by phagocytosis. Although osteoblasts showed high expression of α5β1-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 α5β1 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.

Chronic obstructive asthma is characterized by airway fibrosis. Hypoxia and connective tissue growth factor (CTGF) play important roles in airway fibrosis. Preadipocyte factor-1 (Pref-1) participates in adipocyte differentiation and liver fibrosis. Herein, we investigated the role of Pref-1 in airway fibrosis in chronic obstructive asthma. We found that Pref-1 was overexpressed in lung tissues from chronic obstructive asthma patients compared to normal subjects. Extracellular matrix proteins were inhibited by Pref-1 small interfering (si)RNA in airway fibroblasts from chronic obstructive asthma patients. Furthermore, ovalbumin induced prominent Pref-1 expression and fibronectin coexpression. Hypoxia induced Pref-1 upregulation and its release into medium of WI-38 cells. Hypoxia-induced CTGF expression was inhibited by Pref-1 siRNA. We also found that Pref-1-stimulated fibrotic protein expressions were reduced by ATN-161, curcumin, U0126, and c-Jun siRNA in WI-38. Furthermore, ATN161 inhibited Pref-1-induced ERK phosphorylation, and ITGA5 siRNA inhibited c-Jun phosphorylation. Moreover, expression of CTGF, Fibronectin, α-SMA, and ERK and c-Jun phosphorylation were all increased in fibroblasts from patients with chronic obstructive asthma. Taken together, these results suggest that Pref-1 participates in airway fibrosis and hypoxia-induced CTGF expression via the integrin receptor α5β1/ERK/AP-1 pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Endosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility., (© 2021. The Author(s).)

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5β1 integrins in INVs., (© 2021 Larocque et al.)