Integrin α5 regulates motility of human monocyte-derived Langerhans cells during immune response.

Langerhans cells (LCs) are mainly present in the epidermis and mucosa, and have important roles during skin infection. Migration of LCs to lymph nodes is essential for antigen presentation. However, due to the difficulties in isolating and culturing human LCs, it is not fully understood how LCs move and interact with the extracellular matrix (ECM) through their adhesion molecules such as integrin, during the immune responses. In this study, we aimed to investigate LC motility, cell shape and the role of integrin under inflammatory conditions using monocyte-derived Langerhans cells (moLCs) as a model. As a result, lipopolysaccharide (LPS) stimulation increased adhesion on fibronectin coated substrate and integrin α5 expression in moLCs. Time-lapse imaging of moLCs revealed that stimulation with LPS elongated cell shape, whilst decreasing their motility. Additionally, this decrease in motility was not observed when pre-treated with a neutralising antibody targeting integrin α5. Together, our data suggested that activation of LCs decreases their motility by promoting integrin α5 expression to enhance their affinity to the fibronectin, which may contribute to their migration during inflammation.

Macrophage depletion damages hematopoiesis partially through inhibition of cell homing and expansion after hematopoietic cell transplantation.

Bone marrow macrophages (Mφ) are essential components of the bone marrow niche that regulate the function of hematopoietic stem cells. Poor graft function and inhibition of hematopoietic production can result from abnormal macrophage function; however, the underlying mechanism is unclear. Clodronate liposomes (Clo-Lip) have been used widely to deplete macrophages and study their functions. Our previous results showed that Clod-Lip-mediated clearance of macrophages plays a vital role in regulating hematopoietic reconstruction after allogeneic hematopoietic cell transplantation (HCT). In this study, using an isogenic hematopoietic stem cell transplantation model, we found that Clod-Lip-mediated clearance of macrophages suppressed hematopoietic reconstruction by inhibiting the homing process of hematopoietic cells. We also demonstrated that macrophage depletion inhibited the direct supportive effect of macrophages on hematopoietic stem and progenitor cells and erythroid differentiation but promoted the production of megakaryocytic progenitors ex vivo. We showed that macrophages increase CD49e expression on hematopoietic stem and progenitor cells (HSPCs). However, CD49e inhibitors did not support the proliferative effect of macrophages on hematopoietic cells. In contrast, macrophage E-selectin/ intercellular cell adhesion molecule-1 (ICAM-1) may be involved in directly regulating HSPCs. In conclusion, macrophage depletion with Clo-Lip partially disrupts bone marrow hematopoiesis after HCT by impeding donor cell homing and macrophage-HSPCs interactions.

The novel protein complex FAPα/ITGA5 is involved in the bone destruction of apical periodontitis.

The bacteria that invade the periapical tissue of teeth can directly damage tissue cells such as periapical fibroblasts, leading to an inflammatory response in the periapical tissue and ultimately resulting in bone destruction. We investigated the role of fibroblast activation protein α (FAPα) and integrin α5 (ITGA5) in periapical bone destruction. This study found that FAPα and ITGA5 were highly expressed in human tissues from patients with chronic apical periodontitis. Osteoclast differentiation decreased when FAPα or ITGA5 was silenced and inhibited. The results of protein molecular docking showed that FAPα had good binding affinity to ITGA5, and its free energy was -14.5 kcal/mol. Immunofluorescence staining and co-immunoprecipitation showed that FAPα and ITGA5 formed protein complexes in the inflammatory microenvironment. In conclusion, this study proved that FAPα and ITGA5 participate in the regulation of osteoclast differentiation by forming protein complexes in the inflammatory microenvironment, which then regulates the occurrence and development of chronic apical periodontitis.

Protective effects of macrophage-specific integrin α5 in myocardial infarction are associated with accentuated angiogenesis.

Macrophages sense changes in the extracellular matrix environment through the integrins and play a central role in regulation of the reparative response after myocardial infarction. Here we show that macrophage integrin α5 protects the infarcted heart from adverse remodeling and that the protective actions are associated with acquisition of an angiogenic macrophage phenotype. We demonstrate that myeloid cell- and macrophage-specific integrin α5 knockout mice have accentuated adverse post-infarction remodeling, accompanied by reduced angiogenesis in the infarct and border zone. Single cell RNA-sequencing identifies an angiogenic infarct macrophage population with high Itga5 expression. The angiogenic effects of integrin α5 in macrophages involve upregulation of Vascular Endothelial Growth Factor A. RNA-sequencing of the macrophage transcriptome in vivo and in vitro followed by bioinformatic analysis identifies several intracellular kinases as potential downstream targets of integrin α5. Neutralization assays demonstrate that the angiogenic actions of integrin α5-stimulated macrophages involve activation of Focal Adhesion Kinase and Phosphoinositide 3 Kinase cascades.

Regulation of integrin α5ß1-mediated Staphylococcus aureus cellular invasion by the septin cytoskeleton.

Staphylococcus aureus, a Gram-positive bacterial pathogen, is an urgent health threat causing a wide range of clinical infections. Originally viewed as a strict extracellular pathogen, accumulating evidence has revealed S. aureus to be a facultative intracellular pathogen subverting host cell signalling to support invasion. The majority of clinical isolates produce fibronectin-binding proteins A and B (FnBPA and FnBPB) to interact with host integrin α5ß1, a key component of focal adhesions. S. aureus binding of integrin α5ß1 promotes its clustering on the host cell surface, triggering activation of focal adhesion kinase (FAK) and cytoskeleton rearrangements to promote bacterial invasion into non-phagocytic cells. Here, we discover that septins, a component of the cytoskeleton that assembles on membranes, are recruited as collar-like structures with actin to S. aureus invasion sites engaging integrin α5ß1. To investigate septin recruitment to the plasma membrane in a bacteria-free system, we used FnBPA-coated latex beads and showed that septins are recruited upon activation of integrin α5ß1. SEPT2 depletion reduced S. aureus invasion, but increased surface expression of integrin α5 and adhesion of S. aureus to host cells. Consistent with this, SEPT2 depletion increased cellular protein levels of integrin α5 and ß1 subunits, as well as FAK. Collectively, these results provide insights into regulation of integrin α5ß1 and invasion of S. aureus by the septin cytoskeleton.

Platelet factor 4 induces bone loss by inhibiting the integrin α5-FAK-ERK pathway.

BACKGROUND: The effect of platelet factor 4 (PF4) on bone marrow mesenchymal stem cells (BMMSCs) and osteoporosis is poorly understood. Therefore, this study aimed to evaluate the effects of PF4-triggered bone destruction in mice and determine the underlying mechanism. METHODS: First, in vitro cell proliferation and cell cycle of BMMSCs were assessed using a CCK8 assay and flow cytometry, respectively. Osteogenic differentiation was confirmed using staining and quantification of alkaline phosphatase and Alizarin Red S. Next, an osteoporotic mouse model was established by performing bilateral ovariectomy (OVX). Furthermore, the PF4 concentrations were obtained using enzyme-linked immunosorbent assay. The bone microarchitecture of the femur was evaluated using microCT and histological analyses. Finally, the key regulators of osteogenesis and pathways were investigated using quantitative real-time polymerase chain reaction and Western blotting. RESULTS: Human PF4 widely and moderately decreased the cell proliferation and osteogenic differentiation ability of BMMSCs. Furthermore, the levels of PF4 in the serum and bone marrow were generally increased, whereas bone microarchitecture deteriorated due to OVX. Moreover, in vivo mouse PF4 supplementation triggered bone deterioration of the femur. In addition, several key regulators of osteogenesis were downregulated, and the integrin α5-focal adhesion kinase-extracellular signal-regulated kinase (ITGA5-FAK-ERK) pathway was inhibited due to PF4 supplementation. CONCLUSIONS: PF4 may be attributed to OVX-induced bone loss triggered by the suppression of bone formation in vivo and alleviate BMMSC osteogenic differentiation by inhibiting the ITGA5-FAK-ERK pathway.

Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis.

BACKGROUND: TP53, encoding the tumor suppressor p53, is frequently mutated in various cancers, producing mutant p53 proteins (mutp53) which can exhibit neomorphic, gain-of-function properties. The latter transform p53 into an oncoprotein that promotes metastatic tumor progression via downstream effectors such as ENTPD5, an endoplasmic reticulum UDPase involved in the calnexin/calreticulin cycle of N-glycoprotein biosynthesis. Elucidating the mechanisms underlying the pro-metastatic functions of the mutp53-ENTPD5 axis is crucial for developing targeted therapies for aggressive metastatic cancer. METHODS: We analyzed pancreatic, lung, and breast adenocarcinoma cells with p53 missense mutations to study the impact of mutp53 and ENTPD5 on the N-glycoproteins integrin-α5 (ITGA5) and integrin-ß1 (ITGB1), which heterodimerize to form the key fibronectin receptor. We assessed the role of the mutp53-ENTPD5 axis in integrin-dependent tumor-stroma interactions and tumor cell motility using adhesion, migration, and invasion assays, identifying and validating therapeutic intervention targets. We employed an orthotopic xenograft model of pancreatic ductal adenocarcinoma to examine in vivo targeting of mutp53-ENTPD5-mediated ITGA5 regulation for cancer therapy. RESULTS: Mutp53 depletion diminished ITGA5 and ITGB1 expression and impaired tumor cell adhesion, migration, and invasion, rescued by ENTPD5. The mutp53-ENTPD5 axis maintained ITGA5 expression and function via the calnexin/calreticulin cycle. Targeting this axis using ITGA5-blocking antibodies, α-glucosidase inhibitors, or pharmacological degradation of mutp53 by HSP90 inhibitors, such as Ganetespib, effectively inhibited ITGA5-mediated cancer cell motility in vitro. In the orthotopic xenograft model, Ganetespib reduced ITGA5 expression and metastasis in an ENTPD5-dependent manner. CONCLUSIONS: The mutp53-ENTPD5 axis fosters ITGA5 and ITGB1 expression and tumor cell motility through the calnexin/calreticulin cycle, contributing to cancer metastasis. ITGA5-blocking antibodies or α-glucosidase inhibitors target this axis and represent potential therapeutic options worth exploring in preclinical models. The pharmacologic degradation of mutp53 by HSP90 inhibitors effectively blocks ENTPD5-ITGA5-mediated cancer cell motility and metastasis in vivo, warranting further clinical evaluation in p53-mutant cancers. This research underscores the significance of understanding the complex interplay between mutp53, ENTPD5, and the calnexin/calreticulin cycle in integrin-mediated metastatic tumor progression, offering valuable insights for the development of potential therapeutic strategies.

γ-Bungarotoxin impairs the vascular endothelial barrier function by inhibiting integrin α5.

γ-bungarotoxin (γ-BGT) is an RGD motif-containing protein, derived from the venom of Bungarus multicinctus, leading to acute death in mice. These RGD motif-containing proteins from snake venom belonging to the disintegrin family can interfere with vascular endothelial homeostasis by directly binding cell surface integrins. Targeting integrins that generate vascular endothelial dysfunction may contribute to γ-BGT poisoning, however, the underlying mechanisms have not been investigated in detail. In this study, the results showed that γ-BGT played a role in -promoting the permeability of the vascular endothelial barrier. Depending on its selective binding to integrin α5 in vascular endothelium (VE), γ-BGT initiated downstream events, including focal adhesion kinase dephosphorylation and cytoskeleton remodeling, resulting in the intercellular junction interruption. Those alternations facilitated paracellular permeability of VE and barrier dysfunction. Proteomics profiling identified that as a downstream effector of the integrin α5 / FAK signaling pathway cyclin D1 partially mediated the cellular structural changes and barrier dysfunction. Furthermore, VE-released plasminogen activator urokinase and platelet-derived growth factor D could serve as potential diagnostic biomarkers for γ-BGT-induced vascular endothelial dysfunction. Our results indicate the mechanisms through which γ-BGT as a novel disintegrin directly interacts with the VE, with consequences for barrier dysfunction.

Elevated α5 integrin expression on myeloid cells in motor areas in amyotrophic lateral sclerosis is a therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a fatal disease affecting upper and lower motor neurons. Microglia directly interact with motor neurons and participate in the progression of ALS. Single-cell mass cytometry (CyTOF) analysis revealed prominent expression of α5 integrin in microglia and macrophages in a superoxide dismutase-1 G93A mouse model of ALS (SOD1G93A). In postmortem tissues from ALS patients with various clinical ALS phenotypes and disease duration, α5 integrin is prominent in motor pathways of the central and peripheral nervous system and in perivascular zones associated with the blood-brain barrier. In SOD1G93A mice, administration of a monoclonal antibody against α5 integrin increased survival compared to an isotype control and improved motor function on behavioral testing. Together, these findings in mice and in humans suggest that α5 integrin is a potential therapeutic target in ALS.

Astaxanthin inhibits integrin α5 expression by suppressing activation of JAK1/STAT3 in Helicobacter pylori­stimulated gastric epithelial cells.

Integrins act as cell­matrix adhesion molecules involved in cell attachment to the extracellular matrix and generate signals that respond to cancer metastasis. Integrin α5ß1 is a heterodimer with α5 and ß1 subunits and mediates cell adhesion and migration of cancer cells. Integrins are transcriptionally regulated by the Janus kinase (JAK)/STAT signaling pathways. Our previous study revealed that Helicobacter pylori increased the levels of reactive oxygen species (ROS), which activate JAK1/STAT3 in gastric cancer AGS cells in vitro. Astaxanthin (ASX) has been reported to be an effective antioxidant and anticancer nutrient. The present study investigated whether ASX suppresses H. pylori­induced integrin α5 expression, cell adhesion and migration and whether ASX reduces ROS levels and suppresses phosphorylation of JAK1/STAT3 in gastric cancer AGS cells stimulated with H. pylori. The effect of ASX was determined using a dichlorofluorescein fluorescence assay, western blot analysis, adhesion assay and wound­healing assay in AGS cells stimulated with H. pylori. The results demonstrated that H. pylori increased the expression levels of integrin α5, without affecting integrin ß1, and increased cell adhesion and migration of AGS cells. ASX reduced ROS levels and suppressed JAK1/STAT3 activation, integrin α5 expression, cell adhesion and migration of H. pylori­stimulated AGS cells. In addition, both a JAK/STAT inhibitor, AG490, and an integrin α5ß1 antagonist, K34C, suppressed cell adhesion and migration in H. pylori­stimulated AGS cells. AG490 inhibited integrin α5 expression in AGS cells stimulated with H. pylori. In conclusion, ASX inhibited H. pylori­induced integrin α5­mediated cell adhesion and migration by decreasing the levels of ROS and suppressing JAK1/STAT3 activation in gastric epithelial cells.

Novel TCF21high pericyte subpopulation promotes colorectal cancer metastasis by remodelling perivascular matrix.

OBJECTIVE: Haematogenous dissemination is a prevalent route of colorectal cancer (CRC) metastasis. However, as the gatekeeper of vessels, the role of tumour pericytes (TPCs) in haematogenous metastasis remains largely unknown. Here, we aimed to investigate the heterogeneity of TPCs and their effects on CRC metastasis. DESIGN: TPCs were isolated from patients with CRC with or without liver metastases and analysed by single-cell RNA sequencing (scRNA-seq). Clinical CRC specimens were collected to analyse the association between the molecular profiling of TPCs and CRC metastasis. RNA-sequencing, chromatin immunoprecipitation-sequencing and bisulfite-sequencing were performed to investigate the TCF21-regulated genes and mechanisms underlying integrin α5 on TCF21 DNA hypermethylation. Pericyte-conditional Tcf21-knockout mice were constructed to investigate the effects of TCF21 in TPCs on CRC metastasis. Masson staining, atomic force microscopy, second-harmonic generation and two-photon fluorescence microscopy were employed to observe perivascular extracellular matrix (ECM) remodelling. RESULTS: Thirteen TPC subpopulations were identified by scRNA-seq. A novel subset of TCF21high TPCs, termed 'matrix-pericytes', was associated with liver metastasis in patients with CRC. TCF21 in TPCs increased perivascular ECM stiffness, collagen rearrangement and basement membrane degradation, establishing a perivascular metastatic microenvironment to instigate colorectal cancer liver metastasis (CRCLM). Tcf21 depletion in TPCs mitigated perivascular ECM remodelling and CRCLM, whereas the coinjection of TCF21high TPCs and CRC cells markedly promoted CRCLM. Mechanistically, loss of integrin α5 inhibited the FAK/PI3K/AKT/DNMT1 axis to impair TCF21 DNA hypermethylation in TCF21high TPCs. CONCLUSION: This study uncovers a previously unidentified role of TPCs in haematogenous metastasis and provides a potential diagnostic marker and therapeutic target for CRC metastasis.

The members of the miR-148/152 family inhibit cancer stem cell-like properties in gastric cancer via negative regulation of ITGA5.

BACKGROUND: The role of microRNA (miRNA) in modulating the function of cancer stem cells through diverse signaling pathway has been evidenced. We here identified a role of microRNA (miRNA) family, specifically miR-148/152, in gastric cancer and delineated its functional effects on gastric cancer stem cells. METHODS: Bioinformatics analysis was conducted to analyze expression of integrin α5 (ITGA5) which was verified through expression determination in clinical tissue samples. Next, the upstream regulatory factors of ITGA5 were determined. CD44+EpCAM (high) cells sorted from AGS cells subjected to gain-of-function experiments, followed by evaluation of their capacity of colony formation, generation of tumorosphere, cell migration and viability in vitro and xenograft tumor formation in vivo. RESULTS: ITGA5 was elevated in gastric cancer tissues and confirmed as a target gene of the miR-148/152 family members. The miR-148/152 family members were downregulated in gastric cancer tissues and cells. Decreased expression of miR-148/152 family members was also detected in gastric cancer stem cells. However, the raised expression led to reduced colony formation, tumorosphere, cell migration, cell viability, and drug resistance of CD44+EpCAM (high) AGS cells in vitro, and tumorigenesis in vitro. ITGA5 overexpression reversed the effect of the miR-148/152 family members. CONCLUSIONS: This study demonstrates that the miR-148/152 family members may prevent gastric cancer stem cell-like properties by targeting ITGA5, which can serve as an appealing target for gastric cancer treatment.

N-Acetyl-L-cysteine facilitates tendon repair and promotes the tenogenic differentiation of tendon stem/progenitor cells by enhancing the integrin α5/ß1/PI3K/AKT signaling.

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 α5ß1 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.

Actomyosin-mediated cellular tension promotes Yap nuclear translocation and myocardial proliferation through α5 integrin signaling.

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state results in the loss of regenerative potential of the mammalian heart shortly after birth. Nonmuscle myosin IIB (NM IIB)-mediated actomyosin contractility regulates cardiomyocyte cytokinesis in the embryonic heart, and NM IIB levels decline after birth, suggesting a role for cellular tension in the regulation of cardiomyocyte cell cycle activity in the postnatal heart. To investigate the role of actomyosin contractility in cardiomyocyte cell cycle arrest, we conditionally activated ROCK2 kinase domain (ROCK2:ER) in the murine postnatal heart. Here, we show that α5/ß1 integrin and fibronectin matrix increase in response to actomyosin-mediated tension. Moreover, activation of ROCK2:ER promotes nuclear translocation of Yap, a mechanosensitive transcriptional co-activator, and enhances cardiomyocyte proliferation. Finally, we show that reduction of myocardial α5 integrin rescues the myocardial proliferation phenotype in ROCK2:ER hearts. These data demonstrate that cardiomyocytes respond to increased intracellular tension by altering their intercellular contacts in favor of cell-matrix interactions, leading to Yap nuclear translocation, thus uncovering a function for nonmuscle myosin contractility in promoting cardiomyocyte proliferation in the postnatal heart.

Protein kinase B (AKT) upregulation and Thy-1-αvß3 integrin-induced phosphorylation of Connexin43 by activated AKT in astrogliosis.

BACKGROUND: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvß3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. METHODS: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). RESULTS: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. CONCLUSIONS: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.

Regulatory roles of fibronectin and integrin α5 in reorganization of the actin cytoskeleton and completion of adipogenesis.

Cellular differentiation is characterized by changes in cell morphology that are largely determined by actin dynamics. We previously showed that depolymerization of the actin cytoskeleton triggers the differentiation of preadipocytes into mature adipocytes as a result of inhibition of the transcriptional coactivator activity of megakaryoblastic leukemia 1 (MKL1). The extracellular matrix (ECM) influences cell morphology via interaction with integrins, and reorganization of the ECM is associated with cell differentiation. Here we show that interaction between actin dynamics and ECM rearrangement plays a key role in adipocyte differentiation. We found that depolymerization of the actin cytoskeleton precedes disruption and degradation of fibrillar fibronectin (FN) structures at the cell surface after the induction of adipogenesis in cultured preadipocytes. A FN matrix suppressed both reorganization of the actin cytoskeleton into the pattern characteristic of adipocytes and terminal adipocyte differentiation, and these inhibitory effects were overcome by knockdown of integrin α5 (ITGα5). Peroxisome proliferator-activated receptor γ was required for down-regulation of FN during adipocyte differentiation, and MKL1 was necessary for the expression of ITGα5. Our findings suggest that cell-autonomous down-regulation of FN-ITGα5 interaction contributes to reorganization of the actin cytoskeleton and completion of adipocyte differentiation.

Fibronectin-Integrin α5 Signaling in Vascular Complications of Type 1 Diabetes.

Vascular complications are a major cause of illness and death in patients with type 1 diabetes (T1D). Diabetic vascular basement membranes are enriched in fibronectin (FN), an extracellular matrix protein that amplifies inflammatory signaling in endothelial cells through its main receptor, integrin α5ß1. Binding of the integrin α5 cytoplasmic domain to phosphodiesterase 4D5 (PDE4D5), which increases phosphodiesterase catalytic activity and inhibits antiinflammatory cAMP signaling, was found to mediate these effects. Here, we examined mice in which the integrin α5 cytoplasmic domain is replaced by that of α2 (integrin α5/2) or the integrin α5 binding site in PDE4D is mutated (PDE4Dmut). T1D was induced via injection of streptozotocin and hyperlipidemia induced via injection of PCSK9 virus and provision of a high-fat diet. We found that in T1D and hyperlipidemia, the integrin α5/2 mutation reduced atherosclerosis plaque size by ∼50%, with reduced inflammatory cell invasion and metalloproteinase expression. Integrin α5/2 T1D mice also had improved blood-flow recovery from hindlimb ischemia and improved biomechanical properties of the carotid artery. By contrast, the PDE4Dmut had no beneficial effects in T1D. FN signaling through integrin α5 is thus a major contributor to diabetic vascular disease but not through its interaction with PDE4D.

Fibronectin and Integrin α5 play overlapping and independent roles in regulating the development of pharyngeal endoderm and cartilage.

Craniofacial skeletal elements are derived from cranial neural crest cells (CNCCs), which migrate along discrete paths and populate distinct pharyngeal arches, structures that are separated by the neighboring endodermal pouches (EPs). Interactions between the CNCCs and the endoderm are critical for proper craniofacial development. In zebrafish, integrin α5 (Itga5) functions in the endoderm to regulate formation of specifically the first EP (EP1) and the development of the hyoid cartilage. Here we show that fibronectin (Fn), a major component of the extracellular matrix (ECM), is also required for these developmental processes, and that the penetrance of defects in mutants is temperature-dependent. fn1a-/- embryos exhibited defects that are similar to, but much more severe than, those of itga5-/- embryos, and a loss of integrin av (itgav) function enhanced both endoderm and cartilage defects in itga5-/- embryos, suggesting that Itga5 and Itgav cooperate to transmit signals from Fn to regulate the development of endoderm and cartilage. Whereas the endodermal defects in itga5; itga5v-/- double mutant embryos were comparable to those of fn1a-/- mutants, the cartilage defects were much milder. Furthermore, Fn assembly was detected in migrating CNCCs, and the epithelial organization and differentiation of CNCC-derived arches were impaired in fn1a-/- embryos, indicating that Fn1 exerts functions in arch development that are independent of Itga5 and Itgav. Additionally, reduction of itga5 function in fn1a-/- embryos led to profound defects in body axis elongation, as well as in endoderm and cartilage formation, suggesting that other ECM proteins signal through Itga5 to regulate development of the endoderm and cartilage. Thus, our studies reveal that Fn1a and Itga5 have both overlapping and independent functions in regulating development of the pharyngeal endoderm and cartilage.

Pericyte-to-endothelial cell signaling via vitronectin-integrin regulates blood-CNS barrier.

Endothelial cells of blood vessels of the central nervous system (CNS) constitute blood-CNS barriers. Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment. Notably, the abluminal surfaces of CNS capillaries are ensheathed by pericytes and astrocytes. However, extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular matrix protein secreted by CNS pericytes, as a regulator of blood-CNS barrier function via interactions with its integrin receptor, α5, in endothelial cells. Genetic ablation of vitronectin or mutating vitronectin to prevent integrin binding, as well as endothelial-specific deletion of integrin α5, causes barrier leakage in mice. Furthermore, vitronectin-integrin α5 signaling maintains barrier integrity by actively inhibiting transcytosis in endothelial cells. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability.

FGA inhibits metastases and induces autophagic cell death in gastric cancer via inhibiting ITGA5 to regulate the FAK/ERK pathway.

This study aims to investigate the expression levels of fibrinogen α chain (FGA) in human gastric cancer (GC) tissues and cell lines, clarify its role in gastric cancer progression, and explore its underlying mechanism. Bioinformatics analysis, Immunoblot, Immunohistochemical (IHC), and quantitative PCR assays were performed to assess the expression of FGA in human gastric cancer tissues and cell lines. CCK-8 and colony formation assays were performed to detect its role in the proliferation of gastric cancer cells. Wound healing, transwell, and Immunofluorescence were performed to detect its effects on gastric cancer cell motility and epithelial-mesenchymal transition (EMT) processes. Luciferase and CHIP assays were performed to confirm the transcriptional regulation of FGA on ITGA5. Immunoblot assays and double-label RFP-GFP-LC3 immunofluorescence analysis were conducted to detect its effects on gastric cancer cell autophagy and FAK/ERK pathway, and in vivo tumor growth assays were further performed. We found the low expression of FGA in human gastric cancer tissues and cell lines. FGA suppressed gastric cancer cell proliferation, motility, and EMT process, and stimulated cell autophagy. We further found that FGA suppressed the expression of Integrin-α5 (ITGA5) and inhibited the FAK/ERK pathway, therefore suppressing the progression of gastric cancer. The in vivo assays further confirmed that FGA suppressed tumor growth of gastric cancer cells in the BALB/c nude mice (18-22 g, female, 8-week-old) through suppressing ITGA5-mediated FAK/ERK pathway in mice. We demonstrated the mechanism underlying FGA suppressing gastric cancer progression, and therefore we thought FGA could serve as a tumor suppressor protein in gastric cancer.