A flexible loop in the paxillin LIM3 domain mediates its direct binding to integrin ß subunits.

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance, and haemostasis but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, FA assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of FA sites, with the cytoplasmic tails of the integrin ß1 and ß3 subunits. The binding interface resides in paxillin's LIM3 domain, where based on the NMR structure and functional analyses, a flexible, 7-amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin ß3 compromises cell adhesion and motility of murine fibroblasts. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin ß3 function.

ROS-mediated ITGB5 promotes tongue squamous cell carcinoma metastasis through epithelial mesenchymal transition and cell adhesion signal pathway.

PURPOSE: Integrin ß5 (ITGB5) is an integrin ß subunit member widely expressed in the human bodies, especially in cancer cells and tissues, which is a key factor in promoting tumor metastasis. In this study we investigated the differential expression of ITGB5 in tongue squamous cell carcinoma (TSCC), especially in those with lymph node metastasis, and revealed the possible mechanism. METHODS: The expression of ITGB5 in TSCC was analyzed by database and verified by immunohistochemistry through 135 TSCC patients' tissue sections from Sun Yat-sen Memorial Hospital and Guangzhou First People's Hospital. The relationship between ITGB5 and lymph node metastasis or prognosis was analyzed retrospectively. The effects of ITGB5 on TSCC cells were examined through knocking down or overexpression and its possible regulator and signal pathway were explored. RESULTS: The expression of ITGB5 in TSCC was higher than that in adjacent tissue, and the expression in patients with lymph node metastasis was higher than that in patients without lymph node metastasis. The high expression of ITGB5 predicted a worse prognosis. Knock down of ITGB5 suppressed invasion and migration of TSCC cells, while overexpression of ITGB5 contributed to invasion and migration. Reactive oxygen species (ROS) regulated epithelial mesenchymal transition (EMT), and we further verified that ROS enhanced the expression of ITGB5 to promote the metastasis of TSCC. Mechanistically, ITGB5 functions through cell adhesion signal pathway. CONCLUSION: The increased expression of ITGB5 in tongue squamous cell carcinoma with lymph node metastasis may be a potential target for evaluating lymph node metastasis and worse prognosis of tongue squamous cell carcinoma. Scavenge of ROS or knock down of ITGB5 may be the strategies to overcome metastasis of TSCC.

Trafficking circuit of CD8+ T cells between the intestine and bone marrow governs antitumour immunity.

Immunotherapy elicits a systemic antitumour immune response in peripheral circulating T cells. However, the T cell trafficking circuit between organs and their contributions to antitumour immunity remain largely unknown. Here we show in multiple mouse leukaemia models that high infiltration of leukaemic cells in bone marrow (BM) stimulates the transition of CD8+CD44+CD62L+ central memory T cells into CD8+CD44-CD62L- T cells, designated as inter-organ migratory T cells (TIM cells). TIM cells move from the BM to the intestine by upregulating integrin ß7 and downregulating C-X-C motif chemokine receptor 3 during leukaemogenesis. Upon immunogenic chemotherapy, these BM-derived TIM cells return from the intestine to the BM through integrin α4-vascular cell adhesion molecule 1 interaction. Blocking C-X-C motif chemokine receptor 3 function boosts the immune response against leukaemia by enhancing T cell trafficking. This phenomenon can also be observed in patients with leukaemia. In summary, we identify an unrecognized intestine-BM trafficking circuit of T cells that contributes to the antitumour effects of immunogenic chemotherapy.

The integrin receptor beta7 subunit mediates airway remodeling and hyperresponsiveness in allergen exposed mice.

BACKGROUND: Fibroblast differentiation to a myofibroblast phenotype is a feature of airway remodeling in asthma. Lung fibroblasts express the integrin receptor α4ß7 and fibronectin induces myofibroblast differentiation via this receptor. OBJECTIVES: To investigate the role of the ß7 integrin receptor subunit and α4ß7 integrin complex in airway remodeling and airway hyperresponsiveness (AHR) in a murine model of chronic allergen exposure. METHODS: C57BL/6 wild type (WT) and ß7 integrin null mice (ß7 -/-) were sensitized (days 1,10) and challenged with ovalbumin (OVA) three times a week for one or 4 weeks. Similar experiments were performed with WT mice in the presence or absence of α4ß7 blocking antibodies. Bronchoalveolar (BAL) cell counts, AHR, histological evaluation, soluble collagen content, Transforming growth factor-ß (TGFß) and Interleukin-13 (IL13) were measured. Phenotype of fibroblasts cultured from WT and ß7 -/- saline (SAL) and OVA treated mice was evaluated. RESULTS: Eosinophil numbers were similar in WT vs ß7-/- mice. Prolonged OVA exposure in ß7-/- mice was associated with reduced AHR, lung collagen content, peribronchial smooth muscle, lung tissue TGFß and IL13 expression as compared to WT. Similar findings were observed in WT mice treated with α4ß7 blocking antibodies. Fibroblast migration was enhanced in response to OVA in WT but not ß7 -/- fibroblasts. α-SMA and fibronectin expression were reduced in ß7-/- fibroblasts relative to WT. CONCLUSIONS: The ß7 integrin subunit and the α4ß7 integrin complex modulate AHR and airway remodeling in a murine model of allergen exposure. This effect is, at least in part, explained by inhibition of fibroblast activation and is independent of eosinophilic inflammation.

Synthetic integrin antibodies discovered by yeast display reveal αV subunit pairing preferences with ß subunits.

Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVß3, αVß5, αVß6, αVß8, and α5ß1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6 = ß8 > ß3 > ß1 = ß5.

CRISPR/Cas9-mediated knock out of ITGB6 in human OSCC cells reduced migration and proliferation ability.

BACKGROUND: The treatment of oral squamous cell carcinoma (OSCC) remains challenging and survival rates have not been improved significantly over the past decades. Integrins have been recognized driving the cancer progression and high expression levels cause poor outcomes in patients afflicted with OSCC. Integrin αvß6 and its subunit integrin beta 6 (ITGB6) were discovered to enhance the invasiveness by providing beneficial effects on downstream pathways promoting the cancer progression. The objective of this study was to establish a CRISPR/Cas9-mediated knock out of ITGB6 in the human OSCC cell line HN and investigate the effects on the migration and proliferation ability. METHODS: ITGB6 knock out was performed using the CRISPR/Cas9-system, RNPs, and lipofection. Monoclonal cell clones were achieved by limiting dilution and knock out verification was carried out by sanger sequencing and FACS on protein level. The effects of the knock out on the proliferation and migration ability were evaluated by using MTT and scratch assays. In addition, in silico TCGA analysis was utilized regarding the effects of ITGB6 on overall survival and perineural invasion. RESULTS: In silico analysis revealed a significant impact of ITGB6 mRNA expression levels on the overall survival of patients afflicted with OSCC. Additionally, a significantly higher rate of perineural invasion was discovered. CRISPR/Cas9-mediated knock out of ITGB6 was performed in the OSCC cell line HN, resulting in the generation of a monoclonal knock out clone. The knock out clone exhibited a significantly reduced migration and proliferation ability when compared to the wildtype. CONCLUSIONS: ITGB6 is a relevant factor in the progression of OSCC and can be used for the development of novel treatment strategies. The present study is the first to establish a monoclonal CRISPR/Cas9-mediated ITGB6 knockout cell clone derived from an OSCC cell line. It suggests that ITGB6 has a significant impact on the proliferative and migratory capacity in vitro.

Circ_0008571 modulates the phenotype of vascular smooth muscle cells by targeting miR-145-5p in intracranial aneurysms.

BACKGROUND: The dysfunction of human vascular smooth cells (hVSMCs) is significantly connected to the development of intracranial aneurysms (IAs). By suppressing the activity of microRNAs (miRNAs), circular RNAs (circRNAs) participate in IA pathogenesis. Nevertheless, the role of hsa_circ_0008571 in IAs remains unclear. METHODS: circRNA sequencing was used to identify circRNAs from human IA tissues. To determine the function of circ_0008571, Transwell, wound healing, and cell proliferation assays were conducted. To identify the target of circ_0008571, the analyses of CircInteractome and TargetScan, as well as the luciferase assay were carried out. Furthermore, circ_0008571 knockdown and over-expression were performed to investigate its functions in IA development and the underlying molecular mechanisms. RESULTS: Both hsa_circ_0008571 and Integrin beta 8 (ITGB8) were downregulated, while miR-145-5p transcription was elevated in the aneurysm wall of IAs patients compared to superficial temporal artery tissues. In vitro, cell migration and growth were dramatically suppressed after hsa_circ_0008571 overexpression. Mechanistically, has_circ_0008571 could suppress miR-145-5p activity by direct sponging. Moreover, we found that ITGB8 expression and the activation of the TGF-ß-mediated signaling pathway were significantly enhanced. CONCLUSION: The hsa_circ_0008571-miR-145-5p-ITGB8 axis plays an essential role in IA progression.

ITGB5 facilitates gastric cancer metastasis by promoting TGFBR2 endosomal recycling.

TGFBR2, a key regulator of the TGFß signaling pathway, plays a crucial role in gastric cancer (GC) metastasis through its endosomal recycling process. Despite its importance, the mechanisms governing this process remain unclear. Here, we identify integrin ß5 (ITGB5) as a critical mediator that promotes TGFBR2 endosomal recycling. Our study reveals elevated expression of ITGB5 in GC, particularly in metastatic cases, correlating with poor patient outcomes. Knockdown of ITGB5 impairs GC cell metastasis both in vitro and in vivo. Mechanistically, ITGB5 facilitates epithelial-mesenchymal transition mediated by TGFß signaling, thereby enhancing GC metastasis. Acting as a scaffold, ITGB5 interacts with TGFBR2 and SNX17, facilitating SNX17-mediated endosomal recycling of TGFBR2 and preventing lysosomal degradation, thereby maintaining its surface distribution on tumor cells. Notably, TGFß signaling directly upregulates ITGB5 expression, establishing a positive feedback loop that exacerbates GC metastasis. Our findings shed light on the role of ITGB5 in promoting GC metastasis through SNX17-mediated endosomal recycling of TGFBR2, providing insights for the development of targeted cancer therapies.

Sevoflurane blocks KLF5-mediated transcriptional activation of ITGB2 to inhibit macrophage infiltration in hepatic ischemia/reperfusion injury.

BACKGROUND: Sevoflurane (Sevo) preconditioning and postconditioning play a protective role against injury induced by hepatic ischemia/reperfusion (I/R). At the same time, the involvement of macrophage infiltration in this process and the precise mechanisms are unclear. Here, we designed this research to elucidate the protective effects of Sevo against hepatic I/R injury and the molecules involved. METHODS: The alleviating effect of Sevo on the liver injury was analyzed by liver function analysis, hematoxylin and eosin staining, Masson trichrome staining, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling, western blot analysis and an enzyme-linked immunosorbent assay. An in vitro cell model was developed using alpha mouse liver 12 (AML12) cells, and the cell model was treated with oxygen-glucose deprivation and reoxygenation and Sevo. Multiple bioinformatics databases were used to screen transcriptional regulators related to hepatic I/R injury and the targets of Krueppel-like factor 5 (KLF5). KLF5 expression was artificially upregulated alone or with integrin beta-2 (ITGB2) knockdown to substantiate their involvement in Sevo-mediated hepatoprotection. RESULTS: Sevo protected the liver against I/R injury by reducing cell apoptosis and inflammatory response. KLF5 was upregulated in liver tissues following I/R injury, whereas KLF5 overexpression aggravated macrophage infiltration and liver injury induced by I/R injury. KLF5 bound to the promoter of ITGB2 to enhance ITGB2 transcription. Knockdown of ITGB2 reversed the aggravation of injury caused by KLF5 overexpression in mice and AML12 cells. CONCLUSIONS: Sevo blocked KLF5-mediated transcriptional activation of ITGB2, thereby inhibiting macrophage infiltration in hepatic I/R injury.

The protective roles of integrin α4ß7 and Amphiregulin-expressing innate lymphoid cells in lupus nephritis.

Type 2 innate lymphoid cells (ILC2s) have emerged as key regulators of the immune response in renal inflammatory diseases such as lupus nephritis. However, the mechanisms underlying ILC2 adhesion and migration in the kidney remain poorly understood. Here, we revealed the critical role of integrin α4ß7 in mediating renal ILC2 adhesion and function. We found that integrin α4ß7 enables the retention of ILC2s in the kidney by binding to VCAM-1, E-cadherin, or fibronectin on structural cells. Moreover, integrin α4ß7 knockdown reduced the production of the reparative cytokine amphiregulin (Areg) by ILC2s. In lupus nephritis, TLR7/9 signaling within the kidney microenvironment downregulates integrin α4ß7 expression, leading to decreased Areg production and promoting the egress of ILC2s. Notably, IL-33 treatment upregulated integrin α4ß7 and Areg expression in ILC2s, thereby enhancing survival and reducing inflammation in lupus nephritis. Together, these findings highlight the potential of targeting ILC2 adhesion as a therapeutic strategy for autoimmune kidney diseases.

High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

The ADAM17 inhibitor ZLDI-8 sensitized hepatocellular carcinoma cells to sorafenib through Notch1-integrin ß-talk.

ZLDI-8 is an A disintegrin and metalloproteinase domain 17 (ADAM17) inhibitor that suppresses the shedding of Notch1 to the Notch1 intracellular domain (NICD). In previous studies, we found that ZLDI-8 was able to sensitize HCC to sorafenib, but the mechanism of action remains unclear. The sensitizing effects of ZLDI-8 were tested both in vitro and in vivo. EMT-related factors, sorafenib sensitivity-related proteins and ECM-related gene expression were assessed using immunohistochemistry, RTPCR and Western blotting. Knockdown assays were conducted to determine the relationship between the Notch and Integrin pathways. CoIP assays, nuclear and cytoplasmic fractionation and immunofluorescence colocalization were applied to explore the interaction between the Notch and Integrin pathways. Appropriate statistical analysis methods were used to assess the significance of the experimental results and to ensure the scientific validity and reliability of the experimental design. We found that ECM- and EMT-related proteins were downregulated after ZLDI-8 treatment (P<0.05). ZLDI-8 significantly downregulated Integrinß1 and Integrinß3 in HCC in vitro and in vivo (P<0.05), possibly through Foxc2-dependent regulation. Mechanistically, interfering with the expression of both Integrin-linked kinase (ILK) and the NICD may downregulate the expression of proteins targeted by sorafenib, thereby sensitizing cells to sorafenib. The retroregulation of Integrinß by ILK may occur through the interaction between the NICD and ILK and may be the result of the translocation of the complexus. Our study indicates that blocking the Notch pathway may affect Integrinß through crosstalk between the Notch1 and Integrinß/ILK signaling pathways, thus providing a potential therapeutic strategy for HCC.

Super-enhancer-driven LIF promotes the mesenchymal transition in glioblastoma by activating ITGB2 signaling feedback in microglia.

BACKGROUND: The mesenchymal (MES) subtype of glioblastoma (GBM) is believed to be influenced by both cancer cell-intrinsic alterations and extrinsic cellular interactions, yet the underlying mechanisms remain unexplored. METHODS: Identification of microglial heterogeneity by bioinformatics analysis. Transwell migration, invasion assays, and tumor models were used to determine gene function and the role of small molecule inhibitors. RNA sequencing, chromatin immunoprecipitation, and dual-luciferase reporter assays were performed to explore the underlying regulatory mechanisms. RESULTS: We identified the inflammatory microglial subtype of tumor-associated microglia (TAM) and found that its specific gene integrin beta 2 (ITGB2) was highly expressed in TAM of MES GBM tissues. Mechanistically, the activation of ITGB2 in microglia promoted the interaction between the SH2 domain of STAT3 and the cytoplasmic domain of ITGB2, thereby stimulating the JAK1/STAT3/IL-6 signaling feedback to promote the MES transition of GBM cells. Additionally, microglia communicated with GBM cells through the interaction between the receptor ITGB2 on microglia and the ligand ICAM-1 on GBM cells, while an increased secretion of ICAM-1 was induced by the proinflammatory cytokine leukemia inhibitory factor (LIF). Further studies demonstrated that inhibition of cyclin-dependent kinase 7 substantially reduced the recruitment of SNW1 to the super-enhancer of LIF, resulting in transcriptional inhibition of LIF. We identified notoginsenoside R1 as a novel LIF inhibitor that exhibited synergistic effects in combination with temozolomide. CONCLUSIONS: Our research reveals that the epigenetic-mediated interaction of GBM cells with TAM drives the MES transition of GBM and provides a novel therapeutic avenue for patients with MES GBM.

ITGB6 promotes pancreatic fibrosis and aggravates the malignant process of pancreatic cancer via JAK2/STAT3 signaling pathway.

Integrin ß6 (ITGB6) is upregulated in multiple tumor types and elevated ITGB6 levels have been detected in patients with chronic pancreatitis. However, the role of ITGB6 in pancreatic fibrosis and cancer remains to be elucidated. In the present study, ITGB6 expression was assessed using western blotting and qRT-PCR. Besides, cell proliferation, cycling, migration, and invasion were evaluated using CCK-8, flow cytometry, wound healing, and transwell assays, respectively. The expression of fibrosis and JAK2/STAT3 signaling markers was detected by western blotting and immunofluorescence analysis. Moreover, nude mice were subcutaneously injected with co-cultured cell suspensions to establish an in vivo model. The results showed that ITGB6 was highly expressed in pancreatic cancer tissues and TGF-ß-induced pancreatic stellate cells (PSCs). Inhibition of ITGB6 expression in PSCs resulted in clear inhibition of activated PSC proliferation, migration, and fibrogenesis. Additionally, reduced ITGB6 expression inhibits the JAK2/STAT3 signaling pathway. Interestingly, activators of the JAK2/STAT3 signaling pathway reversed the effects of ITGB6 disruption on PSCs. Activated PSCs notably promoted the proliferation, invasion, and migration of pancreatic cancer cells in a co-culture assay. In contrast, activated PSCs with low ITGB6 expression failed to significantly affect the malignancy of pancreatic cancer cells. Moreover, in vivo results showed that interference with ITGB6 inhibited the activation of PSCs and promoted the development of pancreatic cancer. Silencing ITGB6 inhibited the proliferation, migration, and fibrosis-like effects of activated PSCs and indirectly inhibited the metastasis and malignant process of pancreatic cancer by inhibiting the JAK2/STAT3 signaling pathway. Therefore, ITGB6 is a potential candidate target for pancreatic cancer prevention and treatment.

Phospho-eIF4E stimulation regulates coronavirus entry by selective expression of cell membrane-residential factors.

The eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation on serine 209. In a recent study, by two rounds of TMT relative quantitative proteomics, we found that phosphorylated eIF4E (p-eIF4E) favors the translation of selected mRNAs, and the encoded proteins are mainly involved in ECM-receptor, focal adhesion, and PI3K-Akt signaling. The current paper is focused on the relationship between p-eIF4E and the downstream host cell proteins, and their presumed effect on efficient entry of PEDV. We found that the depletion of membrane-residential factor TSPAN3, CD63, and ITGB2 significantly inhibited viral invasion of PEDV, and reduced the entry of pseudotyped particles PEDV-pp, SARS-CoV-pp, and SARS-CoV-2-pp. The specific antibodies of TSPAN3, CD63, and ITGB2 blocked the adsorption of PEDV into host cells. Moreover, we detected that eIF4E phosphorylation was increased at 1 h after PEDV infection, in accordance with the expression of TSPAN3, CD63, and ITGB2. Similar trends appeared in the intestines of piglets in the early stage of PEDV challenge. Compared with Vero cells, S209A-Vero cells in which eIF4E cannot be phosphorylated showed a decrease of invading PEDV virions. MNK kinase inhibitor blocked PEDV invasion, as well as reduced the accumulation of TSPAN3, CD63, and ITGB2. Further study showed that the ERK-MNK pathway was responsible for the regulation of PEDV-induced early phosphorylation of eIF4E. This paper demonstrates for the first time the connections among p-eIF4E stimulation and membrane-residential host factors. Our findings also enrich the understanding of the biological function of phosphorylated eIF4E during the viral life cycle.IMPORTANCEThe eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation. In our previous study, several host factors susceptible to a high level of p-eIF4E were found to be conducive to viral infection by coronavirus PEDV. The current paper is focused on cell membrane-residential factors, which are involved in signal pathways that are sensitive to phosphorylated eIF4E. We found that the ERK-MNK pathway was activated, which resulted in the stimulation of phosphorylation of eIF4E in early PEDV infection. Phospho-eIF4E promoted the viral invasion of PEDV by upregulating the expression of host factors TSPAN3, CD63, and ITGB2 at the translation level rather than at the transcription level. Moreover, TSPAN3, CD63, or ITGB2 facilitates the efficient entry of coronavirus SARS-CoV, SARS-CoV-2, and HCoV-OC43. Our findings broaden our insights into the dynamic phosphorylation of eIF4E during the viral life cycle, and provide further evidence that phosphorylated eIF4E regulates selective translation of host mRNA.

Acidity-induced ITGB6 promote migration and invasion of lung cancer cells by epithelial-mesenchymal transition and focal adhesion.

Non-small cell lung cancer (NSCLC) is a prevalent tumor and acidic tumor microenvironment provides an energy source driving tumor progression. We previously demonstrated significantly upregulated Integrin ß6 (ITGB6) in NSCLC cells. This study was designed to investigate the role of ITGB6 in NSCLC metastasis and explore the potential mechanisms. The expression of ITGB6 was evaluated in patients with NSCLC. Migration and invasion assays were utilized to investigate the role of ITGB6, and ChIP-qPCR and dual-luciferase reporter experiments preliminarily analyzed the relationship between ETS proto-oncogene 1 (ETS1) and ITGB6. Bioinformatics analysis and rescue models were performed to explore the underlying mechanisms. The results demonstrated that ITGB6 was upregulated in NSCLC patients and the difference was even more pronounced in patients with poor prognosis. Functionally, acidity-induced ITGB6 promoted migration and invasion of NSCLC cells in vitro, and epithelial-mesenchymal transition (EMT) and focal adhesion were the important mechanisms responsible for ITGB6-involved metastasis. Mechanistically, we revealed ETS1 enriched in the ITGB6 promoter region and promoted transcription to triggered the activation of subsequent signaling pathways. Moreover, ChIP-qPCR and dual-luciferase reporter experiments demonstrated that ETS1 played an important role in directly mediating ITGB6 expression. Furthermore, we found ITGB6 was responsible for the acidic microenvironment-mediated migration and invasion processes in NSCLC by performing rescue experiments with ITGB6 knockdown. Our findings indicated acidic microenvironment directly induced ETS1 to regulate the expression of ITGB6, and then the highly expressed ITGB6 further mediate EMT and activates the downstream focal adhesion pathways, eventually promotes the invasion and migration in NSCLC progression and metastasis.

PTP1B mediates the inhibitory effect of MFGE8 on insulin signaling through the ß5 integrin.

Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvß5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin ß5 complexes with the insulin receptor beta (IRß) in skeletal muscle, resulting in dephosphorylation of IRß and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between ß5 and IRß impacts IRß phosphorylation status. We show in in vitro and in vivo in skeletal muscle in mice that antibody-mediated blockade of the ß5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRß resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The ß5-PTP1B complex is recruited by MFGE8 to IRß leading to termination of canonical insulin signaling. ß5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and ß5 in regulating insulin signaling.

Integrin ß5 subunit regulates hyperglycemia-induced vascular endothelial cell apoptosis through FoxO1-mediated macroautophagy.

BACKGROUND: Hyperglycemia frequently induces apoptosis in endothelial cells and ultimately contributes to microvascular dysfunction in patients with diabetes mellitus (DM). Previous research reported that the expression of integrins as well as their ligands was elevated in the diseased vessels of DM patients. However, the association between integrins and hyperglycemia-induced cell death is still unclear. This research was designed to investigate the role played by integrin subunit ß5 (ITGB5) in hyperglycemia-induced endothelial cell apoptosis. METHODS: We used leptin receptor knockout (Lepr-KO) ( db / db ) mice as spontaneous diabetes animal model. Selective deletion of ITGB5 in endothelial cell was achieved by injecting vascular targeted adeno-associated virus via tail vein. Besides, we also applied small interfering RNA in vitro to study the mechanism of ITGB5 in regulating high glucose-induced cell apoptosis. RESULTS: ITGB5 and its ligand, fibronectin, were both upregulated after exposure to high glucose in vivo and in vitro . ITGB5 knockdown alleviated hyperglycemia-induced vascular endothelial cell apoptosis and microvascular rarefaction in vivo.In vitro analysis revealed that knockdown of either ITGB5 or fibronectin ameliorated high glucose-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). In addition, knockdown of ITGB5 inhibited fibronectin-induced HUVEC apoptosis, which indicated that the fibronectin-ITGB5 interaction participated in high glucose-induced endothelial cell apoptosis. By using RNA-sequencing technology and bioinformatic analysis, we identified Forkhead Box Protein O1 (FoxO1) as an important downstream target regulated by ITGB5. Moreover, we demonstrated that the excessive macroautophagy induced by high glucose can contribute to HUVEC apoptosis, which was regulated by the ITGB5-FoxO1 axis. CONCLUSION: The study revealed that high glucose-induced endothelial cell apoptosis was positively regulated by ITGB5, which suggested that ITGB5 could potentially be used to predict and treat DM-related vascular complications.

Integrin ß8 prevents pericyte-myofibroblast transition and renal fibrosis through inhibiting the TGF-ß1/TGFBR1/Smad3 pathway in diabetic kidney disease.

Diabetic kidney disease (DKD) is one of the leading causes to develop end-stage kidney disease worldwide. Pericytes are implicated in the development of tissue fibrosis. However, the underlying mechanisms of pericytes in DKD remain largely unknown. We isolated and cultured primary pericytes and rat mesangial cells (HBZY-1). Western blot and qRT-PCR analysis were used to explore the role and regulatory mechanism of Integrin ß8/transforming growth factor beta 1 (TGF-ß1) pathway. We also constructed pericyte-specific Integrin ß8 knock-in mice as the research objects to determine the role of Integrin ß8 in vivo. We discovered that reduced Integrin ß8 expression was closely associated with pericyte transition in DKD. Overexpressed Integrin ß8 in pericytes dramatically suppressed TGF-ß1/TGF beta receptor 1 (TGFBR1)/Smad3 signaling pathway and protected glomerular endothelial cells (GECs) in vitro. In vivo, pericyte-specific Integrin ß8 knock-in ameliorated pericyte transition, endothelium injury and renal fibrosis in STZ-induced diabetic mice. Mechanistically, Murine double minute 2 (MDM2) was found to increase the degradation of Integrin ß8 and caused TGF-ß1 release and activation. Knockdown MDM2 could partly reverse the decline of Integrin ß8 and suppress pericytes transition. In conclusion, the present findings suggested that upregulated MDM2 expression contributes to the degradation of Integrin ß8 and activation of TGF-ß1/TGFBR1/Smad3 signaling pathway, which ultimately leads to pericyte transition during DKD progression. These results indicate MDM2/Integrin ß8 might be considered as therapeutic targets for DKD.

Cell softness reveals tumorigenic potential via ITGB8/AKT/glycolysis signaling in a mice model of orthotopic bladder cancer.

BACKGROUND: Bladder cancer, characterized by a high potential of tumor recurrence, has high lifelong monitoring and treatment costs. To date, tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types. Nonetheless, the existence of soft tumor cells in bladder tumors remains elusive. Thus, our study aimed to develop a micro-barrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells. METHODS: The stiffness of bladder cancer cells was determined by atomic force microscopy (AFM). The modified microfluidic chip was utilized to separate soft cells, and the 3D Matrigel culture system was to maintain the softness of tumor cells. Expression patterns of integrin ß8 (ITGB8), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were determined by Western blotting. Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59 (TRIM59). The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models. RESULTS: Using our newly designed microfluidic approach, we identified a small fraction of soft tumor cells in bladder cancer cells. More importantly, the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens, in which the number of soft tumor cells was associated with tumor relapse. Furthermore, we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells. Simultaneously, we detected a remarkable up-regulation in ITGB8, TRIM59, and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts. CONCLUSIONS: The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness. Meanwhile, the soft tumor cells become more sensitive to chemotherapy after stiffening, that offers new insights for hampering tumor progression and recurrence.