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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Exploring the role of ITGB6: fibrosis, cancer, and other diseases.

Integrin ß6 (ITGB6), a member of the integrin family of proteins, is only present in epithelial tissues and frequently associates with integrin subunit αv to form transmembrane heterodimers named integrin αvß6. Importantly, ITGB6 determines αvß6 expression and availability. In addition to being engaged in organ fibrosis, ITGB6 is also directly linked to the emergence of cancer, periodontitis, and several potential genetic diseases. Therefore, it is of great significance to study the molecular-biological mechanism of ITGB6, which could provide novel insights for future clinical diagnosis and therapy. This review introduces the structure, distribution, and biological function of ITGB6. This review also expounds on ITGB6-related diseases, detailing the known biological effects of ITGB6.

Characterization of tumor microenvironment in glioblastoma multiforme identifies ITGB2 as a key immune and stromal related regulator in glial cell types.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor characterized by inter and intra-tumor heterogeneity and complex tumor microenvironment. To uncover the molecular targets in this milieu, we systematically identified immune and stromal interactions at the glial cell type level that leverages on RNA-sequencing data of GBM patients from The Cancer Genome Atlas. The perturbed genes between the high vs low immune and stromal scored patients were subjected to weighted gene co-expression network analysis to identify the glial cell type specific networks in immune and stromal infiltrated patients. The intramodular connectivity analysis identified the highly connected genes in each module. Combining it with univariable and multivariable prognostic analysis revealed common vital gene ITGB2, between the immune and stromal infiltrated patients enriched in microglia and newly formed oligodendrocytes. We found following unique hub genes in immune infiltrated patients; COL6A3 (microglia), ITGAM (oligodendrocyte precursor cells), TNFSF9 (microglia), and in stromal infiltrated patients, SERPINE1 (microglia) and THBS1 (newly formed oligodendrocytes, oligodendrocyte precursor cells). To validate these hub genes, we used external GBM patient single cell RNA-sequencing dataset and this identified ITGB2 to be significantly enriched in microglia, newly formed oligodendrocytes, T-cells, macrophages and adipocyte cell types in both immune and stromal datasets. The tumor infiltration analysis of ITGB2 showed that it is correlated with myeloid dendritic cells, macrophages, monocytes, neutrophils, B-cells, fibroblasts and adipocytes. Overall, the systematic screening of tumor microenvironment components at glial cell types uncovered ITGB2 as a potential target in primary GBM.

CircItgb5 promotes synthetic phenotype of pulmonary artery smooth muscle cells via interacting with miR-96-5p and Uba1 in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare but fatal cardiopulmonary disease mainly characterized by pulmonary vascular remodeling. Aberrant expression of circRNAs has been reported to play a crucial role in pulmonary vascular remodeling. The existing literature predominantly centers on studies that examined the sponge mechanism of circRNAs. However, the mechanism of circRNAs in regulating PAH-related protein remains largely unknown. This study aimed to investigate the effect of circItgb5 on pulmonary vascular remodeling and the underlying functional mechanism. MATERIALS AND METHODS: High-throughput circRNAs sequencing was used to detect circItgb5 expression in control and PDGF-BB-treated pulmonary arterial smooth muscle cells (PASMCs). Localization of circItgb5 in PASMCs was determined via the fluorescence in situ hybridization assay. Sanger sequencing was applied to analyze the circularization of Itgb5. The identification of proteins interacting with circItgb5 was achieved through a RNA pull-down assay. To assess the impact of circItgb5 on PASMCs proliferation, an EdU assay was employed. Additionally, the cell cycle of PASMCs was examined using a flow cytometry assay. Western blotting was used to detect biomarkers associated with the phenotypic switch of PASMCs. Furthermore, a monocrotaline (MCT)-induced PAH rat model was established to explore the effect of silencing circItgb5 on pulmonary vascular remodeling. RESULTS: CircItgb5 was significantly upregulated in PDGF-BB-treated PASMCs and was predominately localized in the cytoplasm of PASMCs. In vivo experiments revealed that the knockdown of circItgb5 attenuated MCT-induced pulmonary vascular remodeling and right ventricular hypertrophy. In vitro experiments revealed that circItgb5 promoted the transition of PASMCs to synthetic phenotype. Mechanistically, circItgb5 sponged miR-96-5p to increase mTOR level and interacted with Uba1 protein to activate the Ube2n/Mdm2/ACE2 pathway. CONCLUSIONS: CircItgb5 promoted the transition of PASMCs to synthetic phenotype by interacting with miR-96-5p and Uba1 protein. Knockdown of circItgb5 mitigated pulmonary arterial pressure, pulmonary vascular remodeling and right ventricular hypertrophy. Overall, circItgb5 has the potential for application as a therapeutic target for PAH.

Upregulation of ITGB6 in primary palmar hyperhidrosis.

BACKGROUND: The regulatory effect of integrin ß6 (ITGB6) on sweat gland cells in primary palmar hyperhidrosis (PPH) remains unclear. OBJECTIVES: This study investigated the involvement of ITGB6 in the pathogenesis of PPH. MATERIAL AND METHODS: Sweat gland tissues were collected from PPH patients and healthy volunteers. The expression levels of ITGB6 in sweat gland tissues were detected with quantitative polymerase chain reaction (qPCR), western blot and immunohistochemical staining. Sweat gland cells were extracted from PPH patients, and identified with immunofluorescence staining of CEA and CK7. The expression of aquaporin 5 (AQP5) and Na-K-Cl cotransporter 1 (NKCC1) in primary sweat gland cells that overexpress ITGB6 were also detected. Through a series of bioinformatic methods, differentially expressed genes in sweat gland tissues were examined and validated via comparing PPH samples and controls. The key proteins and biological functions enriched in PPH were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. RESULTS: The ITGB6 was upregulated in sweat gland tissues of PPH patients compared to that of healthy volunteers. The CEA and CK7 were positively expressed in sweat gland cells extracted from PPH patients. The overexpression of ITGB6 upregulated AQP5 and NKCC1 protein expression in the sweat gland cells of PPH patients. A total of 562 differentially expressed mRNAs were identified using high-throughput sequencing (394 upregulated, 168 downregulated), which were mainly active in the chemokine and Wnt signaling pathways. After verification with qPCR and western blot, the overexpression of ITGB6 significantly upregulated CXCL3, CXCL5, CXCL10, and CXCL11, and downregulated Wnt2 mRNA and protein expression in sweat gland cells. CONCLUSIONS: The ITGB6 is upregulated in PPH patients. It may be involved in the pathogenesis of PPH by upregulating AQP5, NKCC1, CXCL3, CXCL5, CXCL10, and CXCL11, and downregulating Wnt2 expression in sweat glands.

Integrin ß6 mediates epithelial-mesenchymal transition in diabetic kidney disease.

The progression of diabetic kidney disease (DKD) is associated with increased fibronectin (FN) levels in proximal tubular epithelial cells. Bioinformatics analysis showed that integrin ß6 and cell adhesion function were significantly changed in the cortices of db/db mice. Remodelling of cell adhesion is one of the core changes during epithelial-mesenchymal transition (EMT) in DKD. Integrin is a family of transmembrane proteins that regulates cell adhesion and migration, and extracellular FN is the major ligand of integrin ß6. We found that the expression of integrin ß6 was elevated in the proximal tubules of db/db mice and FN-induced renal proximal tubule cells. The levels of EMT were also significantly increased in vivo and in vitro. In addition, FN treatment activated the Fak/Src pathway, increased the expression of p-YAP, and then upregulated the Notch1 pathway in diabetic proximal tubules. Knockdown of integrin ß6 or Notch1 reduced the EMT aggravation induced by FN. Furthermore, urinary integrin ß6 was significantly increased in DKD patients. Our findings reveal a critical role of integrin ß6 in regulating EMT in proximal tubular epithelial cells and identify a novel direction for the detection and treatment of DKD.