ITGB1 Expression Research Articles

Selection and induction? Using ITGB4 to track the role of selection and constriction-induced changes in phenotype and 3D genome structure shifts after constricted migration

Metastatic cancer cells squeeze through small pores, often smaller than their nucleus, in order to reach a secondary site of proliferation. This constriction exerts forces on the nucleus, leading to deformations. Our previous work showed that A375 melanoma cells that have passed through 10 rounds of constricted migration exhibit stable differences in migratory phenotype, gene expression, and 3D genome structure (Golloshi et al. EMBO Reports, 2022). We are now investigating what combination of selection and induction forces lead to this stable difference. We have identified a cell surface protein, Integrin-β4 (ITGB4), as a marker whose RNA and protein expression level closely associates with cellular constricted migration proficiency over the course of sequential rounds of constriction. Sorting cells for expression of ITGB4 revealed a subpopulation of cells that, before constriction, already possess some of the phenotypic and 3D genome structure characteristics of proficient migrators. We next carefully tracked ITGB4 protein levels over a timecourse of sequential constricted migration, compared to unconstricted migration, in both the initial A375 population and a clonal population. We found evidence for both selection of ITGB4 expressing cells and also induction of ITGB4 expression after multiple constrictions. Intriguingly, this careful timecourse revealed that it consistently takes 3-4 rounds of constriction before a dramatic and stable shift in cell migratory phenotype. To investigate the molecular underpinnings of this lag, we collected nucleus images, Hi-C chromosome contact data, and gene expression data after 1 and 4 rounds of constriction. We propose that stable phenotypic differences after sequential constricted migration are the product of selection from a continuously regenerated heterogeneity combined with changes induced by the cumulative impact of multiple rounds of cell and nucleus constriction.

Propofol-mediated circ_0000735 downregulation restrains tumor growth by decreasing integrin-β1 expression in non-small cell lung cancer.

Propofol, an intravenous anesthetic agent, exerts an anti-tumor peculiarity in multifarious tumors. Circular RNA hsa_circ_0000735 (circ_0000735) is involved in non-small cell lung cancer (NSCLC) progression. The purpose of this study is to investigate whether propofol can curb NSCLC progression via regulating circ_0000735 expression. Cell viability, proliferation, apoptosis, and invasion were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine, flow cytometry, and transwell assays. Evaluation of protein levels was performed using western blotting or immunohistochemistry. Detection of circ_0000735 in tissue samples and cells was carried out using a real-time quantitative polymerase chain reaction. The molecular mechanisms associated with circ_0000735 were predicted by bioinformatics analysis and verified by dual-luciferase reporter assays. The relationship between propofol and circ_0000735 in vivo was verified by xenograft models. The results showed that circ_0000735 was overexpressed in NSCLC samples and cells. Propofol treatment overtly decreased circ_0000735 expression in NSCLC cells and repressed NSCLC cell viability, proliferation, invasion, and facilitated NSCLC cell apoptosis, but these effects mediated by propofol were counteracted by circ_0000735 overexpression. Circ_0000735 functioned as a miR-153-3p sponge and regulated integrin-β1 (ITGB1) expression via adsorbing miR-153-3p. ITGB1 overexpression reversed circ_0000735 silencing-mediated effects on NSCLC cell viability, proliferation, invasion, and apoptosis. In conclusion, propofol restrained NSCLC growth by downregulating circ_0000735, which functioned as a miR-153-3p sponge and regulated ITGB1 expression via adsorbing miR-153-3p. This study provides evidence to support that propofol curbs NSCLC progression by regulating circRNA expression.

3D-Printed Porous Tantalum Scaffold Improves Muscle Attachment via Integrin-β1-Activated AKT/MAPK Signaling Pathway.

3D-printed porous titanium (Ti) alloy scaffolds have been reported for facilitating muscle attachment in our previous study. However, the anti-avulsion ability needs to be improved. In this study, we used 3D-printed porous tantalum (Ta) scaffolds to improve muscle attachment. The differences in chemical and physical characteristics and muscle adhesion between the two scaffolds were tested and compared in the gene and protein level both in vitro and in vivo. The possible molecular mechanism was analyzed and further proved. The results showed that compared with the porous Ti alloy, porous Ta had better cell proliferation, differentiation, migration, and adhesion via the integrin-β1 (Itgb1)-activated AKT/MAPK signaling pathway in L6 rat myoblasts. When artificially down-regulated the expression of Itgb1, cell adhesion and myogenesis differentiation were affected and the phosphorylation of the AKT/MAPK signaling pathway was suppressed. In rat intramuscular implantation, porous Ta had a significantly higher muscle ingrowth rate (85.63% ± 4.97 vs 65.98% ± 4.52, p < 0.01) and larger avulsion force (0.972 vs 0.823 N/mm2, p < 0.05) than the porous Ti alloy. These findings demonstrate that the 3D-printed porous Ta scaffold is beneficial for further clinical application of muscle attachment.

Post-warming culture of human vitrified blastocysts with prolactin improves trophoblast outgrowth

BackgroundHuman embryos express the prolactin (PRL) receptor at the morula and blastocyst stages. Treatment with PRL from cleavage to the blastocyst stage improves blastocyst outgrowth on fibronectin-coated dishes. However, whether post-warming PRL treatment of blastocysts cultured without PRL could improve outgrowth competence remains unknown. Furthermore, the optimal time for post-warming PRL treatment remains to be ascertained. This study investigated the effects of PRL treatment during recovery culture on human blastocyst outgrowth and its related genes.MethodsIn total, 374 discarded vitrified blastocysts were randomly allocated to two groups, to be cultured with (n = 208) or without PRL (control; n = 166) for 120 min for recovery, and then plated on fibronectin-coated dishes. The expression level of PRL-interacting genes, blastocyst adhesion rate, outgrowth area, distance of trophoblast migration, and outgrowth degeneration were examined.ResultsThe mRNA expression of ezrin, radixin, and moesin, which regulate cell adhesion and invasion by controlling actin reorganization during epithelial-to-mesenchymal transition (EMT), was stimulated by PRL treatment for 120 min. The expression of EMT-related genes, transforming growth factor β1, snail1, and twist1 was also promoted following treatment with PRL for 120 min. PRL-treated blastocysts also exhibited augmented expression of cadherin 2 and transcriptional repression of cadherin 1. Higher mRNA expression of integrin-based focal adhesion-related genes, ITGA5 and ITGB1, was observed after treatment with PRL for 120 min than in the non- and shorter-treatment groups. PRL treatment for 120 min did not alter the rate of blastocyst adhesion to fibronectin-coated dishes 96 h after the outgrowth culture assay. However, multiple linear regression analysis revealed that the outgrowth area was significantly increased in PRL-treated blastocysts. The migration distance of trophoblast cells was significantly increased and degeneration rate was significantly decreased after PRL treatment. Furthermore, a more beneficial effect of PRL treatment on blastocyst outgrowth was observed when the blastocysts were vitrified on day 5 than when they were vitrified on day 6.ConclusionsPost-warming culture of human vitrified blastocysts with PRL for 120 min promoted trophoblast outgrowth in vitrified human blastocysts. Furthermore, PRL treatment may reduce outgrowth degeneration by increasing resistance to apoptosis during trophoblast migration.

Impacts of sodium butyrate on intestinal mucosal barrier and intestinal microbial community in a weaned piglet model.

Butyrate is thought to enhance intestinal mucosal homeostasis, but the detailed mechanism remains unclear. Therefore, further investigation on the mechanism of butyrate regulation of intestinal mucosal homeostasis was performed. This study used weaned piglets with similar intestinal metabolic function to humans as a research model. The dietary supplemented 0.2% sodium butyrate group (0.2% S) and negative control group (CON) were established to detect the effects of butyrate on growth performance, intestinal tissue morphology, mucosal barrier function, and intestinal microbial community structure in weaned piglets. There was an increase in average daily gain (ADG) during three different experimental periods and a reduction in average daily feed intake (ADFI) and feed-to-gain ratio (F:G) during days 1-35 and days 15-35 in 0.2% S compared with CON (P > 0.05). Furthermore, villus height in the ileum and duodenum was increased, and crypt depths in the colon and jejunum were reduced in both groups (P < 0.05). Moreover, the ratio of villus height and crypt depth (V/C) in 0.2% S both in the ileum and jejunum was significantly increased (P < 0.05) compared with CON. The relative mRNA expression of PKC, MUC1, CLDN1, and ITGB1 was upregulated in the ileum of 0.2% S compared with CON (P < 0.05). The digesta samples of 0.2% S, both in the ileum (P < 0.05) and colon, contained greater intestinal bacterial abundance and diversity of probiotics, including Lactobacillus, Streptococcus, Megasphaera, and Blautia, which promoted amino acid metabolism and energy production and conversion in the colon and the synthesis of carbon-containing biomolecules in the ileum. In summary, dietary supplementation with 0.2% sodium butyrate was shown to have a tendency to improve the growth performance of weaned piglets and enhance intestinal mucosal barrier function via altering the gut microbiota.

ALKBH5 activates FAK signaling through m6A demethylation in ITGB1 mRNA and enhances tumor-associated lymphangiogenesis and lymph node metastasis in ovarian cancer.

Background: Lymph node (LN) metastasis is common in patients with epithelial ovarian cancer (EOC) and is associated with poor prognosis. Tumor-associated lymphangiogenesis is the first stage of LN metastasis. Research on lymphangiogenesis and lymph node metastases can help develop new anti-LN-targeted therapies. Aberrant N6-methyladenosine (m6A) modifications have been reported to be linked to LN metastasis in several cancers, however, their role in EOC lymphangiogenesis and LN metastasis remains unclear. Methods: m6A levels in EOC tissues with or without LN metastases were evaluated by dot blot analysis. Real-time polymerase chain reaction (PCR) and immunofluorescence were used to examine the expression of m6A-related enzymes. Additionally, in vitro and in vivo functional studies were performed to discover the importance of the AlkB homolog 5 (ALKBH5) gene in EOC lymphatic metastasis. To identify the downstream target genes regulated by ALKBH5, we performed RNA pulldown, RNA-binding protein immunoprecipitation-quantitative PCR, co-immunoprecipitation, m6A-modified RNA immunoprecipitation-quantitative PCR, and luciferase reporter assays. Results: m6A modification was reduced in ovarian cancers with LN metastases. ALKBH5 overexpression increased tumor-associated lymphangiogenesis and LN metastasis both in vitro and in vivo. ALKBH5 overexpression also reversed the m6A modification in ITGB1 mRNA and suppressed the YTHDF2 protein-mediated m6A-dependent ITGB1 mRNA degradation, which resulted in increased expression of ITGB1 and phosphorylation of the focal adhesion kinase (FAK) and Src proto-oncogene proteins, thereby increasing LN metastasis. Furthermore, hypoxia induced the expression of hypoxia inducible factor 1 subunit alpha, which increased ALKBH5 expression and enhanced LN metastasis in EOC. Conclusions: The ALKBH5/m6A-ITGB1/FAK signalling axis is important in ovarian cancer lymphangiogenesis and LN metastasis. Antibodies that block ITGB1 and FAK kinase-inhibitors are promising anti-metastatic agents.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Novel role for integrin β4 in asthmatic children infected with Mycoplasma pneumoniae

Objectives: The regulatory role of integrin β4 (ITGB4) in asthmatic children infected with Mycoplasma pneumoniae (MP) was analyzed, and its potential molecular mechanisms and function were studied.Methods: We collected epidemiological data from 70,455 asthmatic children. The immunofluorescence assay was used to test 9 index IgMs against respiratory tract pathogens for 736 serum samples. Then, 98 children with severe asthma were treated via fiberoptic bronchoscope examination. During the surgery, the patients’ lavage fluid was collected. Additionally, differences in transforming growth factor-beta (TGF-β) expression between the MP-infected and noninfected groups were examined. Experiments were performed using white blood cell counting methods and flow cytometry for 98 asthmatic children.Results: We tested 736 specimens, and the percentages of MP, RSV, and ADV infections were 50.27%, 11.68%, and 10.05%, respectively. The percentage of eosinophils was increased significantly in the AS-I-MP group, and their TGF-β expression levels were increased, which was related to tissue fibrosis. Furthermore, MP infection exacerbated the decreasing trend of ITGB4 expression in patients’ blood compared with the noninfected group.Conclusions: There might be a chain reaction from MP infection to an increase in ITGB4, a decrease in TGF-β, a large accumulation of eosinophils and the development of asthma in children.

KCNF1 promotes lung cancer by modulating ITGB4 expression

Lung cancer continues to be the leading cause of cancer death in the United States. Despite recent advances, the five-year survival rate for lung cancer compared to other cancers still remains fairly low. The discovery of molecular targets for lung cancer is key to the development of new approaches and therapies. Electrically silent voltage-gated potassium channel (KvS) subfamilies, which are unable to form functional homotetramers, are implicated in cell-cycle progression, cell proliferation and tumorigenesis. Here, we analyzed the expression of KvS subfamilies in human lung tumors and identified that potassium voltage-gated channel subfamily F member 1 (KCNF1) was up-regulated in non-small cell lung cancer (NSCLC). Silencing of KCNF1 in NSCLC cell lines reduced cell proliferation and tumor progression in mouse xenografts, re-established the integrity of the basement membrane, and enhanced cisplatin sensitivity. KCNF1 was predominately localized in the nucleoplasm and likely mediated its functions in an ion-independent manner. We identified integrin β4 subunit (ITGB4) as a downstream target for KCNF1. Our findings suggest that KCNF1 promotes lung cancer by enhancing ITGB4 signaling and implicate KCNF1 as a novel therapeutic target for lung cancer.

The Role of Integrin Beta-1 in the Chemoresistance of Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive disease that has a poor prognosis with a survival rate of 30-40%. The bone marrow microenvironment provides protection to AML cells from chemotherapy and thus confers cell adhesion-mediated drug resistance (CAM-DR) and promotes relapse. Integrins are key anchor molecules in CAM-DR of leukemia cells; they are heterodimeric adhesion molecules on the cell surface consisting of an alpha and a beta unit. Upregulated integrin alpha 4 and 6 expression is associated with poor survival in leukemias. We hypothesize that the targeting of integrin beta 1 (ITGB1, β1, or CD29) will also affect the integrin alpha units as they are heterodimer partners. This could be a superior approach to overcome CAM-DR in AML than just targeting the individual alpha units. METHODS: ITGB1 gene was knocked out via CRISPR-Cas9 using 2 guide RNAs in AML cell lines and expression of integrin subunits was profiled by flow cytometry. AML cell lines were treated in vitro with the anti-human ITGB1 monoclonal antibody OS2966 in combination with Ara-C chemotherapy and we determined effects on cell viability by flow cytometry and on cell signaling using RNA-sequencing. Using human patient-derived AML xenograft mouse models, we tested if blockade of ITGB1 combined with chemotherapy can overcome CAM-DR in vivo. Mice were treated with control total Ig (10 mg/kg), OS2966 (10 mg/kg), total Ig + Ara-C (600 mg/kg), or OS2966 + Ara-C twice a week for two weeks. RESULTS: Analysis of patient-derived AML cells at diagnosis and relapse, and of AML cell lines, showed high expression of ITGB1 and varying levels of other alpha and beta units. Loss-of-function studies of ITGB1 using CRISPR/Cas9-mediated knockout of ITGB1 in MLL-rearranged and FLT3-ITD+ AML cell lines MV4-11 and MOLM-13, and in AML cell line U937, revealed that surface expression of integrin alphas 3, 4, 5 and 6 were markedly decreased as determined by flow cytometry analysis. ITGB2 expression was high in all 3 AML cell lines and not affected by ITGB1 deletion. Remarkably, ITGB3 was downregulated upon ITGB1 deletion in 2 out of 3 AML cell lines, whereas the 3rd AML cell line did not express any ITGB3. As a result of the ITGB1 deletion, AML cells showed less adhesion to murine bone marrow stroma (OP9) cells and to extracellular matrix proteins. We found that inhibition of surface ITGB1 on human AML using OS2966 de-adhered the AML cells from OP9 cells. Moreover, ITGB1 blockade using OS2966 synergized with Ara-C in vitro and significantly decreased cell viability compared to single agent controls in human AML cell lines U937 and MV4-11. RNA sequencing data suggest that the NFkB pathway is significantly dysregulated in the combination treatment Ara-C and OS2966 compared to Ara-C alone. To test ITGB1 inhibition in vivo, a patient-derived AML sample, HEM279 (Fig. 1A), and AML cell line, U937 (Fig. 1B), were engrafted into NSG-SGM3 mice. Leukemic mice were treated with single agents including control total Ig, OS2966, or Ara-C; and with total Ig in combination with Ara-C or OS2966. In both xenograft models, the combination treatment of OS2966 with Ara-C significantly prolonged survival of leukemic mice compared to Ara-C only: HEM279: MST 115 +/- 15.4 days vs 73.1 +/- 5.2 days post-AML injection; p=0.0005; U937: MST 170 days vs 32 days post-AML injection; p=0.0004; 50% of the mice engrafted with U937 and treated with OS2966 with Ara-C survived until day 290 post-AML injection, which is when the experiment was terminated. CONCLUSIONS: Integrin alpha subunit expression is dependent on ITGB1 expression, supporting our hypothesis that targeting integrin beta 1 affects alpha integrin units. ITGB1 inhibition with OS2966 combined with chemotherapy significantly decreases cell viability of AML cell lines in vitro and prolongs survival of xenograft mice in vivo. Our findings will allow for better understanding of the role of ITGB1 in drug resistance of AML and of ITGB1 as a preclinical target in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The effects of follicle stimulating hormone (FSH)-induced controlled ovarian hyperstimulation and nutrition on implantation-related gene expression in caruncular tissues of non-pregnant sheep

Disturbances at the conceptus-maternal interface can have detrimental effects on pregnancy outcome. Additionally, changes in body condition and exogenously administered gonadotropins could affect ovarian and uterine function, including cell proliferation and ovulation rates, and alter endometrial receptivity. In ruminants, endometrial caruncles maintain placental function via interaction with fetal chorionic cotyledons. Here, the effects of feeding regimens on the expression of selected genes known to be involved in uterine receptivity were investigated in the caruncles of control and FSH-superovulated ewes. Sheep were grouped according to their diet: control fed (CF), overfed (OF) or underfed (UF), and were either superovulated with FSH (SOV) or untreated (CON, naturally cycling) (n = 3–5/group). Caruncular samples for the assessment of the transcript levels of 11 target genes were collected at either the early (day 5) or mid-luteal (day 10) phases of the luteal lifespan, resulting in 12 groups of animals. The day of the estrous cycle affected the expression of ITGAV, ITGB3, FGF10 and IGFBP3 mRNA. There was lower expression of MUC1, and higher expression of FGF10, ITGB3 and FN1, on day 10 in CF_SOV animals. Compared with CF, expression of integrins (ITGB3, ITGA5 and ITGA4) was higher in OF and UF, and higher transcript levels of HGF and IGFBP3 in UF animals on day 10. Expression of ITGA5, ITGB1, -3, -5 and MUC1 was greater in OF_SOV than CF_SOV at day 10. In conclusion, it appears that imbalanced nutrition, by altering the expression of genes responsible for intercellular communication, cell adhesion, and encoding for growth factors, could affect the uterine responsiveness to exogenously applied hormonal stimulation and, likely, uterine receptivity.

ODP560 Thyroid Hormones membrane receptor, Integrin αvβ3, as a potential prognostic marker of chemotherapy response in breast cancer

Abstract Despite the development of novel therapies, breast cancer (BC) tumors are treated with conventional therapy eventually. Due to tumor heterogeneity, it is necessary to define new biomarkers to identify groups with different molecular characteristic and on this basis improve the treatment decision. The impact of thyroid status in BC growth has previously been studied. In particular, alterations of thyroid function during chemotherapy for BC treatment have been found and it has been demonstrated that T3 induces chemosensitization of BC cells. However, the relationship between thyroid hormones (THs) membrane receptor, integrin αvβ3, and BC response to chemotherapy remains unclear. The aim of this study is to evaluate the relationship between THs and integrin αvβ3 expression as prognostic value in BC patients. The mRNA expression of ITGB3 in the TCGA-PanCancer Atlas BC dataset (1084 patients) was associated with worse overall survival. Moreover, ITGB3 mRNA expression was significantly correlated with genes encoding multidrug resistance transporter proteins MDR1 and BCRP, (Pearson, R score &amp;gt;0.3 and p&amp;lt;0,05). The diagnostic role of ITGB3 in BC was assessed by receptor operating characteristic (ROC) curve analysis. ITGB3 mRNA levels were higher in non-responder patients treated with anthracycline (AUC=0,673, p=1.8e-10) and taxanes (AUC=0.675, p=1.9e-07). To confirm the association between the integrinαvβ3-mediated effects and multidrug resistance protein transporters, we conducted in vitro assays ofMDA-MB-231 cells treated with physiological concentrations of THs and evaluated their action on MDR1 protein levels and Rhodamine efflux as an indicator of the transporter activity. We found that THs induce MDR1 protein expression and transporter activation and this effect is blocked by the presence of the integrin selective inhibitor cilengitide. Furthermore, in MDA-MB-231 doxorubicin-resistant cells THs induce MDR1 and BCRP greater protein expression than in MDA-MB-231 parental cells and this effect is abrogated by cilengitide. Therefore, we found that integrin β3 is correlated with worse overall survival and response to anthracyclines and taxanes pointing out its role as acancer biomarker with potential clinical utility. In patients with high integrin β3 mRNA expression, we found a significant correlation with proteins involved in drug transport such as MDR1 and BCRP. We also demonstrated that THs through integrin αvβ3 modulate the expression and activity of drug transporters. These results pointout the role of THs, and their membrane receptor, in BC response to treatments and introduce a new biomarker with potential clinical significance. Presentation: No date and time listed

Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4.

Natriuretic peptide receptor 1 (NPR1) serves as a modulator of vascular endothelial homeostasis. Interactions between monocytes and endothelial cells may initiate endothelium dysfunction, which is known as an early hallmark of atherosclerosis. In this study, we performed RNA-sequencing analysis for the aorta of Npr1 knockout (Npr1+/−) mice and found that differentially expressed genes were significantly related to cell adhesion. This result was supported by an increased expression of intercellular adhesion molecule 1 (ICAM-1) in the aortic endothelium of Npr1+/− mice. Moreover, we observed that the knockdown of NPR1 increased ICAM-1 expression and promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs). NPR1 overexpression decreased ICAM-1 expression and inhibited the adhesion of monocytes to HUVECs treated by TNF-α (a cell adhesion inducer). Further analysis showed that adhesion-related genes were enriched in the focal adhesion signaling pathway, in which integrin beta 4 (Itgb4) was determined as a key gene. Notably, ITGB4 expression increased in vascular endothelium of Npr1+/− mice and in NPR1-knockdown HUVECs. The deficiency of ITGB4 decreased ICAM-1 expression and attenuated monocyte adhesion to NPR1-knockdown endothelial cells. Additionally, a reduced NPR1 and an increased ITGB4 expression level were found in an atherosclerosis mouse model. In conclusion, our findings demonstrate that NPR1 deficiency increases vascular endothelial cell adhesion by stimulating ITGB4 expression, which may contribute to the development of atherosclerosis.

Airway epithelial ITGB4 deficiency induces airway remodeling in a mouse model

Airway epithelial cells (AECs) with impaired barrier function contribute to airway remodeling through the activation of epithelial-mesenchymal trophic units (EMTUs). Although the decreased expression of ITGB4 in AECs is implicated in the pathogenesis of asthma, how ITGB4 deficiency impacts airway remodeling remains obscure. This study aims to determine the effect of epithelial ITGB4 deficiency on the barrier function of AECs, asthma susceptibility, airway remodeling, and EMTU activation. AEC-specific ITGB4 conditional knockout mice (ITGB4-/-) were generated and an asthma model was employed by the sensitization and challenge of house dust mite (HDM). EMTU activation-related growth factors were examined in ITGB4-silenced primary human bronchial epithelial cells of healthy subjects after HDM stimulation. Dexamethasone, the inhibitors of JNK phosphorylation or FGF2 were administered for the identification of the molecular mechanisms of airway remodeling in HDM-exposed ITGB4-/- mice. ITGB4 deficiency in AECs enhanced asthma susceptibility and airway remodeling by disrupting airway epithelial barrier function. Aggravated airway remodeling in HDM-exposed ITGB4-/- mice was induced through the enhanced activation of EMTU mediated by Src homology domain 2-containing protein tyrosine phosphatase 2/c-Jun N-terminal kinase/Jun N-terminal kinase-dependent transcription factor/FGF2 (SHP2/JNK/c-Jun/FGF2) signaling pathway, which was partially independent of airway inflammation. Both JNK and FGF2 inhibitors significantly inhibited the aggravated airway remodeling and EMTU activation in HDM-exposed ITGB4-/- mice. Airway epithelial ITGB4 deficiency induces airway remodeling in a mouse model of asthma through enhanced EMTU activation that is regulated by the SHP2/JNK/c-Jun/FGF2 pathway.

ARRDC3 inhibits liver fibrosis and epithelial-to-mesenchymal transition via the ITGB4/PI3K/Akt signaling pathway

Objective The effect of ARRDC3 has not been reported in liver fibrosis. Our study aimed to explore the molecular mechanisms by which ARRDC3 attenuates liver fibrosis. Methods The vectors pcDNA-ARRDC3 (which promotes ARRDC3 expression) and si-ITGB4 (which blocks IGTB4 expression) and their negative controls were constructed. The rat liver fibrosis model was established by intraperitoneal injection of CCl4 with or without intraperitoneal injection of pcDNA-ARRDC3. ELISA was used to detect the concentrations of γ-GGT, ALT, AST, and ALP in serum. HE, Masson’s trichome, and Sirius red staining were used to observe the pathological changes in liver tissue. LX-2 cells were treated with TGF-β, and pcDNA-ARRDC3 or si-ITGB4RNA was transfected to promote ARRDC3 expression or knock down ITGB4 expression. Western blotting was used to detect the expression levels of proteins. Results ARRDC3 effectively reduced liver injury, improved liver function, and decreased collagen production and deposition in the CCl4-induced rat fibrosis model. The studies showed that overexpressed ARRDC3 remarkably reduced the expression of E-cadherin and collagen-related protein and increased the expression of mesenchymal markers and EMT-related transcription factors, consequently inhibiting the activity of the ITGB4/PI3K/Akt signaling pathway. Conclusion Our study shows that ARRDC3 could ameliorate CCl4-induced liver fibrosis and EMT progression via the ITGB4/PI3K/Akt signaling pathway, which provides a meaningful reference for the clinical targeted treatment of liver fibrosis.

FOXO1 inhibits FSL-1 regulation of integrin β6 by blocking STAT3 binding to the integrin β6 gene promoter.

Integrin β6 (ITGB6), an epithelial-specific receptor, is downregulated in the gingival epithelium of periodontitis and is associated with inflammation response and periodontitis development. However, the transcriptional regulatory mechanism of ITGB6 downregulation in the human gingival epithelium remains unclear. Fibroblast-stimulating lipopeptide-1 (FSL-1), an oral biofilm component, promotes an epithelial cell-driven proinflammatory response in periodontitis partially by suppressing ITGB6 expression. The aim of the current study was to investigate the transcriptional regulatory mechanism of ITGB6 inhibition by FSL-1 in human epithelial cells (HaCaT and primary human gingival epithelial cells), and to delineate the transcriptional mechanism of ITGB6 suppression in periodontitis. We found that FSL-1 inhibited ITGB6 transcription through increasing forkhead box protein O1 (FOXO1) expression and inhibiting signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, FOXO1 bound to STAT3 directly, leading to decreased STAT3 phosphorylation induced by FSL-1. Consequently, the binding of phosphorylated STAT3 to the ITGB6 promoter was decreased, and ITGB6 transcription was therefore downregulated following FSL-1 stimulation. The reciprocal action of STAT3 and FOXO1 on ITGB6 downregulation was also confirmed by the immunostaining of the inflammatory epithelium associated with periodontitis. Our findings suggest that the interaction of FOXO1–STAT3 may be a useful signal target for the treatment of periodontitis.

A network pharmacology integrated pharmacokinetics strategy to investigate the pharmacological mechanism of absorbed components from crude and processed Zingiberis Rhizoma on deficiency-cold and hemorrhagic syndrome

Ethnopharmacological relevanceZingiberis Rhizoma (ZR) and Zingiberis Rhizoma Carbonisata (ZRC), as two forms of ginger-based herbal drugs used in China for at least 2000 years, have been recorded in Chinese Pharmacopoeia and applied for specific indications in traditional Chinese medicine (TCM). Aim of the studyThe present study aimed to explore the underlying therapeutic and processing mechanism of the absorbed components of ZR and ZRC on deficiency-cold and hemorrhagic syndrome (DCHS) using network pharmacological technique combined with pharmacokinetics strategy. Materials and methodsIn this study, a rapid and sensitive approach was conceived to simultaneously determine the seven components (zingiberone, 6-gingerol, 8-gingerol, 6-shogaol, 6-paradol, diacetyl-6-gingerol and 10-gingerol) in rat serum by HPLC-DAD-MS. The network pharmacological technique was employed to evaluate the effect of the absorbed components of ZR and ZRC on DCHS. Also, the vitro experiments were carried out to validate the functions of the seven compounds on coagulation and other major haematological effects. ResultsThe values of intra-assay and inter-assay precision were determined to be less than 7.44%, with an accuracy value ranging from 83.64% to 107.99%. Analysis of rat plasma revealed that the extraction recoveries and matrix effects of the seven analytes were >85.76%. The method for validation following oral administration of ZR and ZRC to rats was proved to be a success in the pharmacokinetic study of the seven ingredients. Pharmacokinetics showed that ZR processing could enhance the absorption and utilization of 6-shogaol, 6-paradol and diacetyl-6-gingerol, meanwhile reduce the absorption of 6-gingerol, 8-gingerol, and 10-gingerol. Through the pathway enrichment analysis, it was found that the significant biological process of ZR and ZRC on DCHS was primarily associated with complement, coagulation cascades and platelet activation pathways. The vitro experiments indicated that zingiberone, 6-paradol and diacetyl-6-gingerol had a hemostatic effect by upregulating the expression of one or more targets such as TNF-α, FⅩa, FⅫ, FⅧ, ICAM-1, vWF and ITGB3. While 6-gingerol, 6-shogaol, 8-gingerol and 10-gingerol played a critical role in promoting blood circulation by increasing the expression of TM and/or PORC, and/or reducing the expression of ITGB3. ConclusionIn brief, network pharmacological technique in combination with pharmacokinetics strategy provided an applicable method for pharmacological mechanism study of ZR and ZRC, which, also, could be used as reference for quality control of the two drugs. In a broader sense, this combined strategy might even be valuable in uncovering the therapeutic and processing mechanism of Chinese herbs on a systematic level.

Role of β1-integrin in promoting cell motility and tamoxifen resistance of human breast cancer MCF-7 cells.

The mechanism of acquired resistance of tamoxifen in endocrine therapy of breast cancer is not fully understood. In this study, we investigated the genomic changes in acquired tamoxifen-resistant cell lines. Tamoxifen-resistantsubclones (MCF-7R) derived from parent MCF-7 cells, which is an ER(+) breast cancer cell line, cultured with 4-hydrotamoxifen more than 6 months were used to obtain genomic alterations. Cell growth, microarray, and quantitative real-time PCR (q-RTPCR) assays were conducted. Additionally, the ITGB1 function was investigated in MCF-7R cells and MCF-7R ITGB1-silenced subclones using MTT and Transwell assays. Online pathway analysis was performed to assess the genetic characteristics of tamoxifen resistance. The gene expression profile of the tamoxifen-resistant cell line was considerably changed compared to the tamoxifen-sensitive cell line. Of 4102 genes with altered expressions, 1986 genes were upregulated, whereas 2116 were downregulated. The ITGB1 expression in MCF-7R cells was higher than that in MCF-7 cells. Interestingly, ITGB1 silencing partially rescued the sensitivity of MCF-7R cells to tamoxifen and reduced their motility. The activation of the β1-integrin signaling pathway was probably responsible for this phenomenon. Our data confirm the presence of alterations in the genes of tamoxifen-resistance breast cancer cells. ITGB1 probably partially contributes to tamoxifen resistance and cell motility via the β1-integrin signaling pathway. Thus, ITGB1 may be a potential target for the improvement of anti-hormone therapy reaction in ER(+) breast cancer patients.

P04.01.B High impact of ITGB1 on Pi3K/AKT expression in medulloblastoma

Abstract Medulloblastoma, an embryonal tumor of the cerebellum, is one of the most frequent malignant brain tumors. Despite the increasing use of genetic variation in treatment stratification, high-risk patients characterized by light meningeal spread, TP53 mutations, or MYC amplification still have poor survival. Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is one of the most important intracellular pathways, which can be considered as a master regulator for cancer. In tissue samples obtained from medulloblastoma patients, the significant upregulation of PI3K/AKT was associated with a lifting expression level of integrin β1(ITGB1). To understand the underlying mechanism, we investigated the effect of ITGB1 on the PI3K/AKT pathway in medulloblastoma cell lines. Transfection of this ITGB1 reduced proliferation and invasion of several medulloblastoma cell lines and inhibit epithelial-mesenchymal transition. In addition, knocking down ITGB1 expression can significantly inhibit the activation of PI3K/AKT signaling pathway. In conclusion, ITGB1 may selectively activation the pathophysiological effect of aberrant PI3K/AKT expression and serve as a targeted approach for medulloblastoma therapy.

Gene Expression Patterns of Osteopontin Isoforms and Integrins in Malignant Melanoma.

Osteopontin (OPN) is a multifunctional glycoprotein that physiologically interacts with different types of integrins. It is considered to be a possible prognostic biomarker in certain tumor types; however, various splicing isoforms exist, which have not been investigated in melanoma. We aimed to define the relative expression pattern of five OPN isoforms and clarify the prognostic significance of the splice variants in melanoma. We also aimed to investigate the expression pattern of eight integrins in the same tumors. Gene expression analyses revealed that the relative expression of OPNa, OPNb, and OPNc is significantly higher in metastatic tumors compared to primary lesions (p < 0.01), whereas the expression of OPN4 and OPN5 was low in both. The more aggressive nodular melanomas had higher expression levels compared to the superficial spreading subtype (p ≤ 0.05). The relative expression of the eight tested integrins was low, with only the expression of ITGB3 being detectable in nodular melanoma (Medianlog2 = 1.274). A positive correlation was found between Breslow thickness and the expression of OPNc variant, whereby thicker tumors (>4 mm) had significantly higher expression (p ≤ 0.05). The Breslow thickness was negatively correlated with the expression of OPN4, and similarly with ITGA2. OPNc also exhibited significant positive correlation with the presence of metastasis. Our data show that high expression of OPNa, OPNb, and especially OPNc and low expression of OPN4 and ITGA2 are associated with an advanced stage of tumor progression and poor prognosis in melanoma.