Integrin Β1 Subunit Research Articles

Identification of the minimal N-glycosylation on integrin α5β1 required for its inhibitory effect on EGFR signaling and cell proliferation

The N-glycosylation of integrin α5β1 is involved in multiple cell biological functions. Our group previously reported that the N-glycosylation of the Calf-1,2 domain on α5 subunit (S3-5,10-14) was important for its inhibitory effect on EGFR signaling through regulating α5-EGFR complex formation. In this follow-up study, we provide evidence that the N-glycosylation on integrin β1 subunit suppress cell growth by promoting its association with EGFR under fibronectin (FN)-coated conditions. Expression of wild-type (WT) β1, but not the N-glycosylation mutant S4-6 β1, which contains fewer N-glycans, inhibited EGFR signaling and cell proliferation after cell adhesion to FN. Furthermore, consistent restoration of the N-glycans on sites 1-3 of β1 reinstated the inhibitory effects. Mechanistically, the N-glycosylation mutant of β1 (S4-6+1-3) inhibited the EGFR response upon EGF stimulation via facilitating the α5β1-EGFR complex formation. Moreover, we identified the N-glycosylation of sites 10-14 on α5 and 1-3 on β1 were most important for EGFR signaling. Taken together, these data indicate that α5S3-5+10-14β1S4-6+1-3 mutant represents the minimal N-glycosylation required for its regulation on EGFR signaling and cell proliferation, providing a plausible mechanism for the crosstalk between with α5β1 and EGFR.

Platelet physiology and immunology: pathogenesis and treatment of classical and non-classical fetal and neonatal alloimmune thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a devastating disorder affecting approximately 0.5–1.5/1,000 live neonates. It occurs due to maternal immune responses against paternally inherited human platelet antigens (HPAs), resulting in low platelet counts, severe bleeding (e.g., intracranial hemorrhage; ICH), intrauterine growth restriction (IUGR), and miscarriage. There are currently 37 known HPAs; and 15 HPAs are located on the integrin β3 subunit. Fetomaternal incompatibilities in this protein have been most frequently reported, in which HPA-1 system accounts for more than 75% of FNAIT cases. The data from our animal models and from human anti-HPA-1a demonstrate that maternal anti-β3 antibodies have an anti-angiogenic effect, and that anti-angiogenesis, not thrombocytopenia, may be the key cause of ICH, suggesting that fetal platelet transfusion may have limited clinical benefits. Anti-β3 antibodies may also damage antigen positive trophoblasts via natural killer (NK) cells, causing placental dysfunction and miscarriage. Notably, anti-GPIbα alloantibody (e.g., anti-HPA-2) may induce platelet cell-based thrombin generation and thrombosis in placenta, leading to miscarriage. The consequences of anti-angiogenesis and pathology in placenta have not been adequately explored but may cause symptoms beyond thrombocytopenia and bleeding disorders, termed non-classical FNAIT. Meanwhile, maternal intravenous IgG (IVIG) transfusion is likely able to block pathogenic antibody transport across placenta, and ameliorate thrombocytopenia in fetal reticuloendothelial system (RES), although it is currently unclear whether IVIG has equal efficacy for all anti-HPAs or other platelet antigens such as CD36. Further research is required to define standard treatment protocols and explore new treatment options, such as anti-HPA-1a prophylaxis, anti-neonatal Fc receptor (FcRn) and anti-NK therapies. In this review, we summarize the current state of literature comprising platelet versatilities and hemostasis, and integrate new discoveries related to FNAIT etiological factors in order to develop better diagnostic and therapeutic strategies against this life-threatening disease.

Impact of Fucosylation on Self-Assembly of Prostate and Breast Tumor Spheroids by Using Cyclo-RGDfK(TPP) Peptide and Image Object Detection.

IntroductionCore fucosylation of N-glycans on the integrin β1 subunit is essential for the functional activity of the integrin. The binding of α5β1 integrin with the tripeptide Arg-Gly-Asp (RGD) motif within the extracellular matrix protein fibronectin may be influenced by the α-1,6-fucose core or α-1,2-fucose and α-1,3/4-fucose peripheral N-glycan profiles. Here, we investigated whether fucosylation impacts the formation of matrix-free 3D multicellular tumor spheroids (MCTS) from human triple negative breast MDA-MB231 cell line, prostate PC3 and DU145 cell lines and DU145 gemcitabine resistant (GemR) variant by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method.MethodsMicroscopic imaging, lectin histochemistry, flow cytometry, WST-1 cell viability assay and You Only Look Once version 2 (YOLOv2) training object detection using cyclic learning rates were used to evaluate the formation of MCTS, morphologic changes, and the expression levels of α-1,6-fucose and α-1,2-fucose linkages on the cell surface.ResultsDU145 prostate cancer cells expressed higher α-1,6-fucose than α-1,2-fucose linkages on their cell surface, as determined by lectin cytochemistry and flow cytometry. Blockage of the α-1,6- and α-1,2-fucose linkages with Aspergillus oryzae lectin (AOL) and Ulex Europaeus agglutinin I (UEA I) one hour before the addition of cyclic-RGDfK(TPP) peptide to the monolayer of the cancer cells resulted in a statistically significant dose-dependent reduction in spheroid volumes using threshold diameters of 40 and 60 µm. Application of a 40 µm threshold diameter measurements of spheroids resulted in fewer false-positive ones compared to the 60 µm diameter threshold previously used in our studies. A state-of-the-art, image object detection system YOLOv2 was used to automate the analysis of spheroid measurements and volumes. The results showed that YOLOv2 corroborated manual spheroid detection and volume measurements with high precision and accuracy.ConclusionFor the first time, the findings demonstrate that α-1,6- and α-1,2-fucose linkages of N-glycans on the cell surface receptors facilitate cyclo-RGDfK(TPP)-mediated self-assembly of cancer cells to form 3D multicellular tumor spheroids.

A disintegrin and metalloproteinase 12 prevents heart failure by regulating cardiac hypertrophy and fibrosis.

A disintegrin and metalloproteinase (ADAM)12 is considered to promote cardiac dysfunction based on the finding that a small-molecule ADAM12 inhibitor, KB-R7785, ameliorated cardiac function in a transverse aortic constriction (TAC) model by inhibiting the proteolytic activation of heparin-binding-EGF signaling. However, this compound has poor selectivity for ADAM12, and the role of ADAM12 in cardiac dysfunction has not yet been investigated using genetic loss-of-function mice. We revealed that ADAM12 knockout mice showed significantly more advanced cardiac hypertrophy and higher mortality rates than wild-type mice 4 wk after TAC surgery. An ADAM12 deficiency resulted in significantly more expanded cardiac fibrosis accompanied by increased collagen-related gene expression in failing hearts. The results of a genome-wide transcriptional analysis suggested a strongly enhanced focal adhesion- and fibrosis-related signaling pathway in ADAM12 knockout hearts. The loss of ADAM12 increased the abundance of the integrinβ1 subunit and transforming growth factor (TGF)-β receptor types I and III, and this was followed by the phosphorylation of focal adhesion kinase, Akt, mammalian target of rapamycin, ERK, and Smad2/3 in the heart, which resulted in cardiac dysfunction. The present results revealed that the loss of ADAM12 enhanced focal adhesion and canonical TGF-β signaling by regulating the abundance of the integrinβ1 and TGF-β receptors.NEW & NOTEWORTHY In contrast to a long-believed cardio-damaging role of a disintegrin and metalloproteinase (ADAM)12, cardiac hypertrophy was more severe, cardiac function was lower, and mortality was higher in ADAM12 knockout mice than in wild-type mice after transverse aortic constriction surgery. The loss of ADAM12 enhanced focal adhesion- and fibrosis-related signaling pathways in the heart, which may compromise cardiac function. These results provide insights for the development of novel therapeutics that target ADAM12 to treat heart failure.

Corrigendum. Decreased PECAM1-mediated TGF-β1 expression in the mid-secretory endometrium in women with recurrent implantation failure.

Study question Is recurrent implantation failure (RIF) associated with decreased expression of platelet and endothelial cell adhesion molecule 1 (PECAM1) and transforming growth factor β1 (TGF-β1) in the endometrium during the implantation window? Summary answer The present study demonstrates that the expression of PECAM1 and TGF-β1 is significantly decreased in the mid-secretory endometrium in women with RIF, which may account for embryo implantation failure. What is known already RIF has become a bottleneck issue that hampers the improvement of pregnancy rates in IVF-embryo transfer (IVF-ET). The causes of RIF are complex and may involve the dysregulation of various growth factors, metabolites, and inflammatory cytokines. At present, the precise pathogenesis of RIF has not been elucidated. Study design, size, duration This was a prospective case-control study. Endometrial tissue samples were obtained from January 2014 to December 2016 from two groups of women who had undergone IVF (RIF group, 22 women who underwent ≥3 ETs including a total of ≥4 good-quality embryos without pregnancy, control group, 18 women who conceived in their first treatment cycle). At the same time, samples were obtained from 18 women with infertility secondary to tubal factor in the early proliferative, late proliferative and mid-secretory phases of the menstrual cycle (n = 6 per group). Samples used for isolation of primary human endometrial epithelial cells and stromal cells (HEECs and HESCs) were collected in December 2017 from six women with infertility secondary to tubal factor. Participants/materials, setting, methods We investigated gene expression using integrative whole genome expression microarray analysis, including differentially expressed gene screening, principal component analysis, and functional enrichment analysis. RT-qPCR, western blotting, immunohistochemistry, immunofluorescence co-localization analysis and short hairpin RNA (shRNA) plasmid transfection in Ishikawa cell line, HEECs and HESCs were used to investigate the expression of PECAM1 and TGF-β1. Main results and the role of chance Integrative data mining of whole-genome expression profiles identified cell adhesion as a key regulator in RIF. Database retrieval and literature review screened several novel cell adhesion-related genes that might participate in embryo implantation, which include PECAM1, intercellular adhesion molecule 2 (ICAM2), integrin subunit β2 (ITGB2), selectin P (SELP) and TEK receptor tyrosine kinase (TEK). Among these targets, the mRNA and protein levels of PECAM1 were significantly lower in the RIF group than those in the control group. During the menstrual cycles of women with secondary infertility, the protein expression level of PECAM1 was the lowest in early proliferative phase, slightly increased in late proliferative phase and was the highest in mid-secretory phase. While the expression level of HOXA10, an endometrial receptivity marker, kept at a low level in early proliferative phase and increased in late proliferative phase, then maintained at a high level in the mid-secretory phase. Furthermore, TGF-β1, mediated by PECAM1, was also decreased significantly in the RIF group. Using shRNA-based approach, we demonstrated that the depletion of PECAM1 significantly decreased the expression of TGF-β1 in Ishikawa cells, as well as in primary HEECs and HESCs. These results indicated that PECAM1 and TGF-β1 might play a pivotal role in modulating endometrial receptivity. Limitations reasons for caution Although we have shown that PECAM1 and TGF-β1 were down-regulated in the women with RIF, the molecular mechanism of the effect of the factors on the endometrial receptivity remain unclear. Wider implications of the findings Our findings provide insight into the contribution of PECAM1 and TGF-β1 in regulating implantation, which could be used to develop potential therapeutic methods for RIF. Study funding/competing interest(s) This work was supported by grants from the National Natural Science Foundation of China (Nos. 81771656 and 81370763), Special fund for clinical research of the Chinese Medical Association (No. 16020480664), and the Merck Serono China Research Fund for Fertility Agreement. The authors have no competing interests.

An Authentic Murine Model of Fetal/Neonatal Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally-inherited antigens present on the surface of fetal platelets. The human platelet alloantigen, HPA-1a (also known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism residing within the ~50 amino acid plexin/semaphorin/integrin (PSI) domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein (GP)IIIa), is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa. Neither human polyclonal nor mouse monoclonal anti-HPA-1a antibodies, however, recognize murine GPIIIa due to amino acid differences both within and surrounding the HPA-1a alloepitope. As a result, there are currently no authentic mouse models of FNAIT capable of recapitulating the human alloimmune response to this clinically important platelet alloantigen. We have recently shown that humanizing residues 30, 32, 33, and 39 within the PSI domain, as well as amino acid 470 within the linearly distant but conformationally close Epidermal Growth Factor (EGF) 1 domain, of murine GPIIIa, is sufficient to re-create the epitope recognized by most maternal anti-HPA-1a alloantibodies. We therefore used CRISPR/Cas9 gene editing technology to generate transgenic mice whose platelets express human residues A30P32L33D39Q470 on a murine GPIIIa backbone - hereafter referred to as APLDQ mice. Intraperitoneal injection of an anti-HPA-1a mAb induced severe thrombocytopenia in these APLDQ, but not wild-type, mice. Furthermore, platelets from APLDQ mice, when introduced into wild-type mice, elicited a strong polyclonal immune response that was specific for, and importantly restricted to, the epitopes created by these humanized residues, demonstrating that the APLDQ humanized form of murine GPIIIa is immunogenic in mice. Wild-type female mice pre-immunized with APLDQ platelets, when bred with APLDQ male mice, gave birth to severely thrombocytopenic pups, many of whom exhibited an accompanying bleeding phenotype. Notably, administration of intravenous immunoglobulin G (IVIG) into pregnant female mice at days 10 and 17 lowered the concentration of anti-APLDQ alloantibodies in both the maternal and fetal circulation, and importantly normalized the platelet count in the pups. Taken together, these data establish a novel murine model of FNAIT that recapitulates many of the clinically important features of FNAIT. Availability of APLDQ humanized transgenic mice should pave the way for the pre-clinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans. Disclosures No relevant conflicts of interest to declare.

Breast cancer cells expressing cancer-associated sialyl-Tn antigen have less capacity to develop osteolytic lesions in a mouse model of skeletal colonization.

Breast cancer is one of the most prevalent malignancies in women, and approximately 75-80% of patients with advanced breast cancer develop bone metastasis. Expression of the cancer-associated carbohydrate antigen sialyl-Tn (STn) in breast cancer is associated with a poor prognosis; however, involvement of STn in the development of metastatic bone lesions remains unclear. We investigated whether STn expression on breast cancer cells influences intraosseous tumor growth and bone response in mice models of skeletal colonization. STn-positive (STn+) breast cancer cells were generated by stable transfection of an expression vector encoding ST6GaLNAc I into the breast cancer cell line MDA-MB-231. Parental MDA-MB-231 cells not expressing STn antigen were used as STn-negative (STn-) breast cancer cells. Contrary to expectations, STn expression attenuated the development of destructive bone lesions in the in vivo mice models. An in vitro study demonstrated that STn expression impaired adhesion of MDA-MB-231cells to bone marrow stromal cells. This finding in vitro was also confirmed by another breast cancer cell line MCF-7. Cell adhesion to fibronectin and type I collagen was also impaired in STn+ MDA-MB-231 cells compared to that in STn- MDA-MB-231 cells, suggesting integrin dysfunction. Given that the integrin β1 subunit is the main carrier of the STn epitope, it is likely that changes in glycan structure impaired the adhesive capacity of β1 integrin in the bone environment, leading to attenuation of tumor cell engraftment. In conclusion, breast cancer cells expressing STn antigen had less capacity for skeletal colonization, possibly due to impaired adhesive capability.

Current Anti-HPA-1a Standard Antibodies React with the β3 Integrin Subunit but not with αIIbβ3 and αvβ3 Complexes.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) results from maternal alloantibodies (abs) reacting with fetal platelets expressing paternal human platelet antigens (HPAs), mostly HPA-1a. Anti-HPA-1a abs, are the most frequent cause of severe thrombocytopenia and intracranial hemorrhage (ICH). Titration of anti-HPA-1a in maternal serum using standard National Institute for Biological Standards and Control (NIBSC) 03/152 is one diagnostic approach to predict the severity of FNAIT. Recently, we found three anti-HPA-1a subtypes reacting with the β3 subunit independently or dependently from complexes with αIIb and αv. Endothelial cell-reactive anti-αvβ3 abs were found predominantly in cases with ICH. Our aim was to assess whether available standard material represents all anti-HPA-1a subtypes. In this study, anti-HPA-1a sera (NIBSC 03/152) and human monoclonal antibodies (moabs) against HPA-1a (moabs 26.4 and 813) were evaluated using transfected cell lines expressing αIIbβ3, αvβ3 or monomeric cβ3. Flow cytometry analyses with well-characterized murine moabs recognizing αIIbβ3, αvβ3, or β3 alone demonstrated that AP3 reacts compound-independently, whereas compound-dependent moabs Gi5 and 23C6 reacted only with complexes. NIBSC 03/152, moabs 26.4, and 813 against HPA-1a reacted like AP3, same results were obtained with monomeric cβ3 in immunoblotting. Antigen capture assay targeting endothelial cells showed anti-HPA-1a reactivity disappearance after cβ3 beads adsorption. Furthermore, in contrast to anti-HPA-1a abs from ICH cases, none of NIBSC 03/152, 26.4, and 813 inhibited tube formation. These results suggest that current anti-HPA-1a standard material contains only the anti-β3 subtype. The absence of anti-αvβ3 makes NIBSC 03/152 less suitable as standard to predict the severity of FNAIT.

Integrin heterodimer α9 β1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state.

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α9 and β1 subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII-like repeats. Therefore, this study re-evaluated the existence of integrin α9 β1 actively functioning on the plasma membrane of porcine SSCs using full-length native tenascin C with FNIII-like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α9 β1 on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α9 β1 significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α9 and β1 subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.

Activation of αvβ3 Integrin Alters Fibronectin Fibril Formation in Human Trabecular Meshwork Cells in a ROCK-Independent Manner.

PurposeFibronectin fibrillogenesis is an integrin-mediated process that may contribute to the pathogenesis of primary open-angle glaucoma (POAG). Here, we examined the effects of αvβ3 integrins on fibrillogenesis in immortalized TM-1 cells and human trabecular meshwork (HTM) cells.MethodsTM-1 cells overexpressing wild-type β3 (WTβ3) or constitutively active β3 (CAβ3) integrin subunits were generated. Control cells were transduced with an empty vector (EV). Deoxycholic acid (DOC) extraction of monolayers, immunofluorescence microscopy, and On-cell western analyses were used to determine levels of fibronectin fibrillogenesis and fibronectin fibril composition (EDA+ and EDB+ fibronectins) and conformation. αvβ3 and α5β1 Integrin levels were determined using fluorescence-activated cell sorting (FACS). Cilengitide and an adenovirus vector expressing WTβ3 or CAβ3 integrin subunits were used to examine the role of αvβ3 integrin in HTM cells. The role of the canonical α5β1 integrin–mediated pathway in fibrillogenesis was determined using the fibronectin-binding peptide FUD, the β1 integrin function-blocking antibody 13, and the Rho kinase (ROCK) inhibitor Y27632.ResultsActivation of αvβ3 integrin enhanced the assembly of fibronectin into DOC-insoluble fibrils in both TM-1 and HTM cells. The formation of fibronectin fibrils was dependent on α5β1 integrin and could be inhibited by FUD. However, fibrillogenesis was unaffected by Y27632. Fibrils assembled by CAβ3 cells also contained high levels of EDA+ and EDB+ fibronectin and fibronectin that was stretched.Conclusionsαvβ3 Integrin signaling altered the deposition and structure of fibronectin fibrils using a β1 integrin/ROCK-independent mechanism. Thus, αvβ3 integrins could play a significant role in altering the function of fibronectin matrices in POAG.

Integrin αvβ3 enhances replication of porcine epidemic diarrhea virus on Vero E6 and porcine intestinal epithelial cells

The entry mechanism of porcine epidemic diarrhea virus (PEDV) remains unclear, especially the virus receptor. Our previous study revealed a potential correlation between integrin αvβ3 and PEDV infection. In the current study, the effect of overexpression, silencing, antibody inhibition, and co-expression with porcine aminopeptidase N (pAPN) of integrin αvβ3 on PEDV infection was investigated and analyzed in African green monkey Vero E6 cells and porcine intestinal epithelial cells (IECs) using the classical strain CV777 and variant strain HM2017 of PEDV. Integrin αvβ3 significantly enhanced the replication of the classical and variant strains of PEDV in Vero E6 cells and IECs. The integrin αv and β3 subunits were both involved in the enhancement of PEDV infection, the Arg–Gly–Asp peptides targeting integrin αvβ3 significantly inhibited replication of PEDV in Vero E6 cells, and co-expression of integrin αvβ3 with pAPN significantly enhanced replication of PEDV in Vero E6 and BHK-21 cells. These results demonstrate that integrin αvβ3 enhances PEDV replication in Vero E6 cells and IECs. These data provide novel insights into the entry mechanism of PEDV.

Identification of differentially expressed genes between primary lung cancer and lymph node metastasis via bioinformatic analysis

Lung cancer (LC), with its high morbidity and mortality rates, is one of the most widespread and malignant neoplasms. Mediastinal lymph node metastasis (MLNM) severely affects postoperative survival of patients with LC. Additionally, the molecular mechanisms of LC with MLNM (MM LC) remain not well understood. To identify the key biomarkers in its carcinogenesis and development, the datasets GSE23822 and GSE13213 were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified, and the Database for Annotation, Visualization and Integrated Discovery was used to perform functional annotations of DEGs. Search Tool for the Retrieval of Interacting Genes and Cytoscape were utilized to obtain the protein-protein interaction (PPI) network, and to analyze the most significant module. Subsequently, a Kaplan-Meier plotter was used to analyze overall survival (OS). Additionally, one co-expression network of the hub genes was obtained from cBioPortal. A total of 308 DEGs were identified in the two microarray datasets, which were mainly enriched during cellular processes, including the Gene Ontology terms ‘cell’, ‘catalytic activity’, ‘molecular function regulator’, ‘signal transducer activity’ and ‘binding’. The PPI network was composed of 315 edges and 167 nodes. Its significant module had 11 hub genes, and high expression of actin β, MYC, arginine vasopressin, vesicle associated membrane protein 2 and integrin subunit β1, and low expression of NOTCH1, synaptojanin 2 and intersectin 2 were significantly associated with poor OS. In summary, hub genes and DEGs presented in the present study may help identify underlying targets for diagnostic and therapeutic methods for MM LC.

FRK suppresses human glioma growth by inhibiting ITGB1/FAK signaling

Fyn-related kinase (FRK), a member of the Src-related tyrosine kinase family, functions as a tumor suppressor in several malignancies. We previously showed that FRK overexpression inhibited the growth of glioma cells. However, it is unknown whether FRK is equally effective against intracranial glioma in vivo, and the mechanism by which FRK influences glioma cell growth remains unclear. In this study, we found that tumor volume was reduced by about one-third in mice with FRK overexpression, which showed improved survival relative to controls. Immunofluorescence analysis revealed that FRK overexpression inhibited glioma cell proliferation and induced their apoptosis. Importantly, in vitro we further found that FRK decreased the expression of integrin subunit β1 (ITGB1) at both the mRNA and protein levels. FRK also inhibited transactivation by ITGB1, resulting in the suppression of its target proteins AKT and focal adhesion kinase (FAK). ITGB1 overexpression promoted glioma cell growth and partially reduced FRK-induced growth suppression. These results indicate that FRK inhibits human glioma growth via regulating ITGB1/FAK signaling and provide a potential therapeutic target for the treatment of glioma.

A role for gangliosides and β1-integrin in the motility of olfactory ensheathing glia.

Olfactory ensheathing glia (OEG) are found in the olfactory mucosa, nerve and bulb, and provide in vivo ensheathment for the unmyelinated olfactory axons within the central and peripheral nervous system domains. OEG cells are able to migrate long distances within the neuropil of the central nervous system. Because gangliosides such as 9-O-acetyl GD3 have crucial regulatory roles in neuronal migration during development, we analyzed whether OEG in organotypical cultures are revealed by anti-9-O-acetyl GD3 and/or gangliosides are recognized by the A2B5 antibody (G-A2B5), and whether these gangliosides are involved in OEG migration. Our results showed that all OEG migrating out of a section of olfactory bulb onto a laminin substrate bound to the 9-O-acetyl GD3 and A2B5 antibodies, and that 2',3'-cyclic nucleotide phosphodiesterase (CNPase) colocalized with 9-O-acetyl GD3 and with G-A2B5. Additionally, we showed that the immune blockade of 9-O-acetyl GD3 or G-A2B5 reduced the migration of OEG on laminin, and that 9-O-acetyl GD3 and G-A2B5 colocalized with the β1-integrin subunit. We also confirmed the phenotype of in-vitro-grown OEG cells derived from adult rats, showing that they express CNPase, and also α-smooth muscle actin, which is not expressed by Schwann cells. Our data showed that the gangliosides 9-O-acetyl GD3 and G-A2B5 participate in the migratory activity of OEG cells, and that the β1-integrin subunit colocalizes with these gangliosides. These results suggest a new role for β1-integrin and gangliosides in the polarized migration of OEG cells, and provide new information on the molecules controlling OEG motility and behavior.

Orphan nuclear receptor COUP-TFII is an oncogenic gene in renal cell carcinoma.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) may be an oncogenic gene in renal cell carcinoma (RCC). However, the direct association between COUP-TFII expression and patient survival has not been investigated in patients with RCC, and the molecular oncogenesis of COUP-TFII in RCC remains unclear. The mRNA expression levels of COUP-TFII in the tumors of 283 patients with RCC were determined by RT-qPCR. The remaining 266 patients were categorized into low- and high-expression groups according to the cut off value generated by receiver operating curve (ROC) analysis. The function of COUP-TFII in RCC cells was tested by knockdown experiments in vitro. In the present study, it was revealed that the mRNA expression levels of COUP-TFII were significantly higher in tumors compared with those in adjacent non-cancerous tissues, and that the overexpression of COUP-TFII was strongly associated with poor patient survival. It was further demonstrated that knockdown of COUP-TFII suppressed proliferation, and induced apoptosis and cell cycle arrest in RCC cells in vitro. This also resulted in the activation of the mitochondria-mediated apoptosis pathway, impaired migration and invasion of RCC cells through epithelial-mesenchymal transition in vitro, and suppressed tumor growth in vivo. In addition, it was revealed that the induction of cell migration and invasion by COUP-TFII was mediated, at least in part, by integrin subunit β1. In summary, the present study indicated that COUP-TFII is an oncogenic gene in RCC, and a potential therapeutic target for the treatment of the disease.

Osteoblasts grown on microroughened titanium surfaces regulate angiogenic growth factor production through specific integrin receptors

Cellular attachment and response to biomaterials are mediated by integrin receptor binding to extracellular matrix proteins adsorbed onto the material surface. Osteoblasts interact with their substrates via several integrin complexes including fibronectin-binding α5β1 and collagen-binding α1β1 and α2β1. Knockdown of α2 or β1 integrin subunits inhibits the production of factors that promote an osteogenic microenvironment, including osteocalcin, osteoprotegerin, and TGFβ1. Osteoblasts also secrete several angiogenic growth factors such as VEGF-A (VEGF165), FGF-2, and angiopoietin 1, which are regulated by titanium surface topography and surface energy. Here, we examined whether signaling through integrin receptor complexes regulates production and secretion of angiogenic factors during osteoblast differentiation on microtextured Ti surfaces. To do this, integrin subunits α1, α2, α5, and β1 were stably silenced in MG63 osteoblast-like cells cultured on grit-blasted/acid-etched hydrophobic Ti (SLA) or on hydrophilic SLA (modSLA). VEGF-A production increased in response to Ti surface topography and energy in integrin α2, α5, and β1 silenced cells but decreased in α1-silenced cells. FGF-2 decreased on modSLA substrates in both α1 and α2-silenced cells but was unchanged in response to silencing of either α5 or β1. In integrin α1, α2, and β1-silenced cells, Ang-1 increased on modSLA but α5-silencing did not affect Ang-1 production during surface mediated differentiation. These results suggest that signaling through specific integrin receptor complexes during osteoblast differentiation on microstructured Ti substrates, regulates the production of angiogenic factors by those cells, and this is differentially regulated by surface hydrophilicity. Statement of SignificanceSuccessful implantation of synthetic biomaterials into bone depends on the biological process known as osseointegration. Osseointegration is a highly regulated communication of cells that orchestrates the migration of progenitor cells towards the implant site and promotes the deposition and mineralization of extracellular matrix proteins within the implant microenvironment, to tightly join the implant to native bone. In this process, angiogenesis functions as the initiation site of progenitor cell migration and is necessary for matrix deposition by providing the necessary nutrients for bone formation. In the present study, we show a novel regulation of specific angiogenic growth factors by integrin receptor complexes. This research is important to develop biomaterials that promote and maintain osseointegration through proper vascularization and prevent implant failure in patients lacking sufficient angiogenesis.

Conjugation of the T1 sequence from CCN1 to fibrin hydrogels for therapeutic vascularization.

Hydrogel matrices with angiogenic properties are much desirable for therapeutic vascularization strategies, namely to provide vascular supply to ischemic areas, transplanted cells, or bioengineered tissues. Here we report the pro-angiogenic effect of fibrin (Fb) functionalization with the T1 sequence from the angiogenic inducer CCN1, forseeing its use in the injured brain and spinal cord. Fibrin functionalization with 40 μM of T1 peptide effectively improved cellular sprouting of human brain microvascular endothelial cells (hCMEC/D3) in the absence of vascular endothelial growth factor (VEGF), without impacting the viscoelastic properties of Fb, cell viability, or proliferation. The pro-angiogenic effect of immobilized T1 was potentiated in the presence of VEGF and partially mediated through α6β1 integrin. The tethering of T1 also enhanced sprouting of human cord blood-derived outgrowth endothelial cells (OEC). Still, to elicit such effect, a higher input T1 concentration was required (60 μM), in line with the lower protein levels of α6 and β1 integrin subunits found in OEC comparing to hCMEC/D3, prior to embedment in Fb gel. Finally, the ability of T1-functionalized Fb in inducing cappilary invasion in vivo was assessed using the CAM assay, which evidenced a significant increase in the number of newly formed vessels at sites of implantation of T1-functionalized Fb, in the absence of soluble angiogenic factors. Overall these results demonstrate the potential of T1 peptide-presenting gels for use in therapeutic vascularization approaches. Considering T1 neurite-extension promoting capability and pro-angiogenic properties, T1-functionalized Fb hydrogels are particularly promising for application in the injured central nervous system.

Abstract 69: Integrin α6β4 associates with plectin and vimentin in focal adhesions to enhance breast cancer cell invasion

Abstract Integrin α6β4 binds plectin to associate with intermediate filaments such as vimentin. While vimentin and integrin α6β4 are associated with the aggressiveness of triple negative breast cancer (TNBC), the biological importance of their association is not clear. Here, we utilized naturally occurring Epidermolysis Bullosa mutations in the integrin β4 subunit that are deficient in plectin binding to investigate the association between integrin α6β4 and vimentin. Using integrin β4-deficient BT549 cells, we generated cell lines that expressed integrin β4 wild type (β4-wt), plectin binding mutants (β4-R1225H or β4-R1281W), 1355-1662 deletion mutant (β4-Δ1355-1662; which retains plectin binding but deletes the signaling domain) or an empty control vector (EV). Migration assay and three-dimensional (3D) culture were performed to test whether integrin α6β4 signaling and its association with plectin contribute to the migratory and invasive phenotypes. Compared to EV cells, β4-wt increases cell migration and 3D invasive growth, but the β4-R1225H and β4-R1281W could not. CRISPR knockout of plectin also blocked β4-wt 3D cell growth. Utilizing total internal reflection fluorescence (TIRF) microscopy, we found that integrin β4-wt and β4-Δ1355-1662 were recruited to focal adhesions (FAs, indicated by paxillin co-staining) on laminin-1 plus fibronectin matrices, but not when plated on laminin-1 or fibronectin alone. Integrin β4-R1225H and β4-R1281W did not localize to FAs. Similarly, integrin β4-wt in plectin knockout cells could not aggregate in FAs. Furthermore, we observed that integrin αv and β1 colocalized with integrin β4 in FAs. Proximity ligation assay (PLA) indicated that the close interaction of integrin β1 and plectin. Interestingly, depletion of integrin β1 significantly reduced integrin β4 accumulation in FAs. Further immunostaining showed that integrin β4, plectin, and vimentin aggregated in FAs in the central area of the cell where FAs predominantly formed. Depletion of vimentin did not impact the recruitment of integrin β4 in FAs or cell proliferation, but significantly suppressed cell migration and 3D growth in β4-wt cells. Integrin β4-plectin-vimentin complexes were also found in MDA-MB-231 cells where they co-localized with paxillin and connected with stress fibers. In summary, our study demonstrates the essential roles of plectin binding and vimentin in promoting the migratory and invasive phenotypes by integrin α6β4 signaling in TNBC. Our study further highlights a novel function of plectin binding and integrin β1 in enhancing integrin β4 dynamics. Citation Format: Lei Qi, Teresa Knifley, Min Chen, Kathleen L. O’Connor. Integrin α6β4 associates with plectin and vimentin in focal adhesions to enhance breast cancer cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 69.

Efficiency of different fragment lengths of the ubiquitous chromatin opening element HNRPA2B1-CBX3 in driving human CD18 gene expression within self-inactivating lentiviral vectors for gene therapy applications

Patients with leukocyte adhesion deficiency type 1 (LAD1) suffer from life-threatening bacterial infections due to mutations in the common β2 integrin subunit (CD18/ITGB2 gene).We tested different fragments of the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1-CBX3 locus for their efficiency in driving the human CD18 gene expression and compared it with that of an elongation factor 1 alpha promoter (EF1αL, 1169 bp; EF1αS 248 bp) and a murine stem cell virus (MSCV) promoter within the context of the same lentiviral vector backbone. These vectors were tested in vitro for the human CD18 gene expression on the surface of CD34+ hematopoietic stem cells (HSCs) isolated from both moderate and severe LAD1 patients. Among the promoters tested in the patients' CD34+ HSCs, only U631 bp, U652 bp, U1262 bp, 5′ 2.2 kb A2UCOE and EF1αS resulted in higher percentage of CD18+CD34+ cells comparable to that of the MSCV promoter. The U655 bp, U723 bp, U1296 bp, U2598 bp and EF1αL promoters resulted in comparatively lower numbers of CD18+CD34+ cells. This study would be useful in investigating the human CD18 gene expression in an ex vivo experiment to demonstrate the phenotypic correction of LAD1 in a pre-clinical model.

Comprehensive Evaluation of Surface Potential Characteristics on Mesenchymal Stem Cells' Osteogenic Differentiation.

The surface electric potential of biomaterials has been extensively proven to play a critical role in stem cells' fate. However, there are ambiguous reports on the relation of stem cells' osteogenic capacity to surface potential characteristics (potential polarity and intensity). To address this, we adopted a surface with a wide potential range and both positive/negative polarity in a comprehensive view to get insight into surface potential-regulating cellular osteogenic differentiation. Tb xDy1- xFe2 alloy/poly(vinylidene fluoride-trifluoroethylene) magnetoelectric films were prepared, and the film could provide controllable surface potential characteristics with positive or negative polarity and potential (ϕME) intensity variation from 0 to ±120 mV as well as keep the surface chemical composition and microstructure unchanged. Cell culture results showed that osteogenic differentiation of mesenchymal stem cells on both positive and negative potential films was obviously upregulated when the /ϕME/ intensities were set from 0-55 mV. Differently, the highest upregulated osteogenic differentiation on the positive potential films corresponded to the /ϕME/ intensity from 35-55 mV and was better than that on the negative potential films whereas the highest on the negative potential films corresponded to the /ϕME/ intensity from 0-35 mV and was better than that on the positive potential films. This fact could illustrate why previous reports appeared ambiguously; i.e., the comparative result in osteogenic differentiation between the positive and negative potential films strongly depends on the selection of surface potential intensity. On the basis of assaying of the exposed functional sites (RGD and PHSRN) of the adsorbed fibronectin (FN) and the expression of cellular integrin α5 and β1 subunits, the difference in the behavior between the positive and negative potential films was attributed to the distinct conformation of adsorbed fibronectin (FN) and the opposite changing trend with /ϕME/ for the two films, which triggers the osteogenesis-related FAK/ERK signaling pathway to a different extent. This study could provide new cognition for the in-depth understanding of the regulation mechanism underlying surface potential characteristics in cell behaviors.