MAB1974 Sigma-AldrichAnti-Integrin β3 Antibody, clone BB10

Integrin-mediated force application induces a conformational change in latent TGF-β1 that leads to the release of the active form of the growth factor from the extracellular matrix (ECM). Mechanical activation of TGF-β1 is currently understood as an acute process that depends on the contractile force of cells. However, we show that ECM remodeling, preceding the activation step, mechanically primes latent TGF-β1 akin to loading a mechanical spring. Cell-based assays and unique strain devices were used to produce a cell-derived ECM of controlled organization and prestrain. Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-β1 activation after cell contraction or direct force application using magnetic microbeads. The release of active TGF-β1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM. The finding that ECM prestrain regulates the bioavailability of TGF-β1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.

The majority of women with ovarian cancer are diagnosed in late stages, and the mortality rate is high. The use of biomarkers as prognostic factors may improve the treatment and clinical outcome of these patients. We performed an external validation of the potential biomarkers CLU, ITGB3, CAPG, and PRAME to determine if the expression levels are relevant to use as prognostic factors.We analysed the gene expression of CLU, ITGB3, CAPG, and PRAME in 30 advanced staged serous adenocarcinomas with quantitative real-time polymerase chain reaction (QPCR) and the protein levels were analysed in 98 serous adenocarcinomas with western blot for semiquantitative analysis. Statistical differences in mRNA and protein expressions between tumours from survivors and tumours from deceased patients were evaluated using the Mann-Whitney U test.The gene and protein ITGB3 (Integrin beta 3) were significantly more expressed in tumours from survivors compared to tumours from deceased patients, which is in concordance with our previous results. However, no significant differences were detected for the other three genes or proteins CLU, CAPG, and PRAME.The loss of ITGB3 expression in tumours from deceased patients and high expression in tumours from survivors could be used as a biomarker for patients with advanced serous tumours.

Integrin alpha6beta4-mediated adhesion interactions play key roles in keratinocyte and epithelial tumor cell biology. In order to evaluate how alpha6beta4 adhesion interactions contribute to these important cellular processes, the authors generated soluble versions of the integrin by recombinant expression of the subunit ectodomains fused to a human immunoglobulin G (IgG) Fc constant domain. Coexpression of the appropriate subunits enabled dimerization, secretion and purification of stable Fc-containing alpha6beta4 heterodimers. The soluble proteins exhibited the same metal ion and ligand dependency in their binding characteristics as intact alpha6beta4. Using these reagents in combination with anti-beta4 antibodies, the authors identified two distinct functional epitopes on the beta4 subunit. They demonstrated the involvement of one epitope in adhesion interactions and the other in regulating adhesion-independent growth in alpha6beta4-expressing tumor cell lines. The availability of these soluble integrin reagents and the data provided herein help to further delineate the structure-function relationships regulating alpha6beta4 signaling biology.

The mortality rate for patients with ovarian carcinomas is high and the available prognostic factors are insufficient. The use of biomarkers may contribute to better prediction and survival for these patients. We aimed to study the gene and protein expressions for 7 potential biomarkers, to determine if it is possible to use them as prognostic factors. Genes selected from our previous microarray analysis (2006), CLU, ITGB3, TACC1, MUC5B, CAPG, PRAME and TROAP, were analyzed in 19 of the tumors with quantitative real-time polymerase chain reaction (QPCR). We found that CLU and ITGB3 were more expressed in tumors from survivors and PRAME and CAPG were more expressed in tumors from deceased patients. None of the other 3 genes were significantly differently expressed. The protein expressions of CLU, ITGB3, PRAME and CAPG were analyzed in 43 of the tumors with western blot for semiquantitative analysis. We established that the mRNA and protein expressions correlated and that all 4 proteins were significantly differently expressed. Further, immunohistochemistry (IHC) was used to localize the expression of the proteins in the tumor samples. According to our results, the 4 biomarkers CLU, ITGB3, PRAME and CAPG may be used as prognostic factors for patients with stage III serous ovarian adenocarcinomas.

The localization of five integrin subunit proteins was studied in human erythroleukemia (HEL) cells spreading on various culture substrata in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and the absence of serum. The cells readily adhered on fibronectin, but TPA was needed for adherence on vitronectin and for the spreading of the cells on both substrata. Indirect immunofluorescence microscopy showed that in the spread cells cultured on vitronectin or fibronectin for 2 hours, beta 1, beta 3, alpha 5, and alpha IIb integrin subunits were localized at focal adhesions as identified by talin-immunoreactivity. The alpha v integrin immunoreactivity was initially found at the focal adhesions when the cells were cultured on vitronectin, but was also found later in cells cultured on fibronectin. The alpha IIb integrin immunoreactivity disappeared from focal adhesions within 24 hours. The alpha 5 and beta 1 integrin immunoreactivities disappeared from the focal adhesions in cells cultured on vitronectin, but not in cells cultured on fibronectin. When the cells were plated on glass substratum in the presence of TPA, they spread much slower than on vitronectin or fibronectin, but some cells showed focal adhesions after only 8 hours in culture. In this case, the alpha v and beta 3 integrin subunits were found at focal adhesions. After TPA treatment, HEL cells deposited thrombospondin-immunoreactive material onto their culture substratum, but synthesis of fibronectin, vitronectin, fibrinogen, or von Willebrand factor was not detected. Thus, the results suggest that TPA would activate several integrin receptors in HEL cells and also stimulate the secretion of thrombospondin, which might be used as an adhesion ligand for the integrin vitronectin receptor alpha v/beta 3 complex.