Beta1 Integrin Expression Research Articles

Immunophenotypic Identification and Characterization of Tumor Cells and Infiltrating Cell Populations in Meningiomas

Meningiomas are primary tumors of the central nervous system composed of both neoplastic and other infiltrating cells. We determined the cellular composition of 51 meningioma samples by multiparameter flow cytometric (MFC) immunophenotyping and investigated the potential relationship between mRNA and protein expression levels of neoplastic cells. For immunophenotypic, morphologic, and cytogenetic characterization of individual cell populations, a large panel of markers was used together with phagocytic/endocytic functional assays and MFC sorting. Overall, our results revealed coexistence of CD45(-) neoplastic cells and CD45(+) immune infiltrating cells in all meningiomas. Infiltrating cells included tissue macrophages, with an HLA-DR(+)CD14(+)CD45(+)CD68(+)CD16(-/+)CD33(-/+) phenotype and high phagocytic/endocytic activity, and a small proportion of cytotoxic lymphocytes (mostly T CD8(+) and natural killer cells). Tumor cells expressed multiple cell adhesion proteins, tetraspanins, HLA-I/HLA-DR molecules, complement regulatory proteins, cell surface ectoenzymes, and growth factor receptors. Noteworthy, the relationship between mRNA and protein levels was variable, depending on the proteins evaluated and the level of infiltration by immune cells. In summary, our results indicate that MFC immunophenotyping provides a reliable tool for the characterization of the patterns of protein expression of different cell populations coexisting in meningioma samples, with a more accurate measure of gene expression profiles of tumor cells at the functional/protein level than conventional mRNA microarray, independently of the degree of infiltration of the tumor by immune cells.

Abstract 4426: Is development of hepatocellular carcinoma driven by liver homing signals?

Abstract There are a limited number of models to study hepatocellular carcinoma (HCC). Hepa 1-6 cell line can be used to develop tumors when injected directly in the liver of C57/BL6 mice. We isolated cells from a tumor which developed in the liver following the intrasplenic injection of Hepa 1-6 cells and found that the daughter cells led to more systematic development of tumors. Studies were performed to test the characteristics of the 2 cell lines. In vivo tumorigenicity was assessed by injecting cells in the spleen. Minimal cell dose and liver specificity were assessed at 28 days. Tumors (≥0,5mm) were counted and alpha-fetoprotein (AFP) mRNA measured by relative RT-PCR. Cell lines were compared in vitro for their alpha-1 integrin (ITGa1), beta1-integrin (ITGb1), AFP and Epithelial Cell Adhesion Molecule (EpCAM) mRNA expression. Apoptosis was measured following exposure to cisplatinum [25µg/ml]. Invasiveness and motility were assessed using a modified wound healing assay and a double layered COL1 hemisphere invasion assay. Cell doubling time (CDT) was measured by cell count. Intrahepatic tumours developed more quickly (21d vs. 70d) and more often (66% (4/6) vs. 15% (1/7)) with the daughter cell line. Tumors first appeared 21 days after intrasplenic injection and increased steadily thereafter: this was associated with increased AFP expression in liver homogenates. The minimal cell concentration required to give raise to visible tumors was 10K with the daughter cell line while no tumor was observed at 28d with the parental cell line (1M cells). No extra hepatic tumours were found at any point and with any cell concentration. However, subcutaneous injection induced hepatic tumours in every animal only when using the more tumorigenic daughter cell line (100% 3/3 at 28d). In vitro, the daughter cell line showed increased resistance to apoptosis when exposed to COL1, did not show increase proliferation, but were less motile and less invasive. Cell doubling time was longer with the daughter cell line then with the parental one (45.5h±2.7h vs 34.6h±0.4h; p<0.05). There was no significant difference in the levels of the pro-apoptotic Bid, Bak and Bad proteins and in the Bcl-xL anti-apoptotic protein, ERK1/2 and AKT between the 2 cell lines. On the other hand, the more aggressive cell line showed almost a tenfold increased expression of EpCAM (9.77±2.71 vs 1.05±0.24), and lower expressions of ITGa1 (0.05±0.03 vs 1.06±0.24) and ITGb1 (0.36±0.14 vs 1.03±0.16), all these differences being significant (p<0.05). In conclusion, we observed that HCC cells derived from the same clone can have strikingly different tumorigenic potential. Increased expression of the epithelial cell adhesion molecule was associated with a high potential for intrahepatic tumor development despite a less aggressive phenotype in vitro. These results suggest that liver homing signals might be important for the establishment/survival of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4426. doi:1538-7445.AM2012-4426

Abstract 1654: The low-penetrance breast cancer risk allele MCS5A functions in T-cells through downregulation of the E3-ubiquitin ligase Fbxo10, to alter mammary epithelial cell differentiation

Abstract Genome-wide association studies (GWAS) have identified ∼50 low-penetrance, high-population frequency breast cancer susceptibility variants, of which the vast majority are non-protein coding. Mechanisms through which such variants modulate breast cancer risk are largely unknown. The non-coding mammary carcinoma susceptibility locus 5a (MCS5A/Mcs5a) is associated with breast cancer risk in both women and rats (Samuelson et al., 2007 PNAS). We use a comparative genetics approach to functionally characterize this locus. Using mammary gland and bone marrow transplantation assays in congenic rat models, we showed that the Mcs5a resistance allele acts through a non-mammary cell-autonomous immune mechanism. We found that Mcs5a is associated with altered T-cell homeostasis and functions, most notably a 38% and 89% higher abundance of gammadelta T-cells in the spleen and mammary gland, respectively (Smits et al., 2011 Breast Cancer Res). On the molecular genetic level, the non-protein coding Mcs5a resistance allele was found to strongly associate with ∼2-fold and ∼4-fold lower Fbxo10 transcript levels in thymic and splenic T-cells, respectively, but not in other immune cell types or mammary gland (Smits et al., 2011 Nucleic Acids Res). Fbxo10 is an E3-ubiquitin ligase gene, transcriptionally initiating from within Mcs5a. Using flow cytometric analysis of the rat mammary epithelium with antibodies against CD24, CD29, CD31, CD45, cytokeratin (CK) 14, CK19, as well as staining with phalloidin, we identified the luminal and basal/myoepithelial cell populations (Sharma et al., 2011 PLoS ONE). Quantitative analysis of these populations revealed that in a window of cancer susceptibility to carcinogen exposure (∼50-90 days of age), mammary glands from Mcs5a resistant congenic rats have a 47% lower frequency of basal/myoepithelial (CD24medCD29high) cells. Loss of CD29 (Integrin-beta1) expression in the mouse mammary epithelium has been implicated in mammary cancer resistance. To investigate the function of Fbxo10, we generated an Fbxo10 knockout mouse model, which recapitulated both the gammadelta T-cell and the mammary epithelial cell differentiation phenotypes. We hypothesize that Mcs5a controls Fbxo10 expression levels in the thymus, resulting in higher abundance of gammadelta T-cells, especially in the mammary epithelium, which contributes to resistance to carcinoma development through increased immune surveillance and/or altered mammary epithelial cell differentiation. Fbxo10 and its downstream ubiquitination proteins represent novel breast cancer prevention targets within the immune system. This study exemplifies the necessity of mammalian genetic models for deciphering mechanisms through which low-penetrance variants affect cancer susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1654. doi:1538-7445.AM2012-1654

321 HDAC BLOCKADE COUNTERACTS RESISTANCE DEVELOPMENT CAUSED BY CHRONIC MTOR INHIBITION

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2012321 HDAC BLOCKADE COUNTERACTS RESISTANCE DEVELOPMENT CAUSED BY CHRONIC MTOR INHIBITION Jasmina Makarevic, Igor Tsaur, Eva Weich, Axel Haferkamp, and Roman Blaheta Jasmina MakarevicJasmina Makarevic Frankfurt, Germany More articles by this author , Igor TsaurIgor Tsaur Frankfurt, Germany More articles by this author , Eva WeichEva Weich Frankfurt, Germany More articles by this author , Axel HaferkampAxel Haferkamp Frankfurt, Germany More articles by this author , and Roman BlahetaRoman Blaheta Frankfurt, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.381AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES mTOR-targeting is an attractive approach to prostate cancer therapy. However, the benefit of long-term application has not been evaluated in detail. Growth and invasive behavior of prostate cancer cells under chronic exposure to the mTOR-inhibitor temsirolimus was, therefore, investigated. Further investigation was designed to determine whether additionally targeting histone deacetylase (HDAC) by an HDAC-inhibitor (valproic acid, VPA) might counteract undesired modification of the tumor caused by chronic use of temsirolimus. METHODS Prostate cancer cells were either treated with temsirolimus over 12 months, starting at 1 nM and increasing stepwise to 5 μM (PC3TEM), or only treated with medium (PC3). 3 days before experiment begin, temsirolimus containing culture medium was replaced by a temsirolimus free medium and tumor cells analyzed thereafter, or PC3TEM (versus PC3) then re-incubated with low-dosed (10 nM) temsirolimus to evaluate drug responsiveness. Growth, proliferation, adhesion to matrix proteins or endothelial cells and invasion of PC3 versus PC3TEM were investigated. Expression of cell cycle regulating proteins (cdk1, cdk2, cdk4, cyclin A,B,D3,E, p21, p27), mTOR related intracellular signaling (Akt, mTOR, rictor, raptor, p70S6k) and the expression profile of alpha and beta integrin subtypes were also evaluated. Additionally, HDAC was blocked in PC3TEM cells and the consequences on invasive growth investigated. RESULTS PC3 TEM accumulated in the G2/M-phase, accompanied by cdk1, cdk2, cyclin B elevation and reduction of p21 and p27. Application of 10 nM temsirolimus to PC3TEM did not diminish cell growth and proliferation, whereas proliferative activity in PC3 was significantly diminished by 10 nM temsirolimus. PC3TEM cell adhesion and invasion was enhanced by 10 nM temsirolimus, compared to temsirolimus treated PC3 control cells. The target proteins Akt, mTOR, rictor (but not raptor) and p70S6k were all elevated (total and activated) in PC3TEM compared to PC3 cells. Distinct modifications were also seen with respect to alpha and beta integrin expression. Additional application of VPA blocked growth and adhesion of PC3TEM cells, reverted integrin modulation and also deactivated proteins of the mTOR signaling pathway. CONCLUSIONS Chronic use of the mTOR inhibitor temsirolimus causes prostate cancer cell resistance. HDAC-inhibition counteracts this process, possibly by cross-communicating with the mTOR signaling axis. Specific targeting of HDAC may, therefore, enhance the benefit of an mTOR-inhibitor based regimen. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e130 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jasmina Makarevic Frankfurt, Germany More articles by this author Igor Tsaur Frankfurt, Germany More articles by this author Eva Weich Frankfurt, Germany More articles by this author Axel Haferkamp Frankfurt, Germany More articles by this author Roman Blaheta Frankfurt, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 17143: RASSF5, a New Mediator of Angiogenesis, Mediates Integrin Endocytosis in Endothelial Cells and Vessel Stability in vivo

Integrins are critical mediators of angiogenesis. In our previous work, we identified the GTPase Rap1 as a mediator of integrin activation in endothelial cells (EC) and angiogenesis. RASSF5/RAPL is an effector protein of Rap1 promoting integrin activation and adhesion in lymphocytes. However, the role of RASSF5 for integrin regulation in EC and angiogenesis is unclear. Silencing of RASSF5 with siRNA significantly decreased angiogenic sprouting of HUVEC in a 3-dimensional spheroidal system (83 % inhibition) and blocked VEGF-induced invasion (68 % inhibition), while not inhibiting EC proliferation. Surprisingly, while silencing of Rap1 reduced HUVEC adhesion, silencing of RASSF5 unexpectedly significantly increased integrin-dependent adhesion suggesting distinct roles for RASSF5 and Rap1 in integrin regulation in EC. In line with these results, silencing of RASSF5 significantly increased surface expression of beta1-integrin as assessed by FACS, while not affecting total beta1-integrin protein levels in HUVEC. It is conceivable, that the enhanced surface expression of beta1-integrins upon silencing of RASSF5 is caused by a redistribution of the integrin from an intracellular pool to the cell surface. Therefore, we studied the role of RASSF5 for the endocytosis of beta1-integrins in HUVEC. Strikingly, silencing of RASSF5 blocked beta1-integrin endocytosis by 43 ± 6 %. Interestingly, silencing of RASSF5 preferentially reduced the internalization of activated (matrix-bound) alpha5beta1-integrins. Endogenous alpha5-integrin co-immunoprecipitated with overexpressed RASSF5. In addition, silencing of RASSF5 enhanced actin polymerization and Rac1 activity in EC. Strikingly, in vivo silencing of the RASSF5 homologue in the zebrafish embryo with morpholino led to hemorrhages in the newly formed vessels. In conclusion, our results demonstrate that RASSF5 is binding to the alpha5beta1-integrin and mediating integrin internalization and actin cytoskeleton reorganization in EC, thereby promoting angiogenic sprouting and migration. Our data identify a new angiogenic signaling pathway and shed light on the relevance of alpha5beta1-integrin endocytosis for angiogenic functions such as sprouting, migration and vessel stability.

Phenotyping of chondrocytes from human osteoarthritic cartilage: chondrocyte expression of beta integrins and correlation with anatomic injury

Chondrocyte-ECM (extracellular matrix) interactions are believed to play a pivotal role in the development and metabolic homeostasis of articular cartilage. Cell surface adhesion molecules have been reported to modulate chondrocyte binding to ECM (collagen, fibronectin, laminin) and they also act as transducers of critical signals in many biological processes such as growth, differentiation, migration and matrix synthesis. Recently, it has been shown that normal human articular chondrocytes strongly express beta1 integrins, which are constituted by a common chain (beta1) and a variable alphachain, but the behaviour of these molecules in human osteoarthritic cartilage has not been extensively investigated. We studied the expression of beta integrins (beta1-5, alpha1-6, av chains), LFA-1, ICAM-1 and CD44, on freshly isolated chondrocytes obtained from 10 osteoarthritic patients undergoing surgical knee replacement. Chondrocytes were isolated by enzymatic digestion from three zones of each articular cartilage with a differing degree of macroscopic and microscopic damage. Integrin expression and cell cycle analysis were carried out by flowcytometry. Chondrocytes from costal cartilages of 5 human foetuses were also studied. Chondrocytes from osteoarthritic cartilage expressed high levels of beta1 integrin and, at different percentages, all the alphachains. The alphachain most frequently expressed was alpha1, foilowed by alpha3, alpha5, alpha2, alphav. Integrin expression decreased from the least to the most damaged zone of articular cartilage and cell cycle analysis showed that proliferating chondrocytes (S phase) were prevalent on the latter zone. beta2, beta3, beta2, beta5, CD44, LFA-1/ICAM-1 complex were very low expressed. Fetal chondrocytes strongly expressed beta1 and beta5 chains. These data provide evidence to show that integrin expression on human chondrocytes changes in osteoarthritis and suggest that perturbations of chondrocyte-ECM signalling occur in the development of the disease. The different pattern of expression of beta1 and beta5 chains on adult and fetal chondrocytes leads to speculate that integrins play a key role in control of cartilage morphogenesis and differentiation.

Id1-induced inhibition of p53 facilitates endothelial cell migration and tube formation by regulating the expression of beta1-integrin

The Id1 protein is critical for endothelial cell angiogenesis, and this function is particularly relevant to cancer development, cardiovascular disease, and wound healing. We hypothesized that Id1 enhanced migration and tubulogenesis by controlling the expression and function of p53. In this study, we examined cell migration following Id1 overexpression and silencing endothelial cells. The results showed that overexpression of Id1 enhanced cell migration and increased beta1-integrin expression, but inhibition of beta1-integrin blocked motility even in clones overexpressing Id1, suggesting that Id1 regulated motility through beta1-integrin. Further analysis revealed that p53, whose expression and distribution is regulated by Id1, was critical for cell migration, and may be involved in regulating the expression of beta1-integrin. Inhibiting p53 function using PFT-α, a functional inhibitor of p53, increased the expression of beta1-integrin and promoted cell migration even in Id1-silencing endothelial cells, demonstrating that the Id1 knockdowns induced inhibition of endothelial cell migration and the expression of beta1-integrin were controlled by p53. In addition, Id1-p53 pathway regulated the cytoskeleton formation and tubulogenesis. These results demonstrate that Id1-induced beta1-integrin expression in endothelial cells and the function of Id1 in cell migration and tubulogenesis are dependent on p53.

Abstract 2330: Role of RET finger protein in the integrin beta1 expression in cancer cells and its significance in cancer patients

Abstract Integrins play central roles in the biology by regulating cell adhesion to the extracellular matrix and cell migration, growth, differentiation and apoptosis. Although contribution of integrins on cancer development has been well studied, the mechanism of the regulation of integrins expression remains elusive. Here we report that RET finger protein (RFP) regulates integrin beta1 expression in cancer cells. RNAi-mediated depletion of RFP resulted in decrease of integrin beta1 expression and impaired cell migration/invasion. RNA immunoprecipitation assay revealed that RFP interacts with integrin beta1 mRNA, and RFP knockdown caused destabilization of the mRNA. Furthermore, we examined the expression of RFP in human colon cancer and endometrial cancer, and assessed its clinical significance. High levels of RFP expression correlated with poor clinical outcome in colon cancer and endometrial cancer patients. These findings suggest that RFP regulates the cancer cell migration/invasion through the upregulation of integrin beta1 expression by stabilization of its mRNA, and expression of RFP can be a prognostic marker for an unfavorable clinical outcome in patients with colon cancer and endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2011-2330

Abstract 2902: Phosphorylation of 12 lipoxygenase at Tyrosines 19 and 614 regulates its activity during beta4 integrin signaling

Abstract 12-Lipoxygenase (12-LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid 12(S)-HETE, an eicosanoid implicated in tumor angiogenesis, growth, and metastasis. We have demonstrated previously that 12-LOX interacts with integrin beta4 cytoplasmic domain by using yeast two hybrid system. Integrins are involved in metastasis by mediating attachment and migration of cells, as well as through transducing signals. Increased expression of 12-LOX and beta4 integrin have been documented in a number of carcinomas. The purpose of this study is to find out the mechanisms underlying the signaling pathways in 12-LOX mediated cancer progression. The phosphorylation of beta4 integrin, Src, and 12-LOX were analyzed using specific antibodies against phospho-tyrosine, phospho-beta4 integrin, Phospho-Src (pSrc). c-Src activity and association was determined in beta4 integrin immunoprecipitate. The HEK293 cells transiently transfected with wild type or dominant negative (DN) Src were used as in vitro models to study the regulation of 12-LOX activity. The 12-LOX product 12(S)-HETE was analyzed by using an analytical liquid chromatography-tandem mass spectrometry.12-LOX tyrosine phosphorylation sites mutants viz., 19, 295, and 614 were used to study the effect on its activity upon beta4 ligation. We show here that the cytoplasmic domain of beta4 integrin, can interact specifically with pSrc after beta4 integrin ligation in A431 cells. Herein we report that beta4 integrin ligation resulted in significant increase in 12(S)-HETE level in A431 cells, compared to that of unligated beta4 integrin. An increase in Src kinase activity was found in beta4 integrin ligation of A431 cells and inhibition of Src kinase by PP2 or DN Src significantly reduced 12(S)-HETE production. The ligation of beta4 integrin induces src activation, which resulted in tyrosine phosphorylation of 12-LOX. The residues that are important for 12-LOX activity and tyrosine phosphorylation were mapped to Y19 and Y614. We examined that 12-LOX mutants (Y19F and Y614F), showed 70% less enzymatic activity, compared to wild type 12-LOX. Furthermore, we showed that the 12-LOX activity modulated by these residues impacts migration in HUVEC cells. To our knowledge, this is the first report to show that Src kinase activity is required for beta4 integrin mediated regulation of 12-LOX, which may in turn regulate metastatic potential of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2011-2902

Abstract 428: Positive correlation of beta1 integrin expression and prognosis in clinically localized prostate cancer

Abstract Introduction and objective: Integrins are transmembrane glycoprotein receptors that regulate cell-matrix interactions, functioning as sensors of the environment. They also act as cell adhesion molecules, being responsible for the maintenance of the epithelial phenotype. Some studies have reported correlation between alterations of integrins expression and carcinogenesis, specially β1 integrin, but their role in prostate cancer is unclear. Our aim is to study the expression of β1 integrin and evaluate its association with recurrence of prostate cancer after surgical treatment Methods: For this case-control study, we selected 111 patients with localized tumor submitted to radical prostatectomy by the same surgeon. 60 of these patients have not presented recurrence after a median follow-up of 123 months. Recurrence was defined as a PSA level above 0.4ng/ml. Integrin expression was evaluated by immunohistochemistry in a Tissue Microarray containing two samples from each tumor. We employed a semiquantitative analysis and considered a case as positive when the expression was strong and diffusely present. We correlated expression with recurrence employing the χ2 test and p<0.05 was considered significant. Results: Positive expression of β1 integrin was found in 79 out of 100 evaluated cases, ten cases were lost in the tissue microarray. Correlating expression with recurrence we found in the univariate analysis that negative expression of β1 integrin was statistically associated with recurrence (p=0,042) and time to recurrence after radical prostatectomy, when expression of β1 is negative, the odds of recurrence was two times higher than that observed in positive cases (p=0.026 – IC 95% [1.09; 3.83]. In multivariate analysis, including PSA and Gleason, β1 integrin remained independently associated with recurrence p=0.01. Conclusions: In the present study we have shown that a down regulation of β1integrin expression was significantly correlated with recurrence after radical prostatectomy for localized prostate cancer, making this integrin a potential candidate as a marker of prognosis. Larger series will be evaluated in order to confirm our initial findings Figure Kaplan Meier curve for recurrence-free survival according to β1expression in 111primaryPCa submitted to RP Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 428. doi:10.1158/1538-7445.AM2011-428

Atomic force microscope studies demonstrate enhanced beta1‐integrin adhesion as a factor assocatiated with age‐related increases in vascular smooth muscle stiffness

Beta1-integrin, an important vascular smooth muscle cell-extracelluar matrix (VSMC-ECM) adhesion molecule, shows enhanced VSMC expression associated with age-related increases in aortic stiffness. We investigated whether active beta 1-integrin adhesion to the ECM protein collagen type 1 (COL1) and fibronectin (FN) was enhanced in VSMC from old and young male monkey thoracic aortas using the atomic force microscopic (AFM). As the abundance of beta1-integrin is significantly higher in old compared to young monkeys, we hypothesized that enhanced beta 1-integrin adhesion could provide a mechanism for accentuating aortic stiffness by strengthening VSMC-ECM connections. As evidence of increased beta 1-integrin expression in old vs young VSMC, AFM probes functionalized with FN or COL1 exhibited significantly increased adhesion to the beta1-integrin in old vs young VSMC. In old vs young, COL1 binding probability was 70±1% vs 63±1% and adhesion force was 72±1.3 pN vs 50±2 pN whereas for FN binding probability was 43±1% vs 33±1% and adhesion force 61±1.4 pN vs 39 ± 1 pN. Our results support the involvement of beta1-integrin as a potential factor in the increased stiffness of VSMC observed during aging that could contribute to enhanced stiffness of the aortic wall. (NIH R01HL102472 and P01HL095486).

Pro-apoptotic role of integrin β3 in glioma cells

Malignant gliomas are the most destructive type of brain cancer. In order to gain a better understanding of the molecular mechanisms of glioma cell death and survival, we previously established an alkylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant variant of C6 rat glioma cells. Proteomic analysis indicated a significant down-regulation of integrin beta 3 (ITGB3) in the BCNU-resistant C6R cells. Re-expression of ITGB3 in C6R cells restored the BCNU sensitivity. In U87MG, U373MG, and T98G human glioma cells, there was a positive correlation between ITGB3 expression and the sensitivity to BCNU and etoposide, suggesting an important role of ITGB3 in glioma cell death. Over-expression of ITGB3 cDNA significantly increased the sensitivity of the human glioma cells to the anticancer drug-induced apoptosis. Nitric oxide showed an additive effect on the anticancer drug-induced glioma cell death by increasing ITGB3 expression. Subsequent dissection of signaling pathways indicated that extracellular signal-regulated kinase and unligated integrin-mediated cell death pathway may be involved in the pro-apoptotic role of ITGB3 in glioma cells. These results implicate ITGB3 in glioma cell death/survival and drug resistance.

Zona pellucida glycoprotein 3 (pZP3) and integrin β2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test

Brilliant cresyl blues (BCB) staining test is a useful tool in assessing the competence of cumulus-oocyte-complexes (COCs) in several mammalian species. It is mostly used to select gametes after they are recovered from the ovary or before and after IVM to isolate those oocytes that reach developmental competency. However, there is evidence that double exposure to BCB test may lead to impaired fertilization or even have a toxic effect on cells. The aim of the present study was to investigate the expression pattern of sperm-egg interaction molecules in oocytes after single and double exposure to BCB test. Follicles were dissected from porcine ovaries after slaughter and aspirated COCs were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The BCB test was applied to COCs before and after IVM. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH; BCB test), real-time quantitative PCR reaction methods, western blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoprotein 3 (pZP3), and integrin beta 2 (ITGB2), as well as the levels of pZP3 and ITGB2 proteins. In the control group, assessment of the expression of the investigated genes was performed before and after IVM without BCB test. We observed a significantly higher level of pZP3 mRNA in oocytes after single exposure to BCB test compared to control before and after IVM (P < 0.001), and to double staining (P < 0.05). The level of ITGB2 mRNA was also increased in gametes after single exposure to BCB test as compared to control before and after IVM (P < 0.001, P < 0.01, respectively), and double staining (P < 0.05). Western blot analysis demonstrated a higher level of pZP3 protein in oocytes after single staining with BCB as compared to control both before and after IVM (P < 0.001, P < 0.05, respectively) and double staining (P < 0.05). Confocal microscopic observations have revealed the same pattern of increased level of pZP3 and ITGB2 expression after single exposure to BCB test. In both cases we detected specific cytoplasmic localization of both proteins. The ITGB2 protein has zona pellucida and membrane localization in control oocytes before IVM. After IVM and after single exposure to BCB, ITGB2 was also strongly detected in the cytoplasm. In both cases, after double exposure to BCB both proteins were detected only partially in the cytoplasm. Our results suggest that (i) single exposure to BCB increased the expression of sperm-oocyte interaction genes, (ii) double exposure to BCB leads to only partial expression of pZP3 and ITGB2 in oocyte cytoplasm, (iii) the BCB staining test itself may be a cause of specific pZP3 translocation from the zona pellucida to the cytoplasm, and that (iv) in vitro maturation of oocytes may increase ITGB2 expression and translocation from the zona pellucida to the cytoplasm.

Human Spinal Cord Injury Causes Specific Increases in Surface Expression of Beta Integrins on Leukocytes

Spinal cord injury (SCI) activates circulating leukocytes that migrate into the injured cord and bystander organs using adhesion molecule-mediated mechanisms. These cells cause oxidative damage, resulting in secondary injury to the spinal cord, as well as injury to bystander organs. This study was designed to examine, over a 6-h to 2-week period, changes in adhesion molecule surface expression on human peripheral leukocytes after SCI (9 subjects), using as controls 10 uninjured subjects and 6 general trauma patients (trauma controls, TC). Both the percentage of cells expressing a given adhesion molecule and the average level of its expression was quantified for both circulating neutrophils and monocytes. The percentage of neutrophils and monocytes expressing the selectin CD62L was unchanged in TC and SCI patients after injury compared to uninjured subjects. Concurrently, the amount of surface CD62L on neutrophils was decreased in SCI and TC subjects, and on monocytes after SCI. The percentage of neutrophils expressing α4 decreased in TC, but not in SCI, subjects. Likewise, the percentage of neutrophils and monocytes expressing CD11d decreased markedly in TC subjects, but not after SCI. In contrast, the mean surface expression of α4 and CD11d by neutrophils and monocytes increased after SCI compared with uninjured and TC subjects. The percentage of cells and surface expression of CD11b were similar in neutrophils of all three groups, whereas CD11b surface expression increased after SCI in monocytes. In summary, unlike changes found after general trauma, the proinflammatory stimulation induced by SCI increases the surface expression of adhesion molecules on circulating neutrophils and monocytes before they infiltrate the injured spinal cord and multiple organs of patients. Integrins may be excellent targets for anti-inflammatory treatment after human SCI.

MicroRNA-124 suppresses oral squamous cell carcinoma motility by targeting ITGB1

Alterations in the levels of molecules which interact with the extracellular matrix, such as integrins, are associated with invasion of oral squamous cell carcinomas (OSCC). The molecular mechanisms underlying dysregulation of integrin expression in OSCC, however, remain unclear. Here, we show that microRNA-124, a small non-coding RNA down-regulated in OSCC, is able to downregulate expression of integrin beta-1 (ITGB1) by interacting with its 3′ untranslated region. Over-expression of miR-124 attenuates endogenous ITGB1 expression and reduces the adherence and motility of OSCC cells, suggesting disruption of miR-124-mediated repression of ITGB1 may be a key factor in OSCC progression.

Suppression of inflammatory responses of endothelial cells by shear stress: Role of integrin signalling

Inflammatory responses of endothelial cells (EC) are conditioned by local haemodynamic forces, but the mechanisms underlying mechanotransduction remain uncertain. We cultured human EC in capillaries, and conditioned them by shear stress before treatment with tumour necrosis factor-alpha (TNF), and analysis of recruitment of flowing leukocytes. Shear conditioning of several types of human EC for 24h powerfully suppressed responses to TNF. This shear-induced reduction in sensitivity to TNF was less effective when expression of beta1-integrins (but not beta3-integrins) was suppressed using small interfering RNA before application of shear.

Flow cytometric sorting and analysis of human epidermal stem cell candidates

ESC (epidermal stem cells) play a central role in the regeneration of human epidermis. These cells are also responsible for wound healing and neoplasm formation. Efficient isolation of ESC allows their use in medicine and pharmacy as well as in basic science. Cultured keratinocytes and ESC may be used as biological dressing in burn injuries, chronic wounds and hereditary disorders. Therefore, the isolation and characterization of ESC have been goals in biomedical science. Here, we present a flow cytometric method for the isolation and analysis of human ESC candidates. The strategy presented for the isolation of ESC combines previously proposed enzymatic digestion and FACS-sorting of the obtained cell suspension that utilizes morphological features, integrin-beta1 expression and Rh123 (Rhodamine 123) accumulation of the cells. We also performed a flow cytometric analysis of sorted cells using a cell tracer.

Involvement of integrin up‐regulation in RANKL/RANK pathway of chondrosarcomas migration

Invasion of tumor cells is the primary cause of therapeutic failure in malignant chondrosarcomas treatment. Receptor activator of nuclear factor-kappaB ligand (RANKL) and its receptor, RANK, play a key roles in osteoclastogenesis and tumor metastasis. We found that the RANKL and RANK expression in human chondrosarcoma tissues was higher than that in normal cartilage. We also found that RANKL directed the migration and increased cell surface expression of beta1 integrin in human chondrosarcoma cells (JJ012 cells). Pretreatment of JJ012 cells with MAPK kinase (MEK) inhibitors, PD98059 or U0126, inhibited the RANKL-induced migration and integrin expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited RANKL-induced cells migration and integrin up-regulation. Taken together, these results suggest that the RANKL acts through MEK/ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activation of beta1 integrin and contributing to the migration of human chondrosarcoma cells.

Integrin &amp;beta;4 was downregulated on the airway epithelia of asthma patients

The shedding of airway epithelial cells and loss of epithelial functional homeostasis are major pathological characteristics of asthma; however, the mechanism underlying these pathologies remains obscure. Our previous work showed that there were three variation sites in 5' flanking region of integrin beta4 in asthma patients, which was correlated with decreased expression of integrin beta4 in peripheral leukocytes. Integrin beta4 is an important structural adhesion molecule on airway epithelia to keep the structural adhesion of epithelial cells. In this work, we further demonstrated that integrin beta4 expression was downregulated in airway epithelia of asthma patients. To probe the relationship between imbalanced expression of integrin beta4 and dysfunction of the airway epithelial cells in asthma, integrin beta4 was silenced in human bronchial epithelium cells (16HBE14O) by integrin beta4 small-interfering RNA lentivirus vector. Upon silencing of integrin beta4, 16HBE14O cells showed reduced proliferation and wound repair. Most cells were shown to be arrested in G1 phase after integrin beta4 silencing, and increased apoptosis was induced in the integrin beta4-silenced cells. In summary, our results provided compelling evidence that integrin beta4 was involved in the structural integrity and functional homeostasis of airway epithelial cells. It is likely that downregulation of integrin beta4 on asthma airway epithelia contributes to the structural disruption and dysfunction of airway epithelial cells.

Overexpression of P-selectin glycoprotein ligand-1 enhances adhesive properties of endothelial progenitor cells through Syk activation

P-selectin glycoprotein ligand-1 (PSGL-1) not only functions as an anchor molecule to capture monocytes and other leukocytes to endothelial cells in ischemic tissue by its interaction with P-selectin, but also transduces signals to initiate firm adhesion. Endothelial progenitor cells are derived from monocytes and play a very important role in neovascularization. Transplantation of endothelial progenitor cells is a promising therapeutic strategy to improve treatment of ischemic disease such as myocardial and cerebral infarction; however, its efficacy is now limited by the fact that few of the transplanted cells adhere to and accumulate in the ischemic tissue. In this study we aimed to investigate whether the overexpression of PSGL-1 gene promotes endothelial progenitor cells adhesion activity and explore the underlying mechanisms. We found that after transfection with human PSGL-1 gene, endothelial progenitor cells exhibited higher affinity to activated human umbilical vein endothelial cells or recombined P-selectin/ICAM-1 monolayer. The overexpression of PSGL-1-enhanced beta2-integrin expression on endothelial progenitor cells surface, and this effect was Syk dependent. The specific Syk inhibitor abolished the elevating effect of overexpression of PSGL-1 on surface beta2-integrin expression and the adhesive affinity of endothelial progenitor cells. These results suggested that Syk plays a key role in signal transduction downstream of PSGL-1 in endothelial progenitor cells, and the overexpression of PSGL-1 improves endothelial progenitor cells adhesive properties through enhanced activation of Syk and following integrin activation.