Expression Of Integrin Alpha Research Articles

Abstract 215: Aminoflavone exhibits in vivo efficacy on cancer stem cells in a spontaneous estrogen-dependent breast cancer murine model

Abstract Stem-cell populations have been identified in a range of solid tumors and possess the capacity to self renew. It is thought that these cancer stem cells (CSCs) play an important role in cancer establishment, progression, and resistance to current treatments. CSCs have been identified in primary breast cancer tissues and cell lines derived from metastatic tumors. Traditional cancer therapies are effective at debulking some tumors but often fail to produce long-term clinical remissions, presumably due to their inability to eradicate the cancer stem cell population. Therefore, novel treatments aimed at targeting this population can potentially enhance treatment of both primary and metastatic tumors. Aminoflavone (AF; NSC 686288) is a novel anticancer agent. Previous work from our research group demonstrated that AF is a ligand of the aryl hydrocarbon receptor (AhR) and displays cytotoxicity in the MCF-7 breast cancer cell line. A prodrug formulation of AF (AFP464, NSC710464), recently entered phase II clinical trials. Moreover, we previously found that AF disrupts MCF-7 mammosphere formation via AhR signaling activation to inhibit alpha 6-integrin expression. To investigate the efficacy of AF in vivo, we used an estrogen dependent, Tamoxifen-sensitive spontaneous M05 model system involving 1-year-old virgin female BALB/c mice developed in our animal facility. The M05 tumor is a semi-differentiated adenocarcinoma that expresses estrogen and progesterone receptors. After AF treatment (12 mg/kg, i.p., QD × 5 days) tumor size and growth rate decreased substantially compared to control animals. Cytometric analyses of cells obtained from AF-treated and control animals revealed that Lin (-)/ CD29 (Hi)/CD24 (+) cancer stem cells were lower in animals treated with AF than in the vehicle control group. We also observed a diminished capacity for mammosphere formation in cells obtained from tumors treated with AF. These data suggest AF diminishes the quantity of breast cancer stem cells as well as their integrity and capacity to form mammospheres in vitro and in vivo. These findings shed additional insight into the molecular mechanism of anticancer action for this novel anticancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens. Citation Format: Mariana Callero, Damian Berardi, Laura Todaro, Marina Simian, Eileen J. Brantley, Ubaldo Soto, Andrea Loaiza-Perez. Aminoflavone exhibits in vivo efficacy on cancer stem cells in a spontaneous estrogen-dependent breast cancer murine model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2014-215

Mo1214 Long-Term Safety and Efficacy of Golimumab in Patients With Moderately to Severely Active Ulcerative Colitis: Results From the PURSUIT-SC Maintenance Study Extension

Mo1214 Long-Term Safety and Efficacy of Golimumab in Patients With Moderately to Severely Active Ulcerative Colitis: Results From the PURSUIT-SC Maintenance Study Extension

OP010 AlphaE integrin expression as a predictive biomarker for induction of clinical remission by etrolizumab: Analysis of a phase II trial in moderate-to-severely active ulcerative colitis

OP010 AlphaE integrin expression as a predictive biomarker for induction of clinical remission by etrolizumab: Analysis of a phase II trial in moderate-to-severely active ulcerative colitis

Integrin alpha 6: A novel therapeutic target in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC), the most common subtype of esophageal cancer in East Asian countries, is a devastating disease characterized by distinctly high incidence and mortality rates. Our previous expression profile analysis showed that integrin alpha 6 (ITGA6) is highly expressed in ESCC tissues. To validate cell surface expression of ITGA6 as a novel target in ESCC, we investigated ITGA6 expression in tumor tissue samples and cell lines of ESCC and found that ITGA6 was upregulated in these cells. In vitro knockdown of ITGA6 in ESCC cells resulted in inhibition of cell proliferation, invasion and colony formation. In addition, we demonstrated that ITGA6 associates with integrin beta 4 (ITGB4), and that this heterodimer complex is upregulated in both ESCC tissues and cell lines. Moreover, our biodistribution results in an ESCC xenograft model indicated that ITGA6 is a possible target for antibody-related diagnostic and therapeutic modalities in ESCC. Thus, our findings suggest that ITGA6 plays an important role in tumorigenesis in ESCC and represents a potential therapeutic target in the treatment of ESCC.

Adhesion Proteins - An Impact on Skeletal Myoblast Differentiation

Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro.

Abstract 1944: Evaluation of the prognostic value of miRNA-181b and its target identification and validation in colorectal cancers.

Abstract Background: microRNAs (miRNAs) are 19 to 24 nucleotide sequences that regulate cell proliferation, differentiation, and apoptosis. miRNA-181b is involved in tumor initiation and progression of several human malignancies including colorectal cancers (CRCs). Thus, we sought to determine the prognostic value of miRNA-181b in CRCs. Methods: Expression of miRNA-181b was quantified, by quantitative PCR (q-RT-PCR), in normal (benign colonic epithelia) tissues and in their corresponding CRC archival tissue samples collected from 345 patients. The expression levels were correlated with patient overall survival. Putative molecular targets of miRNA-181b were identified by in silico analysis. miRNA-181b mediated, post-transcriptional regulation of these targets was assessed in CRC cell lines (SW480, HCT116 p53-/- and HCT116 +/+) by transfection studies. The targets were validated by measuring their mRNA expression levels, by use of qRT-PCR, in CRC tissues that exhibited elevated levels of miRNA-181b as well as by cloning techniques to assess a direct interaction between miRNA-181b and 3’UTRs of its target genes using luciferase assays. Results: Survival analysis indicated that increased expression of miRNA-181b was associated with poor prognosis for CRCs (log rank, p=0.004). Fourteen putative molecular targets of miRNA-181b were identified by in silico analysis, and miRNA-181b mediated post-transcriptional regulation of these targets was validated in CRC cell lines by q-RT-PCR. Integrin alpha-3 was up-regulated, and BCL2LII and TNFα were down-regulated upon overexpression of miRNA-181b in vitro. Also, these targets were validated in CRC tissues that exhibited elevated miRNA-181b levels. The direct interaction of miRNA-181b with the target gene 3’UTRs was validated with luciferase assays. Conclusions: These findings demonstrate that miRNA-181b regulates expression of Integrin alpha-3, BCL2LII, and TNFα; and is a prognostic marker for colorectal cancers. Citation Format: Liselle Bovell, Balananda-Dhurjati K. Putcha, Divya Devadasan, Sejong Bae, William E. Grizzle, Upender Manne. Evaluation of the prognostic value of miRNA-181b and its target identification and validation in colorectal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2013-1944

Expression of Integrin Alpha-3 and Beta-4 Subunits on the Process of Peri-Implant Epithelium Formation

Integrin, a component of the hemidesmosome, plays a role for epithelial cell migration and adhesion. This study investigated the process of peri-implant epithelium (PIE) formation after implantation, and compared it to the process of oral mucosa healing after tooth extraction. At the healing site of extraction socket without implant, the original junctional epithelium (JE) had disappeared at week 2, and the oral epithelium (OE) with integrin-α3 positive basal cells extending from the sides of the wound, then joined in the middle of the extraction socket. On the other hand in implant group, newly formed epithelium with integrin-α3 positive cells from the OE extended apically 1 week after implantation. After 3 weeks, basal cells of the new epithelium consisted of those with integrin-α3 positive but β4 negative. Finally, after 4 weeks, integrin-β4 was expressed at the implant-PIE interface. These findings suggest that integrin α3β1 plays a role in cell migration during PIE formation from OE. Furthermore, after the completion of PIE constitution, integrin α6β4 contributes to the attachment to titanium.

Synthetic Peptide-Acrylate Surface for Self-Renewal of Human Retinal Progenitor Cells

Human retinal progenitor cells (hRPCs), isolated from fetal retina, require extracellular matrix proteins such as fibronectin or laminin for successful attachment and self-renewal in vitro. Here we have shown that a novel synthetic vitronectin-mimicking surface supports self-renewal and multipotency of hRPCs in a chemically defined culture system. The morphology, adhesion, and proliferation of hRPC were equivalent on a novel vitronectin-mimicking surface (Synthemax) compared to a fibronectin-coated surface. When evaluated using real-time polymerase chain reaction, Western blotting, and flow cytometry, both surfaces maintained self-renewal of hRPCs, as shown by similar expression levels of Sox2, Nestin, cMyc, Klf4, and Pax6, with no change in integrin beta1 and integrin alpha5 expression. We suggest that the use of synthetic, xeno-free surfaces such as Synthemax will be useful for basic research studies, as well as development of translational strategies aimed at using stem cell transplantation to treat disease.

Role of Integrin Switch and Transforming Growth Factor Beta 3 in Hypoxia-Induced Invasion Inhibition of Human Extravillous Trophoblast Cells1

The physiological hypoxic condition favors the angiogenesis in the placenta. However, it remains unclear how hypoxia regulates the invasion of human extravillous trophoblast cells. In the present study, we first showed that alpha5 integrin expression increased and alpha1 integrin expression decreased in human extravillous trophoblast cells cultured in 1% oxygen as compared with control cells cultured in 8% oxygen. Further data showed that the neutralizing antibody against alpha5 integrin increased the invasion of human extravillous trophoblast cells and the neutralizing antibody against alpha1 integrin inhibited the invasion of human extravillous trophoblast cells. Human extravillous trophoblast cells cultured in 1% oxygen showed reduced invasive capacity, which can be effectively blocked by alpha5 integrin neutralizing antibody. Moreover, human extravillous trophoblast cells exposed to 1% oxygen demonstrated increased expression of transforming growth factor-beta3 (TGFB3), and recombinant human TGFB3 inhibited the invasion of human extravillous trophoblast cells in a dose-dependent manner. The neutralizing antibodies against alpha5 integrin and TGFB3 markedly abrogated hypoxia-induced invasion inhibition in human extravillous trophoblast cells. These data indicate that hypoxia may inhibit the invasion of human extravillous trophoblast cells through inducing the integrin switch from alpha1 integrin to alpha5 integrin and promoting TGFB3 expression.

A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells

Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20μg/mL of TSP-HepI peptide for 18h enhanced cell migration (p<0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC.

Abstract LB-198: Ryl hydrocarbon receptor agonist Aminoflavone disrupts mammosphere formation via alpha 6 integrin inhibition

Abstract Introduction: Emerging evidence has suggested that cancer stem cells (CSCs) play a prominent role in the initiation and dissemination of a myriad of malignancies including breast cancer. CSCs have the capacity to self-renew within heterogeneous tumor cells. Currently available therapeutic options for breast cancer are incapable of suppressing CSC growth. Alpha 6 integrin contributes to CSC growth while aryl hydrocarbon receptor (AhR) activation impedes CSC proliferation. AhR agonist Aminoflavone, a synthetic flavonoid, is currently undergoing clinical evaluation for the treatment of breast cancer. We hypothesize that Aminoflavone alters the integrity and formation of cell suspension clusters initiated by breast cancer stem cells (mammospheres) by modulating alpha 6 integrin expression in an AhR-dependent manner Experimental Procedures: Mammospheres derived from MCF-7 breast cancer cells were exposed to Aminoflavone at varying times. We also treated mammospheres with Tamoxifen an anticancer agent frequently used in the treatment of breast cancer. Following Aminoflavone treatment, cells were analyzed microscopically to evaluate alterations in mammosphere structure. In addition, the expression of molecular targets associated with CSC growth in addition to alpha 6 integrin were evaluated by Western blotting and quantitative RT-PCR analysis. Results: We observed that Aminoflavone, but not Tamoxifen, disaggregates MCF-7 mammospheres. In contrast, Aminoflavone did not affect mammospheres produced from AhR defective MCF-7 (AHR100) cells. Aminoflavone abrogated the ability of secondary mammospheres to form. In addition, alpha-6-integrin expression was abolished following Aminoflavone treatment in MCF-7 but not AHR100 cells. The expression of several genes whose products regulate alpha-6 integrin expression were also modulated following Aminoflavone treatment in MCF-7 cells. Conclusion: These data suggest that Aminoflavone disrupts mammosphere formation via AhR signaling activation that results in alpha 6 integrin inhibition. These findings support continued efforts to develop novel AhR ligands with efficacy against breast CSCs as a viable therapeutic strategy for breast cancer. 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 LB-198. doi:1538-7445.AM2012-LB-198

Abstract 4344: Ionizing radiation induces adhesion of breast cancer cells to fibronectin through upregulation of integrin alpha5beta1

Abstract Metastatic cancer is marked by the loss of integrity of extracellular matrix (ECM). Adhesion of tumor cells to the ECM mediated by integrins on the surface of the tumor cells is a key step in the initial phase of metastasis. Since ionizing radiation (IR), which is commonly used for cancer therapy can induce alterations of the cell surface, we investigated the effect of IR on the adhesion of breast cancer cells to ECM proteins. Results showed that in MDA-MB-231 breast cancer cell line, IR induced significant increase in both integrin expression and integrin-mediated adhesion to fibronectin, laminin, collagen I and IV. Soluble Arg-Gly-Asp peptide fragments or function-blocking antibodies directed against integrin alpha5beta1 inhibited IR-induced adhesion of MDA-MB-231 cells to fibronectin, indicating that IR-induced adhesion is caused by up-regulation of alpha5beta1 integrin fibronectin receptors. In contrast to MDA-MB-231 cells harboring mutant p53 (R280K), MCF-7 cells (WT p53) were unable to increase adhesion and up-regulation of alpha5beta1 integrin. Interestingly, knockdown of mutant p53 abrogated IR-induced integrin alpha5 integrin expression and adhesion to fibronectin. Our results indicate that mutant p53 coordinates breast cancer cell adhesion through up-regulation of alpha5beta1 integrin. This study suggests that alpha5beta1 integrin might be an effective target to improve the efficacy of radiotherapy in mutant p53 expressing cancers. 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 4344. doi:1538-7445.AM2012-4344

The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130Cas, which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1–Rho-GTPase interaction in metastasis, we disrupted Cav1–Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130Cas phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130Cas/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.

Differential Expression of Alpha2 Integrin In Human Bone Marrow and Cord Blood CD34+CD38- Progenitors and Stem Cells Reconstituting NOD/SCID-IL2Rgammacnull Mice.

AbstractAbstract 2609Human primitive stem cells reside in the CD34+CD38- fraction in cord blood and bone marrow. However, there is a high level of heterogeneity in these cell fractions, and the phenotype of the rare primitive stem cells remains poorly defined. We have studied expression of integrin alpha2 chain, a member of a family of beta1 integrin cell adhesion receptors, in CD34+CD38- cells in adult bone marrow and cord blood. >90% of bone marrow CD34+CD38- cells and >95% of CD34+CD38-CD90+ cells, enriched in long-term in vivo reconstituting stem cells (Majeti et al., Cell Stem Cell 1:635, 2007) expressed the integrin alpha2 chain. In contrast, in cord blood CD34+CD38- and the CD34+CD38-CD90+ subpopulation, the integrin alpha2 chain was expressed only in 37.1+/− 5.3% and 32.2+/− 4.9% of the cells (mean+/− SD), respectively. To determine whether integrin alpha2 expression could be used to identify functionally distinct stem and progenitor cell populations in cord blood and bone marrow, we isolated CD34+CD38- integrin alpha2+ and alpha2- cells by flow cytometry and analyzed these by in vivo transplantation into immunodeficient NOD/SCID-IL2Rgammacnull (NSG) mice and by in vitro progenitor cell assays (long-term culture initiating cell, LTC-IC, and colony assays). Transplantation of cord blood CD34+CD38- integrin alpha2+ cells resulted in significantly higher level of human CD45+ (p<0.05), myeloid (p<0.01) and CD34+ (p<0.05) cell engraftment at 16–18 weeks after transplantation than integrin alpha2- cells (reconstitution/300 cells in age and sex matched recipients, Figure 1). In contrast, there were no differences in reconstitution at 12 weeks in mice transplanted with cord blood CD34+CD38- alpha2+ and alpha2- cells. Because of lower engraftment capacity of adult bone marrow cells in immunodeficient mice, bone marrow CD34+CD38- cells were analyzed after intra-bone transplantation. After 12 weeks only few mice transplanted with the CD34+CD38- integrin alpha2+ cells but none transplanted with the corresponding alpha2- cells were reconstituted with human CD45+ or myeloid cells at a level '0.1%. The LTC-IC progenitors within the CD34+CD38- populations, assayed after 6 weeks culture on stroma, in adult bone marrow were highly enriched in the alpha2+ as compared with alpha2- fraction (mean +/− SD 96.7+/− 57.0 and 0.2+/− 0.6 colonies/50 cells, respectively), whereas in cord blood they resided equally in both integrin alpha2+ and alpha2- cell fractions (mean +/− SD 77.4+/− 59.4 and 108.8+/− 96.0 colonies/50 cells, respectively). The lineage committed CFU-GM and BFU-E progenitors in adult bone marrow were within integrin CD34+CD38- alpha2+ and alpha2- fractions (mean +/− SD 8.5+/− 5.3 and 14.9+/− 16.2 colonies/100 cells, respectively), whereas in cord blood they were significantly enriched in the alpha2- fraction (mean +/− SD 9.3+/− 5.5 and 22.0+/− 6.7 colonies/100 cells in alpha2+ and alpha2- fractions, respectively). Taken together, our results show expression of integrin alpha2 receptor in most of the primitive cord blood long-term in vivo reconstituting stem cells, with a gradual loss of the integrin alpha2 receptor during maturation to short term in vivo reconstituting stem cells, LTC-IC and lineage committed progenitors. Furthermore, our findings show distinct ontogeny-related differences in the expression of the integrin alpha2 receptor in the functionally defined primitive and lineage-committed CD34+CD38- progenitors, indicating differences in cellular interactions of cord blood and bone marrow progenitors with the hematopoietic niches. [Display omitted] Disclosures:Ekblom:Bristol-Myers Squibb: Honoraria.

Nm23-H1 suppresses hepatocarcinoma cell adhesion and migration on fibronectin by modulating glycosylation of integrin beta1

BackgroundNm23 gene was isolated as a metastatic suppressor gene. The antimetastatic effect of Nm23 has been an enigma for more than 10 years. Little is known about its molecular mechanisms. In this study we overexpressed Nm23-H1 in H7721 cells and observed reduction of cell adhesion, migration and extension of actin stress fibers in cells stimulated by fibronectin (Fn).MethodspcDNA3/Nm23-H1 was introduced into H7721 cells, and expression of Nm23-H1 was monitored by RT-PCR and western blot. Cell adhesion, actin extension and wound-induced migration assays were done on dishes coated with fibronectin. Phosphorylation of focal adhesion kinase (FAK) and total amount of integrin alpha5 and beta1 in Nm23-H1 transfected cells and control cells were measured by western blot. Flow cytometry was used to detect expression of surface alpha5 and beta1 integrin. N-glycosylation inhibitor tunicamycin was used to deglycosylate the integrin beta1 subunit.ResultsOverexpression of nm23-H1 in H7721 cells reduced cell adhesion, migration and extension of actin stress fibers on dishes coated with Fn. Phosphorylation of FAK in Nm23-H1 transfected cells was also attenuated. Integrin alpha5 and beta1 gene messages were unaltered in nm23-H1 overexpressed cells as detected by RT-PCR. However, while cell surface integrin alpha5 was unchanged, surface expression of beta1 integrin was downregulated. Western blot also showed that the total amounts of integrin alpha5 and beta1 were unaltered, but the level of mature integrin beta1 isoform was decreased significantly. Furthermore, partially glycosylated precursor beta1 was increased, which indicated that the impaired glycosylation of integrin beta1 precursor might contribute to the loss of cell surface integrin beta1 in nm23-H1 overexpressed cells.ConclusionThese results suggest that by modulating glycosylation of integrin beta1, nm23-H1 down-regulates integrin beta1 subunit on cell surface and mediates intracellular signaling and subsequent suppression of the invasive process, including cell adhesion and migration.

Age-related differences in integrin expression in peripheral blood lymphocytes

Alpha integrins play an important role in cell to cell and cell to extra-cellular matrix interactions required for an effective T-lymphocyte-mediated immune response, however little is known about age related differences in expression of alpha integrins on T-cells in humans. We here measured alpha-4 (α4) integrin (CD49d) expression on T-lymphocytes via peripheral blood sampling, comparing parameters between cohorts of young and old adults. No age-related differences were found for the absolute numbers of T-cells, although the percentage of CD4+ T-cells in older adults was significantly greater and the percentage of CD8+ T-cells lower than in younger cohorts. Percentage and absolute numbers of CD3+ T-cells co-expressing CD49d were significantly lower in older adults compared to younger cohorts, and the percentage of gated CD4+ and CD8+ cells that co-labelled positively for CD49d was also reduced in this group. There were no age-related differences in circulating levels of cytokines (Type I interferons) that are known to regulate cell surface integrin expression. Reduced expression of alpha integrins on T-cells may be an early indicator of the loss of homeostatic control that occurs with ageing, contributing to diminished effector T-cell responses during senescence.

Immunocytochemical Studies of Adhesion Molecules on Mouse UNK Cells and Their Extracellular Matrix Ligands During Mouse Pregnancy

Uterine natural killer (uNK) cells are the dominant lymphocytes of pregnant mammals' uterus. Studies have identified four differentiation stage of mouse uNK cells based on Dolichos biflorus lectin cytochemistry, and their distribution showed preferential domain in the uterus through out the pregnancy. This work was done to investigate the expression of alpha5, alpha6, and beta7 integrins on uNK cells and their ligands distribution. Section of mouse uterus from sixth to seventeenth gestational days were submitted to immunocytochemistry and positive reactions for alpha5, alpha6, and beta7 integrins were found on uNK from eighth to tenth gestational days but not after twelfth gestational days. Fibronectin reactions were seemed from sixth to tenth gestational days around uNK from the myometrium and endometrium close to the myometrium. No reaction for fibronectin was seen in the decidualized and nondecidualized endometrium near the placenta. Laminin reaction was seen just in the antimesometrial side. beta7 integrin seems to be the active receptor to bind with VCAM-1 or MAdCAM-1 of endothelial cells, promoting the uNK cross through the vessels. The absence of laminin in an uNK domain suggests these cells are not dependent of laminin and alpha6 integrin for their establishment. However, fibronectin seems to support uNK migration, proliferation, differentiation, and survival in the uterus by binding with alpha5 integrin. The loss of alpha5 integrin ligation by the down regulation of fibronectin could inhibits these events and further studies are need to investigate whether unligated alpha5 can actively and initiate apoptosis, maybe in a caspase 8-dependent way that has been called integrin-mediated death.

Effect of ovarian stimulation with human menopausal gonadotropin and recombinant follicle stimulating hormone on the expression of integrins alpha(3), beta(1) in the rat endometrium during the implantation period

Objective To investigate the effect of exogenous ovarian stimulation with human menopausal gonadotropin (hMG) and recombinant follicle stimulating hormone (rFSH) on the expression of integrins alpha(3), beta(1) in the rat endometrium during implantation. Study design Following three successive normal estrous cycles the animals were divided into five groups: Group I ( n = 10, control group) received no medication; Group II ( n = 10) received 10 units of hMG; Group III ( n = 10) received 20 units of hMG; Group IV ( n = 10) received 10 units of rFSH; Group V ( n = 10) received 20 units of rFSH at midday of middiestrous. The rats were then mated with fertile males. The animals were sacrificed on the day of implantation. The uterine horns were placed in fixative and paraffin blocks of the tissue were cut in 5 μm sections. The tissues were stained with primary antibodies; monoclonal anti-integrin alpha(3) and monoclonal anti-integrin beta(1) using immunohistochemical methods. The staining intensities of alpha(3) and beta(1) integrins were calculated separately for epithelium and stroma in each group. Results Staining intensities of alpha(3) and beta(1) integrins in both the epithelium and the stroma were significantly lower in the treatment groups than the control group ( p < 0.05). Conclusion Ovarian stimulation by low and high doses of HMG and rFSH may have an effect on endometrial receptivity, possibly via a decrease in expression of integrins in the endometrium during the implantation period.

Integrin α5β1 is necessary for regulation of radial migration of cortical neurons during mouse brain development

During cerebral cortex development, post-mitotic neurons interact with radial glial fibers and the extracellular environment to migrate away from the ventricular region and form a correct laminar structure. Integrin receptors are major mediators of cell–cell and cell–extracellular matrix interactions. Several integrin heterodimers are present during formation of the cortical layers. The α5β1 receptor is expressed in the neural progenitors of the ventricular zone during cerebral cortex formation. Using in utero electroporation to introduce short hairpin RNAs in the brain at embryonic day 15.5, we were able to inhibit acutely the expression of α5 integrin in the developing cortex. The knockdown of α5 integrin expression level in neural precursors resulted in an inhibition of radial migration, without perturbing the glial scaffold. Moreover, the same inhibitory effect on neuronal migration was observed after electroporation of a Cre recombinase expression plasmid into the neural progenitors of conditional knockout mice for α5 integrin. In both types of experiments, the electroporated cells expressing reduced levels of α5 integrin accumulated in the premigratory region with an abnormal morphology. At postnatal day 2, ectopic neurons were observed in cortical layer V, while a deficit of neurons was observed in cortical layer II–IV. We show that these neurons do not express a layer V-specific marker, suggesting that they have not undergone premature differentiation. Overall, these results indicate that α5β1 integrin functions in the regulation of neural morphology and migration during cortical development, playing a role in cortical lamination.

Glucocorticoids increase α5 integrin expression and adhesion of synovial fibroblasts but inhibit ERK signaling, migration, and cartilage invasion

In rheumatoid arthritis (RA), integrins mediate cell adhesion, migration, and invasion, and their expression is regulated by cytokines and growth factors. The aim of this study was to investigate whether hormones such as cortisol or other steroids can influence integrin expression and function in the synovial cells of patients with RA. We performed immunofluorescence and fluorescence-activated cell sorting analyses to quantify surface integrin levels. Adhesion and migration assays were performed to study the function of synovial fibroblasts (SFs). ERK activation was measured by cellular activation of a signaling enzyme-linked immunosorbent assay. Invasion of SFs into cartilage was determined in the SCID mouse coimplantation model of RA in vivo. In RA, expression of integrin subunits alpha5, alphav, and beta1 was higher at the site of invasion compared with the sublining zone. Testosterone and 17beta-estradiol had no influence on integrin levels, but cortisol up-regulated expression of the alpha5 subunit in a time-dependent and dose-dependent manner. In addition, cortisol increased the adhesion of SFs to fibronectin and inhibited ERK signaling upon integrin activation or upon stimulation with tumor necrosis factor. Small interfering RNA or a neutralizing antibody to alpha5 integrin increased SF migration, indicating that up-regulated alpha5 integrin is responsible for an immobile phenotype. In addition, in the SCID mouse model, SF invasion into cartilage was attenuated by glucocorticoid treatment in vivo. Glucocorticoids increase integrin expression and the adhesion of cells to fibronectin, inhibit ERK signaling, and down-regulate the invasiveness of SFs in vivo. This study demonstrates that an important antiinflammatory aspect of glucocorticoids is regulating the expression and function of alpha5 integrin.