Integrin Alpha Subunits Research Articles

Inhibition of TGF-β receptor I by siRNA suppresses the motility and invasiveness of T24 bladder cancer cells via modulation of integrins and matrix metalloproteinase

Urinary bladder transitional-cell carcinoma is still challenging because the mechanisms underlying the tumor progression are still largely unknown. Transforming growth factor beta1 (TGF-beta1) is considered a crucial molecule in the tumorigenesis of urinary bladder carcinoma. Many studies have indicated that it is also associated with epithelial-mesenchymal transition, angiogenesis, migration and metastases in many types of malignant tumors. We blocked the TGF-beta signal pathway in T24 human bladder cancer cells with a siRNA (TsiRNA), which targets the TGF-beta type I receptor and evaluated the effects of TGF-beta1 and TsiRNA on the cell motility and invasiveness by Matrigel migration assay, wound-healing assay and Matrigel invasion assay. RT-PCR and Western blotting analysis were used to examine the effects of TGF-beta1 and TsiRNA on the expression of TGFBRI and genes, which are related to tumor migration and invasion. While exogenous TGF-beta1 enhanced the migration and invasion of T24 cells, TsiRNA significantly suppressed them. RT-PCR and Western blotting analysis revealed that TsiRNA could downregulate both the expression of alpha3, beta1 and alpha2 integrin subunits and the activity of matrix metalloproteinase 9 enhanced by exogenous TGF-beta1. Our study suggested that inhibition of TGF-beta1 signaling pathway by siRNA could be beneficial in the treatment of patients with metastatic bladder cancer.

Extracellular Matrix Lumican Deposited on the Surface of Neutrophils Promotes Migration by Binding to β2 Integrin

During inflammation, circulating polymorphonuclear neutrophils (PMNs) receive signals to cross the endothelial barrier and migrate through the extracellular matrix (ECM) to reach the injured site. Migration requires complex and poorly understood interactions of chemokines, chemokine receptors, ECM molecules, integrins, and other receptors. Here we show that the ECM protein lumican regulates PMN migration through interactions with specific integrin receptors. Lumican-deficient (Lum(-/-)) mice manifest connective tissue defects, impaired innate immune response, and poor wound healing with reduced PMN infiltration. Lum(-/-) PMNs exhibit poor chemotactic migration that is restored with exogenous recombinant lumican and inhibited by anti-lumican antibody, confirming a role for lumican in PMN migration. Treatment of PMNs with antibodies that block beta(2), beta(1), and alpha(M) integrin subunits inhibits lumican-mediated migration. Furthermore, immunohistochemical and biochemical approaches indicate binding of lumican to beta(2), alpha(M), and alpha(L) integrin subunits. Thus, lumican may regulate PMN migration mediated by MAC-1 (alpha(M)/beta(2)) and LFA-1 (alpha(L)/beta(2)), the two major PMN surface integrins. We detected lumican on the surface of peritoneal PMNs and not bone marrow or peripheral blood PMNs. This suggests that PMNs must acquire lumican during or after crossing the endothelial barrier as they exit circulation. We also found that peritoneal PMNs do not express lumican, whereas endothelial cells do. Taken together these observations suggest a novel endothelial lumican-mediated paracrine regulation of neutrophils early on in their migration path.

Expression of Integrin α6β1 Enhances Tumorigenesis in Glioma Cells

The integrin alpha6beta1 and its main ligand laminin-111 are overexpressed in glioblastoma, as compared with normal brain tissue, suggesting they may be involved in glioblastoma malignancy. To address this question, we stably expressed the alpha6 integrin subunit in the U87 cell line via retroviral-mediated gene transfer. We show that cell surface expression of the alpha6beta1 integrin led to dramatic changes in tumor U87 cell behavior, both in vitro and in vivo. Nude mice receiving either subcutaneous or intracerebral inoculation of alpha6beta1-expressing cells developed substantially more voluminous tumors than mice injected with control cells. The difference in tumor growth was associated with a marked increase in vascularization in response to alpha6beta1 integrin expression and may also be related to changes in the balance between cell proliferation and survival. Indeed, expression of alpha6beta1 enhanced proliferation and decreased apoptosis of U87 cells both in the tumor and in vitro. Additionally, we demonstrate that alpha6beta1 is implicated in glioblastoma cell migration and invasion and that laminin-111 might mediate dissemination of alpha6beta1-positive cells in vivo. Our results highlight for the first time the considerable role of the integrin alpha6beta1 in glioma progression.

The integrin 9 1 on hematopoietic stem and progenitor cells: involvement in cell adhesion, proliferation and differentiation

Hematopoietic stem and progenitor cells can interact with their microenvironment via integrins which are adhesion receptors consisting of alpha and beta subunits. Current knowledge suggests that the integrin subunits alpha4 and alpha6 expressed on hematopoietic stem and progenitor cells have distinct roles in retaining stem cells in the bone marrow. The aim of our study was to gain insight into the expression and functions of the integrin subunits alpha7-alpha11 within the endosteal stem cell niche. Human osteoblasts isolated from trabecular bone and hematopoietic stem and progenitor cells purified from umbilical cord blood or bone marrow aspirates were analyzed for the expression of integrin alpha7-alpha11 chains by reverse transcriptase polymerase chain reaction. The involvement of the integrin alpha9beta1 in hematopoietic stem and progenitor cell adhesion, proliferation and differentiation was analyzed in functional assays. Transcripts for all investigated integrin chains were found in primary osteoblasts. Highly purified hematopoietic stem and progenitor cells, however, expressed only transcripts encoding integrin subunits alpha7 and alpha9. Flow cytometric analysis verified extracellular expression of the integrin alpha9beta1 on hematopoietic stem and progenitor cells. Cell-cell adhesion assays with osteoblasts and dye-labeled CD34(+) hematopoietic stem and progenitor cells in the presence of function-blocking antibodies revealed a role of integrin alpha9 in hematopoietic stem and progenitor cell adhesion to osteoblasts. Furthermore, the addition of anti-integrin alpha9 antibodies significantly inhibited proliferation and in vitro differentiation of CD34(+) hematopoietic stem and progenitor cells. The integrin alpha9beta1 has been identified as a new member of the integrin beta1-subfamily expressed on human hematopoietic stem and progenitor cells. The functional studies strongly suggest that integrin alpha9beta1 contributes to adhesion and differentiation of hematopoietic stem and progenitor cells in the endosteal stem cell niche.

Inhibition of endothelial progenitor cell glycogen synthase kinase-3β results in attenuated neointima formation and enhanced re-endothelialization after arterial injury

Endothelial progenitor cells (EPCs) are circulating pluripotent vascular cells capable of enhancing re-endothelialization and diminishing neointima formation following arterial injury. Glycogen synthase kinase (GSK)-3beta is a protein kinase that has been implicated in the regulation of progenitor cell biology. We hypothesized that EPC abundance and function could be enhanced with the use of an inhibitor of GSK-3beta (GSKi), thereby resulting in improved arterial repair. Human EPCs were expanded ex vivo, treated with a specific GSKi, and then assessed for both yield and functional characteristics by in vitro assays for adherence, apoptosis, and survival. In vivo functionality of treated human EPCs was assessed in immune-tolerant mice subjected to femoral artery wire injury. Re-endothelialization was assessed at 72 h and neointima formation at 7 and 14 days following injury. GSKi treatment resulted in an improvement in the yield of EPCs and a reduction in apoptosis in cells derived from both healthy controls and patients with coronary artery disease. Treatment also increased vascular endothelial growth factor secretion, up-regulated expression of mRNA for the alpha-4 integrin subunit, and improved adhesion, an effect which could be abrogated with an alpha-4 integrin blocking antibody. EPCs without or with ex vivo GSKi treatment enhanced re-endothelialization 72 h following injury as well as reduced neointima formation at 7 days (e.g. endothelial coverage: 7.2 +/- 1.7% vs. 70.7 +/- 5.8% vs. 87.2 +/- 4.1%; intima to media ratios: 1.05 +/- 0.19 vs. 0.39 +/- 0.08 vs. 0.14 +/- 0.02; P < 0.05 for all comparisons), an effect that was persistent at 14 days. GSKi improves the functional profile of EPCs and is associated with improved re-endothelialization and reduced neointima formation following injury.

A role for decorin in controlling proliferation, adhesion, and migration of murine embryonic fibroblasts

The proteoglycan decorin putatively inhibits cell adhesion and cell migration on various extracellular matrix substrates through interactions with beta(1) integrins. This study, therefore, examined the adhesive, migration, and proliferative characteristics of decorin knockout (Dcn(-/-)) murine embryonic fibroblasts compared to wild-type controls on collagen-coated, fibronectin-coated, and uncoated tissue culture plates. The Dcn(-/-) cells showed significantly greater proliferation than wild-type controls on all substrates. The Dcn(-/-) cells also showed significantly greater adhesion to both collagen and fibronectin; both cell types showed greater adhesion to collagen. The addition of exogenous decorin had a differential effect on adhesion to collagen between cell types, but not on fibronectin. For collagen, blocking either alpha(2) or beta(1) integrin subunits significantly reduced adhesion for Dcn(-/-) cells; whereas for fibronectin, blocking either the alpha(5) or beta(1) integrin subunits reduced adhesion for both cell types. Decorin and the alpha(5)beta(1) integrin may have lesser roles in adhesion to fibronectin than previously presumed. Finally, compared to wild-type cells, Dcn(-/-) cells showed greater migration on both uncoated and collagen substrates. This study demonstrates that decorin affects the biology of various integrins that participate in cell proliferation, adhesion, and migration on various substrates.

Functional blocking of specific integrins inhibit colonic cancer migration

For more effective oncological management of disseminated colorectal cancer, therapies must be devised that target the different individual stages of metastasis development. Recent work showed that integrin subunits alpha2, alpha6 and beta4 are involved in the colorectal cancer cell extravasation process. By means of Immunocytochemistry and Western blotting, it was shown that all three integrins are expressed not only in human colorectal cancer cells (HT29) but also in rat colonic cancer cells (DHDK12). Using in vivo models and intravital video microscopy techniques, it was shown that functional blocking of these integrin subunits by specific antibodies produced a significant reduction in cancer cell extravasation and migration. In conclusion, integrin subunits alpha2, alpha6 and beta4 are expressed in unrelated colorectal cancer cell strains and appear to play a key role in cancer cell migration.

Activation and deactivation of periventricular white matter phagocytes during postnatal mouse development

Brain microglia are related to peripheral macrophages but undergo a highly specific process of regional maturation and differentiation inside the brain. Here, we examined this deactivation and morphological differentiation in cerebral cortex and periventricular subcortical white matter, the main "fountain of microglia" site, during postnatal mouse development, 0-28 days after birth (P0-P28). Only macrophages in subcortical white matter but not cortical microglia exhibited strong expression of typical activation markers alpha5, alpha6, alphaM, alphaX, and beta2 integrin subunits and B7.2 at any postnatal time point studied. White matter phagocyte activation was maximal at P0, decreased linearly over P3 and P7 and disappeared at P10. P7 white matter phagocytes also expressed high levels of IGF1 and MCSF, but not TNFalpha mRNA; this expression disappeared at P14. This process of deactivation followed the presence of ingested phagocytic material but correlated only moderately with ramification, and not with the extent of TUNEL+ death in neighboring cells, their ingestion or microglial proliferation. Intravenous fluosphere labeling revealed postnatal recruitment and transformation of circulating leukocytes into meningeal and perivascular macrophages as well as into ramified cortical microglia, but bypassing the white matter areas. In conclusion, this study describes strong and selective activation of postnatally resident phagocytes in the P0-P7 subcortical white matter, roughly equivalent to mid 3rd trimester human fetal development. This presence of highly active and IGF1- and MCSF-expressing phagocytes in the neighborhood of vulnerable white matter could play an important role in the genesis of or protection against axonal damage in the fetus and premature neonate.

Reorganization of the integrin alpha2 subunit controls cell adhesion and cancer cell invasion in prostate cancer.

The mechanisms of invasion and metastasis are poorly understood. Our previous studies demonstrated that cancer cell invasion may result from reorganization of membrane molecules, thereby initiating signaling pathways. To increase our understanding on how cancer cells govern metastases we studied the established LNCaP prostate cancer progression model. Herein we show that the bone metastatic derivative cell line, C4-2B, displays changes in adhesion to collagen type I and invasion into collagen type I. Moreover, we found that these changes were concomitant with activation of the FAK/src/paxillin/Rac/JNK signaling pathway and increased activity of matrix metalloproteinases (MMPs)-2 and -9. Inhibition of src and JNK resulted in inhibition of adhesion and invasion, and deactivation of the signaling molecules in the identified pathway as well as reduced activity of MMPs. Additionally, we found a pivotal role for the integrin alpha2 subunit since lateral redistribution and clustering were responsible for activation of the downstream signaling and function blocking of the integrin alpha2 subunit resulted in poor adhesion and inhibition of invasion. In conclusion, our results suggest that invasion of prostate cancer cells can be ascribed to reorganization and clustering of integrin alpha2 subunits, resulting in activation of associated FAK/src/paxillin/Rac/JNK, leading to increased activity of MMPs and thus invasion.

Rabs and cancer cell motility

The Rab family of small GTPases functions in regulating vesicular transport in all eukaryotes. In the past few years, several important reports have linked some members of the Rab family to intriguing mechanistic aspects of cancer cell migration and invasiveness. Rab5 and Rab21 associate with alpha-integrin subunits and modulate their endosomal traffic and subcellular localization. Expression of the latter enhances adhesion and migration of certain cancer cell types. Rab25 has been functionally linked to tumor progression and the invasiveness of some epithelial cancers. Rab25 promotes invasive migration of cells in three-dimensional microenvironments by associating with alpha5beta1 integrin, and directing its recycling to dynamic ruffling protrusions at the migrating cell front. Acting directly, or through its effector, the Rab-coupling protein, Rab25 could potentially engage both integrin and epidermal growth factor receptor and enhance their oncogenic recycling and signaling. Tumor invasiveness may also be modulated by Rab8-mediated exocytic traffic of MT1-matrix metalloproteinase, with the latter's activity likely influenced by interaction with the mammalian suppressor of Sec4 (Mss4), a Rab8 guanine nucleotide exchange factor, and integrin. We discuss highlights in the recent literature that point towards a role for Rab-mediated membrane traffic in cancer cell migration and invasion.

An N-Glycosylation Site on theβ-Propeller Domain of the Integrin α5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification byβ1,4-N-Acetylglucosaminyltransferase III

Recently we reported that N-glycans on the beta-propeller domain of the integrin alpha5 subunit (S-3,4,5) are essential for alpha5beta1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type alpha5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the alpha5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type alpha5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the alpha5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.

Urokinase receptor mediates lung fibroblast attachment and migration toward provisional matrix proteins through interaction with multiple integrins.

Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibits a dependence on the urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the urokinase-type plasminogen activator cellular receptor (u-PAR)-integrin interactions and to determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR colocalizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin, and collagens. Inhibitory antibody studies have revealed that human lung fibroblasts utilize alpha(v)beta(5) to attach to vitronectin, predominantly alpha(5)beta(1) (and alpha(v)beta(3)) to attach to fibronectin, and alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) to attach to collagen. Blocking studies with alpha-integrin subunit decoy peptides and u-PAR neutralizing antibodies indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these decoy peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple alpha-integrins but with a preference for alpha(3). Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of fibroblast invasion would be expected to lead to amelioration of fibroproliferative diseases of the lung.

Identification of integrins α6 and β7 as c‐Jun‐ and transformation‐relevant genes in highly invasive fibrosarcoma cells

Understanding the mechanisms of tumor cell invasion is essential for our attempts to prevent cancer deaths. We screened by DNAmicroarrays the c-Jun- and transformation-related gene expression changes in S-adenosylmethionine decarboxylase (AdoMetDC)-overexpressing mouse fibroblasts that are highly invasive in vivo, and their derivatives expressing a tetracycline-inducible dominant-negative mutant of c-Jun (TAM67) or c-Jun shRNA. Among the small set of target genes detected were integrins alpha6 and beta7, cathepsin L and thymosin beta4, all upregulated in the AdoMetDC-transformed cells and downregulated upon reversal of transformation by TAM67 or c-Jun shRNA. The upregulation of integrin alpha6 subunit, pairing with integrin beta1, endowed the transformed cells with the capability to attach to basement membrane laminin and to spread. Further, inhibition of integrin alpha6 or beta1 function with neutralizing antibodies blocked the invasiveness of AdoMetDC-transformants and human HT-1080 fibrosarcoma cells in three-dimensional Matrigel. Moreover, immunohistochemical analyses showed strong integrin alpha6 staining in high-grade human fibrosarcomas. Our data show that c-Jun can regulate all three key steps of invasion: cell adhesion (integrin alpha6), basement membrane/extracellular matrix degradation (cathepsin L) and cell migration (thymosin beta4). In addition, this is the first study to associate integrin beta7, known as a leukocyte-specific integrin binding to endothelial/epithelial cell adhesion molecules, with the transformed phenotype in cells of nonleukocyte origin. As tumor cell invasion is a prerequisite for metastasis, the observed critical role of integrin alpha6beta1 in fibrosarcoma cell invasion/spreading allures testing antagonists to integrin alpha6beta1, alone or combined with inhibitors of cathepsin L and thymosin beta4, as chemotherapeutic agents.

β1‐integrins shedding in a guinea‐pig model of chronic asthma with remodelled airways

A hallmark of airway remodelling in asthma is subepithelial fibrosis, but its relation with airway dysfunction is still controversial. To describe airway functional abnormalities and subepithelial remodelling induced by repetitive antigenic challenges. Nine inhaled antigenic challenges were applied every 10 days to guinea-pigs sensitized to ovalbumin (OVA). Antigen-induced airway hyperresponsiveness (AI-AHR) to histamine and its immunohistopathological relationship was evaluated at the first, third and ninth OVA challenges. From the first challenge on, OVA induced acute transient bronchoobstruction followed by development of AI-AHR. A progressive rise in baseline Penh (a bronchoobstruction index) and granulocyte airway infiltration was also observed. After the ninth OVA challenge, the amount of extracellular matrix in the subepithelial region (SER) of bronchi and bronchioles was increased. Immunohistochemistry analysis showed that this SER fibrosis was associated to beta1-integrin subunit overexpression, even in acellular areas. Immunoelectronmicroscopy corroborated the location of beta1-integrin in extracellular matrix, essentially in types l and II collagen fibres. Presence of alpha1- and alpha2-integrin subunits in these areas was also corroborated. AI-AHR was correlated with degree of SER increment, cell infiltration, and beta1-integrin expression. Our data suggested that beta1-integrin shedding produced by repetitive allergen challenges in guinea-pigs was associated with collagen deposition in SER of bronchi and bronchioles, along with inflammatory cells infiltration and AI-AHR development.

In Vivo reorganization of alpha 1 integrin in developing skeletal muscle

Integrins play an important role in muscle development. In developing chick skeletal muscle In Vitro, the integrin subunit alpha 1 has been shown to reorganize from a punctate to a periodic distribution along the AI junction subsequent to sarcomere assembly. Also subsequent to sarcomere assembly, laminin, a major component of the basement membrane, has been observed with a periodicity along the myofibril. Analysis In Vitro has shown that the periodic distribution of laminin and alpha 1 integrin are correlated both spatially and temporally and suggests a role for alpha 1 integrin in the establishment of the basement membrane. To confirm the distribution of these proteins in muscle development In Vivo, sectioned skeletal muscle from embryonic chick days 16‐20 was examined. Initially muscle sections were immunofluorescently labeled with antibodies to alpha 1 integrin and muscle specific proteins. Data indicated that alpha 1 integrin reorganized from a punctate to a periodic distribution beginning at day 16. By day 18 all myofibers examined were observed to have alpha 1 integrin periodically distributed along the sarcomeres. The temporal and spatial distribution of laminin in relation to alpha 1 integrin at embryonic days 16‐18 will be examined to determine the correlation of these two proteins in In Vivo muscle development.

Effects of natalizumab, an α4 integrin inhibitor, on the development of Hartley guinea pigs

Natalizumab is a humanized monoclonal immunoglobulin G4 antibody directed against the human alpha4 integrin subunit, disrupting interaction with its ligands. Natalizumab inhibits the interaction of alpha4 integrins with fibronectin, vascular cell adhesion molecule-1, and mucosal addressin cellular adhesion molecule-1, which are of potential importance in development. Two studies were undertaken to evaluate the effects of natalizumab on embryo/fetal development in guinea pigs. In the first study, pregnant guinea pigs were treated with intravenous injections of 3, 10, or 30 mg/kg natalizumab or vehicle every other day from gestational day (GD) 4 to 30. In the second study, females were treated on alternate days starting at least 28 days prior to mating through GD 30. Fetal examinations and histopathologic examination of the liver, heart, thymus, spleen, and intestinal tract were performed following maternal euthanasia on GD 59-62. Natalizumab had no significant effect on embryo/fetal development in either study. Exposure to natalizumab during organogenesis did not result in treatment-related external, visceral, or skeletal variations or malformations or histopathologic changes. No fetotoxicity or teratogenic effects were attributable to natalizumab in these studies.

Expression of integrin-α3 mRNA in meningiomas and its correlation with proliferation and invasion

To investigate the expression of integrin-alpha3 mRNA in meningiomas and its correlation with proliferation and invasion, the expression of integrin-alpha(3) subunit was detected by using in situ hybridization in patients with meningiomas (36 cases) and normal dura (2 cases) and arachnoid tissues (2 cases). SABC immunohistochemical assay was used to study the expression of Ki-67 nuclear antigen. The results showed that the expression of integrin-alpha(3) mRNA in benign, atypical and malignant meningiomas was 2.52+/-0.362, 1.75+/-0.316 and 1.42+/-0.633, respectively. The expression levels of integrin-alpha(3) mRNA was significantly lower in atypical or malignant meningiomas than those in benign meningiomas (P<0.05, P<0.01, respectively). The expression of integrin-alpha(3) mRNA in those with invasive biological behavior was lower than that in those without invasion (1.63+/-0.462 vs. 2.61+/-0.526, P<0.01). Moreover, the expression of integrin-alpha(3) mRNA was inversely associated with Ki-67 labeling index in all cases. It suggested that integrin-alpha(3) subunit participated in the modulatory process of growth of meningiomas. The proliferation activity and malignant grade of meningiomas were increased with the decreased expression of integrin-alpha(3) subunit, and the down-regulation of integrin-alpha(3) mRNA was associated with the invasive biological behaviors in meningiomas.

Reduction of Mouse Egg Surface Integrin Alpha9 Subunit (ITGA9) Reduces the Egg's Ability to Support Sperm-Egg Binding and Fusion1

The involvement of egg integrins in mammalian sperm-egg interactions has been controversial, with data from integrin inhibitor studies contrasting with evidence from knockouts showing that specific integrin subunits are not essential for fertility. An alpha(4)/alpha(9) (ITGA4/ITGA9) integrin subfamily member has been implicated in fertilization but not extensively examined, so we tested the following three hypotheses: 1) an ITGA4/ITGA9 integrin participates in sperm-egg interactions, 2) short-term acute knockdown by RNA interference of integrin subunits would result in a fertilization phenotype differing from that of chronic depletion via knockout, and 3) detection of a fertilization phenotype is sensitive to in vitro fertilization (IVF) assay conditions. We show that mouse and human eggs express the alpha(9) integrin subunit (ITGA9). RNA interference-mediated knockdown resulted in reduced levels of Itga9 mRNA and surface protein in mouse eggs. RNA interference attempts to knockdown ITGA9's likely beta partner, beta(1) (ITGB1), resulted in reduced Itgb1 mRNA but no reduction in ITGB1 surface protein. Therefore, studies using a function-blocking anti-ITGB1 antibody tested the hypothesis that ITGB1 participates in gamete interactions. Analyses of sperm-egg interactions with Itga9-knockdown eggs and anti-ITGB1 antibody-treated eggs in IVF assays using specific sperm:egg ratios revealed the following: 1) a reduction, but not complete loss, of sperm-egg binding and fusion was observed and 2) the reduction of sperm-egg binding and fusion was not detected in inseminations with high sperm:egg ratios. These data demonstrate that ITGA9 and ITGB1 participate in sperm-egg interactions but clearly are not the only molecules involved. This also shows that careful design of IVF parameters allows detection of deficiencies in gamete interactions.

Mechanism of betulinic acid inhibition of collagen biosynthesis in human endometrial adenocarcinoma cells

Collagen as a ligand for integrin receptors plays important role in the integrin - dependent regulation of cellular metabolism. Since betulinic acid (BA) evokes anticancer activity, its effect on collagen biosynthesis was studied in cultured endometrial adenocarcinoma cells. Confluent cells were treated with different concentrations of BA for 24 hours. It was found that BA inhibit collagen biosynthesis ([3H] proline incorporation assay). The mechanism of this phenomenon was found at the level of insulin-like growth factor-I receptor (IGF-IR) and alpha2 integrin signalling (Western immunoblot analysis). The expressions of IGF-I receptor and alpha2 integrin subunit as well as integrin activated focal adhesion kinase (FAK) were decreased in the cells treated with BA. It was accompanied by a parallel decrease in the expression of Sos protein and phosphorylated MAP-kinases (ERK1, ERK2) and up - regulation of NF-kappaB. The data suggest that BA-dependent inhibition of collagen biosynthesis in cultured human endometrial adenocarcinoma cells undergoes through alpha2 integrin and IGF-IR signaling that activate NF-kappaB, potent inhibitor of collagen gene expression.

Importance of N-Glycosylation on .ALPHA.5.BETA.1 Integrin for Its Biological Functions

N-Glycosylation of proteins is conserved in eukaryotes, which is one of the most abundant post-translational modification reactions, and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structure (glycan) are associated with many physiological and pathological events, including cell adhesion, migration, cell growth, cell differentiation and tumor invasion. Glycosylation reactions are catalyzed by the action of glycosyltransferases, which add sugar chains to various glycans on glycoproteins, glycolipids and proteoglycans. Here, we focus mainly on the modification of N-glycans with N-acetylglucosaminyltransferase III (GnT-III), N-acetylglucosaminyltransferase V (GnT-V) and alpha2,6 sialyltransferase (ST6GalI) to address the important roles of N-glycans in integrin-meditaed cell adhesion and migration. In addition, we also discuss the potential roles of N-glycosylation sites on integrin alpha5 subunit.