Down-regulation of integrin alpha(v)beta(3) expression and integrin-mediated signaling in glioma cells by adenovirus-mediated transfer of antisense urokinase-type plasminogen activator receptor (uPAR) and sense p16 genes

Interaction between the extracellular matrix and integrin receptors on cell surfaces leads not only to cell adhesion but also to intracellular signaling events that affect cell migration, proliferation, and survival. The vitronectin receptor alpha(v)beta(3) integrin is of key importance in glioma cell biology. The expression of urokinase-type plasminogen activator receptor (uPAR) was recently shown to co-regulate with the expression of alpha(v)beta(3) integrin. Moreover, restoration of the p16 protein in glioma cells inhibits the alpha(v)beta(3) integrin-mediated spreading of those cells on vitronectin. Thus we hypothesized that adenovirus-mediated down-regulation of uPAR and overexpression of p16 might down-regulate the expression of alpha(v)beta(3) integrin and the integrin-mediated signaling in glioma cells, thereby defeating the malignant phenotype. In this study, we used replication-deficient adenovirus vectors that contain either a uPAR antisense expression cassette (Ad-uPAR) or wild-type p16 cDNA (Ad-p16) and a bicistronic adenovirus construct in which both the uPAR antisense and p16 sense expression cassettes (Ad-uPAR/p16) are inserted in the E1-deleted region of the vector. Infecting the malignant glioma cell line SNB19 with Ad-uPAR, Ad-p16, or Ad-uPAR/p16 in the presence of vitronectin resulted in decreased alpha(v)beta(3) integrin expression and integrin-mediated biological effects, including adhesion, migration, proliferation, and survival Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.     

The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility

Integrin affinity is modulated by intracellular signaling cascades, in a process known as "inside-out" signaling, leading to changes in cell adhesion and motility. Protein kinase C (PKC) plays a critical role in integrin-mediated events; however, the mechanism that links PKC to integrins remains unclear. Here, we report that PKCepsilon positively regulates integrin-dependent adhesion, spreading, and motility of human glioma cells. PKCepsilon activation was associated with increased focal adhesion and lamellipodia formation as well as clustering of select integrins, and it is required for phorbol 12-myristate 13-acetate-induced adhesion and motility. We provide novel evidence that the scaffolding protein RACK1 mediates the interaction between integrin beta chain and activated PKCepsilon. Both depletion of RACK1 by antisense strategy and overexpression of a truncated form of RACK1 which lacks the integrin binding region resulted in decreased PKCepsilon-induced adhesion and migration, suggesting that RACK1 links PKCepsilon to integrin beta chains. Altogether, these results provide a novel mechanistic link between PKC activation and integrin-mediated adhesion and motility.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Alpha 6.beta 1 integrin (laminin receptor) is down-regulated by tumor necrosis factor alpha and interleukin-1 beta in human endothelial cells.

Endothelial cells from human umbilical vein (HEC) express several distinct integrin complexes that mediate the interaction with the basal membrane components. In this paper we show that treatment with tumor necrosis factor alpha (TNF alpha) down-regulates the expression of the laminin receptor alpha 6.beta 1 integrin in cultured HEC. After 48 h of treatment with TNF alpha, the level of expression of the alpha 6.beta 1 complex reached 20% of the control value. The down-regulation of the alpha 6.beta 1 integrin is caused by a decreased expression of the alpha 6 subunit, whereas the synthesis of the beta 1 subunit remains constant. Northern blot analysis shows that the decreased level of alpha 6 subunit synthesis is caused by down-regulation of alpha 6 mRNA in TNF alpha-treated HEC. TNF alpha treatment does not alter the expression of alpha 2, alpha 3, and alpha 5 integrins, also present on endothelial cell surface, thus showing that this cytokine has a selective action on distinct integrin complexes. Down-regulation of alpha 6.beta 1 correlates with pronounced reduction in adhesion of TNF alpha-treated HEC to laminin, but not to fibronectin-coated culture dishes. In addition to TNF alpha, interleukin-1 beta also decreases the expression of the alpha 6.beta 1 integrin and reduces adhesion to laminin, thus suggesting that this regulation plays an important role in inflammation.     

Integrin regulation by vascular endothelial growth factor in human brain microvascular endothelial cells: role of alpha6beta1 integrin in angiogenesis

The precise role of vascular endothelial growth factor (VEGF) in regulating integrins in brain microvascular endothelial cells is unknown. Here, we analyzed VEGF effects on integrin expression and activation in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and ribonuclease (RNase) protection assays, we observed that VEGF up-regulated the mRNA expression of alpha(6) integrin in HBMECs. VEGF significantly increased alpha(6)beta(1) integrin expression, but not alpha(6)beta(4) integrin expression in these cells. Specific down-regulation of alpha(6) integrin expression by small interfering RNA (siRNA) oligonucleotides inhibited both the capillary morphogenesis of HBMECs and their adhesion and migration. Additionally, VEGF treatment resulted in activation of alpha(6)beta(1) integrins in HBMECs. Functional blocking of alpha(6) integrin with its specific antibody inhibited the VEGF-induced adhesion and migration as well as in vivo angiogenesis, and markedly suppressed tumor angiogenesis and breast carcinoma growth in vivo. Thus, VEGF can modulate angiogenesis via increased expression and activation of alpha(6)beta(1) integrins, which may promote VEGF-driven tumor angiogenesis in vivo.     

Expression of the vacuolar H+-ATPase 16-kDa subunit results in the Triton X-100-insoluble aggregation of beta1 integrin and reduction of its cell surface expression

Vacuolar H(+)-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H(+)-ATPase via one of its transmembrane domains, TMD4, strongly associates with beta(1) integrin, affecting beta(1) integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of beta(1) integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose alpha(5)beta(1) integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of beta(1) integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of beta(1) integrin (premature form) and, consequently, the level of the 130-kDa form of beta(1) integrin (mature form). Further experiments showed that the normal levels of association between the premature beta(1) integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of beta(1) integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits beta(1) integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional beta(1) integrin.     

Transforming growth factors beta(1) (TGF-beta(1)) and TGF-beta(2) promote glioma cell migration via Up-regulation of alpha(V)beta(3) integrin expression

The migratory behaviour of malignant gliomas relies on the interaction of integrins with extracellular matrix (ECM) components. Transforming growth factor-beta(1) (TGF-beta(1)) potently stimulates glioma cell motility whereas TGF-beta(2) is known for its immunosuppressive properties. Here, we show that both TGF-beta(1) and TGF-beta(2) promote migration of glioma cells. In parallel, TGF-beta(1) and TGF-beta(2) induce alpha(V) and beta(3) intergrin mRNA expression and enhance cell surface expression of alpha(V)beta(3) integrin. TGF-beta-mediated promotion of migration is abrogated by echistatin, a Arg-Gly-Asp (RGD) peptide antagonist of alpha(V)beta(3) integrin, and by a neutralizing anti-alpha(V)beta(3) integrin antibody. Taken together, we report a novel mechanism by which TGF-beta modulates cell ECM interactions and promotes glioma cell motility.     

Expression of chicken integrin beta 1 subunit in rat PC12 cells

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin. These results indicate that chicken integrin beta 1 may functionally substitute for beta 1 subunit of rat integrins in PC12 cells. Apparently, the structure of the integrin beta 1 subunit is highly conserved in the evolution of these species.     

ADAM15 suppresses cell motility by driving integrin alpha5beta1 cell surface expression via Erk inactivation

Human ADAM15 is unique among the A disintegrin and metalloprotease domain (ADAM) family because of the integrin binding motif Arg-Gly-Asp (RGD) within its disintegrin domain. Integrin alpha5beta1 has been reported to bind to ADAM15 in an RGD-dependent manner, but the biological significance of the interaction between ADAM15 and alpha5beta1 is unknown. To characterize the effects of ADAM15 on alpha5beta1-mediated cell adhesion and migration and elucidate the potential mechanism, CHO cells which express endogenous integrin alpha5beta1 were transfected with human ADAM15 cDNA. ADAM15 overexpression led to enhanced cell adhesion and decreased migration on fibronectin, which were suppressed by down-regulation of integrin alpha5. Overexpression of ADAM15 not only increased the cell surface expression of integrin alpha5 but also resulted in a more clustered staining of alpha5 on cell surface, while the beta1 subunit remained unchanged. Unexpectedly, results from immunoprecipitation and immunofluorescence indicated that ADAM15 and alpha5beta1 integrin did not interact directly in CHO cells. We found that ADAM15 expression decreased the phosphorylation of Erk1/2. Consistently, down-regulation of Erk1/2 phosphorylation by MEK inhibitor PD98059 or siRNA against Erk1/2 enhanced the expression of alpha5 on cell surface. By using a B16F10 pulmonary metastasis model, we revealed that overexpression of ADAM15 significantly reduced the number of metastatic nodules on the lung. Taken together, this study reveals for the first time that ADAM15 could drive alpha5 integrin expression on cell surface via down-regulation of phosphorylated Erk1/2. This presents a novel mechanism by which ADAM15 regulates cell-matrix adhesion and migration.     

Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.     

The effect of gamma-tocopherol on proliferation, integrin expression, adhesion, and migration of human glioma cells

The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. gamma-tocopherol at 50 microM concentration exerted more inhibitory effect than alpha-tocopherol at the same concentration on glioma cell proliferation. Integrin alpha5 and beta1 protein levels were increased upon both alpha- and gamma-tocopherol treatments. In parallel, an increase in the alpha5beta1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where gamma-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin alpha5 and beta1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the alpha5beta1 heterodimer. Cell migration is stimulated by gamma-tocopherol. It is concluded that alpha5 and beta1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events.     

Immunohistochemical localization of beta1 and beta4 integrins in mouse endometrium during implantation and early pregnancy

Implantation presents the remarkable synchronisation between the development of embryo and differentiation of endometrium. Cell-cell adhesion is an important phenomenon taking place during blastocyst implantation in uterine membrane. We think that the investigation of existence and the level of integrins in women can be a guide for treatment of infertility. Our purpose in this study was to show expression beta1 and beta4 integrins on gestational days 4, 6, 12 by immunohistochemical methods and to investigate whether beta4 integrin is a useful marker for receptivity. beta1 and beta4 integrin were exhibited on surface epithelium on gestational day 4. On the other hand, strong beta4 immunoreactivity was detected on surface epithelium and glandular cells on gestational day 12 but no beta1 reactivity was present in the surface epithelium and glandular cells on day 12. In conclusion, both beta1 and beta4 integrins may have a role in implantation process because positive immunoreactivity was seen on apical membrane of surface epithelium on day 4 when implantation occurred. The localization to apical pole of surface epithelium suggest a role for beta1, beta4 integrins in initial embryo and endometrium interaction. It does not seem that beta1 integrin has a role supporting pregnancy since expression of beta4 on surface epithelium and glandular epithelium disappeared on day 12. beta4 integrin expression increasing on day 12 of pregnancy leads us to think a possible functional role supporting pregnancy.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

The integrin beta1 subunit cytoplasmic tail forms oligomers: a potential role in beta1 integrin clustering

Integrins are alpha/beta heterodimeric cell surface receptors devoid of enzymatic activity. Signal transduction therefore requires the association of cytosolic and cytoskeletal proteins with the integrin subunit intracellular regions. This association is initiated upon ligand binding to the integrin receptor and includes clustering of the integrins and recruitment of focal adhesion-associated proteins. Whether integrin clustering is solely dependent on ligand binding to the integrin extracellular parts or involves also interactions between the intracellular tails of integrins is so far unknown. To investigate intracellular events in integrin clustering, we have used peptides corresponding to the integrin beta1 cytoplasmic region. Loading of cells with the peptides results in a decreased cell adhesion and in an inhibition of cell spreading in agreement with the previously reported dominant negative effect of the beta1 integrin cytoplasmic tail on integrin clustering. Direct protein-protein interaction studies by surface plasmon resonance demonstrate that integrin beta1 cytoplasmic peptides self-associate in contrast to integrin beta3 cytoplasmic tails. Size exclusion chromatography and SDS-PAGE analysis of the peptides further show that the integrin beta1 cytoplasmic parts form oligomers and that they assume alpha helical conformation to the extent of about 13% and that this fraction is increased upon aggregation. Thus self-association of the integrin beta1 subunit cytoplasmic regions may be central to beta1 integrin clustering.     

Regulation of cell adhesion receptors by transforming growth factor-beta. Concomitant regulation of integrins that share a common beta 1 subunit

Cell adhesion to extracellular matrices is mediated by a set of heterodimeric cell surface receptors called integrins that might be the subject of regulation by growth and differentiation factors. We have examined the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of the very late antigens or alpha beta 1 group of integrins in human cell lines. The six known members of this family share a common beta 1 subunit but have distinct alpha subunits that confer selective affinity toward type I collagen, fibronectin, laminin, and other as yet unknown cell adhesion proteins. Using a panel of specific antibodies and cDNA probes, we show that in WI-38 lung fibroblasts TGF-beta 1 elevates concomitantly the expression of alpha 1, alpha 2, alpha 3, alpha 5, and beta 1 integrin subunits at the protein and/or mRNA level, their assembly into the corresponding alpha beta 1 complexes, and their exposure on the cell surface. The rate of synthesis of total alpha subunits relative to beta 1 subunit is higher in TGF-beta 1-treated cells than in control cells. The characteristically slow (t1/2 approximately 10 h) rate of beta 1 conversion from precursor form to mature glycoprotein in untreated cells increases markedly (to t1/2 approximately 3 h) in response to TGF-beta 1. The results suggest that in WI-38 fibroblasts the beta 1 subunit is synthesized in excess over alpha subunits, and assembly of beta 1 subunits with rate-limiting alpha subunits is required for transit through the Golgi and exposure of alpha beta 1 complex on the cell surface. TGF-beta 1 does not induce the synthesis of integrin subunits that are not expressed in unstimulated cells, such as alpha 4 and alpha 6 subunits in WI-38 fibroblasts. However, alpha 4 and alpha 6 subunits can be regulated by TGF-beta in those cells that express them. The results suggest that TGF-beta regulates the expression of individual integrin subunits by parallel but independent mechanisms. By modifying the balance of individual alpha beta 1 integrins, TGF-beta 1 might modulate those aspects of cell migration, positioning, and development that are guided by adhesion to extracellular matrices.     

Beta 1 and beta 3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix.

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of beta 1 and beta 3 integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning beta 1 integrins with little, if any, contribution by the alpha V beta 3 integrin; on the other hand, cell migration over these substrates depended to a large extent on the alpha V beta 3 receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the beta 1 integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the beta 3 receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of beta 1 integrins binding to each matrix component: FN (alpha 5 beta 1, alpha 3 beta 1, alpha V beta 1), LN (alpha 1 beta 1, alpha 7 beta 1), VN (alpha V beta 1), I (alpha 1 beta 1, alpha 2 beta 1), and IV (alpha 1 beta 1). In contrast, the beta 3 integrin, alpha V beta 3, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that beta 1 and beta 3 integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.     

Apoptosis and involution in the mammary gland are altered in mice lacking a novel receptor, beta1,4-Galactosyltransferase I

Receptor-mediated cell-extracellular matrix (ECM) interactions are critical regulators of cell survival, and perturbing these signaling pathways can disrupt cellular differentiation and function in a variety of tissues, including the mammary gland. One such receptor is the cell surface-associated, long isoform of beta1,4-galactosyltransferase I (GalT I). Deletion of long GalT I leads to increased mammary ductal branching morphogenesis [Dev. Biol., 244 (2002) 114]. Here, we show that this expansion in the mammary epithelial (ME) cell compartment is accomplished through decreased apoptosis during pregnancy and involution. Decreased apoptosis during involution is concomitant with delayed alveolar collapse, persistent expression of the milk protein gene alpha-lactalbumin and delayed expression of genes associated with the tissue-remodeling phase of involution. Using 3-dimensional in vitro cultures, we show that the decrease in apoptosis is dependent on laminin 1, a ligand for surface GalT I, suggesting that surface GalT I negatively influences ECM-dependent cell survival, a novel function for an ECM receptor. In the best-studied examples, ECM promotes survival through integrin receptor-mediated activation of focal adhesion kinase (FAK). Aggregation of surface GalT I also activates FAK, therefore, we asked if FAK activation was altered in ME from long GalT I null mice. Activated FAK was appropriately localized to focal adhesions in long GalT I null ME. However, FAK activation was constitutively reduced 4.5-fold in long GalT I nulls relative to wild type. Expression of the integrin beta1 subunit was not affected by loss of long GalT I. Collectively, these results suggest that surface GalT I might negatively regulate ME cell survival by linking integrin-independent FAK activation to apoptotic rather than survival signaling events.     

Novel role of presenilins in maturation and transport of integrin beta 1

Presenilins (PSs) play important roles in modulating the trafficking and maturation of several membrane proteins. However, the target membrane proteins whose trafficking and maturation are regulated by PS are largely unknown. By characterizing PS-deficient fibroblasts, we found that integrin beta1 maturation is promoted markedly in PS1 and PS2 double-deficient fibroblasts and moderately in PS1- or PS2-deficient fibroblasts; in contrast, nicastrin maturation is completely inhibited in PS1 and PS2 double-deficient fibroblasts. Subcellular fractionation analysis demonstrated that integrin beta1 maturation is promoted in the Golgi apparatus. The mature integrin beta1 with an increased expression level was delivered to the cell surface, which resulted in an increased cell surface expression level of mature integrin beta1 in PS1 and PS2 double-deficient fibroblasts. PS1 and PS2 double-deficient fibroblasts exhibited an enhanced ability to adhere to culture dishes coated with integrin beta1 ligands, namely, fibronectin and laminin. The inhibition of gamma-secretase activity enhances neither integrin beta1 maturation nor the adhesion of wild-type cells. Moreover, PS deficiency also promoted the maturation of integrins alpha3 and alpha5 and the cell surface expression of integrin alpha3. Integrins alpha3 and alpha5 were coimmunoprecipitated with integrin beta1, suggesting the formation of the functional heterodimers integrins alpha3beta1 and alpha5beta1. Note that integrin beta1 exhibited features opposite those of nicastrin in terms of maturation and trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus in PS1 and PS2 double-deficient fibroblasts. Our results therefore suggest that PS regulates the maturation of membrane proteins in opposite directions and cell adhesion by modulating integrin maturation.     

Activated Rac1 selectively up-regulates the expression of integrin alpha6beta4 and induces cell adhesion and membrane ruffles of nonadherent colon cancer Colo201 cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin beta4 dependent since an antibody for integrin beta4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin alpha6beta4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of alpha6beta4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated alpha6beta4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin alpha6beta4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.