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PubMed2004

Reversion of the Jun-induced oncogenic phenotype by enhanced synthesis of sialosyllactosylceramide (GM3 ganglioside).

In the mouse fibroblast cell line C3H 10T1/2 and the chicken fibroblast cell line DF1, the ganglioside GM3 is the major glycosphingolipid component of the plasma membrane. Expression of the viral oncoprotein Jun (v-Jun) induces transformed cell clones with greatly reduced levels of GM3 and GM3 synthase (lactosylceramide alpha2,3-sialyltransferase) mRNA in both 10T1/2 and DF1 cell cultures. Compared with nontransformed controls, v-Jun transfectants show enhanced ability of anchorage-independent growth, and their growth rates as adherent cells are increased. When the mouse GM3 synthase gene is transfected with the pcDNA vector into v-Jun-transformed 10T1/2 cells, the levels of GM3 synthase and corresponding mRNA are restored to those of control cells. Reexpression of GM3 correlates with a reduced ability of the cells to form colonies in nutrient agar. Similarly, when the newly cloned chicken GM3 synthase gene is transfected into v-Jun-transformed DF1 with the pcDNA vector, the GM3 synthase level is restored to that of control cells, and the ability of the cells to form agar colonies is reduced. The levels of GM3 in the cell also affect membrane microdomains. The complex of GM3 with tetraspanin CD9 and integrin alpha5beta1 inhibits motility and invasiveness. The amounts of this complex are greatly reduced in transformed cells. Expression of GM3 and consequent reversion of the transformed phenotype results in increased levels of that microdomain complex.

Animals↗

PubMed2003

Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering.

We report the isolation and amino acid sequences of six novel dimeric disintegrins from the venoms of Vipera lebetina obtusa (VLO), V. berus (VB), V. ammodytes (VA), Echis ocellatus (EO) and Echis multisquamatus (EMS). Disintegrins VLO4, VB7, VA6 and EO4 displayed the RGD motif and inhibited the adhesion of K562 cells, expressing the integrin alpha5beta1 to immobilized fibronectin. A second group of dimeric disintegrins (VLO5 and EO5) had MLD and VGD motifs in their subunits and blocked the adhesion of the alpha4beta1 integrin to vascular cell adhesion molecule 1 with high selectivity. On the other hand, disintegrin EMS11 inhibited both alpha5beta1 and alpha4beta1 integrins with almost the same degree of specificity. Comparison of the amino acid sequences of the dimeric disintegrins with those of other disintegrins by multiple-sequence alignment and phylogenetic analysis, in conjunction with current biochemical and genetic data, supports the view that the different disintegrin subfamilies evolved from a common ADAM (a disintegrin and metalloproteinase-like) scaffold and that structural diversification occurred through disulphide bond engineering.

Amino Acid Motifs↗

PubMed2005

Integrin alpha4beta1 promotes focal adhesion kinase-independent cell motility via alpha4 cytoplasmic domain-specific activation of c-Src.

The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.

Amino Acid Sequence↗

PubMed2006

Fibronectin induces cell proliferation and inhibits apoptosis in human bronchial epithelial cells: pro-oncogenic effects mediated by PI3-kinase and NF-kappa B.

The extracellular matrix glycoprotein, fibronectin, influences a variety of cellular functions including adhesion, migration, survival, differentiation, and growth. Fibronectin has also been shown to increase the migration and proliferation of human lung carcinoma cells. However, the role of fibronectin in controlling lung airway epithelial cell phenotype remains unknown. Here, we demonstrate that fibronectin stimulates the proliferation of human bronchial epithelial cells (BEAS-2B and 16-HBE). Of note, fibronectin induced the mRNA and protein expression of c-Myc and cyclin D1, while it decreased the expressions of cyclin-dependent kinase inhibitor p21 (WAF-1/CIP1/MDA-6) (p21) and the tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten (PTEN). Fibronectin also stimulated the phosphorylation of the phosphatidylinositol 3 kinase (PI3-K) downstream signal Akt. The inhibitor of PI3-K, Wortmannin, and anti-alpha5beta1 integrin antibodies abrogated the effect of fibronectin on c-Myc, cyclin D1, p21, and PTEN expression. The stimulatory effect of fibronectin was mediated by nuclear factor kappaB (NF-kappaB) since fibronectin induced the expression of the p65 component of NF-kappaB and enhanced NF-kappaB DNA binding. Furthermore, we found that p65 small interfering RNA inhibited the effect of fibronectin on c-Myc, cyclin D1, p21, PTEN expression, and on fibronectin-induced cell proliferation. Finally, we found that fibronectin inhibits apoptosis by reducing DNA fragmentation and inhibiting the activities of caspases 3/7. Taken together, our findings demonstrate that fibronectin stimulates human bronchial epithelial cell growth and inhibits apoptosis through activation of NF-kappaB, which, in turn, increases the expression of c-Myc and cyclin D1 and decreases p21 and PTEN via alpha5beta1 integrin-dependent signals that include PI3-K/Akt. Therefore, alternations in the extracellular matrix composition of the lung, with increased fibronectin, might promote epithelial cell growth and thereby contribute to oncogenesis in certain settings.

Apoptosis↗

PubMed2006

Microfluidic techniques for single-cell protein expression analysis.

BACKGROUND: The analysis of single cells obtained from needle aspirates of tumors is constrained by the need for processing. To this end, we investigated two microfluidic approaches to measure the expression of surface proteins in single cancer cells or in small populations (<50 cells). METHODS: One approach involved indirect fluorescence labeling of cell-surface proteins and channeling of cells in a microfluidic device past a fluorescence detector for signal quantification and analysis. A second approach channeled cells in a microfluidic device over detection zones coated with ligands to surface proteins and measured rates of passage and of retardation based on transient interactions between surface proteins and ligands. RESULTS: The fluorescence device detected expression of integrin alpha5 induced by basic fibroblast growth factor (FGF-2) treatment in MCF-7 cells and that of Her-2/neu in SK-BR-3 cells compared with controls. Experiments measuring passage retardation showed significant differences in passage rates between FGF-2-treated and untreated MCF-7 cells over reaction regions coated with fibronectin and antibody to integrin alpha5beta1 compared with control regions. Blocking peptides reversed the retardation, demonstrating specificity. CONCLUSIONS: Immunofluorescence detection in a microfluidic channel demonstrates the potential for assaying surface protein expression in a few individual cells and will permit the development of future iterations not requiring cell handling. The flow retardation device represents the first application of this technology for assessing cell-surface protein expression in cancer cells and may provide a way for analyzing expression profiles of single cells without preanalytical manipulation.

Antibodies↗

PubMed2006

Fibronectin stimulates non-small cell lung carcinoma cell growth through activation of Akt/mammalian target of rapamycin/S6 kinase and inactivation of LKB1/AMP-activated protein kinase signal pathways.

The Akt/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathway is considered a central regulator of protein synthesis and of cell proliferation, differentiation, and survival. However, the role of the Akt/mTOR/p70S6K pathway in lung carcinoma remains unknown. We previously showed that fibronectin, a matrix glycoprotein highly expressed in tobacco-related lung disease, stimulates non-small cell lung carcinoma (NSCLC) cell growth and survival. Herein, we explore the role of the Akt/mTOR/p70S6K pathway in fibronectin-induced NSCLC cell growth. We found that fibronectin stimulated the phosphorylation of Akt, an upstream inducer of mTOR, and induced the phosphorylation of p70S6K1 and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), two downstream targets of mTOR in NSCLC cells (H1792 and H1838), whereas it inhibited the phosphatase and tensin homologue deleted on chromosome 10, a tumor suppressor protein that antagonizes the phosphatidylinositol 3-kinase/Akt signal. In addition, treatment with fibronectin inhibited the mRNA and protein expression of LKB1 as well as the phosphorylation of AMP-activated protein kinase (AMPKalpha), both known to down-regulate mTOR. Rapamycin, an inhibitor of mTOR, blocked the fibronectin-induced phosphorylation of p70S6K and 4E-BP1. Akt small interfering RNA (siRNA) and an antibody against the fibronectin-binding integrin alpha5beta1 also blocked the p70S6K phosphorylation in response to fibronectin. In contrast, an inhibitor of extracellular signal-regulated kinase 1/2 (PD98095) had no effect on fibronectin-induced phosphorylation of p70S6K. Moreover, the combination of rapamycin and siRNA for Akt blocked fibronectin-induced cell proliferation. Taken together, these observations suggest that fibronectin-induced stimulation of NSCLC cell proliferation requires activation of the Akt/mTOR/p70S6K pathway and is associated with inhibition of LKB1/AMPK signaling.

AMP-Activated Protein Kinase Kinases↗

PubMed2004

Fibronectin stimulates human lung carcinoma cell growth by inducing cyclooxygenase-2 (COX-2) expression.

Tobacco use is the most important risk factor for the development of lung carcinoma. One characteristic shared by tobacco-related lung diseases is altered lung connective tissue content and composition. In particular, tobacco results in increased expression of fibronectin (FN), a matrix glycoprotein implicated in lung development, injury and repair and in tumor cell invasion. We hypothesized that excessive deposition of FN in lung might promote lung carcinoma cell proliferation. Consistent with this hypothesis, we found that FN stimulated human lung carcinoma cell proliferation and diminished apoptosis in vitro, and that this effect was mediated through the integrin alpha5beta1 and associated with upregulation of cyclooxygenase-2 (COX-2) mRNA and protein expression, and increased prostaglandin E2 (PGE2) biosynthesis. The stimulatory effect of FN on COX-2 was blocked by the specific COX-2 inhibitor NS-398 and by inhibitors of protein kinase C (PKC), Calphostin C, and extracellular signal-regulated kinases (Erks), PD98095. Electrophoretic mobility shift assays revealed that FN increased the nuclear binding activity of cyclic AMP response element binding protein (CREB) and CCAAT/enhancer-binding protein (C/EBP), 2 proteins known to play important roles in the regulation of COX-2 promoter activity. Transient transfection assays with wild-type and mutated constructs of the human COX-2 gene promoter revealed that the stimulatory effect of FN was prevented when either the CRE or the NF-IL6 (C/EBP) sites were mutated. Taken together, the results indicate that FN stimulates human lung carcinoma cell proliferation and diminishes apoptosis by inducing COX-2 gene expression and PGE2 biosynthesis. Activation of PKC and Erk and DNA-protein interactions at CRE and NF-IL6 (C/EBP) sites in the COX-2 gene promoter appear to play key roles in this process. This work demonstrates that signaling through specific matrix-binding beta1 integrins (i.e., alpha5beta1) resulting from exaggerated deposition in lung of the matrix glycoprotein fibronectin might promote lung carcinoma cell growth.

Apoptosis↗

PubMed2003

Recombinant expression and characterization of a novel fibronectin isoform expressed in cartilaginous tissues.

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.

Animals↗

PubMed2003

Src kinase has a central role in in vitro cellular internalization of Staphylococcus aureus.

Traditionally recognized as an extracellular pathogen, the Gram-positive bacterium Staphylococcus aureus can also be internalized by a variety of cell types in vitro. Internalization is known to involve binding of the host extracellular protein fibronectin to the bacterium, recognition of the fibronectin-coated bacterium by the fibronectin-binding integrin alpha5beta1 on the host cell surface, and integrin-mediated internalization. Here we examine elements of mammalian cell signalling pathways involved in S. aureus internalization. The mouse fibroblast cell line GD25, in which the gene encoding the beta1 integrin subunit is inactivated, has been complemented with a beta1 integrin cDNA encoding a tyrosine (Y) to phenylalanine (F) mutation in each of the two beta1 integrin intracellular NPXY motifs. This cell line, GD25beta1 A Y783/795F, is defective in migration on fibronectin coated surfaces and intracellular signalling activities involving the tyrosine kinase Src. GD25beta1 A Y783/795F cells have a decreased ability to internalize S. aureus compared to GD25beta1 A cells expressing wild-type beta1 integrins. Furthermore, using mouse embryo fibroblasts in which different members of the Src family kinases are genetically inactivated, we demonstrate that optimal internalization is dependent on expression of Src kinase. Interferon, which has been implicated in repression of the effects of the viral homologue of Src inhibits internalization of S. aureus indicating that internalization may be blocked by inhibitors of Src kinase function. We then demonstrate that Src family kinase specific inhibitors effectively block S. aureus internalization into HeLa cells leading to the conclusion that a function unique to Src is required for optimal internalization of S. aureus in vitro.

Animals↗

PubMed2005

Integrins adhesion molecules and some of their ligands in laryngeal cancer.

In this review data regarding the regular expression and distribution of integrins adhesion molecules as well as some of their ligands in cases of squamous cell carcinoma of the larynx (LSCC) are discussed. Cancer of the larynx is one of the most common malignancies of the head and neck. Upon neoplastic transformation several alterations of the integrins adhesion molecules and extracellular matrix (ECM) components are observed, i.e. the loss of the normal architecture especially depolarization of some integrins, like alpha6beta4, alpha3beta1, alpha2beta1 as well as the presence of alpha6beta1 integrin in neoplastic epithelium. Furthermore, the upregulation of alpha5beta1 integrin is a negative prognostic factor. In cases of laryngeal cancer, disorganization of the basal membrane components (laminins 1 and 5, type IV collagen, tenascin, fibronectin isoforms) which represent integrins ligands, is found. This is due to partial or complete destruction of the basal membrane, thus, permitting extravasation and migration of cancer cells. These phenomena are observed in a greater degree in tumors showing poor prognosis. It seems that damage of these adhesion complexes is important in the neoplastic process. So, further studies are necessary to establish the importance of these markers to early larynx cancer diagnosis and reach the decision on appropriate therapeutic strategies in the management of those patients.

Animals↗

PubMed1996

Autocrine motility factor signals integrin-mediated metastatic melanoma cell adhesion and invasion.

The binding of autocrine motility factor (AMF) to its cell surface receptor, gp78, stimulates tumor cell motility. In this report, we provide evidence that stimulation of gp78 by either AMF or a monoclonal antibody to gp78 (3F3A) increases adhesion and spreading of metastatic murine melanoma (B16a) cells on fibronectin. This gp78-regulated increase is mediated by up-regulation of surface alphaIIbbeta3++ and alpha5beta1 integrin receptors. In addition, AMF treatment of B16a cells increased translocation of alphaIIbbeta3 and alpha5beta1 from the cytoplasm to the cell surface. However, alphaIIbbeta3 and alpha5beta1 demonstrate separate and unique staining patterns at the surface of B16a cells in response to stimulation of gp78. Furthermore, stimulation of B16a cells with AMF increased their invasion through Matrigel. This stimulated invasion was inhibited by antibodies to alphaIIbbeta3 but not by antibodies to alpha5beta1. The increased integrin surface expression and function in response to AMF was blocked by N-benzyl-N-hydroxy-5-phenylpentanamide, an inhibitor of 12-lipoxygenase, and calphostin C, an inhibitor of protein kinase C. The results demonstrate that AMF stimulates integrin-mediated B16a cell adhesion, spreading, and invasion, and these events are regulated by a signaling pathway involving 12-lipoxygenases and protein kinase C.

Animals↗

PubMed2004

ILK is required for the assembly of matrix-forming adhesions and capillary morphogenesis in endothelial cells.

Integrins play a key role in regulating endothelial cell survival, migration and differentiated function during angiogenic blood-vessel remodeling. Integrin-linked kinase (ILK) is a multidomain protein that interacts with the cytoplasmic tail of integrin beta subunits and is thought to participate in integrin-mediated signal transduction. We report here that attenuation of ILK expression in cultured bovine aortic endothelial cells by RNA interference had marked effects on surface distribution of alpha5beta1 integrin and the organization of cell-matrix adhesions characterized by the disappearance of fibrillar (3D-like) adhesions that are rich in alpha5beta1 and paxillin, and associated fibrillar fibronectin matrix. This defect was not caused by a decrease in fibronectin mRNA levels or by intracellular retention of the protein. Adhesion to surface-adsorbed matrix proteins based on beta1 and beta3 integrin was enhanced following ILK depletion, whereas cell spreading, migration and multilayer alignment into capillary-like structures on Matrigel were impaired. We conclude that ILK is an important regulator of the endothelial phenotype and vascular network formation by directing the assembly and/or maturation of alpha5beta1-competent matrix-forming adhesions.

Actins↗

PubMed2001

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

BACKGROUND: It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. RESULTS: The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. CONCLUSIONS: We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

3T3 Cells↗

PubMed2001

Binding properties, cell delivery, and gene transfer of adenoviral penton base displaying bacteriophage.

The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalisation, properties that can be exploited to reengineer prokaryotic viruses for the infection of mammalian cells. We report that filamentous phage displaying either the full-length penton base gene or a central region of 107 amino acids on their surface were able to bind, internalise, and transduce mammalian cells expressing integrin receptors. Both phage bound alphavbeta3, alphavbeta5, alpha3beta1, and alpha5beta1 integrin subtypes. Cell-binding was shown by electron microscopy; internalisation was investigated by immunofluorescence and confirmed by micropanning. As it has been described for adenovirus, pharmacologic disruption of phosphoinositide-30H kinase, but not of myosin light-chain kinase, inhibited phage internalisation. Recombinant phage encoding an eukaryotic expression cassette was able to mediate gene expression in mammalian cells. Taken together, these data open insights for the exploit of recombinant phage for integrin-targeted gene delivery.

Adenoviridae↗

PubMed1998

Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts.

Integrins, which connect the cytoskeleton to the extracellular matrix and mediate a variety of signaling cascades, may transduce mechanical stimuli into biochemical signals. We studied integrin- and matrix-dependent activation of extracellular signal-regulated kinase (ERK2), c-Jun NH2-terminal kinase (JNK1), and p38 in response to 4% static biaxial stretch in rat cardiac fibroblasts. ERK2 and JNK1, but not p38, were rapidly activated by stretch when the fibroblasts were allowed to synthesize their own matrices. When the cells were limited to specific matrix substrates, ERK2 and JNK1 were differentially activated: ERK2 was only activated when the cells were plated on fibronectin, while JNK1 was activated when the cells were plated on fibronectin, vitronectin, or laminin. Plating cells on collagen before stretching did not activate either kinase. Adhesion to all matrices was integrin-dependent because it could be blocked by inhibitors of specific integrins. ERK2 activation could be blocked with a combination of anti-alpha4 and -alpha5 antibodies and an arginine-glycine-aspartic acid (RGD) peptide, while the antibodies or peptide used separately failed to block ERK2 activation. This result suggests that at least two integrins, alpha4beta1 and an RGD-directed, non-alpha5beta1 integrin, activate ERK2 in response to mechanical stimulation. Activation of JNK1 could not be blocked with the inhibitors, suggesting that an RGD-independent integrin or integrins other than alpha4beta1 can activate JNK1 in cells adherent to fibronectin. This study demonstrates that integrins act as mechanotransducers, providing insight into potential mechanisms for in vivo responses to mechanical stimuli.

Animals↗

PubMedDate not supplied

Insulin receptor regulation of cell surface integrins: a possible mechanism contributing to the development of diabetic complications.

Insulin plays a central role in regulating cellular growth in addition to its classic effects to regulate fuel metabolism. In a previous study, we have identified a patient who was homozygous for a deletion of the insulin receptor gene. In our current investigation, we used cultured skin fibroblasts from this patient as a model system in which to investigate the mechanisms whereby insulin regulates cellular growth in vitro. After cell division, skin fibroblasts from normal individuals migrate on the tissue culture plate and appear to be distributed randomly over the surface of the plate. In contrast, the patient's cells grew in clumps. Furthermore, the patient's fibroblasts exhibited a marked increase in the expression of several integrin subunits, especially the alpha5- and beta1-subunits that comprise the fibronectin receptor. Because the cellular growth pattern was restored to normal when cells were cultivated in the presence of blocking antibodies directed against either alpha5- or beta1-integrin subunits, we infer that increased expression of alpha5beta1-integrin may be the cause of the observed abnormality in the growth of the patient's cells in vitro. Furthermore, insulin stimulation led to downregulation of the levels of the alpha5- and beta1-integrin subunits in normal human fibroblasts but not in the patient's cells that lacked insulin receptors. Taken together, these data suggest that insulin's ability to regulate the expression of cell surface integrins may contribute to the mechanisms whereby insulin regulates cell growth. In light of the important role of integrins in mediating interactions between cells and the basement membrane, we suggest that dysregulation of integrin expression might contribute to the abnormalities in the structure of the basement membranes associated with the chronic microvascular complications of diabetes.

Cells, Cultured↗

PubMed2003

A novel mode for integrin-mediated signaling: tethering is required for phosphorylation of FAK Y397.

The common model for integrin mediated signaling is based on integrin clustering and the potential for that clustering to recruit signaling molecules including FAK and src. The clustering model for transmembrane signaling originated with the analysis of the EGF receptor signaling and remains the predominant model. The roles for substrate-bound ligand and ligand occupancy in integrin-mediated signaling are less clear. A kinetic model was established using HT1080 cells in which there was a linear relationship between the strength of adhesion, the proportion of alpha5beta1 integrin that could be chemically cross-linked, and the number of receptor-ligand bonds. This graded signal produced a similarly graded response measured by the level of specific phosphorylation of FAK Y397. FAK Y397 phosphorylation could also be induced by antibody bound to the substrate. In contrast, clustering of alpha5beta1 on suspended cells with either antibody to beta1 or by clustering of soluble ligand bound to alpha5beta1 induced the phosphorylation of FAK Y861 but not Y397. There were no differences in signaling when activating antibodies were compared with blocking antibodies, presence or absence of ligand. Only tethering of alpha5beta1 to the substrate was required for induction of FAK Y397 phosphorylation.

Cell Adhesion↗

PubMed2005

Identification of a novel cell-adhesive protein spatiotemporally expressed in the basement membrane of mouse developing hair follicle.

We used PCR-based cDNA subtraction to screen for genes up-regulated during mouse hair morphogenesis. One gene selected was predominantly expressed at the tip of developing hair follicles and encoded a protein characterized by the presence of twelve tandem repeats of approximately 120 amino acids and a novel N-terminal domain containing an Arg-Gly-Asp cell-adhesive motif. Immunohistochemistry demonstrated that the protein encoded by this gene, named QBRICK, was localized at the basement membrane zone of embryonic epidermis and hair follicles, in which it was more enriched at the tip rather than the stalk region. Cell adhesion assays showed that QBRICK was active in mediating cell-substratum adhesion through integrins containing alphav or alpha8 chain, but not integrin alpha5beta1. Immunohistochemistry showed that QBRICK colocalized with alphav-containing integrins in the interfollicular region, but with the alpha8-containing integrin at the tip region of developing hair follicles. These results, together, indicate that QBRICK is an adhesive ligand of basement membrane distinctively recognized by cells in the embryonic skin and hair follicles through different types of integrins directed to the Arg-Gly-Asp motif.

Amino Acid Motifs↗