High Concentrations Of Fibronectin Research Articles

Inhibition of glioblastoma dispersal by the MEK inhibitor PD0325901

BackgroundDispersal of glioblastoma (GBM) cells leads to recurrence and poor prognosis. Accordingly, molecular pathways involved in dispersal are potential therapeutic targets. The mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway is commonly dysregulated in GBM, and targeting this pathway with MEK inhibitors has proven effective in controlling tumor growth. Since this pathway also regulates ECM remodeling and actin organization − processes crucial to cell adhesion, substrate attachment, and cell motility – the aim of this study was to determine whether inhibiting this pathway could also impede dispersal.MethodsA variety of methods were used to quantify the effects of the MEK inhibitor, PD0325901, on potential regulators of dispersal. Cohesion, stiffness and viscosity were quantified using a method based on ellipsoid relaxation after removal of a deforming external force. Attachment strength, cell motility, spheroid dispersal velocity, and 3D growth rate were quantified using previously described methods.ResultsWe show that PD0325901 significantly increases aggregate cohesion, stiffness, and viscosity but only when tumor cells have access to high concentrations of fibronectin. Treatment also results in reorganization of actin from cortical into stress fibers, in both 2D and 3D culture. Moreover, drug treatment localized pFAK at sites of cell-substratum adhesion. Collectively, these changes resulted in increased strength of substrate attachment and decreased motility, a decrease in aggregate dispersal velocity, and in a marked decrease in growth rate of both 2D and 3D cultures.ConclusionsInhibition of the MAPK/ERK pathway by PD0325901 may be an effective therapy for reducing dispersal and growth of GBM cells.

Abstract 5057: Mena at the nexus of chemotaxis and haptotaxis during tumor progression

Abstract Directed-cell migration is key step in the metastatic cascade, and multiple cues can promote local invasion. The most well studied mode of cell motility for metastasizing tumor cells is chemotaxis, the directional movement of cells attracted to a source of soluble cues. In contrast, haptotaxis, migration of cells on gradients of substrate-bound factors, remains poorly understood. Mena, an actin regulatory protein, plays an important role in cell motility and is upregulated in various cancers, driving migration in response to chemotactic and haptotactic cues. We have recently shown expression of the pro-metastatic isoform of Mena, MenaINV, increases sensitivity to growth factors (EGF, HGF and IGF) via dysregulation of the phosphatase PTP1B, as well as high concentrations of fibronectin (FN) via its interaction with the integrin α5β1. We hypothesized that MenaINV may also promote crosstalk between these two pathways to drive metastasis. First, we show that MenaINV-driven haptotaxis on FN gradients and FN-driven invasion requires crosstalk between α5β1 and EGFR. Indeed, we show evidence that MenaINV expression is associated high phosphorylation of EGFR in high FN environments in vitro and in human breast cancer databases. Second, we find that MenaINV promotes synergy between EGF and FN in vitro and in vivo, leading to hyper-invasive phenotypes that drive more invasion than which each cue alone. Finally, we show that MenaINV-driven haptotaxis, ECM reorganization and 3D invasion requires RCP-dependent recycling of α5β1 and EGFR. Together, our data demonstrate that MenaINV is a shared component of the machinery that mediates both haptotactic responses to FN and chemotactic responses to EGF, two pathways that promote tumor progression. Citation Format: Madeleine J. Oudin, Miles A. Miller, Tatsiana Kosciuk, Joelle Klazen, Alisha Lussiez, Douglas Lauffenburger, James E. Bear, Frank B. Gertler. Mena at the nexus of chemotaxis and haptotaxis during tumor progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5057.

In This Issue

In This Issue

Tumor Cell-Driven Extracellular Matrix Remodeling Drives Haptotaxis during Metastatic Progression.

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here, we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and MENA, an actin regulator, and involves increases in focal complex signaling and tumor cell-mediated extracellular matrix (ECM) remodeling. Compared with MENA, higher levels of the prometastatic MENA(INV) isoform associate with α5, which enables 3-D haptotaxis of tumor cells toward the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MENA(INV) and FN levels were correlated in two breast cancer cohorts, and high levels of MENA(INV) were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM-guided directional migration. Here, we provide new insight into how tumor cell:ECM interactions generate signals and structures that promote directed tumor cell migration, a critical component of metastasis. Our results identify a tumor cell-intrinsic mechanism driven by the actin regulatory protein MENA that promotes ECM remodeling and haptotaxis along FN gradients. Cancer Discov; 6(5); 516-31. ©2016 AACR.See related commentary by Santiago-Medina and Yang, p. 474This article is highlighted in the In This Issue feature, p. 461.

Lamellipodia are crucial for haptotactic sensing and response.

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis - differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Abstract 437: Mena at the nexus of chemotaxis and haptotaxis during tumor progression

Abstract The most well studied mode of cell motility for metastasizing tumor cells is chemotaxis, the directional movement of cells attracted to a source of soluble cues. In contrast, haptotaxis, migration of cells on gradients of substrate-bound factors, remains poorly understood. Due to the high expression of extracellular matrix (ECM) proteins such as collagen and fibronectin (FN) in metastatic tumors, it is possible that haptotaxis along ECM gradients could also be driving invasion. Mena, an actin regulatory protein, plays an important role in cell motility and is upregulated in various cancers, where Mena, and in particular the MenaINV isoform, potentiates chemotactic and invasive responses to EGF. All Mena isoforms bind directly to the α5 subunit of the α5β1 integrin, a FN receptor. We hypothesized that the pro-metastatic effect of MenaINV arises from increases in tumor cell responses to specific chemotactic and haptotactic cues, including EGF and FN. To study haptotaxis, MDA-MB231 breast adenocarcinoma cells were plated on gradients of fluorescently-labeled FN in 2D or in 3D collagen gels, generated within a PDMS microfluidic device. Wild type cells expressing low levels of endogenous Mena failed to respond to a FN gradient, but migrated equally well in all directions irrespective of the FN gradient. Surprisingly, ectopic expression of Mena in these cells conferred the ability to respond to FN gradients, with cells exhibiting haptotactic response towards higher concentrations of FN in 2D and 3D. Interestingly, cells expressing MenaINV were able to haptotax towards FN concentrations 4 times higher then cells expressing Mena, while reorganizing and recruiting both collagen and FN surrounding them. MenaINV-dependent haptotaxis was ablated by deletion of the α5 binding site within Mena or treatment with an α5 function blocking antibody, but was unaffected by inhibition of another FN receptor, αVβ3. Using intravital imaging of mouse mammary tumors, we visualized haptotaxis of MenaINV-expressing cells towards gradients of fluorescently labelled FN released locally from implanted microsccale devices. Tumor cells expressing MenaINV lacking the α5 binding site did not haptotax, displayed decreased motility and metastasis to the lung. This was also accompanied by a striking change in the organization of the ECM surrounding the tumor. While investigating the mechanism of haptotaxis, we found that MenaINV-driven invasive responses to FN were also dependent on EGFR signalling and mediated by the phosphatase PTP1B. Finally, using a combination of in vitro and in vivo invasion assays, we show that MenaINV expression promoted synergistic invasive responses to combinations of EGF and FN and also caused increased phosphorylation of Paxilin and FAK at focal adhesions. Together, our data demonstrate that MenaINV is a shared component of the machinery that mediates both haptotactic responses to FN and chemotactic responses to EGF, two pathways that promote tumor progression. Citation Format: Madeleine J. Oudin, Oliver Jonas, Tatiana Kosciuk, Liliane C. Broye, Jeff Wyckoff, Miles A. Miller, Alisha Lussiez, Sreeja Asokan, Robert Langer, Douglas Lauffenburger, James E. Bear, Frank B. Gertler. Mena at the nexus of chemotaxis and haptotaxis during tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 437. doi:10.1158/1538-7445.AM2015-437

The ultrastructure of fibronectin fibers pulled from a protein monolayer at the air-liquid interface and the mechanism of the sheet-to-fiber transition

Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ultrastructural characterization is then contrasted with previous FRET studies that characterized the molecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio- or biomimetic polymers.

Two-dimensional motility of a macrophage cell line on microcontact-printed fibronectin.

The ability of macrophages to migrate to sites of infection and inflammation is critical for their role in the innate immune response. Macrophage cell lines have made it possible to study the roles of individual proteins responsible for migration using molecular biology, but it has not been possible to reliably elicit the motility of macrophage cell lines in two dimensions. In the past, measurements of the motility of macrophage cell lines have been largely limited to transwell assays which provide limited quantitative information on motility and limited ability to visualize cell morphology. We used microcontact printing to create polydimethylsiloxane (PDMS) surfaces functionalized with fibronectin that otherwise support little macrophage adhesion. We used these surfaces to measure macrophage migration in two dimensions and found that these cells migrate efficiently in a uniform field of colony-stimulating factor-1, CSF-1. Knockdown of Cdc42 led to a nonstatistically significant reduction in motility, whereas chemical inhibition of PI3K activity led to a complete loss of motility. Inhibition of the RhoA kinase, ROCK, did not abolish the motility of these cells but caused a quantitative change in motility, reducing motility significantly on high concentrations of fibronectin but not on low concentrations. This study illustrates the importance of studying cell motility on well controlled materials to better understand the exact roles of specific proteins on cell migration. © 2014 Wiley Periodicals, Inc.

Raised Vaginal Fluid Fibronectin Level Indicates Premature Rupture of Membrane

Background: Premature rupture of membrane (PROM) is one of the common complications of pregnancy that has major impact on fetal and neonatal outcome. It is the commonest clinical event where a normal pregnancy becomes suddenly a high-risk one for mother and fetus or neonate. Objective: The study was undertaken to investigate whether raised fibronectin level in vaginal fluid may indicate premature rupture of membrane. Materials and Methods: This cross sectional study was conducted in the department of Obstetrics and Gynecology in Sir Salimullah Medical College & Mitford Hospital, Dhaka during the period of January 2006 to December 2007. A total of 114 pregnant women with gestational age 28th week up to 40th week were included. Sixty were PROM (Group I) and 54 were non-PROM (Group II) subjects. Fibronectin in vaginal fluid was measured by an immunochemical reaction by nephelometer. Statistical analysis was done by SPSS version 10.0. Results: The PROM patients had significantly higher concentration of fibronectin (225.77 ± 115.18 ng/mL) compared to that in non-PROM subjects (8.04 ± 16.17 ng/mL) (p < 0.001). Conclusion: It can be concluded that in cases of unequivocal rupture or intactness of the membranes, the result of the fibronectin test corresponds well with the clinical situation. So fibronectin is a sensitive test for detection of amniotic fluid in the vagina. DOI: http://dx.doi.org/10.3329/jemc.v2i2.12841 J Enam Med Col 2012; 2(2): 74-79

Improved interaction of osteoblast-like cells with apatite–nanodiamond coatings depends on fibronectin

New apatite (AP)/nanodiamond (ND) coating has been developed to improve physical and biological properties of stainless steel (SS) versus single AP coating. Homogeneously electrodeposited AP-ND layer demonstrates increased mechanical strength, interlayer cohesion and ductility. In the absence of serum, osteoblast-like MG63 cells attach well but poorly spread on both AP and AP-ND substrata. Pre-adsorption with serum or fibronectin (FN) improves the cellular interaction-an effect that is better pronounced on the AP-ND coating. In single protein adsorption study fluorescein isothiocyanate-labeled FN (FITC-FN) shows enhanced deposition on the AP-ND layer consistent with the significantly improved cell adhesion, spreading and focal adhesions formation (in comparison to SS and AP), particularly at low FN adsorption concentrations (1μg/ml). Higher FN concentrations (20μg/ml) abolish this difference suggesting that the promoted cellular interaction of serum (where FN is low) is caused by the greater affinity for FN. Moreover, it is found that MG63 cells tend to rearrange both adsorbed and secreted FN on the AP-ND layer suggesting facilitated FN matrix formation.

Syndecan-1 regulates cell migration and fibronectin fibril assembly

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.

Fibronectin stimulates Escherichia coli phagocytosis by microglial cells

Microglia express Toll-like receptors (TLRs) that recognize invading pathogens as well as endogenous proteins such as fibronectin under nonphysiological conditions. Here, we demonstrated that fibronectin stimulates murine microglia in culture in a dose-dependent manner: microglial cells secreted proinflammatory cytokines and chemokines and increased phagocytosis of Escherichia coli DH5alpha and E. coli K1 strains. Low levels of fibronectin exerted a synergistic effect on the release of proinflammatory compounds by microglia co-stimulated with agonists for TLR1/2 (Pam(3)CSK(4)) or TLR9 (CpG DNA), but not in combination with the TLR4 agonist lipopolysaccharide (LPS). Phagocytosis of bacterial strains was moderately enhanced when microglia was co-stimulated with high concentrations of fibronectin and one pathogen-derived TLR agonist. In conclusion, fibronectin increased proinflammatory and phagocytotic functions in microglia and partially synergized with microbial TLR agonists.

The changes of extracellular matrix molecules in the resected medial rectus muscle of patients with concomitant exotropia

In order to have a better understanding about the role of the extracellular matrix molecules in the intermittent and constant exotropia, we measured the amounts of fibronectin and proteoglycan in the resected medial rectus muscles of patients with concomitant exotropia. Thirty-one exotropic patients (including intermittent exotropia 17 cases and constant exotropia 14 cases) and 21 normal were chosen. In exotropic group, there were 7 cases with positive family history. Tissues of the medial rectus muscles were obtained from patients with concomitant exotropia during resection surgery and the specimen of medial rectus muscle from 21 normal persons was as control. All the tissues were weighted and then pulverized to obtain supernatant for enzyme linked immunosorbent assay (ELISA). The total amounts of fibronectin and proteoglycan were measured. The correlation of fibronectin and proteoglycan with age, gender, positive family history in different groups of exotropia was analyzed. The amount of fibronectin in the resected medial rectus muscle of patients with concomitant exotropia was significantly lower than that of normal individual (P < 0.01), and the difference of proteoglycan amount between patients and the normal control was not significant (P > 0.05). Patients with intermittent exotropia showed significantly higher concentration of fibronectin than those with constant exotropia (P < 0.01), and the amount of proteoglycan had no significant difference between two groups (P > 0.05). The amounts of proteoglycan decreased significantly with the advance of age (r = -0.8712, P < 0.01), while the amounts of fibronectin had no correction with age (r = -0.1718, P > 0.05). Neither gender nor positive family was correlated with the amounts of proteoglycan and fibronectin (P > 0.05). The change of fibronectin may be related to the concomitant exotropia and the development from intermittent to constant exotropia. An attention should be paid to the roles of fibronectin in the development of strabismus.

Differential effects of matrix and growth factors on endothelial and fibroblast motility: Application of a modified cell migration assay

Cell migration is crucial in virtually every biological process and strongly depends on the nature of the surrounding matrix. An assay that enables real-time studies on the effects of defined matrix components and growth factors on cell migration is not available. We have set up a novel, quantitative migration assay, which enables unharmed cells to migrate along a defined matrix. Here, we used this so-called barrier-assay to define the contribution of fibronectin (FN) and Collagen-I (Col-I) to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and lysophosphatidic acid (LPA)-induced cell migration of endothelial cells (EC) and fibroblasts. In EC, both FN and Col-I stimulated migration, but FN-induced motility was random, while net movement was inhibited. Addition of bFGF and VEGF overcame the effect of FN, with VEGF causing directional movement. In contrast, in 3T3 fibroblasts, FN stimulated motility and this effect was enhanced by bFGF. This motility was more efficient and morphologically completely different compared to LPA stimulation. Strikingly, directional migration of EC was not paralleled by higher amounts of stable microtubules (MT) or an increased reorientation of the microtubule-organizing centre (MTOC). For EC, the FN effect appeared concentration dependent; high FN was able to induce migration, while for fibroblasts both low and high concentrations of FN induced motility. Besides showing distinct responses of the different cells to the same factors, these results address contradictive reports on FN and show that the interplay between matrix components and growth factors determines both pattern and regulation of cell migration. J. Cell. Biochem. 99: 1536-1552, 2006. (c) 2006 Wiley-Liss, Inc.

Lower expression of the α2,3-sialylated fibronectin glycoform and appearance of the asialo-fibronectin glycoform are associated with high concentrations of fibronectin in human seminal plasma with abnormal semen parameters

Sialic acid isoforms expressed on glycoconjugates are known to be involved in different biological functions. The aim of this study was to compare alpha2,3- and/or alpha2,6-linked sialic acid expression on fibronectin in the seminal plasma of male partners from infertile couples. The data obtained were analyzed in relation to normal and abnormal semen parameters, as well as to fibronectin concentrations. Fibronectin concentrations were determined by ELISA using a specific monoclonal antibody. The relative degree of sialic acid linked to fibronectin glycans either by alpha2,3 or alpha2,6 isomeric linkage was estimated by lectin-fibronectin ELISA utilizing lectins from Maackia amurensis and Sambucus nigra, respectively. High seminal fibronectin concentration was more frequently associated with abnormal semen parameters. The glycans of seminal plasma fibronectin, in contrast to blood plasma fibronectin, contained sialic acid more frequently attached by alpha2,3 than by alpha2,6 isomeric linkage. The relative amounts of both alpha2,3- and alpha2,6-linked sialic acid fibronectin isoforms were lower in the seminal plasma of men suspected of infertility. Seminal fibronectin showed an oncofetal type of sialylation and the distribution of hypo- and asialylated fibronectin glycoforms were associated with abnormal semen parameters and with high concentrations of fibronectin.

Bacteria-associated fibronectin does not enhance Chlamydia trachomatis infectivity in vitro

The molecules responsible for mediating attachment of pathogenic bacteria to their host cells are essential determinants of the pathogen's success. Staphylococci, streptococci, and gonococci are known to utilize fibronectin to mediate adhesion and/or entry into eukaryotic cells. It has been shown that Chlamydia trachomatis binds fibronectin via a heparan sulfate-lyase sensitive molecule on the surface of the bacteria. Because heparan sulfate-lyase treated Chlamydia are markedly reduced in infectivity, we hypothesized that fibronectin might be acting as an intermediate to form a molecular bridge between heparan sulfate-like molecules on the surface of Chlamydia and integrins on the host cell. To test this question, fibronectin-deficient C. trachomatis were used in a series of infectivity assays whereby exogenous fibronectin was added to the inoculum. At concentrations up to 100 ug/ml fibronectin did not modulate C. trachomatis infectivity. However, at high fibronectin concentrations (1 mg/ml) bacterial infectivity was reduced. These data suggest that, unlike several other microbial pathogens, C. trachomatis does not utilize fibronectin to mediate infectivity in vitro.

Differential Effects of Fibronectin Fragment on Proteoglycan Metabolism by Intervertebral Disc Cells: A Comparison With Articular Chondrocytes

This in vitro study used the alginate bead culture system to probe for differences in the effects of fibronectin fragment on cell proliferation and proteoglycan metabolism by different populations of intervertebral disc cells and articular chondrocytes. To compare the effects of fibronectin fragment on cell proliferation, and proteoglycan synthesis and degradation by cells from the nucleus pulposus, the anulus fibrosus, and articular cartilage. In articular cartilage, the administration of fibronectin fragment stimulates cartilage degeneration. Fibronectin fragment levels were increased in human intervertebral discs with increased disc degeneration. Fibronectin fragment injected into the central region of the rabbit intervertebral disc induced a progressive degeneration of that disc. Bovine tails and metacarpophalangeal joints from 14- to 18-month-old animals were used. Alginate beads containing cells isolated from intervertebral discs and articular cartilage were cultured with (1-100 nmol/L) or without (control) fibronectin fragment in the presence of 10% fetal bovine serum. In these cultures, deoxyribonucleic acid and proteoglycan contents, as well as the rate of proteoglycan synthesis were determined. Proteoglycan degradation was measured in cultures with or without 10 nmol/L fibronectin fragment. In articular chondrocytes, fibronectin fragment strongly suppressed proteoglycan synthesis and stimulated proteoglycan degradation; the total proteoglycan content was diminished in a dose-dependent manner. Compared to articular chondrocytes, nucleus pulposus cells responded to fibronectin fragments in a similar, although less pronounced manner. On the other hand, anulus fibrosus cells treated with fibronectin fragment did not show any significant effects on proteoglycan degradation. A slight but statistically significant up-regulation of proteoglycan synthesis was observed at 10 nmol/L fibronectin fragment in outer anulus fibrosus cells. However, total proteoglycan content was decreased significantly at high concentrations of fibronectin fragment. Fibronectin fragment has different effects on cell proliferation, proteoglycan synthesis, degradation, and accumulation by articular chondrocytes and intervertebral disc cells. The different effects of fibronectin fragment in those different cell types suggest metabolic differences between these cells, and may further suggest differences in pathways of fibronectin fragment signaling as well as a differential need of these cells to be involved in tissue remodeling in which both anabolic and catabolic pathways might be altered.

Integrin-dependent PLC-γ1 phosphorylation mediates fibronectin-dependent adhesion

Although integrin engagement initiates signaling events such as focal-adhesion kinase (FAK) and Src kinase activation, the role of phosphoinositide turnover in cell adhesion is less clear. To assess PLC-gamma1 function in this process, Plcg1(-/-) fibroblasts (Null) were compared with the same fibroblasts in which PLC-gamma1 was re-expressed (Null+). Following plating on fibronectin, Null cells displayed a significantly impaired rate of adhesion compared with Null+ cells. This defect was detected at low concentrations of fibronectin; at high fibronectin concentrations, the Null and Null+ cells displayed equivalent adhesion characteristics. The differences were not due to PLC-gamma1-dependent changes in integrin subunit expression, nor was integrin receptor clustering impaired with the absence of PLC-gamma1. Experiments with site-specific antibodies and PLC-gamma1 mutants showed that fibronectin selectively increased phosphorylation of Tyr783 and that mutagenesis of this residue, but not Tyr771 or Tyr1253, abrogated fibronectin-dependent adhesion. The SH2 domains of PLC-gamma1 were also required for maximal adhesion on fibronectin. Adhesion to fibronectin induced PLC-gamma1 tyrosine phosphorylation that was inhibited by a Src-kinase inhibitor, but not an epidermal-growth-factor-receptor kinase inhibitor. Moreover, in cells null for Src family members, but not in cells null for FAK family members, integrin-dependent PLC-gamma1 tyrosine phosphorylation was greatly reduced. Finally, the data demonstrated that PLC-gamma1 co-immunoprecipitated with Src following fibronectin-induced integrin activation, and this association did not depend on FAK expression.

The GTPase Rap1 Regulates Phorbol 12-Myristate 13-Acetate-stimulated but Not Ligand-induced β1Integrin-dependent Leukocyte Adhesion

Leukocyte migration from bloodstream to tissue requires rapid, coordinated regulation of integrin-dependent adhesion and de-adhesion. In a previous study we demonstrated that inhibition of protein geranylgeranylation inhibited phorbol ester-stimulated avidity modulation of beta(1) integrin in several leukocyte cell lines. Both RhoA and Rap1 require post-translational modification by geranylgeranylation for full function. In this report we identify Rap1, not RhoA, as a critical geranylgeranylated protein mediating phorbol ester-stimulated beta(1) and beta(2) integrin-dependent adhesion of Jurkat cells. Overexpression of the Rap1-specific GTPase-activating protein, SPA-1, or inactivated form of Rap1 (N17Rap1) blocked phorbol ester-stimulated adhesion of Jurkat cells to fibronectin (alpha(4)beta(1)) and ICAM-1 (alpha(L)beta(2)). With high concentrations of fibronectin as ligand, Jurkat cells adhered spontaneously without phorbol ester stimulation. Unlike the phorbol ester-stimulated adhesion, adhesion induced by high density ligand was not dependent upon Rap1 activation or actin cytoskeleton reorganization. Thus, the "inside-out" adhesion signal induced by phorbol ester and the "outside-in" signal induced by high density ligand involve different pathways.

Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases.

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha 5 beta 1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.