Integrin-dependent Cell Migration Research Articles

Integrin activating molecule-talin1 promotes skin fibrosis in systemic sclerosis.

Integrin-dependent cell adhesion and migration play important roles in systemic sclerosis (SSc). The roles of integrin activating molecules including talins and kindlins, however, are unclear in SSc. We aimed to explore the function of integrin activating molecules in SSc. Transcriptome analysis of skin datasets of SSc patients was performed to explore the function of integrin-activating molecules including talin1, talin2, kindlin1, kindlin2 and kindlin3 in SSc. Expression of talin1 in skin tissue was assessed by multiplex immunohistochemistry staining. Levels of talin1 in serum were determined by ELISA. The effects of talin1 inhibition were analyzed in human dermal fibroblasts by real-time PCR, western blot and flow cytometry. We identified that talin1 appeared to be the primary integrin activating molecule involved in skin fibrosis of SSc. Talin1 was significantly upregulated and positively correlates with the modified Rodnan skin thickness score (mRSS) and the expression of pro-fibrotic biomarkers in the skin lesions of SSc patients. Further analyses revealed that talin1 is predominantly expressed in the dermal fibroblasts of SSc skin and promotes fibroblast activation and collagen production. Additionally, talin1 primarily exerts its effects through integrin β1 and β5 in SSc. Overexpressed talin1 is participated in skin fibrosis of SSc, and talin1 appears to be a potential new therapeutic target for SSc.

Cancer Biology of the EndoplasmicReticulum Lectin Chaperones Calreticulin, Calnexin and PDIA3/ERp57.

The lectin chaperones calreticulin (CALR) and calnexin (CANX), together with their co-chaperone PDIA3, are increasingly implicated in studies of human cancers in roles that extend beyond their primary function as quality control facilitators of protein folding within the endoplasmic reticulum (ER). Led by the discovery that cell surface CALR functions as an immunogen that promotes anti-tumour immunity, studies have now expanded to include their potential uses as prognostic markers for cancers, and in regulation of oncogenic signaling that regulate such diverse processes including integrin-dependent cell adhesion and migration, proliferation, cell death and chemotherapeutic resistance. The diversity stems from the increasing recognition that these proteins have an equally diverse spectrum of subcellular and extracellular localization, and which are aberrantly expressed in tumour cells. This review describes key foundational discoveries and highlight recent findings that further our understanding of the plethora of activities mediated by CALR, CANX and PDIA3.

SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPαΔCYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPαΔCYT>LDLR−/−) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.

Talin1 controls dendritic cell activation by regulating TLR complex assembly and signaling.

Talin critically controls integrin-dependent cell migration, but its regulatory role in skin dendritic cells (DCs) during inflammatory responses has not been investigated. Here, we show that talin1 regulates not only integrin-dependent Langerhans cell (LC) migration, but also MyD88-dependent Toll-like receptor (TLR)-stimulated DC activation. Talin1-deficient LCs failed to exit the epidermis, resulting in reduced LC migration to skin-draining lymph nodes (sdLNs) and defective skin tolerance induction, while talin1-deficient dermal DCs unexpectedly accumulated in the dermis despite their actomyosin-dependent migratory capabilities. Furthermore, talin1-deficient DCs exhibited compromised chemotaxis, NFκB activation, and proinflammatory cytokine production. Mechanistically, talin1 was required for the formation of preassembled TLR complexes in DCs at steady state via direct interaction with MyD88 and PIP5K. Local production of PIP2 by PIP5K then recruited TIRAP to the preassembled complexes, which were required for TLR signalosome assembly during DC activation. Thus, talin1 regulates MyD88-dependent TLR signaling pathways in DCs through a novel mechanism with implications for antimicrobial and inflammatory immune responses.

Desmocollin-2 promotes intestinal mucosal repair by controlling integrin-dependent cell adhesion and migration

The intestinal mucosa is lined by a single layer of epithelial cells that forms a tight barrier, separating luminal antigens and microbes from underlying tissue compartments. Mucosal damage results in a compromised epithelial barrier that can lead to excessive immune responses as observed in inflammatory bowel disease. Efficient wound repair is critical to reestablish the mucosal barrier and homeostasis. Intestinal epithelial cells (IEC) exclusively express the desmosomal cadherins, Desmoglein-2 and Desmocollin-2 (Dsc2) that contribute to mucosal homeostasis by strengthening intercellular adhesion between cells. Despite this important property, specific contributions of desmosomal cadherins to intestinal mucosal repair after injury remain poorly investigated in vivo. Here we show that mice with inducible conditional knockdown (KD) of Dsc2 in IEC (Villin-CreERT2; Dsc2 fl/fl) exhibited impaired mucosal repair after biopsy-induced colonic wounding and recovery from dextran sulfate sodium-induced colitis. In vitro analyses using human intestinal cell lines after KD of Dsc2 revealed delayed epithelial cell migration and repair after scratch-wound healing assay that was associated with reduced cell–matrix traction forces, decreased levels of integrin β1 and β4, and altered activity of the small GTPase Rap1. Taken together, these results demonstrate that epithelial Dsc2 is a key contributor to intestinal mucosal wound healing in vivo.

Focal adhesion kinase–dependent activation of the early endocytic protein Rab5 is associated with cell migration

Focal adhesion kinase (FAK) is a central regulator of integrin-dependent cell adhesion and migration and has recently been shown to co-localize with endosomal proteins. The early endocytic protein Rab5 controls integrin trafficking, focal adhesion disassembly, and cell migration and has been shown to be activated upon integrin engagement by mechanisms that remain unclear. Because FAK is a critical regulator of integrin-dependent signaling and Rab5 recapitulates FAK-mediated effects, we evaluated the possibility that FAK activates Rab5 and contributes to cell migration. Pulldown assays revealed that Rab5-GTP levels are decreased upon treatment with a pharmacological inhibitor of FAK, PF562,271, in resting A549 cells. These events were associated with decreased peripheral Rab5 puncta and a reduced number of early endosome antigen 1 (EEA1)-positive early endosomes. Accordingly, as indicated by FAK inhibition experiments and in FAK-null fibroblasts, adhesion-induced FAK activity increased Rab5-GTP levels. In fact, expression of WT FAK and FAK/Y180A/M183A (open conformation), but not FAK/Arg454 (kinase-dead), augmented Rab5-GTP levels in FAK-null fibroblasts and A549 cells. Moreover, expression of a GDP-bound Rab5 mutant (Rab5/S34N) or shRNA-mediated knockdown of endogenous Rab5 prevented FAK-induced A549 cell migration, whereas expression of WT or GTP-bound Rab5 (Rab5/Q79L), but not Rab5/S34N, promoted cell migration in FAK-null fibroblasts. Mechanistically, FAK co-immunoprecipitated with the GTPase-activating protein p85α in a phosphorylation (Tyr397)-dependent manner, preventing Rab5-GTP loading, as shown by knockdown and transfection recovery experiments. Taken together, these results reveal that FAK activates Rab5, leading to cell migration.

SO2 derivatives and As co-exposure promote liver cancer metastasis through integrin αvβ3 activation

Arsenic (As) and sulfur dioxide (SO2) are two environmental pollutants that have been shown to promote the development of human cancer. In recent years, due to increased pollution, humans are often exposed to SO2, in addition to As. Despite the development and implementation of standards for environment and air quality, cases of disease caused by As or SO2 continue to rise alarmingly. It is currently unknown whether simultaneous exposure to As and SO2 results in increased cancer promoting activity. In this study, concentrations of As and SO2 below the limits established by the world health organization (WHO) in force environmental standards (concentrations of As should be lower than 1×10-2 mg/L and SO2 should be lower than 50 μg/m3), were employed to investigate possible, long-term, synergistic effects of As and SO2, by using cell-based assays. We found that co-exposure to these pollutants significantly promotes HepG2 cancer cell migration, while As or SO2 alone have no remarkable effects. Integrins αvβ3 play a key role in this process, as cilengitide, an integrin αvβ3 inhibitor, substantially prevented As and SO2-induced cell migration. MMPs, IL-8, and TGF-β were also involved in the induced cell migration. In summary, combined exposure to As and SO2 promotes integrin-dependent cell migration and may be of relevance for the activation of mechanisms underlying liver cancer progression.

Contribution of Fractalkine to Incorrect Angiogenesis in Preeclamptic Placentas

Disregarding the multiple research in the field of preeclampsia, the exact pathology remains unclear. The shallow invasion of extravillous trophoblast (EVT) in spiral arteries leads to the development of pregnancy‐induced hypertension, nevertheless the etiological factor is unknown. Disturbed differentiation of EVT and incorrect placental angiogenesis seem to be one of the most important starting points for pathogenetic and etiological concepts in preeclampsia. As fractalkine (chemokine CX3CL1) by the activation of its only receptor CX3CR1 stimulates angiogenesis and integrin‐dependent cell migration, the authors ask the question about the possible role of CX3CL1 in placental angiogenesis in preeclampsia. A total number of 58 pregnant patients were included into the study, preeclampsia was diagnosed in 29 patients, and 29 patients were healthy, normotensive term pregnancies. In all women between 30 and 32 week of pregnancy CX3CL1 level was assessed in plasma using ELISA and the expression of CX3CR1 and angiogenesis score (V/EVTI: vascular/extravascular tissular index) were assessed in placental tissues obtained after delivery. Quantitative immunohistochemistry was applied for assessment of both CX3CR1 expression and V/EVTI in placental sections. For this second purpose, morphometric measurements of the placental microvasculature were performed after immunohistochemistry of endothelial cells with CD‐34 antibody. The average duration of pregnancy in preeclamptic group was shorter by 5,3 weeks than in normotensive pregnancies. CXC3CL1 plasma concentration was significantly lower in preeclamptic patients (mean 279 pg/ml ± 243 vs mean 846 pg/ml ± 418), whereas CX3CR1 expression was significantly (p < 0.05) higher in preeclamptic group. A negative correlation between CX3CL1 level and CX3CR1 expression was observed. Moreover, the V/EVTI value was lowered in preeclampsia (p < 0.05). In conclusion, disturbed placental angiogenesis in preeclampsia may be associated with low plasma fractalkine level.Support or Funding InformationWUM grant: 2M2‐W1‐18This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

68Ga-DOTA-E[c(RGDfK)]2 PET Imaging of SHARPIN-Regulated Integrin Activity in Mice.

Shank-associated RH domain-interacting protein (SHARPIN) is a cytosolic protein that plays a key role in activation of nuclear factor κ-light-chain enhancer of activated B cells and regulation of inflammation. Furthermore, SHARPIN controls integrin-dependent cell adhesion and migration in several normal and malignant cell types, and loss of SHARPIN correlates with increased integrin activity in mice. Arginyl-glycyl-aspartic acid (RGD), a cell adhesion tripeptide motif, is an integrin recognition sequence that facilitates PET imaging of integrin upregulation during tumor angiogenesis. We hypothesized that increased integrin activity due to loss of SHARPIN protein would affect the uptake of αvβ3-selective cyclic, dimeric peptide 68Ga-DOTA-E[c(RGDfK)]2, where E[c(RGDfk)]2 = glutamic acid-[cyclo(arginyl-glycyl-aspartic acid-D-phenylalanine-lysine)], both in several tissue types and in the tumor microenvironment. To test this hypothesis, we used RGD-based in vivo PET imaging to evaluate wild-type (wt) and SHARPIN-deficient mice (Sharpincpdm , where cpdm = chronic proliferative dermatitis in mice) with and without melanoma tumor allografts. Methods:Sharpincpdm mice with spontaneous null mutation in the Sharpin gene and their wt littermates with or without B16-F10-luc melanoma tumors were studied by in vivo imaging and ex vivo measurements with cyclic-RGD peptide 68Ga-DOTA-E[c(RGDfK)]2 After the last 68Ga-DOTA-E[c(RGDfK)]2 peptide PET/CT, tumors were cut into cryosections for autoradiography, histology, and immunohistochemistry. Results: The ex vivo uptake of 68Ga-DOTA-E[c(RGDfK)]2 in the mouse skin and tumor was significantly higher in Sharpincpdm mice than in wt mice. B16-F10-luc tumors were detected 4 d after inoculation, without differences in volume or blood flow between the mouse strains. PET imaging with 68Ga-DOTA-E[c(RGDfK)]2 peptide at day 10 after inoculation revealed significantly higher uptake in the tumors transplanted into Sharpincpdm mice than in wt mice. Furthermore, tumor vascularization was increased in the Sharpincpdm mice. Conclusion:Sharpincpdm mice demonstrated increased integrin activity and vascularization in B16-F10-luc melanoma tumors, as demonstrated by RGD-based in vivo PET imaging. These data indicate that SHARPIN, a protein previously associated with increased cancer growth and metastasis, may also have important regulatory roles in controlling the tumor microenvironment.

Abstract 3149: Collagen XIII induces β1-integrin activation and triple-negative breast cancer metastasis

Abstract Increased collagen expression and deposition is a risk factor for breast cancer development. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse model show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. However, function of collagen XIII in cancer development and progression largely remains to be determined. We found that collagen XIII expression is significantly induced during breast cancer development, especially in triple negative breast cancer. Increased collagen XIII mRNA level is associated with poor prognosis. Importantly, silencing collagen XIII expression in MDA MB 231 cells slow down xenograft growth and significantly reduced cancer metastasis in mice. Using gain- and loss-of function approaches, we showed that collagen XIII enhanced cell migration, invasion, and mammosphere/tumorsphere formation. We also found that collagen XIII induced integrin-β1 activation, and subsequently enhanced TGF-β signaling. Blocking integrin-β1 activation with AIIB2 antibody significantly reduced collagen XIII-induced cell migration and mammosphere formation. These findings demonstrate a novel function of collagen XIII in promoting cancer metastasis through enhancing β1 integrin-dependent cell migration and invasion Citation Format: Hui Zhang, Tricia Fredericks, Ren Xu. Collagen XIII induces β1-integrin activation and triple-negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3149.

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways.

Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor β1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2β1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.

P3.02-092 CD151-Integrin-C-Kit Axis Plays an Important Role in the Pathogenesis of Non-Small Cell Lung Cancer

CD151, a master regulator of laminin-binding integrins (α3β1、α6β1 and α6β1), assembles these integrins into complexes called tetraspanin-enriched microdomains. Hence, CD151 is well positioned to modulate integrin-dependent cell spreading, migration, signaling, and adhesion strengthening. Now, it has been shown to be involved in tumor progression. The molecular mechanism of CD151 in cancer is based on its ability to organize distribution and function of interacting proteins, ie, laminin-binding integrins (α3β1、α6β1 and α6β1), receptors for growth factors (HGFR, EGFR).

A Novel α9 Integrin Ligand, XCL1/Lymphotactin, Is Involved in the Development of Murine Models of Autoimmune Diseases.

The integrin α9β1 is a key receptor involved in the development of autoimmune diseases. However, the detailed mechanism for the association of α9β1 integrin with its ligands remains unclear. In this study, we introduce XCL1/lymphotactin, a member of the chemokine family, as a novel ligand for α9 integrin. Using α9 integrin-overexpressing NIH3T3 cells and endogenously α9 integrin-expressing human rhabdomyosarcoma cells, the interaction between XCL1 and α9 integrin was confirmed by pull-down assays. XCL1 enhanced α9 integrin-dependent cell migration of these cells, thus acting on α9 integrin as a chemoattractant. We also analyzed the in vivo function of XCL1 in the development of anti-type II collagen Ab-induced inflammatory arthritis (CAIA) in BALB/c mice and experimental autoimmune encephalomyelitis in C57BL/6 mice, because α9 integrin is involved in these autoimmune disease models. In CAIA, recombinant XCL1 aggravated the disease and this exacerbation was inhibited by an anti-α9 integrin Ab. An XCL1-neutralizing Ab produced in this study also ameliorated CAIA. Furthermore, the XCL1-neutralizing Ab abrogated the disease progression in experimental autoimmune encephalomyelitis. Therefore, to our knowledge this study provides the first in vitro and in vivo evidence that the interaction between XCL1 and α9 integrin has an important role for autoimmune diseases.

Induction of Antibodies Directed Against Branched Core O-Mannosyl Glycopeptides-Selectivity Complimentary to the ConA Lectin.

Mammalian protein O-mannosylation, initiated by attachment of α-mannopyranose to Ser or Thr residues, comprise a group of post-translational modifications (PTMs) involved in muscle and brain development. Recent advances in glycoproteomics methodology and the "SimpleCell" strategy have enabled rapid identification of glycoproteins and specific glycosylation sites. Despite the enormous progress made, the biological impact of the mammalian O-mannosyl glycoproteome remains largely unknown to date. Tools are still needed to investigate the structure, role, and abundance of O-mannosyl glycans. Although O-mannosyl branching has been shown to be of relevance in integrin-dependent cell migration, and also plays a role in demyelinating diseases, such as multiple sclerosis, a broader understanding of the biological roles of branched O-mannosyl glycans is lacking in part due to the paucity of detection tools. In this work, a glycopeptide vaccine construct was synthesized and used to generate antibodies against branched O-mannosyl glycans. Glycopeptide microarray screening revealed high selectivity of the induced antibodies for branched glycan core structures presented on different peptide backbones, with no cross-reactivity observed with related linear glycans. For comparison, microarray screening of the mannose-binding lectin concanavalin A (ConA), which is commonly used in glycoproteomics workflows to enrich tryptic O-mannosyl peptides, showed that the ConA lectin did not recognize branched O-mannosyl glycans. The binding preference of ConA for short linear O-mannosyl glycans was rationalized in terms of molecular structure using crystallographic data augmented by molecular modeling. The contrast between the ConA binding specificity and that of the new antibodies indicates a novel role for the antibodies in studies of protein O-mannosylation.

Integrins and Cell Metabolism: An Intimate Relationship Impacting Cancer.

Integrins are important regulators of cell survival, proliferation, adhesion and migration. Once activated, integrins establish a regulated link between the extracellular matrix and the cytoskeleton. Integrins have well-established functions in cancer, such as in controlling cell survival by engagement of many specific intracellular signaling pathways and in facilitating metastasis. Integrins and associated proteins are regulated by control of transcription, membrane traffic, and degradation, as well as by a number of post-translational modifications including glycosylation, allowing integrin function to be modulated to conform to various cellular needs and environmental conditions. In this review, we examine the control of integrin function by cell metabolism, and the impact of this regulation in cancer. Within this context, nutrient sufficiency or deprivation is sensed by a number of metabolic signaling pathways such as AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and hypoxia-inducible factor (HIF) 1, which collectively control integrin function by a number of mechanisms. Moreover, metabolic flux through specific pathways also controls integrins, such as by control of integrin glycosylation, thus impacting integrin-dependent cell adhesion and migration. Integrins also control various metabolic signals and pathways, establishing the reciprocity of this regulation. As cancer cells exhibit substantial changes in metabolism, such as a shift to aerobic glycolysis, enhanced glucose utilization and a heightened dependence on specific amino acids, the reciprocal regulation of integrins and metabolism may provide important clues for more effective treatment of various cancers.

Schwann Cells Increase Prostate and Pancreatic Tumor Cell Invasion Using Laminin Binding A6 Integrin.

Human pancreatic and prostate cancers metastasize along nerve axons during perineural invasion. The extracellular matrix laminin class of proteins is an abundant component of both myelinated and non-myelinated nerves. Analysis of human pancreatic and prostate tissue revealed both perineural and endoneural invasion with Schwann cells surrounded or disrupted by tumor, respectively. Tumor and nerve cell co-culture conditions were used to determine if myelinating or non-myelinating Schwann cell (S16 and S16Y, respectively) phenotype was equally likely to promote integrin-dependent cancer cell invasion and migration on laminin. Conditioned medium from S16 cells increased tumor cell (DU145, PC3, and CFPAC1) invasion into laminin approximately 1.3-2.0 fold compared to fetal bovine serum (FBS) treated cells. Integrin function (e.g., ITGA6p formation) increased up to 1.5 fold in prostate (DU145, PC3, RWPE-1) and pancreatic (CFPAC1) cells, and invasion was dependent on ITGA6p formation and ITGB1 as determined by function-blocking antibodies. In contrast, conditioned medium isolated from S16Y cells (non-myelinating phenotype) decreased constitutive levels of ITGA6p in the tumor cells by 50% compared to untreated cells and decreased ITGA6p formation 3.0 fold compared to S16 treated cells. Flow cytometry and western blot analysis revealed loss of ITGA6p formation as reversible and independent of overall loss of ITGA6 expression. These results suggest that the myelinating phenotype of Schwann cells within the tumor microenvironment increased integrin-dependent tumor invasion on laminin.

Lamellipodial tension, not integrin/ligand binding, is the crucial factor to realise integrin activation and cell migration

The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding. This particular system allowed us to selectively analyse key MC proteins and interactions, effectively from the extracellular matrix substrate to intracellular f-actin, and to therewith study mechanobiological aspects of MC engagement also uncoupled from integrin/ligand binding. With this experimental approach, we found that for the initial PIP2-dependent membrane/TLN/f-actin linkage and persistent lamellipodia formation the C-terminal TLN actin binding site (ABS) is dispensable. The establishment of an adequate MC-mediated lamellipodial tension instead depends predominantly on the coupling of this C-terminal TLN ABS to the actomyosin-driven retrograde actin flow force. This lamellipodial tension is crucial for full integrin activation eventually determining integrin-dependent cell migration. In the integrin/ligand-independent condition the frictional membrane resistance participates to these processes. Integrin/ligand binding can also contribute but is not necessarily required.

Abstract 397: The Interaction of Integrin beta1 to Galpha13 Mediates RhoA Inhibition and Cell Migration

Background: Integrin-dependent cell migration is critically important in many physiological and pathological processes such as angiogenesis, inflammation, wound healing, and atherosclerosis. However, the underlying signaling mechanisms remain unclear. Hypothesis: We have recently shown that Ga13 directly interacts with the cytoplasmic domain of integrin b3 subunits in platelets and is a proximal mechanism of integrin outside-in signaling. We further showed that the binding site for Ga13 requires a conserved ExE motif also present in b1 integrins important in adhesion and migration of nearly all vascular cells. We hypothesize that the direct binding of Ga13 to the ExE motif in integrin b1 plays an important role in mediates b1-dependent cell migration. Results: To specifically study the role of b1 integrin in cell migration, we compared cell migration of GD25 cells that are deficient in integrin b1 subunit with cells that re-constitute b1 expression using a scratch wound healing assay and a transwell migration assay. Cell migration was abolished in b1-deficient cells, indicating a critical role for b1 in these assays. To study whether Ga13-integrin interaction is important in cell migration, we constructed b1 mutants that were deficient in binding to Ga13 by mutating the critically important glutamic acid residues to alanine. Co-immunoprecipitation experiments indicate the ExE to alanine mutants abolished Ga13-integrin interaction. The b1-dependent cell migration is dramatically inhibited in the ExE to alanine mutant b1-expressing cells. Importantly, a small peptide based on the ExE motif similarly inhibited cell migration. Furthermore, Ga13 binding-deficient b1 mutant cells showed diminished b1 integrin-dependent Src activation and accelerated RhoA activation during cell spreading on fibronectin, suggesting that the integrin-dependent transient inhibition of RhoA was abolished. Conclusions: Ga13 mediates integrin-dependent cell migration by direct binding to the ExE motif of integrin b1 and mediating c-Src activation and RhoA inhibition.

Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5β1 or αvβ3/β5 integrin antagonists in U87MG glioma cells

BackgroundIntegrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5β1 and αvβ3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5β1 and αvβ3/β5 purified integrins. MethodsSmall antagonists were either selective for α5β1 integrin, for αvβ3/β5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5β1 integrin in the biological assays. ResultsU87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5β1 or αvβ3/β5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5β1 integrin and was inhibited selectively by α5β1 integrin antagonists but increased by αvβ3/β5 integrin antagonists. ConclusionsWe provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions. General significanceOur data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5β1 integrin-dependent cell migration.

Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation.

Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by time-lapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.