Characterization of PINCH-2, a new focal adhesion protein that regulates the PINCH-1-ILK interaction,cell spreading,and migration

Integrin-linked kinase (ILK) is a multidomain protein that plays important roles at cell-extracellular matrix (ECM) adhesion sites. We describe here a new LIM-domain containing protein (termed as PINCH-2) that forms a complex with ILK. PINCH-2 is co-expressed with PINCH-1 (previously known as PINCH), another member of the PINCH protein family, in a variety of human cells. Immunofluorescent staining of cells with PINCH-2-specific antibodies show that PINCH-2 localizes to both cell-ECM contact sites and the nucleus. Deletion of the first LIM (LIM1) domain of PINCH-2 abolished the ability of PINCH-2 to form a complex with ILK. The ILK-binding defective LIM1-deletion mutant, unlike the wild type PINCH-2 or the ILK-binding competent LIM5-deletion mutant, was incapable of localizing to cell-ECM contact sites, suggesting that ILK binding is required for this process. Importantly, the PINCH-2-ILK and PINCH-1-ILK interactions are mutually exclusive. Overexpression of PINCH-2 significantly inhibited the PINCH-1-ILK interaction and reduced cell spreading and migration. These results identify a novel nuclear and focal adhesion protein that associates with ILK and reveals an important role of PINCH-2 in the regulation of the PINCH-1-ILK interaction, cell shape change, and migration.     

Structure of an ultraweak protein-protein complex and its crucial role in regulation of cell morphology and motility

Weak protein-protein interactions (PPIs) (K(D) > 10(-6) M) are critical determinants of many biological processes. However, in contrast to a large growing number of well-characterized, strong PPIs, the weak PPIs, especially those with K(D) > 10(-4) M, are poorly explored. Genome wide, there exist few 3D structures of weak PPIs with K(D) > 10(-4) M, and none with K(D) > 10(-3) M. Here, we report the NMR structure of an extremely weak focal adhesion complex (K(D) approximately 3 x 10(-3) M) between Nck-2 SH3 domain and PINCH-1 LIM4 domain. The structure exhibits a remarkably small and polar interface with distinct binding modes for both SH3 and LIM domains. Such an interface suggests a transient Nck-2/PINCH-1 association process that may trigger rapid focal adhesion turnover during integrin signaling. Genetic rescue experiments demonstrate that this interface is indeed involved in mediating cell shape change and migration. Together, the data provide a molecular basis for an ultraweak PPI in regulating focal adhesion dynamics during integrin signaling.     

PINCH-1 is an obligate partner of integrin-linked kinase (ILK) functioning in cell shape modulation, motility, and survival

PINCH-1 is a widely expressed focal adhesion protein that forms a ternary complex with integrin-linked kinase (ILK) and CH-ILKBP/actopaxin/alpha-parvin (abbreviated as alpha-parvin herein). We have used RNA interference, a powerful approach of reverse genetics, to investigate the functions of PINCH-1 and ILK in human cells. We report here the following. First, PINCH-1 and ILK, but not alpha-parvin, are essential for prompt cell spreading and motility. Second, PINCH-1 and ILK, like alpha-parvin, are crucial for cell survival. Third, PINCH-1 and ILK are required for optimal activating phosphorylation of PKB/Akt, an important signaling intermediate of the survival pathway. Whereas depletion of ILK reduced Ser473 phosphorylation but not Thr308 phosphorylation of PKB/Akt, depletion of PINCH-1 reduced both the Ser473 and Thr308 phosphorylation of PKB/Akt. Fourth, PINCH-1 and ILK function in the survival pathway not only upstream but also downstream (or in parallel) of protein kinase B (PKB)/Akt. Fifth, PINCH-1, ILK and to a less extent alpha-parvin are mutually dependent in maintenance of their protein, but not mRNA, levels. The coordinated down-regulation of PINCH-1, ILK, and alpha-parvin proteins is mediated at least in part by proteasomes. Finally, increased expression of PINCH-2, an ILK-binding protein that is structurally related to PINCH-1, prevented the down-regulation of ILK and alpha-parvin induced by the loss of PINCH-1 but failed to restore the survival signaling or cell shape modulation. These results provide new insights into the functions of PINCH proteins in regulation of ILK and alpha-parvin and control of cell behavior.     

The roles of two distinct regions of PINCH-1 in the regulation of cell attachment and spreading

Cells attach to the extracellular matrix (ECM) through integrins to form focal adhesion complexes, and this process is followed by the extension of lamellipodia to enable cell spreading. PINCH-1, an adaptor protein essential for the regulation of cell-ECM adhesion, consists of five tandem LIM domains and a small C-terminal region. PINCH-1 is known to interact with integrin-linked kinase (ILK) and Ras suppressor protein 1 (Rsu-1); however, the precise mechanism by which this complex regulates cell-ECM adhesion is not fully understood. We report here that the LIM1 domain of PINCH-1, which associates with ILK to stabilize the expression of this protein, is sufficient for cell attachment but not for cell spreading. In contrast, the C-terminal region of PINCH-1, which binds to Rsu-1, plays a pivotal role in cell spreading but not in cell attachment. We also show that PINCH-1 associates with Rsu-1 to activate Rac1 and that Rac1 activation is necessary for cell spreading. Thus, these data reveal how specific domains of PINCH-1 direct two independent pathways: one utilizing ILK to allow cell attachment, and the other recruiting Rsu-1 to activate Rac1 in order to promote cell spreading.     

Binary and ternary complexes of FLNa-Ig21 with cytosolic tails of αMß2 integrin reveal dual role of filamin mediated regulation

BackgroundCytoskeletal protein filamin A is critical for the outside-in signaling of integrins. Although molecular mechanisms of filamin-integrin interactions are not fully understood. Mostly, the membrane distal (MD) part of the cytosolic tail (CT) of β subunit of integrin is known to interact with filamin A domain 21 (FLNa-Ig2). However, binary and ternary complexes of full-length CTs of leucocyte specific ß2 integrins with FLNa-Ig21 are yet to be elucidated.MethodsBinding interactions of the CTs of integrin αMß2 with FLNa-Ig21 are extensively investigated by NMR, ITC, cell-based functional assays and computational docking.ResultsThe αM CT demonstrates interactions with FLNa-Ig21 forming a binary complex. Filamin/αM interface is mediated by sidechain-sidechain interactions among non-polar and aromatic residues involving MP helix of αM and the canonical CD face of FLNa-Ig21. Functional assays delineated an interfacial residue Y1137 of αM CT is critical for in-cell binding to FLNa-Ig2. In addition, full-length ß2 CT occupies two distinct binding sites in complex with FLNa-Ig21. A ternary complex of FLNa-Ig21 with CTs has been characterized. In the ternary complex, αM CT moves away to a distal site of FLNa-Ig21 with fewer interactions.ConclusionOur findings demonstrate a plausible dual role of filamin in integrin regulation. The molecular interactions of the ternary complex are critical for the resting state of integrins whereas stable FLNa-Ig21/αM CT binary complex perhaps be required for the activated state.General significanceFilamin binding to both α and β CTs of other integrins could be essential in regulating bidirectional signaling mechanisms.     

Molecular dissection of PINCH-1 reveals a mechanism of coupling and uncoupling of cell shape modulation and survival

How cells couple and uncouple regulation of cellular processes such as shape change and survival is an important question in molecular cell biology. PINCH-1, a widely expressed protein consisting of five LIM domains and a C-terminal tail, is an essential focal adhesion protein with multiple functions including regulation of the integrin-linked kinase (ILK) level, cell shape, and survival signaling. We show here that the LIM1-mediated interaction with ILK regulates all these three processes. By contrast, the LIM4-mediated interaction with Nck-2, which regulates cell morphology and migration, is not required for the control of the ILK level and survival. Remarkably, a short 15-residue tail C-terminal to LIM5 is required for both cell shape modulation and survival, albeit it is not required for the control of the ILK level. The C-terminal tail not only regulates PINCH-1 localization to focal adhesions but also functions after it localizes there. These findings suggest that PINCH-1 functions as a molecular platform for coupling and uncoupling diverse cellular processes via overlapping but yet distinct domain interactions.     

Chemokine triggered integrin activation and actin remodeling events guiding lymphocyte migration across vascular barriers

Chemokine signals activate leukocyte integrins and actin remodeling machineries critical for leukocyte adhesion and motility across vascular barriers. The arrest of leukocytes at target blood vessel sites depends on rapid conformational activation of their α4 and β2 integrins by the binding of endothelial-displayed chemokines to leukocyte Gi-protein coupled receptors (GPCRs). A universal regulator of this event is the integrin-actin adaptor, talin1. Chemokine-stimulated GPCRs can transmit within fractions of seconds signals via multiple Rho GTPases, which locally raise plasma membrane levels of the talin activating phosphatidyl inositol, PtdIns(4,5)P2 (PIP2). Additional pools of GPCR stimulated Rac-1 and Rap-1 GTPases together with GPCR stimulated PLC and PI3K family members regulate the turnover of focal contacts of leukocyte integrins, induce the collapse of leukocyte microvilli, and promote polarized leukocyte crawling in search of exit cues. Concomitantly, other leukocyte GTPases trigger invasive protrusions into and between endothelial cells in search of basolateral chemokine exit cues. We will review here major findings and open questions related to these sequential guiding activities of endothelial presented chemokines, focusing mainly on lymphocyte-endothelial interactions as a paradigm for other leukocytes.     

Collagen-binding I domain integrins--what do they do?

Collagens are the most abundant proteins in the mammalian body and it is well recognized that collagens fulfill an important structural role in the extracellular matrix in a number of tissues. Inactivation of the collagen alpha 1(I) gene in mice results in embryonic lethality and collagen mutations in humans cause defects leading to disease. Integrins constitute a major group of receptors for extracellular matrix components, including collagens. Currently four collagen-binding I domain-containing integrins are known, namely alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1 and alpha 11 beta 1. Unlike the undisputed role of collagens as structural elements, the biological importance of integrin mediated cell-collagen interactions is far from clear. This is in part due to the limited information available on the most recent additions of the integrin family, alpha 10 beta 1 and alpha 11 beta 1. Future studies using gene inactivation of individual and multiple integrin genes will allow testing of the hypothesis that collagen-binding integrins have redundant functions but will also shed light on their importance in pathological conditions. In this review we will describe what is currently known about the collagen-binding integrins and discuss their biological functions.     

Interplay between integrins and FLK-1 in shear stress-induced signaling

Blood flow can modulate vascular cell functions. We studied interactions between integrins and Flk-1 in transducing the mechanical shear stress due to flow. This application of a step shear stress caused Flk-1. Casitas B-lineage lymphoma (Cbl) activation (Flk-1. Cbl association, tyrosine phosphorylation of the Cbl-bound Flk-1, and tyrosine phosphorylation of Cbl) in bovine aortic endothelial cells (BAECs). The activation of integrins by plating BAECs on vitronectin or fibronectin also induced this Flk-1. Cbl activation. The shear-induced Flk-1. Cbl activation was blocked by inhibitory antibodies for alphavbeta3- or beta1-integrin, suggesting that it is mediated by integrins. Inhibition of Flk-1 by SU1498 also abolished this shear-induced Flk-1. Cbl activation. In contrast to the requirement of integrins for Flk-1. Cbl activation, the Flk-1 blocker SU1498 had no detectable effect on the shear-induced integrin activation, suggesting that integrins and Flk-1 play sequential roles in the signal transduction hierarchy induced by shear stress. Integrins are essential for the mechanical activation of Flk-1 by shear stress but not for the chemical activation of Flk-1 by VEGF.     

Distinct roles of two structurally closely related focal adhesion proteins, alpha-parvins and beta-parvins, in regulation of cell morphology and survival

Proteins at cell-extracellular matrix adhesions (e.g. focal adhesions) are crucially involved in regulation of cell morphology and survival. We show here that CH-ILKBP/actopaxin/alpha-parvin and affixin/beta-parvin (abbreviated as alpha- and beta-parvin, respectively), two structurally closely related integrin-linked kinase (ILK)-binding focal adhesion proteins, are co-expressed in human cells. Depletion of alpha-parvin dramatically increased the level of beta-parvin, suggesting that beta-parvin is negatively regulated by alpha-parvin in human cells. Loss of PINCH-1 or ILK, to which alpha- and beta-parvin bind, significantly reduced the activation of Rac, a key signaling event that controls lamellipodium formation and cell spreading. We were surprised to find that loss of alpha-parvin, but not that of beta-parvin, markedly stimulated Rac activation and enhanced lamellipodium formation. Overexpression of beta-parvin, however, was insufficient for stimulation of Rac activation or lamellipodium formation, although it was sufficient for promotion of apoptosis, another important cellular process that is regulated by PINCH-1, ILK, and alpha-parvin. In addition, we show that the interactions of ILK with alpha- and beta-parvin are mutually exclusive. Overexpression of beta-parvin or its CH(2) fragment, but not a CH(2) deletion mutant, inhibited the ILK-alpha-parvin complex formation. Finally, we provide evidence suggesting that inhibition of the ILK-alpha-parvin complex is sufficient, although not necessary, for promotion of apoptosis. These results identify Rac as a downstream target of PINCH-1, ILK, and parvin. Furthermore, they demonstrate that alpha- and beta-parvins play distinct roles in mammalian cells and suggest that the formation of the ILK-alpha-parvin complex is crucial for protection of cells from apoptosis.     

A role for alphabeta1 integrins in focal adhesion function and polarized cytoskeletal dynamics

alphabeta1 integrins have been implicated in the survival, spreading, and migration of cells and tissues. To explore the underlying biology, we identified conditions where primary beta1 null keratinocytes adhere, proliferate, and display robust alphavbeta6 integrin-induced, peripheral focal contacts associated with elaborate stress fibers. Mechanistically, this appears to be due to reduced FAK and Src and elevated RhoA and Rock activities. Visualization on a genetic background of GFPactin shows that beta1 null keratinocytes spread, but do so aberrantly, and when induced to migrate from skin explants in vitro, the cells are not able to rapidly reorient their actin cytoskeleton toward the polarized movement. As judged by RFPzyxin/GFPactin videomicroscopy, the alphavbeta6-actin network does not undergo efficient turnover. Without the ability to remodel their integrin-actin network efficiently, alphabeta1-deficient keratinocytes cannot respond dynamically to their environment and polarize movements.     

Significance of thymosin β4 and implication of PINCH-1-ILK-α-parvin (PIP) complex in human dilated cardiomyopathy

Myocardial remodeling is a major contributor in the development of heart failure (HF) after myocardial infarction (MI). Integrin-linked kinase (ILK), LIM-only adaptor PINCH-1, and α-parvin are essential components of focal adhesions (FAs), which are highly expressed in the heart. ILK binds tightly to PINCH-1 and α-parvin, which regulates FA assembly and promotes cell survival via the activation of the kinase Akt. Mice lacking ILK, PINCH or α-parvin have been shown to develop severe defects in the heart, suggesting that these proteins play a critical role in heart function. Utilizing failing human heart tissues (dilated cardiomyopathy, DCM), we found a 2.27-fold (p<0.001) enhanced expression of PINCH, 4 fold for α-parvin, and 10.5 fold (p<0.001) for ILK as compared to non-failing (NF) counterparts. No significant enhancements were found for the PINCH isoform PINCH-2 and parvin isoform β-parvin. Using a co-immunoprecipitation method, we also found that the PINCH-1-ILK-α-parvin (PIP) complex and Akt activation were significantly up-regulated. These observations were further corroborated with the mouse myocardial infarction (MI) and transaortic constriction (TAC) model. Thymosin beta4 (Tβ4), an effective cell penetrating peptide for treating MI, was found to further enhance the level of PIP components and Akt activation, while substantially suppressing NF-κB activation and collagen expression--the hallmarks of cardiac fibrosis. In the presence of an Akt inhibitor, wortmannin, we show that Tβ4 had a decreased effect in protecting the heart from MI. These data suggest that the PIP complex and activation of Akt play critical roles in HF development. Tβ4 treatment likely improves cardiac function by enhancing PIP mediated Akt activation and suppressing NF-κB activation and collagen-mediated fibrosis. These data provide significant insight into the role of the PIP-Akt pathway and its regulation by Tβ4 treatment in post-MI.     

Opposing roles of integrin alpha6Abeta1 and dystroglycan in laminin-mediated extracellular signal-regulated kinase activation

Laminin-integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed beta1-containing integrins and dystroglycan but lacked integrin alpha6beta4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the alpha3beta1and alpha6beta1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin alpha6beta1 and not by alpha3beta1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin alpha6beta1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin alpha6 splice variants, alpha6A and alpha6B, whereas the nonresponding cell line expressed only alpha6B. Furthermore, ERK activation was seen in cells transfected with the integrin alpha6A subunit, but not in alpha6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin alpha6Abeta1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.     

Interaction Analyses of 14-3-3ζ, Dok1, and Phosphorylated Integrin β Cytoplasmic Tails Reveal a Bi-molecular Switch in Integrin Regulation

Integrins are hetero-dimeric (α and β subunits) type I transmembrane proteins that facilitate cell adhesion and migration. The cytoplasmic tails (CTs) of integrins interact with a plethora of intra-cellular proteins that are required for integrin bidirectional signaling. In particular, the β CTs of integrins are known to recruit a variety of cytosolic proteins that often have overlapping recognition sites. However, the chronological sequence of β CTs/cytosolic proteins interactions remains to be fully characterized. Previous studies have shown that the scaffold protein 14-3-3ζ binds to phosphorylated β CTs in activated integrins, whereas interactions of Dok-1 with phosphorylated β CTs maintained integrins in the resting state. In this study, we examined the binding interactions between 14-3-3ζ, Dok1, and phosphorylated integrin β2 and β3 CTs. We show that the scaffold protein 14-3-3ζ interacts with the phosphotyrosine binding (PTB) domain of Dok1 even in the absence of the phosphorylated integrin β CTs. The interactions were mapped onto the β-sheet region of the PTB domain of Dok1. Furthermore, we provide evidence that the 14-3-3ζ/Dok1 binary complex is able to bind to their cognate phosphorylated sequence motifs in the integrin β CTs. We demonstrate that Thr phosphorylated pTTT β2 CT or pTST β3 CT can bind to 14-3-3ζ that is in complex with the Dok1 PTB domain, whereas Ser phosphorylated β2 CT or Tyr phosphorylated β3 CT interacted with Dok1 in 14-3-3ζ/Dok1 complex. Based on these data, we propose that 14-3-3ζ/Dok1 complex could serve as a molecular switch providing novel molecular insights into the regulating integrin activation.     

The Rsu-1-PINCH1-ILK complex is regulated by Ras activation in tumor cells

The link between Ras transformation and enhanced cell migration due to altered integrin signaling is well established in tumorigenesis, however there remain gaps in our understanding of its mechanism. The Ras suppressor, Rsu-1, has recently been linked to the IPP (integrin-linked kinase {ILK}, PINCH-1/LIMS1, parvin) focal adhesion complex based on its interaction with the LIM 5 domain of PINCH1. Defining the role of the Rsu1-PINCH1-ILK-parvin complex in tumorigenesis is important because both ILK and PINCH1 are elevated in certain tumors while ectopic expression of Rsu-1 blocks tumorigenesis. Our studies previously identified an alternatively spliced isoform of Rsu-1 in high-grade gliomas. We report here the detection of a truncated (p29) Rsu-1 protein, which correlates with the presence of the alternatively spliced Rsu-1 RNA. This RNA and the respective protein were detected in human tumor cell lines that contain high levels of activated Ras, and inhibitor studies demonstrate that the Mek-ERK pathway regulates expression of this truncated Rsu-1 product. We also show that Rsu-1 co-localizes with ILK at focal contacts and co-immunoprecipitates with the ILK-PINCH1 complex in non-transformed cells, but following Ras transformation the association of Rsu-1 with the PINCH1-ILK complex is greatly reduced. Using a human breast cancer cell line, our in vitro studies demonstrate that the depletion of Rsu-1 full-length protein enhances cell migration coincident with an increase in Rac-GTP while the depletion of the p29 Rsu-1 truncated protein inhibits migration. These findings indicate that Rsu-1 may inhibit cell migration by stabilizing the IPP adhesion complex and that Ras activation perturbs this inhibitory function by modulating both Rsu-1 splicing and association of full-length Rsu-1 with IPP. Hence, our findings demonstrate that Rsu-1 links the Ras pathway with the IPP complex and the perturbations of cell attachment-dependent signaling that occur in the malignant process.     

Interactions of Human Skin Fibroblasts with Monomeric or Fibrillar Collagens Induce Different Organization of the Cytoskeleton

Fibrillar collagens represent the most abundant extracellular matrix components surrounding fibroblasts. Although there is a large heterogeneity in the collagen composition and in the physiological functions of different tissues, interactions between cells and native collagens monomers are mediated by only two integrins, the α1β1 and α2β1 integrins. In tissue, fibroblasts are exposed to collagen polymers, supramolecular assemblies which might play a role on the availability of the cell-binding sites at the surface of the fibrils. We have addressed this issue by investigating the patterns of adhesion structures in normal human skin fibroblasts exposed to collagen monomers or polymers. Our results showed that cell morphology, cell adhesion pattern, actin organization, and distribution of integrin subunits, talin, vinculin, and phosphotyrosine-containing proteins are dependent on the supramolecular organization of the collagens. In particular, compared to monomers, collagen polymers induced a looser organization of the actin network and a linear clustering of integrins, talin, vinculin, and phosphotyrosine-containing proteins. These results emphasize the role of the physical state of collagen on cellular interactions and underline the role of the extracellular matrix in the phenotypic modulation of fibroblasts. Furthermore, our studies suggest the existence of a local heterogeneity in the biological activity of collagen fibrils.     

Immune complex-stimulated neutrophil LTB4 production is dependent on beta 2 integrins

The beta 2 integrins (LFA-1, Mac-1, and p150,95) are critical for many adhesive functions of leukocytes. Although the binding of the IgG- opsonized particles occurs normally in the absence of beta 2 integrins, phagocytosis of IgG-opsonized particles by activated neutrophils (PMN) requires these integrins. This observation suggests a role for beta 2 integrins in phagocytosis subsequent to particle binding. To investigate the mechanism of involvement of beta 2 integrins in IgG- mediated functions, we examined the role of beta 2 integrins in adhesion to immune complex (IC)-coated surfaces. Initial adhesion and spreading on IC-coated surfaces were equivalent in control and beta 2- deficient phagocytes. However, both genetically beta 2-deficient PMN and PMN treated with the anti-beta 2 mAb IB4 subsequently detached from the IC-coated surfaces. To determine whether biochemical consequences of IgG activation were also affected by beta 2 deficiency, LTB4 production in response to Fc receptor ligation was assessed. LTB4 production by beta 2-deficient PMN adherent to IC-coated surfaces was markedly decreased in comparison with control PMN. Importantly, LTB4 production by PMN stimulated with fluid phase heat-aggregated IgG also required the beta 2 integrins, showing that the defect was not a simple consequence of abnormal adhesion. In contrast, superoxide production by IC-adherent PMN was equivalent in control and beta 2-deficient PMN. The initial rises in intracytoplasmic [Ca2+]i in response to aggregated IgG also were unaffected by inhibition of beta 2 integrins. These data show that lack of beta 2 integrins does not inhibit all FcR-dependent signal transduction. Finally, LTB4 production by normal PMN adherent to ICs was inhibited by antibodies to FcRII, but not FcRIII, showing that FcRII ligation was required for this effect. Together these data identify a role for the beta 2 integrins in a signal transduction pathway leading to sustained adhesion and LTB4 production in response to IC. Since both beta 2 integrins and FcRII are required for these effects, the data further suggest cooperation between these receptors in generating PMN activation in response to IC stimulation.     

Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by integrin-dependent cell adhesion.

Shed menstrual endometrium is viable and has the ability to implant and grow in women, who eventually develop endometriosis. Many of the cell-to-cell or cell-to-extracellular matrix (ECM) connections are mediated by integrins. Monocyte chemotactic protein (MCP)-1, a potent chemotactic factor produced in many cell types, is elevated in the peritoneal fluid of women with endometriosis. In this study, we investigated whether endometrial stromal cell (ESC) adhesion itself induces the expression of MCP-1 and whether this process is integrin mediated. ESC were plated on Petri dishes and 24-well plates coated with fibronectin, laminin, collagen IV, poly-L-lysine, or mouse anti-human integrin beta(1) and beta(2) monoclonal antibodies. Adherence of ESC to various ECM substrates, except for poly-L-lysine, a non-integrin-dependent adhesion matrix, induced the expression of MCP-1 at both mRNA and protein levels. Engagement of beta(1)-containing integrins was associated with ESC adhesion and resulted in up-regulation of MCP-1 gene expression and protein secretion. Disruption of the actin cytoskeleton by treating ESC with cytochalasin D completely blocked the increase of MCP-1 induced in response to integrin activation. These findings indicate a novel mechanism of MCP-1 regulation. Cell adhesion to ECM is an important event that leads to stimulation of MCP-1 expression, and this process is mediated by integrins.     

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits αL and β2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g. VLA-4 - fibronectin).     

Molecular dissection of the alpha-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.