Scott D. Blystone

Purified Integrin Adhesion Complexes Exhibit Actin-Polymerization Activity

BACKGROUND Cell adhesion and motility are accomplished through a functional linkage of the extracellular matrix with the actin cytoskeleton via adhesion complexes composed of integrin receptors and associated proteins. To determine whether this linkage is attained actively or passively, we isolated integrin complexes from nonadherent hematopoietic cells and determined their influence on the polymerization of actin. RESULTS We observed that alpha(V)beta3 complexes are capable of dramatically accelerating the rate of actin assembly, resulting in actin fibers tethered at their growing ends by clustered integrins. The ability to enhance actin polymerization was dependent upon Arg-Gly-Asp-ligand-induced beta3 tyrosine phosphorylation, agonist-induced cellular activation, sequestration of Diaphanous formins, and clustering of the receptor. CONCLUSIONS These results suggest that adhesion complexes actively promote actin assembly from their cytosolic face in order to establish a mechanical linkage with the extracellular matrix.

Macrophages of multiple sclerosis patients display deficient SHP-1 expression and enhanced inflammatory phenotype.

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of proinflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild-type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display coexpression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-κB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-κB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-κB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-κB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.

The formin FRL1 (FMNL1) is an essential component of macrophage podosomes.

Podosomes are highly dynamic actin‐rich adhesion structures in cells of myeloid lineage and some transformed cells. Unlike transformed mesenchymal cell types, podosomes are the sole adhesion structure in macrophage and thus mediate all contact with adhesion substrate, including movement through complex tissues for immune surveillance. The existence of podosomes in inflammatory macrophages and transformed cell types suggest an important role in tissue invasion. The proteome, assembly, and maintenance of podosomes are emerging, but remain incompletely defined. Previously, we reported a formin homology sequence and actin assembly activity in association with macrophage β‐3 integrin. In this study we demonstrate by quantitative reverse transcriptase polymerase chain reaction and Western blotting that the formin FRL1 is specifically upregulated during monocyte differentiation to macrophages. We show that the formin FRL1 localizes to the actin‐rich cores of primary macrophage podosomes. FRL1 co‐precipitates with beta‐3 integrin and both fixed and live cell fluorescence microscopy show that endogenous and overexpressed FRL1 selectively localize to macrophage podosomes. Targeted disruption of FRL1 by siRNA results in reduced cell adhesion and disruption of podosome dynamics. Our data suggest that FRL1 is responsible for modifying actin at the macrophage podosome and may be involved in actin cytoskeleton dynamics during adhesion and migration within tissues.

Ligand-dependent Activation of Integrin αvβ3

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as αvβ3. In this study, we examined the activation of αvβ3 resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected αvβ3, adhesion to vitronectin required tyrosine phosphorylation of the β3subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without β3-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.

Tyrosine Phosphorylation of β3 Integrin Provides a Binding Site for Pyk2

Integrins expressed on leukocytes possess the ability to maintain themselves in a non-adhesive state, thus preventing unwarranted adhesion and uncontrolled inflammation. Leukocyte adhesion is regulated through the modulation of integrin receptors such as αVβ3. Firm adhesion to the extracellular matrix and directed cellular motility requires the reorganization of the actin cytoskeleton. The ability of β3 to recruit signaling and scaffolding molecules to propagate αVβ3 -mediated signals is regulated in part by the phosphorylation of the β3 cytoplasmic tail. The identities of integrin-associated signaling molecules within αVβ3 podosomes and in particular the proximal binding partners of the β3 cytoplasmic tail are not completely known. Here we show that αVβ3 ligation induces Pyk2-Tyr-402 phosphorylation and its association with the β3 cytoplasmic tail in a β3-Tyr-747 phosphorylation-dependent manner. Pyk2 binding to the β3 cytoplasmic tail is direct and dependent upon Pyk2-Tyr-402 and β3 -Tyr-747 phosphorylations. These data identify Pyk2 as a phosphorylated β3 binding partner, providing a potential structural and signaling platform to achieve αVβ3 -mediated remodeling of the actin cytoskeleton.

The multiplicity of human formins: Expression patterns in cells and tissues

Formins are actin‐binding proteins conserved across species from plants to humans. The formin family is defined by their common formin homology (FH2) domains. The 15 distinct human formins are involved in a broad range of cellular functions, including cell adhesion, cytokinesis, cell polarity, and cell morphogenesis. Their commonality is actin polymerization activity inherent to FH2 domains. Although still requiring much study, biochemical activity of formins has been carefully described. In contrast, much less is known of their activities in complex living systems. With the diversity of the formin family and the actin structures that they affect, an extensive future of study beckons. In this study, we report the expression level of all 15 formins in 22 different human cell and tissue types using quantitative real‐time PCR. Identification of major themes in formin expression and documentation of expression profiles should facilitate the cellular study of formins.

Heparin Modulates Integrin-Mediated Cellular Adhesion: Specificity of Interactions with α and β Integrin Subunits

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin αIIbβ3, and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (αvβ3, αvβ5, and αIIbβ3). By comparing K562 cells expressing a common α subunit (Kαvβ3, Kαvβ5) with cells expressing a common β subunit (Kαvβ3, KαIIbβ3), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5–7.5 μg/ml consistently enhanced the adhesion of β3expressing cells (Kαvβ3,KαIIbβ3). In contrast, UH at 0.5–7.5 μg/ml inhibited Kαvβ5adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparins actions were mediated by the specific integrins under study. Preincubation of heparin with Kαvβ3cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparins effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparins modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the β subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.

β3 Tyrosine Phosphorylation and αvβ3-mediated Adhesion Are Required for Vav1 Association and Rho Activation in Leukocytes

Integrin αvβ3-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of β3 tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in αvβ3-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with αvβ3 upon cell adhesion to vitronectin and that this association requires β3 tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during αvβ3-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable β3 mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with β3 integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after αvβ3-mediated adhesion, and the association between Vav1 and β3 occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between β3 integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.

A Pyk2-Vav1 complex is recruited to β3-adhesion sites to initiate Rho activation

Integrin alphavbeta3-mediated adhesion of haemopoietic cells to vitronectin results in beta3 tyrosine phosphorylation and Rho activation which is necessary for adhesion. Previously, we have shown that the RhoGEF (Rho guanine-nucleotide-exchange factor) Vav1 could associate indirectly with alphavbeta3 during leucocyte adhesion to vitronectin. In the present study, we have identified the non-receptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with alphavbeta3. The association of Pyk2 and Vav1 with beta3 relies on the presence of Tyr747 in beta3, the primary site of beta3 phosphorylation. However, association of Pyk2 with Vav1 is independent of beta3 tyrosine phosphorylation. Formation of a Pyk2-Vav1 complex occurs upon cell adhesion and Pro717 of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 In vitro. Cells transfected with GFP (green fluorescent protein)-Pyk2-P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhesion. Using siRNA (small interfering RNA) to specifically reduce Pyk2 levels in cells resulted in disrupted association between Vav1 and beta3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of beta3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.

Heparin modulates the conformation and signaling of platelet integrin αIIbβ3.

INTRODUCTION The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbβ3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbβ3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets. METHODS We assessed conformational changes in αIIbβ3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbβ3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the β3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbβ3, and of recombinant fragments of αIIb and β3, by surface plasmon resonance. RESULTS AND CONCLUSIONS Heparin binding results in conformational changes in αIIbβ3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin β3 cytoplasmic domain, but the presence of heparin increased both β3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant β3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbβ3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbβ3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.