Elsevier

Cellular Immunology

Volume 275, Issues 1–2, January–February 2012, Pages 47-54
Cellular Immunology

Small molecule inhibitors of the Pyk2 and FAK kinases modulate chemoattractant-induced migration, adhesion and Akt activation in follicular and marginal zone B cells

https://doi.org/10.1016/j.cellimm.2012.03.002Get rights and content

Abstract

B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells.

Highlights

► We synthesized a novel Pyk2-selective small molecule inhibitor, PF-719. ► Inhibitors of Pyk2 and FAK were used to probe the roles of these kinases in B cells. ► Both Pyk2 and FAK contribute to B cell migration towards CXCL13 and S1P. ► Pyk2 activity is required for B cell adhesion to ICAM-1. ► FAK activity is required for chemoattractant and BCR-induced Akt phosphorylation.

Introduction

B-lymphocytes produce protective antibodies but also play a major role in autoimmune diseases [1]. Antibodies directed against host components, which are produced by self-reactive B cells, can lead to the formation of immune complexes that trigger inflammation. B cells can also act as antigen-presenting cells (APC) for self-reactive T cells that initiate inflammatory responses. Hence drugs that target B cells are becoming important tools for treating autoimmune diseases [2].

Most B cells are “B-2 cells” that circulate via the vasculature and traffic through lymphoid organs. Chemoattractant-induced migration and adhesion are critical for this process [3], [4]. B-2 cells enter lymphoid organs via high endothelial venules, which are coated with immobilized chemokines such as CCL19/SLC and CXCL13/BLC [5]. Chemokine receptor signaling activates integrins, which mediate the adhesive interactions required for B cell extravasation. Once in the lymphoid organ, B-2 cells migrate along a CXCL13 gradient to B-cell rich regions called lymphoid follicles. B-2 cells that encounter antigen within a follicle and become activated then move towards T-cell regions, where T cells provide signals required for differentiation into antibody-producing cells. The egress of B cells from lymphoid organs is driven by sphingosine 1-phosphate (S1P), a lipid chemoattractant that is present at high concentrations in the blood [6]. This allows naïve B cells to recirculate and antibody-producing cells to migrate to the bone marrow and other sites. In patients with autoimmune diseases, CXCL13 is also produced in inflamed tissues. This stimulates the formation of ectopic B cell follicles where localized antibody secretion can amplify inflammatory processes [1].

Marginal zone (MZ) B cells are a subset of non-circulating B cells that make rapid T cell-independent polyclonal antibody responses when activated by microbial components [7]. In mice, MZ B cells reside near the marginal sinus of the spleen, where they can rapidly detect blood-borne microbial- and damage-associated molecules. This localization depends on CXCL13, S1P, and integrin-mediated adhesion [3]. The Ig repertoire of MZ B cells is focused on microbial components and self-antigens [7], [8], and MZ B cells have been linked to autoimmunity [9], [10]. In lupus, Grave’s Disease, and type 1 diabetes, MZ B cells increase in number and activated MZ B cells traffic to inflamed sites [11], [12], [13]. In addition to producing autoantibodies, MZ B cells are efficient APCs [14] that can activate self-reactive T cells [13], [14], [15], [16].

Although B cell depletion with monoclonal antibodies (e.g. anti-CD20, anti-CD22) holds promise as a treatment for autoimmune diseases [2], [17], [18], [19], these antibodies do not efficiently deplete MZ B cells [20], [21], perhaps because they do not effectively penetrate into tissues. Small molecules inhibitors of B cell trafficking or activation could therefore be useful adjuncts to antibody-mediated B cell depletion.

Focal adhesion kinase (FAK) and Pyk2 are related cytoplasmic tyrosine kinases that regulate cell migration and adhesion in many cell types [22], [23], [24], [25]. In B cells, Pyk2 and FAK are targets of B cell receptor (BCR), chemokine receptor, and integrin signaling [26], [27], [28], [29], [30], [31]. B-2 cells from Pyk2-deficient mice exhibit impaired in vitro migration towards CXCL12, CXCL13, and CCL21 and MZ B cells are completely absent in these mice [32]. Although this points to a critical role for Pyk2 in the development of MZ B cells, the role of Pyk2 in normal MZ B cells from adult animals has not been investigated. FAK is important for B cell progenitors to migrate towards CXCL12 and adhere to ligands for the α4β1 integrin (very late antigen-4; VLA-4) [33]. However, the role of FAK in mature B cells is not well characterized.

In addition to being probes for the functions of Pyk2 and FAK in B cells, chemical inhibitors of Pyk2 or FAK enzymatic activity could be useful for modulating B cell trafficking or activation. Highly selective, ATP-competitive small molecule inhibitors of FAK, as well as dual Pyk2/FAK inhibitors, have been described [34], [35], [36], [37], [38]. We have previously reported that a dual Pyk2/FAK kinase inhibitor can attenuate the spreading of A20 B-lymphoma cells in response to BCR signaling and integrin engagement [27]. However, the effects of Pyk2- and FAK-selective inhibitors on the migration and adhesion of primary B-2 and MZ B cells have not been examined. In this report we describe the synthesis of a novel Pyk2-selective inhibitor, PF-719, and show that small molecule inhibitors of Pyk2 and FAK can significantly reduce chemoattractant-induced migration, adhesion, and Akt phosphorylation in both B-2 and MZ B cells.

Section snippets

Cells

Spleens were removed from 6 to 12 week old C57BL/6 mice according to protocols approved by the University of British Columbia Animal Care Committee. Splenic B cells were isolated using the MACS B cell isolation kit (Miltenyi Biotec) to deplete non-B cells [39]. The resulting cells were >98% B cells, as determined by staining with anti-CD19-FITC (BD Pharmingen). The A20 B-lymphoma cell line was obtained from ATCC and cultured in RPMI-1640 supplemented with 10% heat-inactivated FCS, 50 μM

Small molecule inhibitors selectively block chemoattractant-induced phosphorylation of Pyk2 and FAK in B cells

Receptor-induced stimulation of Pyk2 and FAK kinase activity involves the phosphorylation of key tyrosine residues [23], [25]. The initial phosphorylation of Pyk2 on Y402 or FAK on Y397, which is thought to occur by autophosphorylation, allows Src family kinases to bind via their SH2 domains to Pyk2 or FAK. The Src kinases then phosphorylate tyrosine resides in the activation loops of Pyk2 (Y579/Y580) or FAK (Y576/Y577), resulting in maximal Pyk2/FAK kinase activity towards substrates. We have

Discussion

In this report we describe the synthesis of a novel Pyk2-selective inhibitor and have used both this compound and a previously reported FAK-selective inhibitor to identify key roles for Pyk2 and FAK in circulating B-2 cells and in MZ B cells, a unique B cell population that has been implicated in autoimmune diseases. The kinase activities of Pyk2 and FAK are regulated by similar phosphorylation-dependent conformational changes and the high degree of homology between the kinase domains of Pyk2

Declaration of interest

A.G-P., G.EA., and L.B. are employees of Pfizer Global Research and Development, which developed the PF-719 and PF-573228 compounds. The other authors have no potential conflict of interest.

Acknowledgment

This work was supported by a Grant from the Canadian Institutes of Health Research (Grant MOP-68865) to M.R.G.

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