EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 38, No. 3, 191-195, June 2006

The role of the α4 - interaction in regulating leukocyte trafficking

David M. Rose 20 yrs a paradigm has developed in which the control of leukocyte trafficking is modeled after the Department of Medicine mail zip code system; namely, specific combinations University of California/VA Medical Center of adhesion and chemoattractant molecules ex- San Diego, CA, USA pressed both on leukocytes and the vascular en- Correspondence: Tel, 858-552-8585 ext. 5378; dothelium direct the temporal and spatial recruit- Fax, 858-552-7425; E-mail, [email protected] ment of leukocytes around the body (Butcher, 1991; Springer, 1994). A comprehensive review of leuko- Abbreviations: Arf, ADP-ribosylation-factor; FAK, focal adhesion cyte trafficking is beyond the scope of this review, kinase; GIT, G -coupled receptor kinase interactor; PKA, but the reader is directed to recent reviews (Butcher, protein kinase A; PKL, paxillin kinase linker; PML, progressive 1991; Springer, 1994; Luster et al., 2005). Instead, multifocal leukoencephalophathy; PTP-PEST, protein tyrosine this review focuses on recent work defining the role phosphatase-PEST; Pyk2, proline-rich tyrosine kinase of the molecule 4 1 on leukocyte trafficking and in particular the importance of the interaction of the signaling adapter molecule paxillin Abstract with the 4 subunit in regulating 4 1-dependent functions. The movement of leukocytes from the blood into In general, the movement of leukocytes from the peripheral tissues is a central feature of immune blood into tissues involves three sequential steps surveillance, but also contributes to the patho- (Butcher, 1991; Springer, 1994). First, leukocytes genesis of inflammatory and autoimmune diseases. make transient tethering adhesions with the vascular are a family of adhesion and signaling endothelium, which under shear flow conditions molecules made up of paired α and β subunits, and results in leukocyte rolling along the endothelial the integrin α4β1 plays a prominent role in the surface. Second, should the leukocyte encounter an trafficking of mononuclear leukocytes. We have appropriate stimulus, it undergoes activation and establishes firm adhesion to the endothelium. Third, previously described the direct interaction of the the leukocyte migrates across the endothelium into signaling adaptor molecule paxillin with the cy- the underlying tissue. Integrins are a family of cell toplasmic domain of the α4 integrin subunit. This surface adhesion and signaling molecules made up interaction is critical for α4β1 integrin dependent of non-covalently associated and subunits that cell adhesion under shear flow conditions as it play a critical role in leukocyte trafficking (Luster et provides a needed connection to the actin cyto- al., 2005). The integrin 4 1, the focus of this skeleton. Furthermore, the α4-paxillin interaction is review, is rather unique among integrins as it can required for effective α4β1 dependent leukocyte function in all three steps of leukocyte trafficking migration and does so through the temporal and (Berlin et al., 1995; Rose et al., 2002). 4 1 can be spatial regulation of the small GTPase Rac. These found on virtually all circulating mononuclear leu- findings make the α4-paxillin interaction a poten- kocytes, but is not typically seen on unactivated tially attractive therapeutic target in controlling (Rose et al., 2002). Integrin subunits leukocyte trafficking. have relatively short cytoplasmic domains, but they can associate with numerous allowing linkage to the cytoskeleton and formation of sig- Keywords: 4 integrins; autoimmune diseases; leu- kocyte trafficking; paxillin naling complexes (Liu et al., 2000a). Both interac- tions are critical to integrin-dependent cell adhesion and migration. One such integrin-protein interaction Introduction is the rather unique association of the 4 integrin subunit with the signaling adaptor molecule paxillin Leukocyte trafficking is the process by which cir- (Liu et al., 1999). culating leukocytes in blood leave and enter the Paxillin is a wildly expressed 68 kD cytoplasmic peripheral tissues. This event is critical for both im- protein that can be found associated with cell ad- mune surveillance and inflammation. Over the past hesion complexes (Brown and Turner, 2004). It was 192 Exp. Mol. Med. Vol. 38(3), 191-195, 2006 one of the first signaling adaptor molecules de- 2005). Talin has been established to be a key scribed; it has no intrinsic kinase or phosphatase molecule regulating integrin affinity for ligands (a activity yet it can scaffold a number of signaling process termed inside-out signaling) (Tadokoro et molecules such as kinases, other adaptor proteins, al., 2003). However, the role of talin in 4-paxillin and nucleotide exchange factors as well as binding dependent tethering does not appear to involve 4 cytoskeleton proteins (Figure 1). The binding of integrin affinity modulation (Rose et al., 2003). First, these signaling molecules to paxillin is largely cells expressing 4 with disrupted paxillin binding mediated by the presence of 5 leucine-rich LD had no defect in 4 1 integrin affinity for its ligand domains in the N-terminus and 4 zinc finger LIM VCAM-1 (Rose et al., 2003). Second, ligand-in- domains in paxillin’s C-terminus (Brown and Turner, duced conformation changes in 4 1 were not 2004). The 4 integrin binding site on paxillin has altered by disrupting the 4-paxillin interaction nor been partially localized to a 100 amino acid stretch were conformational changes altered by knocking encompassing LD3 and LD4 (Liu et al., 2002). The down talin levels (Alon et al., 2005). Thus, the role of region of the cytoplasmic domain of 4 integrin that the 4-paxillin interaction in mediating leukocyte mediates paxillin binding rests in the nine amino tethering is through establishment of associations acids between Glu983 and Tyr991 (Liu et al., 2000b). with the cytoskeleton to strengthen adhesion under Mutation of this Tyr991 to an Ala disrupts the 4- shear flow conditions. paxillin interaction (Liu et al., 1999). Further, a serine The 4-paxillin interaction is also important in at position 988 can be reversibly phosphorylated in a establishing firm cell adhesion under shear stress PKA-dependent fashion to regulate paxillin binding conditions (Alon et al., 2005). This is in contrast to to 4 (Han et al., 2003). The temporal and spatial static cell adhesion, which is unaltered by disrupting regulation of this serine phosphorylation and asso- the 4-paxillin interaction (Rose et al., 2003). Again, ciated paxillin binding to 4 is critical in effective cell the mechanism underlying this cell adhesion streng- migration (discussed below) (Goldfinger et al., thening under shear flow conditions is associated 2003). with 4-paxillin-dependent connections with the actin cytoskeleton. Further details on the esta- blishment of these connections between the 4 The α4 integrin-paxillin interaction in cell integrin and the actin cytoskeleton are lacking. The adhesion under shear flow 4-paxillin interaction is known to regulate focal adhesion dynamics through FAK and Pyk-2 acti- The 4 1 integrin is one of the few integrins that can vation as well as activation of small GTPases such support tethering and rolling of leukocytes under as Rac, CDC42, and Rho (Rose et al., 2003). But shear flow conditions, a process largely associated these signaling molecules do not appear to be with the family of adhesion molecules (Alon, involved in the early events in adhesion under shear et al., 1995; Rose et al., 2002; Rosen, 2004). While stress (Alon et al., 2005). initial studies with a truncation of the 4 cytoplasmic domain suggested this region was not involved in regulating tethering interactions, it has become clear that such a truncation may have secondarily altered 1 integrin function masking the role of the 4 cytoplasmic domain in supporting tethering adhesion under flow conditions (Kassner et al., 1995). We recently have demonstrated that the 4-paxillin interaction is critical to establishing tethering ad- hesion, and that adhesion strength is mediated thru connections with the actin cytoskeleton (Alon et al., 2005). In particular, paxillin binding to the cyto- skeletal protein talin is central in linking the 4 integrin with the actin cytoskeleton (Alon et al., 2005). Jurkat cells expressing 4Y991A with dis- rupted paxillin binding to 4 had reduced tethering Figure 1. Paxillin associated proteins. Paxillin is comprised of 5 LD bond strength associated with reduced talin in the motifs in its N-terminus and 4 LIM domains in its C-terminus. These re- 4 integrin adhesion complex (Alon et al., 2005). gions serve as primary docking sites for a number of signaling and cy- toskeletal proteins (not all of which are shown in this figure). Binding Furthermore, knocking-down talin expression in partners include kinases such as FAK, Pyk2 and Src as well as phos- Jurkat cells expressing 4 WT integrin impaired phatases such as PTP-PEST. Cytoskeletal proteins binding to paxillin 4-dependent tethering under shear flow (Alon et al., include vinculin and tubulin. α4 integrin-paxillin interaction in leukocyte trafficking 193

Figure 2. Dynamic regulation of paxillin binding to the 4 integrin subunit by serine phosphorylation of 4 regulates spacial activation of the small GTPase Rac in migrating cells. Paxillin associates with the 4 integrin when the serine at position 988 of the 4 cy- toplasmic domain is unphosphorylated. This 4-pax- illin complex recruits the GTPase activating protein GIT1, which subsequently inhibits Arf6 activation and ultimately inhibits Rac activation. Inhibition of Rac prevents lamellipodia formation at the lateral and tail- ing edge of a migrating cell. Phosphorylation of 4 serine 988 inhibits paxillin binding and removes the suppression of Rac activation, ultimately resulting in lamellipodia formation at the leading edge of the mi- grating cell.

The α4 integrin-paxillin interaction in cell, the 4 subunit is phosphorylated resulting in regulating cell migration disruption of the 4-paxillin interaction (Goldfinger et al., 2003) (Figure 2). This results in the removal of After a leukocyte has firmly arrested on the vascular the 4-paxillin-dependent inhibition of Rac activation endothelium, it then migrates laterally to the junction at this site (Nishiya et al., 2005). It has recently been of two or more endothelial cells, where it may proposed that the ultimate activation of Rac by 4 migrate in between these cells into the underlying integrins proceeds through a pathway dependent on tissue. 4 integrins play roles in both these migra- Src activation (Hsia et al., 2005). Src activation tory steps of leukocyte trafficking (Weber and subsequently leads to Rac activation through the Springer, 1998). Recently, much has been learned activation of the Rac exchange factor DOCK180. about the coordinated signaling involved in cell mig- In addition to functioning as a direct adhesion ration, and the role of the 4-paxillin interaction in 4 molecule mediating cell migration, 4 integrins can integrin-dependent cell migration. Cell migration also function as a signaling molecule augmenting involves a dynamic regulation of cell adhesion in cell migration mediated by other integrins in a trans- which new adhesions are formed at the leading dominant fashion (Rose et al., 2001). 4 1 integrins edge of the cell where lamellipodia are sent out as can stimulate L 2 dependent cell migration, and new projections and adhesions are released from this requires the 4-paxillin interaction (Rose et al., the trailing edge of the cell as the body of the cell is 2001; Rose et al., 2003). This transdominant re- propelled forward (Ridley et al., 2003). gulation is mediated through the 4-paxillin depen- Spatial and temporal regulation of the small dent activation of the nonreceptor tyrosine kinases GTPase Rac is central to effective cell migration FAK and Pyk-2 (Rose et al., 2003). Furthermore, the such that Rac is activated at the leading edge of the affinity of 4 1 also plays a role in stimulating cell promoting lamellipodia formation, while Rac L 2-dependent cell migration as 4 1 in the high activation is inhibited at the lateral and trailing edge affinity state favors interactions with VCAM-1 to drive of the cell (Nobes and Hall, 1999). The 4 integrin 2 integrin-dependent migration (Rose et al., 2001). subunit is dephosphorylated at Ser988 at the lateral In addition, high affinity 4 1 preferentially asso- and trailing edge of a migrating cell, which is ciates with paxillin (Hyduk et al., 2004). Thus, while associated with paxillin binding to 4 at these paxillin does not regulate the affinity of 4 1, its locations (Goldfinger et al., 2003) (Figure 2). At association with high affinity 4 1 contributes to the these sites, paxillin binding to 4 blocks lamellipodia cross-talk with 2 integrin to drive cell migration. formation by inhibiting Rac activation (Nishiya et al., 2005). This occurs as paxillin recruits the GTPase activating protein GIT1 (Nishiya et al., 2005). GIT1 Therapeutic potential of targeting the α4 inhibits Arf6, another member of the Ras small integrin-paxillin interaction GTPase family. The inhibition of Arf6 subsequently inhibits the activation of Rac by yet unknown Given the role of the 4 integrins in trafficking of mechanisms. leukocytes, they have been an attractive target in the In contrast, at the leading edge of the migrating treatment of numerous chronic inflammatory and 194 Exp. Mol. Med. Vol. 38(3), 191-195, 2006 autoimmune diseases such as multiple sclerosis, Alon R, Kassner PD, Carr MW, Finger EB, Hemler ME, inflammatory bowel disease, rheumatoid arthritis, Springer TA. The integrin VLA-4 supports tethering and rolling and atherosclerosis (Gonzalez-Amaro, et al., 2005). in flow on VCAM-1. J Cell Biol 1995;128:1243-53 Indeed, inhibition of 4 integrins with function Ambroise Y, Yaspan B, Ginsberg MH, Boger DL. Inhibitors of blocking antibodies and small molecule inhibitors cell migration that inhibit intracellular paxillin/alpha4 binding: has been effective in attenuating many mouse a well-documented use of positional scanning libraries. Chem models of these diseases (Gonzalez-Amaro, et al., Biol 2002;9:1219-26 2005). Further, a humanized function-blocking anti- Berlin C, Bargatze RF, Campbell JJ, von Andrian UH, Szabo body to 4 has been shown to be effective in clinical MC, Hasslen SR, Nelson RD, Berg EL, Erlandsen SL, Butcher trials of patients with multiple sclerosis and in- EC. alpha 4 integrins mediate lymphocyte attachment and roll- flammatory bowel disease (Miller et al., 2003; Ghosh ing under physiologic flow. Cell 1995;30:413-22 et al., 2003). However, given the role of 4 integrins Brown MC, Turner CE. Paxillin: adapting to change. Physiol in normal physiological functions such as he- Rev 2004;84:1315-39 matopoiesis and immune surveillance, the risk of Butcher EC. Leukocyte-endothelial cell recognition: three (or side-effects of 4 integrin blockage has been a more) steps to specificity and diversity. Cell 1991;67:1033-6 concern. The development of progressive multifocal leukoencephalophathy (PML) in three patients treat- Feral CC, Rose DM, Han J, Fox N, Silverman GJ, Kaushansky K, Ginsberg MH. Blocking the alpha 4 integrin-paxillin inter- ed with anti- 4 antibodies has highlighted this action selectively impairs mononuclear leukocyte recruitment concern as PML occurs most commonly due to to an inflammatory site. J Clin Invest 2006;116:715-23 reemergence of a latent JC virus infection in the central nervous system in immune suppressed Ghosh S, Goldin E, Gordon FH, Malchow HA, Rask-Madsen individuals (Yousry et al., 2006). Consequently, a J, Rutgeerts P, Vyhnalek P, Zadorova Z, Palmer T, Donoghue S; Pan-European Study Group. Natalizumab for more selective targeting of 4 integrins to inhibit active Crohn’s disease. N Engl J Med 2003;348:24-32 leukocyte trafficking while sparing its function in normal physiology may be therapeutically beneficial. Goldfinger LE, Han J, Kiosses WB, Howe AK, Ginsberg MH. To this aim, targeting the 4-paxillin interaction may Spatial restriction of alpha4 integrin phosphorylation regulates be one such possibility. A screening of a com- lamellipodial stability and alpha4beta1-dependent migration. J Cell Biol 2003;162:731-41 binatorial library has successfully identified a small molecule inhibitor of the 4-paxillin interaction in Gonzalez-Amaro R, Mittlebrunn M, Sanchez-Madrid F. Ther- vitro that inhibits cell migration mediated by 4 1 apeutic anti-integrin (alpha4 and alphaL) monoclonal anti- (Ambroise et al., 2002). bodies: two-edged swords? 2005;116:289-96 Further support of the proof of principle of Han J, Rose DM, Woodside DG, Goldfinger LE, Ginsberg MH. targeting the 4-paxillin interaction comes from re- beta 1-dependent T cell migration requires cent studies of a novel knock-in mouse in which the both phosphorylation and dephosphorylation of the alpha 4 cy- Y991A mutation that disrupts the 4-paxillin interac- toplasmic domain to regulate the reversible binding of paxillin. tion has been introduced into the mouse genome J Biol Chem 2003;278:34845-53 (Feral et al., 2006). Such mice are born viable with Hsia DA, Lim ST, Bernard-Trifilo JA, Mitra SK, Tanaka S, den no defect in placentation or heart development, Hertog J, Streblow DN, Ilic D, Ginsberg MH, Schlaepfer DD. which occurred in mice with complete ablation Integrin alpha4beta1 promotes focal adhesion kinase-in- of 4 integrin (Feral et al., 2006). Furthermore, these dependent cell motility via alpha4 cytoplasmic domain-specif- ic activation of c-Src. Mol Cell Biol 2005;25:9700-12 mice have no defect in hematopoiesis or immune system development. But importantly, they show Hyduk SJ, Oh J, Xiao H, Chen M, Cybulsky MI. Paxillin se- impairment in the recruitment of mononuclear leuko- lectively associates with constitutive and chemoattrac- cytes to a site of inflammation (Feral et al., 2006). tant-induced high-affinty alpha4beta1 integrins: implications for integrin signaling. Blood 2004;104:2818-24 Thus, targeting the 4-paxillin interaction may have the potential to impair leukocyte trafficking asso- Kassner PD, Alon R, Springer TA, Hemler ME. Specialized ciated with chronic inflammatory diseases, while functional properties of the integrin alpha 4 cytoplasmic sparing 4 integrin function in normal physiology. domain. Mol Biol Cell 1995;6:661-74 Liu S, Calderwood DA, Ginsberg MH. Integrin cytoplasmic do- References main-binding proteins. J Cell Sci 2000a;113:3563-71 Liu S, Ginsberg MH. Paxillin binding to a conserved sequence Alon R, Feigelson SW, Manevich E, Rose DM, Schmitz J, motif in the alpha 4 integrin cytoplasmic domain. J Biol Chem Overby DR, Winter E, Grabovsky V, Shinder V, Matthews BD, 2000b;274:22736-42 Sokolovsky-Eisenberg M, Ingber DE, Benoit M, Ginsberg MH. Alpha4beta1-dependent adhesion strengthening under me- Liu S, Kiosses WB, Rose DM, Slepak M, Salgia R, Griffin JD, chanical strain is regulated by paxillin association with the al- Turner CE, Schwartz MA, Ginsberg MH. A fragment of paxillin pha4-cytoplasmic domain. J Cell Biol 2005;171:1073-84 binds the alpha 4 integrin cytoplasmic domain (tail) and se- α4 integrin-paxillin interaction in leukocyte trafficking 195 lectively inhibits alpha 4-mediated cell migration. J Biol Chem Rose DM, Han J, Ginsberg MH. Alpha4 integrins and the im- 2002;277:20887-94 mune response. Immunol Rev 2002;186:118-24 Liu S, Thomas SM, Woodside DG, Rose DM, Kiosses WB, Rose DM, Liu S, Woodside DG, Han J, Schlaepfer DD, Pfaff M, Ginsberg MH. Binding of paxillin to alpha4 integrins Ginsberg MH. Paxillin binding to the alpha 4 integrin subunit modifies integrin-dependent biological responses. Nature stimulates LFA-1 ( beta 2)-dependent T cell mi- 1999;402:676-81 gration by augmenting the activation of focal adhesion kin- Luster AD, Alon R, von Andrian UH. Immune cell migration in ase/proline-rich tyrosine kinase-2. J Immunol 2003;170: inflammation: present and future therapeutic targets. Nat 5912-8 Immunol 2005;6:1182-90 Rosen SD. Ligands for L-selectin: homing, inflammation, and Miller DH, Khan OA, Sheremata WA, Blumhardt LD, Rice GP, beyond. Annu Rev Immunol 2004;22:129-56 Libonati MA, Willmer-Hulme AJ, Dalton CM, Miszkiel KA, Springer TA. Traffic signals for lymphocyte recirculation and O'Connor PW; International Natalizumab Multiple Sclerosis leukocyte emigration: the multistep paradigm. Cell 1994;76: Trial Group. A controlled trial of natalizumab for relapsing mul- 301-14 tiple sclerosis. N Engl J Med 2003;348:15-23 Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, de Pereda Nishiya N, Kiosses WB, Han J, Ginsberg MH. An alpha4 in- JM, Ginsberg MH, Calderwood DA. Talin binding to integrin be- tegrin-paxillin-Arf-GAP complex restricts Rac activation to the ta tails: a final common step in integrin activation. Science leading of migrating cells. Nat Cell Biol 2005;7:343-52 2003;302:103-6 Nobes CD, Hall A. Rho GTPases control plarity, protrusion, Weber C, Springer TA. Interaction of very late antigen-4 with and adhesion during cell movement. J Cell Biol 1999; VCAM-1 supports transendothelial chemotaxis of monocytes 144:1235-44 by facilitating lateral migration. J Immunol 1998;161:6825-34 Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Yousry TA, Major EO, Ryschkewitsch C, Fahle G, Fischer S, Borisy G, Parsons JT, Horwitz AR. Cell migration: integrating Hou J, Curfman B, Miszkiel K, Mueller-Lenke N, Sanchez E, signals from front to back. Science 2003;302:1704-9 Barkhof F, Radue EW, Jager HR, Clifford DB. Evaluation of pa- Rose DM, Grabovsky V, Alon R, Ginsberg MH. The affinity of tients treated with natalizumab for progressive multifocal integrin alpha(4)beta(1) governs lymphocyte migration. J leukoencephalopathy. N Engl J Med 2006;354:924-33 Immunol 2001;167:2824-30