PLCe1 regulates SDF-1α–induced lymphocyte adhesion and migration to sites of inflammation Marianne Strazzaa, Inbar Azoulay-Alfagutera, Michael Peleda, Alan V. Smrckab, Edward Y. Skolnika,c,d, Shekhar Srivastavad, and Adam Mora,e,f,1 aDepartment of Medicine, New York University School of Medicine, New York, NY 10016; bDepartment of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642; cDepartment of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016; dThe Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute for Biomolecular Medicine, New York University School of Medicine, New York, NY 10016; eDepartment of Pathology, New York University School of Medicine, New York, NY 10016; and fPerlmutter Cancer Center, New York University School of Medicine, New York, NY 10016 Edited by Jason G. Cyster, University of California, San Francisco, CA, and approved January 25, 2017 (received for review August 3, 2016) Regulation of integrins is critical for lymphocyte adhesion to endo- sensitivity, we conclude that PLCe1 plays an essential role in T-cell thelium and migration throughout the body. Inside-out signaling to recruitment to the site of inflammation. These findings suggest that integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). PLCe1 is a potential target in treating inflammatory conditions. Using an RNA-mediated interference screen, we identified phospho- lipase Ce 1(PLCe1) as a crucial regulator of stromal cell-derived factor Results 1 alpha (SDF-1α)-induced Rap1 activation. We have shown that SDF- PLCe1 Is Required for SDF-1α–Induced Rap1 Activation. To analyze 1α-induced activation of Rap1 is transient in comparison with the Rap1 activation, we transfected Jurkat T cells with a GFP-tagged sustained level following cross-linking of the antigen receptor. We version of the Ras-binding domain of Ral guanine nucleotide dis- identified that PLCe1 was necessary for SDF-1α-induced adhesion sociation stimulator (GFP–Ral–GDS–RBD). When Rap1 is GDP using shear stress, cell morphology alterations, and crawling on in- bound (inactive), GFP–Ral–GDS–RBD is cytosolic; however, when tercellular adhesion molecule 1 (ICAM-1)–expressing cells. Structure– Rap1 is GTP bound (active) it binds to the sensor, mainly at plasma function experiments to separate the dual-enzymatic function of membrane. Therefore, GFP–Ral–GDS–RBDactsasanindicatorof PLCe1 uncover necessary contributions of the CDC25, Pleckstrin Rap1 activation that can be analyzed microscopically and quantified homology, and Ras-associating domains, but not phospholipase by fluorescent line analysis (Fig. S1A). Upon stimulation with anti- activity, to this pathway. In the mouse model of delayed type hy- CD3 or SDF-1α, Rap1 was activated at the plasma membrane (Fig. persensitivity, we have shown an essential role for PLCe1 in T-cell 1A). Similar results were also obtained with a cherry-tagged version migration to inflamed skin, but not for cytokine secretion and pro- of the Ras-associating and Pleckstrin homology domains of Rap1- liferation in regional lymph nodes. Our results reveal a signaling GTP–interacting adaptor molecule (cherry–RIAM–RAPH), an al- pathway where SDF-1α induces T-cell adhesion through activation ternative biosensor (Fig. S1B) (7). In primary human T cells, Rap1 of PLCe1, suggesting that PLCe1 is a specific potential target in treat- GST pull-down assay shows that Rap1–GTP is increased nearly ing conditions involving migration of T cells to inflamed organs. twofold (Fig. 1B) upon stimulation with SDF-1α.Wenextques- INFLAMMATION tioned whether the kinetics of Rap1 activation downstream of the IMMUNOLOGY AND Rap1 | adhesion | T cells | PLCe1 | SDF-1α TCR and CXCR4 were distinct. Following anti-CD3 stimulation Rap1-GTP remains elevated throughout the 30 min (Fig. 1C and -cell adhesion contributes to routine immune surveillance and Fig. S2A). In contrast, Rap1-GTP levels following SDF-1α have two Tgenerating inflammatory responses. The interaction between a T distinct peaks (Fig. 1C and Fig. S2A). The differences in kinetics in cell and an antigen presenting cell (APC) requires an adhesion these two cascades suggest differential regulation at the level of complex that is stable long term; relative to each other, this in- teraction is static (1). Here, the signaling cascade leading to integrin Significance activation is initiated through the T-cell receptor (TCR) and coreceptors (2). Alternatively, T-cell trafficking and extravasation We performed a screen to identify unique proteins regulating require a more dynamic, firm adhesion between T cells and T-cell adhesion downstream of the chemokine receptor CXCR4. We endothelial cells of blood vessel walls. Chemokines are largely re- identified that the guanine exchange factor mediating stromal sponsible for orchestrating T-cell trafficking throughout the body. cell-derived factor 1 alpha (SDF-1α)/C-X-C chemokine receptor type The localized expression of chemokines on endothelia, and che- 4 (CXCR4) activation is phospholipase Ce 1(PLCe1). Experiments to mokine receptor expression patterns on T-cell populations, give rise separate the dual-enzymatic function of PLCe1 uncover necessary to a network of T-cell recruitment with signatures for anatomical contributions of the CDC25, Pleckstrin homology, and Ras-asso- compartments or disease states (3). Whereas much work has been ciating domains, but not phospholipase activity, to its guanine aimed at establishing these signatures, the same cannot be said for exchange activity. In the mouse model of contact sensitivity, we elucidating the signaling cascades downstream of chemokine re- have shown a necessary role for PLCe1 in T-cell recruitment to the ceptors. The small GTPase Ras-proximate-1 or Ras-related protein site of secondary challenge, but not for priming after antigen 1 (Rap1) and its effectors are necessary for integrin activation and, sensitization. We uncover a signaling pathway that mediates therefore, T-cell adhesion (4). Rap1 is active in the GTP-bound SDF-1α–induced T-cell adhesion and suggest that PLCe1isa form following interaction with a guanine exchange factor (GEF) potential specific target for future antiinflammatory drugs. and inactive in the GDP-bound form following interaction with a e e GTPase activating protein (GAP). Phospholipase C 1(PLC 1) is a Author contributions: M.S. and A.M. designed research; M.S., E.Y.S., S.S., and A.M. per- protein with dual enzymatic functions of a lipase and a GEF (5, 6). formed research; M.P. and A.V.S. contributed new reagents/analytic tools; M.S., I.A.-A., Here we show that T-cell adhesion induced by the stromal cell- M.P., and A.M. analyzed data; and M.S. and A.M. wrote the paper. derived factor 1 alpha (SDF-1α)/C-X-C chemokine receptor type 4 The authors declare no conflict of interest. (CXCR4) axis involves Rap1 activation, exclusively mediated by This article is a PNAS Direct Submission. PLCe1. We provide evidence that the lipase function of PLCe1 1To whom correspondence should be addressed. Email: [email protected]. does not contribute to the activation of adhesion in this pathway. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Through expanding these studies to a mouse model of contact 1073/pnas.1612900114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1612900114 PNAS | March 7, 2017 | vol. 114 | no. 10 | 2693–2698 Downloaded by guest on September 28, 2021 0.63 to 3.4 ± 0.42 μm(Fig.2A). In contrast, PLCe1 knockdown ABUntreated Anti-CD3 SDF-1 2.5 2.0 ** cells fail to arrest, with average distance traveled per cell de- 1.5 creasing only slightly from 11.0 ± 0.91 to 8.2 ± 1.14 μm (Fig. 2A). 1.0 This result suggests that PLCe1 may play a role in adhesion and GTP-Rap1 0.5 GFP-RalGDS-RBD Fold change in 0 crawling. To further explore this functional defect, an assay of firm C 22 kDa GTP-Rap1 adhesion to ICAM-1 using shear stress conditions was employed 4 22 kDa Total Rap1 Untreated SDF-1 (9–11). While SDF-1α induces an increase in adhesion of control 3 D 0.331 0.03 T cells, there is no observed adhesion in PLCe1 knockdown T cells 2 250 kDa PLC 1 (Fig. S3A and Fig. 2B), providing evidence of an essential role for 1 42 kDa Actin PLCe1inSDF-1α–induced firm adhesion. Calculating the circu- 0 shScr shPLC 1 0 10 20 30 larity index before and after SDF-1α stimulation provides evi- compared to untreated Time (minutes) Fold change in GTP-Rap1 F Anti-CD3 SDF-1 dence that PLCe1 knockdown cells are defective in adopting a E “crawling morphology” and maintain a circularity index (Fig. 2C) Untreated Anti-CD3 SDF-1 Untreated Anti-CD3 SDF-1 1.5 22 kDa and ratio of Ferets near 1 (Fig. S3B). PLCe1-deficient primary 1.0 22 kDa human T cells are also less responsive to SDF-1α (Fig. 2D). PLCe1 0.5 ** ** *** shScr shPLC 1 knockdown did not induce any alteration in the level of surface 4 E 0 * CXCR4 expression (Fig. 2 ). Collectively, these results suggest 1 3 #1 #2 Fold change in ddct that PLCe1 plays an essential role in T-cell firm adhesion induced siScr shScr 2 #1/#2 shScr shPLC 1 α siPLC by SDF-1 . shPLC 1 0 e Anti-CD3 SDF-1 The Lipase Function of PLC 1 Is Not Required to Activate Rap1 compared to untreated Fold change in GTP-Rap1 downstream of SDF-1α. PLCe1 functions as a phospholipase and a e Fig. 1. PLCe1 is required for SDF-1α–induced Rap1 activation. (A) Jurkat T cells GEF. To determine whether the phospholipase activity of PLC 1 transfected with GFP–Ral–GDS–RBD; stimulation with soluble anti-CD3 (5 μg/mL) is induced by SDF-1α stimulation, we examined diacylglycerol or SDF-1α (100 ng/mL).