1521-0103/367/1/9–19$35.00 https://doi.org/10.1124/jpet.118.249706 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 367:9–19, October 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Distinct Roles of Small GTPases Rac1 and Rac2 in Histamine H4 Receptor–Mediated Chemotaxis of Mast Cells Atsuo Kuramasu, Mie Wakabayashi, Makoto Inui, and Kazuhiko Yanai Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan (A.K., M.I.); and Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan (M.W., K.Y.) Received April 9, 2018; accepted July 12, 2018 ABSTRACT Downloaded from Histamine induces chemotaxis of mast cells through the H4 was involved in chemotaxis through the H4 receptor. In- receptor. However, little is known about the precise intracel- hibition of phosphatidylinositol-4,5-bisphosphate 3-kinase lular signaling pathway that mediates this process. In this (PI3K) by LY294002 suppressed the histamine-induced che- study, we identified small GTPases Rac1 and Rac2 as in- motaxis and activation of Rac GTPases, suggesting that PI3K tracellular binding partners of the H4 receptor and character- regulates chemotaxis upstream of Rac activation. Specific ized their roles in H4 receptor signaling. We showed that knockdown of Rac1 and Rac2 by short-hairpin RNA revealed histamine induced Rac GTPase activation via the H4 receptor. that both Rac GTPases are necessary for histamine-induced jpet.aspetjournals.org A Rac inhibitor NSC23766 attenuated chemotaxis of mast migration. Downregulation of Rac1 and Rac2 led to attenuated cells toward histamine, as well as histamine-induced calcium response in calcium mobilization and ERK activation, respec- mobilization and extracellular signal-regulated kinase (ERK) acti- tively. These observations suggested that Rac1 and Rac2 have vation. Histamine-induced migration of mast cells was also distinct and essential roles in intracellular signaling down- sensitive to PD98059, an inhibitor of the mitogen-activated stream of H4 receptor-PI3K in histamine-induced chemo- protein kinase kinase, indicating that the Rac-ERK pathway taxis of mast cells. at ASPET Journals on September 24, 2021 Introduction storage and chemotaxis toward histamine, both of which are dependent on pertussis toxin–sensitive G protein and phos- Directed migration or chemotaxis of mast cells is an pholipase C (PLC) (Hofstra et al., 2003). Histamine also important process for their recruitment to target tissues in induces interleukin (IL)-6 production in mast cells, which various pathophysiological conditions such as inflammation requires both the phosphatidylinositol-4,5-bisphosphate and allergy. Many chemoattractants including eicosanoids, 3-kinase (PI3K) and extracellular signal-regulated ki- antigens, growth factors, chemokines, and others are known to nase (ERK) pathways downstream of the H receptor (Desai induce chemotaxis of mast cells (Halova et al., 2012). Addi- 4 and Thurmond, 2011). Although many H receptor–related tionally, mast cells themselves produce and release various 4 functions have been reported in various cell types, little is attractants such as histamine to attract other mast cells known about the functional relevance of the intracellular and/or their progenitor cells, resulting in mast cell accumula- signaling pathway downstream of H receptor activation. tion in local tissues (Hofstra et al., 2003). 4 Rac GTPases belong to the Rho subfamily of the Ras small G The histamine H receptor is a G protein-coupled receptor 4 protein superfamily. While Rac1 is widely expressed, Rac2 (GPCR) that is predominantly expressed in immune cells expression is restricted to hematopoietic cells. Rac2-deficient such as mast cells, eosinophils, dendritic cells, monocytes, mice show functional abnormality in various cell types including and T lymphocytes, and plays various roles including migration, shape change, actin polymerization, expression mast cells, neutrophils, macrophages, and lymphocytes, suggest- of surface molecules, and regulation of cytokine production ing that Rac1 and Rac2 have distinct roles (Roberts et al., 1999; (Zampeli and Tiligada, 2009; Thurmond, 2015). In mouse Yang et al., 2000; Croker et al., 2002; Pradip et al., 2003). In mast cells, activation of Rac GTPases is involved in various cell bone marrow–derived mast cells (BMMCs), H4 receptor stimulation induces calcium mobilization from intracellular functions such as migration, polarity, adhesion, cell cycle, and transcriptional regulation (Massol et al., 1998; Timokhina et al., 1998; Yang et al., 2000; Gu et al., 2002; Samayawardhena et al., This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science [Grant 19390061]. 2007). Typically, Rac activation in these events occurs down- https://doi.org/10.1124/jpet.118.249706. stream of the cytokine or immunoglobulin E receptor. However, ABBREVIATIONS: BMMC, bone marrow–derived mast cell; BSA, bovine serum albumin; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; GEF, guanine nucleotide exchange factor; GPCR, G protein-coupled receptor; GST, glutathione S-transferase; IL, interleukin; IP3, 1,4,5- triphosphate; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MBP, maltose binding protein; MEK, mitogen-activated protein kinase kinase; mH4RCT, mouse H4 receptor carboxy-terminal tail; PAK1, p21-activated kinase; PBD, p21 binding domain; PH, pleckstrin homology; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PLC, phospholipase C; shRNA, short-hairpin RNA. 9 10 Kuramasu et al. little is known about Rac GTPase activation downstream of penicillin-100 mg/ml streptomycin, and 5 ng/ml recombinant mouse GPCRs such as the histamine H4 receptor in mast cells. IL-3 (BioLegend). After 24 hours of culture, floating cells were trans- Accumulating evidence has indicated that the carboxy- ferred to a new dish, and cell medium was replaced every 5–7 days. terminal cytoplasmic domain of the GPCR interacts with Differentiation into mast cells was confirmed by the expression of various intracellular proteins that may have important Fc«RI and CD117 (c-kit) as assessed by flow cytometry. After 4–12 weeks in culture, the BMMCs were used for the experiments. functions, such as receptor targeting, clustering of receptor Affinity Purification and Nano-Liquid Chromatography– with various effector molecules, and modulation of signaling Tandem Mass Spectrometry. Cleared lysate of Escherichia coli efficiency (Bockaert et al., 2003). Our previous work also expressing GST or GST-fused mouse H4 receptor carboxy-terminal tail showed proteins that interact with the carboxy-terminal [(mH4RCT), i.e., GST-mH4RCT] was bound to Glutathione Sepharose 7 domain of histamine H2 and H3 receptors have essential roles 4B beads (GE Healthcare). BMMCs (6 Â 10 cells) were lysed in lysis in receptor trafficking and signal transduction (Maeda et al., buffer [50 mM Tris-HCl pH 7.4, 100 mM NaCl, 10% glycerol, 1 mM 2008; Xu et al., 2008). dithiothreitol (DTT), 1 mM EGTA, 1 mM EDTA, 0.5% Triton X-100, with Roche Complete protease inhibitor cocktail (Roche, Basel, In this study, to further characterize H4 receptor signaling, we determined proteins that interacted with the carboxy- Switzerland)] at 4°C on a rotator overnight and centrifuged at terminal domain of the H receptor. We showed that Rac1 15,000g for 15 minutes at 4°C. The supernatant was applied to the 4 GST column and the flow-through was then applied to the GST- and Rac2 GTPases physically associated with the H receptor. 4 mH4RCT column. Both columns were washed extensively and bound Furthermore, these proteins were also functionally coupled proteins were eluted with elution buffer (50 mM Tris-HCl pH 7.4, 1 M Downloaded from with H4 receptor regulation of histamine-induced chemotaxis of NaCl, 10% glycerol, 1 mM DTT, 1 mM EGTA, 1 mM EDTA, 0.5% mast cells. Thus, our results indicated that Rac1 and Rac2 have Triton X-100). The proteins were precipitated with ice-cold distinct intracellular roles: Rac1 regulates histamine-induced trichloroacetic acid (final concentration of 10%) and centrifuged at calcium mobilization and Rac2 controls ERK activation. 15,000g for 10 minutes at 4°C. The pellet was washed with ether/ ethanol (1:1) and dissolved in SDS-PAGE sample buffer. The samples were resolved on a 10%–20% gradient SDS-PAGE gel and stained Materials and Methods using the silver stain mass spectrometry kit (Wako). Excised gel bands jpet.aspetjournals.org were reduced with 100 mM DTT and alkylated with 100 mM Reagents and Antibodies. Histamine dihydrochloride, pyril- iodoacetamide. After washing, the gel pieces were incubated with amine maleate, and cimetidine were obtained from Sigma (St. Louis, trypsin overnight at 30°C. Recovered peptides were desalted with MO). JNJ7777120 and NSC23766 were obtained from Tocris Biosci- Ziptip c18 (Millipore). Samples were analyzed by nano-liquid ence (Bristol, United Kingdom). PD98059, LY294002, and phospho- chromatography–tandem mass spectrometry systems (DiNa HPLC; p44/42 mitogen-activated protein kinase (MAPK) (ERK1/2) KYA TECH Corporation, Tokyo, Japan) and QSTAR XL (Applied (Thr202/Tyr204) antibody were obtained from Cell Signaling Tech- Biosystems, Foster City, CA). Mass data acquisitions were piloted by nology (Tokyo, Japan). SP600125 was obtained from Abcam (Tokyo, the Mascot software (Kanno et al., 2007). at