| 1. |
Kenji Morino, Kazufumi Kunimura, Yuki Sugiura, Yoshihiro Izumi, Keisuke Matsubara, Sayaka Akiyoshi, Rae Maeda, Kenichiro Hirotani, Daiji Sakata, Seiya Mizuno, Satoru Takahashi, Takeshi Bamba, Takehito Uruno, Yoshinori Fukui, Cholesterol sulfate limits neutrophil recruitment and gut inflammation during mucosal injury., Frontiers in immunology, 10.3389/fimmu.2023.1131146, 14, 1131146-1131146, 2023.03, During mucosal injury, intestinal immune cells play a crucial role in eliminating invading bacteria. However, as the excessive accumulation of immune cells promotes inflammation and delays tissue repair, it is essential to identify the mechanism that limits the infiltration of immune cells to the mucosal-luminal interface. Cholesterol sulfate (CS) is the lipid product of the sulfotransferase SULT2B1 and suppresses immune reactions by inhibiting DOCK2-mediated Rac activation. In this study, we aimed to elucidate the physiological role of CS in the intestinal tract. We found that, in the small intestine and colon, CS is predominantly produced in the epithelial cells close to the lumen. While dextran sodium sulfate (DSS)-induced colitis was exacerbated in Sult2b1-deficient mice with increased prevalence of neutrophils, the elimination of either neutrophils or intestinal bacteria in Sult2b1-deficient mice attenuated disease development. Similar results were obtained when the Dock2 was genetically deleted in Sult2b1-deficient mice. In addition, we also show that indomethacin-induced ulcer formation in the small intestine was exacerbated in Sult2b1-deficient mice and was ameliorated by CS administration. Thus, our results uncover that CS acts on inflammatory neutrophils, and prevents excessive gut inflammation by inhibiting the Rac activator DOCK2. The administration of CS may be a novel therapeutic strategy for inflammatory bowel disease and non-steroidal anti-inflammatory drug-induced ulcers.. |
| 2. |
Kazufumi Kunimura, Kazuhiko Yamamura, Takeshi Nakahara, Makiko Kido-Nakahara, Takehito Uruno, Yoshinori Fukui, Identification of a functional DOCK8 gene polymorphism associated with atopic dermatitis, Allergy, 10.1111/all.15429, 77, 12, 3670-3672, 2022.07. |
| 3. |
Tatsuguchi T, Uruno T, Sugiura Y, Sakata D, Izumi Y, Sakurai T, Hattori Y, Oki E, Kubota N, Nishimoto K, Oyama M, Kunimura K, Ohki T, Bamba T, Tahara H, Sakamoto M, Nakamura M, Suematsu M, Fukui Y., Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells, INTERNATIONAL IMMUNOLOGY, 10.1093/intimm/dxac002, 34, 5, 277-289, 2022.04, 本研究は、大腸癌臨床検体及びマウスの移植がんモデルを用いて、がん細胞によって産生されるコレステロール硫酸がリンパ球の腫瘍内浸潤を抑制してがんの免疫回避に寄与していることを明らかにした。. |
| 4. |
Matsubara K, Kunimura K, Yamane N, Aihara R, Sakurai T, Sakata D, Uruno T, Fukui Y., DOCK8 deficiency causes a skewing to type 2 immunity in the gut with expansion of group 2 innate lymphoid cells, Biochem Biophys Res Commun., 10.1016/j.bbrc.2021.04.094., 559, 135-140, 2021.06. |
| 5. |
Tetsuya Sakurai, Mutsuko Kukimoto-Niino, Kazufumi Kunimura, Nana Yamane, Daiji Sakata, Ryosuke Aihara, Tomoharu Yasuda, Shigeyuki Yokoyama, Mikako Shirouzu, Yoshinori Fukui, Takehito Uruno*, A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8, Life Science Alliance, 10.26508/lsa.202000873, 4, 4, e202000873-e202000873, 2021.02, DOCK8 is a Cdc42-specific guanine-nucleotide exchange factor that is essential for development and functions of various subsets of leukocytes in innate and acquired immune responses. Although DOCK8 plays a critical role in spatial control of Cdc42 activity during interstitial leukocyte migration, the mechanism remains unclear. We show that the DOCK homology region (DHR)-1 domain of DOCK8 binds specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and is required for its recruitment to the plasma membrane. Structural and biochemical analyses reveal that DOCK8 DHR-1 domain consists of a C2 domain-like core with loops creating the upper surface pocket, where three basic residues are located for stereospecific recognition of phosphoinositides. Substitution of the two basic residues, K576 and R581, with alanine abolished PI(4,5)P2 binding in vitro, ablated the ability of DOCK8 to activate Cdc42 and support leukocyte migration in three-dimensional collagen gels. Dendritic cells carrying the mutation exhibited defective interstitial migration in vivo. Thus, our study uncovers a critical role of DOCK8 in coupling PI(4,5)P2 signaling with Cdc42 activation for immune regulation.. |
| 6. |
Ryosuke Aihara, Kazufumi Kunimura, Mayuki Watanabe, Takehito Uruno, Nana Yamane, Tetsuya Sakurai, Daiji Sakata, Fusanori Nishimura, Yoshinori Fukui, DOCK8 controls survival of group 3 innate lymphoid cells in the gut through Cdc42 activation, International Immunology, 10.1093/intimm/dxaa066, 2020.09, Abstract
Innate lymphoid cells (ILCs) are a family of developmentally related leukocytes that rapidly secrete polarized sets of cytokines to combat infection and promote tissue repair at mucosal barriers. Among them, group 3 ILCs (ILC3s) play an important role in maintenance of the gut homeostasis by producing IL-22, and their development and function critically depend on the transcription factor RORγt. Although recent evidence indicates that RORγt+ ILC3s are reduced in the gut in the absence of the Cdc42 activator DOCK8 (dedicator of cytokinesis 8), the underlying mechanism remains unclear. We found that genetic deletion of Dock8 in RORγt+-lineage cells markedly reduced ILC3s in the lamina propria of the small intestine. By analyzing BrdU incorporation, it was revealed that DOCK8 deficiency did not affect the cell proliferation. Furthermore, when lineage marker-negative (Lin–) α4β7+ CD127+ RORγt– fetal liver cells were cultured with OP9 stromal cells in the presence of stem cell factor (SCF) and IL-7 in vitro, RORγt+ ILC3s normally developed irrespective of DOCK8 expression. However, DOCK8-deficient ILC3s exhibited a severe defect in survival of ILC3s under the condition with or without IL-7. Similar defects were observed when we analyzed Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Thus, DOCK8 acts in cell-autonomous manner to control survival of ILC3s in the gut through Cdc42 activation. . |
| 7. |
Takehito Uruno, S100A4 Protein Is Essential for the Development of Mature Microfold Cells in Peyer’s Patches, Cell Reports, 10.1016/j.celrep.2019.10.091, 29, 9, 2823-2834, 2019.11, Intestinal microfold cells (M cells) in Peyer's patches are a special subset of epithelial cells that initiate mucosal immune responses through uptake of luminal antigens. Although the cytokine receptor activator of nuclear factor-κB ligand (RANKL) expressed on mesenchymal cells triggers differentiation into M cells, other environmental cues remain unknown. Here, we show that the metastasis-promoting protein S100A4 is required for development of mature M cells. S100A4-producing cells are a heterogenous cell population including lysozyme-expressing dendritic cells and group 3 innate lymphoid cells. We found that in the absence of DOCK8, a Cdc42 activator critical for interstitial leukocyte migration, S100A4-producing cells are reduced in the subepithelial dome, resulting in a maturation defect of M cells. While S100A4 promotes differentiation into mature M cells in organoid culture, genetic inactivation of S100a4 prevents the development of mature M cells in mice. Thus, S100A4 is a key environmental cue that regulates M cell differentiation in collaboration with RANKL.. |
| 8. |
Tetsuya Sakurai, Takehito Uruno, Yuki Sugiura, Takaaki Tatsuguchi, Kazuhiko Yamamura, Miho Ushijima, Yuko Hattori, Mutsuko Kukimoto-Niino, Chiemi Mishima-Tsumagari, Mayuki Watanabe, Makoto Suematsu, Yoshinori Fukui, Cholesterol sulfate is a DOCK2 inhibitor that mediates tissue-specific immune evasion in the eye, Science Signaling, 10.1126/scisignal.aao4874, 11, 541, 2018.07, Although immune responses are essential to protect the body from infection, they can also harm tissues. Certain tissues and organs, including the eye, constitute specialized microenvironments that locally inhibit immune reactivity. Dedicator of cytokinesis protein 2 (DOCK2) is a Rac-specific guanine nucleotide exchange factor (GEF) that is predominantly found in hematopoietic cells. DOCK2 plays a key role in immune surveillance because it is essential for the activation and migration of leukocytes. DOCK2 mutations cause severe immunodeficiency in humans. We found that DOCK2-mediated Rac activation and leukocyte migration were effectively inhibited by cholesterol sulfate (CS), but not by cholesterol or other sulfated steroids. CS bound to the catalytic domain of DOCK2 and suppressed its GEF activity. Mass spectrometric quantification revealed that CS was most abundantly produced in the Harderian gland, which provides the lipids that form the oily layer of the tear film. Sulfation of cholesterol is mediated by the sulfotransferases SULT2B1b and, to a lesser extent, SULT2B1a, which are produced from the same gene through alternative splicing. By genetically inactivating Sult2b1, we showed that the lack of CS in mice augmented ultraviolet- and antigen-induced ocular surface inflammation, which was suppressed by administration of eye drops containing CS. Thus, CS is a naturally occurring DOCK2 inhibitor and contributes to the generation of the immunosuppressive microenvironment in the eye.. |
| 9. |
Takehito Uruno, The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production., Frontiers in immunology, 10.3389/fimmu.2018.00243, 9, 243-243, 2018.02, A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab')2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2-Rac axis in PC differentiation and IgG antibody responses.. |
| 10. |
Mayuki Watanabe, Masao Terasawa, Kei Miyano, Toyoshi Yanagihara, Takehito Uruno, Fumiyuki Sanematsu, Akihiko Nishikimi, Jean Francois Cote, Hideki Sumimoto, Yoshinori Fukui, DOCK2 and DOCK5 Act Additively in Neutrophils To Regulate Chemotaxis, Superoxide Production, and Extracellular Trap Formation, JOURNAL OF IMMUNOLOGY, 10.4049/jimmunol.1400885, 193, 11, 5660-5667, 2014.12. |
| 11. |
Kana Ogawa, Yoshihiko Tanaka, Takehito Uruno, Duan Xuefeng, Yosuke Harada, Fumiki Sanematsu, Kazuhiko Yamamura, Masao Terasawa, Akihiko Nishikimi, Jena Francois Cote, Yoshinori Fukui, DOCK5 functions as a key signaling adaptor that links Fc epsilon RI signals to microtubule dynamics during mast cell degranulation, JOURNAL OF EXPERIMENTAL MEDICINE, 10.1084/jem.20131926, 211, 7, 1401-1413, 2014.06. |
| 12. |
Takehito Uruno, Blockade of inflammatory responses by a small-molecule inhibitor of the Rac activator DOCK2., Chemistry & biology, 10.1016/j.chembiol.2012.03.008, 19, 4, 488-97, 2012.04, Tissue infiltration of activated lymphocytes is a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a reversible manner and inhibited its catalytic activity in vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and T cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant.. |
| 13. |
DOCK8 is a Cdc42 activator critical for interstitial dendritic cell migration during immune responses.
To migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42, yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans. In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially.. |
| 14. |
Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1 from their autoinhibited forms.
DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide exchange factor, controls lymphocyte migration through ras-related C3 botulinum toxin substrate (Rac) activation. Dedicator of cytokinesis 2-engulfment and cell motility protein 1 (DOCK2•ELMO1) complex formation is required for DOCK2-mediated Rac signaling. In this study, we identified the N-terminal 177-residue fragment and the C-terminal 196-residue fragment of human DOCK2 and ELMO1, respectively, as the mutual binding regions, and solved the crystal structure of their complex at 2.1-Å resolution. The C-terminal Pro-rich tail of ELMO1 winds around the Src-homology 3 domain of DOCK2, and an intermolecular five-helix bundle is formed. Overall, the entire regions of both DOCK2 and ELMO1 assemble to create a rigid structure, which is required for the DOCK2•ELMO1 binding, as revealed by mutagenesis. Intriguingly, the DOCK2•ELMO1 interface hydrophobically buries a residue which, when mutated, reportedly relieves DOCK180 from autoinhibition. We demonstrated that the ELMO-interacting region and the DOCK-homology region 2 guanine nucleotide exchange factor domain of DOCK2 associate with each other for the autoinhibition, and that the assembly with ELMO1 weakens the interaction, relieving DOCK2 from the autoinhibition. The interactions between the N- and C-terminal regions of ELMO1 reportedly cause its autoinhibition, and binding with a DOCK protein relieves the autoinhibition for ras homolog gene family, member G binding and membrane localization. In fact, the DOCK2•ELMO1 interface also buries the ELMO1 residues required for the autoinhibition within the hydrophobic core of the helix bundle. Therefore, the present complex structure reveals the structural basis by which DOCK2 and ELMO1 mutually relieve their autoinhibition for the activation of Rac1 for lymphocyte chemotaxis.. |
| 15. |
Takehito Uruno, Selective control of type I IFN induction by the Rac activator DOCK2 during TLR-mediated plasmacytoid dendritic cell activation., The Journal of experimental medicine, 10.1084/jem.20091776, 207, 4, 721-30, 2010.03, Plasmacytoid dendritic cells (pDCs) play a key role in antiviral immunity, but also contribute to the pathogenesis of certain autoimmune diseases, by producing large amounts of type I IFNs. Although activation of pDCs is triggered by engagement of nucleotide-sensing toll-like receptors (TLR) 7 and 9, type I IFN induction additionally requires IkappaB kinase (IKK) alpha-dependent activation of IFN regulatory factor (IRF) 7. However, the signaling pathway mediating IKK-alpha activation is poorly defined. We show that DOCK2, an atypical Rac activator, is essential for TLR7- and TLR9-mediated IFN-alpha induction in pDCs. We found that the exposure of pDCs to nucleic acid ligands induces Rac activation through a TLR-independent and DOCK2-dependent mechanism. Although this Rac activation was dispensable for induction of inflammatory cytokines, phosphorylation of IKK-alpha and nuclear translocation of IRF-7 were impaired in Dock2-deficient pDCs, resulting in selective loss of IFN-alpha induction. Similar results were obtained when a dominant-negative Rac mutant was expressed in wild-type pDCs. Thus, the DOCK2-Rac signaling pathway acts in parallel with TLR engagement to control IKK-alpha activation for type I IFN induction. Owing to its hematopoietic cell-specific expression, DOCK2 may serve as a therapeutic target for type I IFN-related autoimmune diseases.. |
| 16. |
Activation of Arp2/3 complex-mediated actin polymerization by cortactin.
Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.. |
