Tatsuo Kinashi

Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus.

Thymocyte trafficking has an important role in thymic selection. Here we show that the Hippo homologue Mst1 is required for thymocyte migration and antigen recognition by LFA-1 and ICAM-1 within the medulla. Using two-photon imaging of thymic tissues, we found that highly motile mature thymocytes arrest and are activated in the vicinity of rare populations of Aire(+) ICAM-1(hi) medullary thymic epithelia in a negatively selecting environment. Notably, Mst1 deficiency or blocking the cell adhesion molecules LFA-1 and ICAM-1 results in inefficient migration and antigen recognition of CD4(+) thymocytes within the medulla. Consistent with these defects, thymocyte selection is impaired in Mst1(-/-) mice, which display T cell-dependent inflammatory infiltrates in multiple organs and develop autoantibodies. Our results suggest that Mst1 has a key role in regulating thymocyte self-antigen recognition in the medulla.

HTLV-1 bZIP factor induces inflammation through labile Foxp3 expression.

Human T-cell leukemia virus type 1 (HTLV-1) causes both a neoplastic disease and inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the proviral DNA and is constitutively expressed in infected cells and ATL cells. HBZ increases the number of regulatory T (Treg) cells by inducing the Foxp3 gene transcription. Recent studies have revealed that some CD4+Foxp3+ T cells are not terminally differentiated but have a plasticity to convert to other T-cell subsets. Induced Treg (iTreg) cells tend to lose Foxp3 expression, and may acquire an effector phenotype accompanied by the production of inflammatory cytokines, such as interferon-γ (IFN-γ). In this study, we analyzed a pathogenic mechanism of chronic inflammation related with HTLV-1 infection via focusing on HBZ and Foxp3. Infiltration of lymphocytes was observed in the skin, lung and intestine of HBZ-Tg mice. As mechanisms, adhesion and migration of HBZ-expressing CD4+ T cells were enhanced in these mice. Foxp3−CD4+ T cells produced higher amounts of IFN-γ compared to those from non-Tg mice. Expression of Helios was reduced in Treg cells from HBZ-Tg mice and HAM/TSP patients, indicating that iTreg cells are predominant. Consistent with this finding, the conserved non-coding sequence 2 region of the Foxp3 gene was hypermethylated in Treg cells of HBZ-Tg mice, which is a characteristic of iTreg cells. Furthermore, Treg cells in the spleen of HBZ-transgenic mice tended to lose Foxp3 expression and produced an excessive amount of IFN-γ, while Foxp3 expression was stable in natural Treg cells of the thymus. HBZ enhances the generation of iTreg cells, which likely convert to Foxp3−T cells producing IFN-γ. The HBZ-mediated proinflammatory phenotype of CD4+ T cells is implicated in the pathogenesis of HTLV-1-associated inflammation.

Dendritic Cells Regulate High-Speed Interstitial T Cell Migration in the Lymph Node via LFA-1/ICAM-1

T lymphocytes vigorously migrate within the paracortex of lymph nodes (LNs) in search of cognate Ags that are presented by dendritic cells (DCs). However, the mechanisms that support T cells to exert the highest motility in a densely packed LN microenvironment are not fully understood. Two-photon microscopy using LN tissue slices revealed that LFA-1 and ICAM-1 were required for high-velocity migration (>10 μm/min) with relatively straight movement. Importantly, ICAM-1 expressed by myeloid lineages, most likely DCs, but not stromal cells or lymphocytes, was sufficient to support the high-velocity migration. Visualizing DCs in the LN from CD11c-EYFP mice showed that T cells traveled over thin dendrites and the body of DCs. Interestingly, DCs supported T cell motility in vitro in chemokine- and ICAM-1–dependent manners. Moreover, an acute lymphopenic environment in the LN significantly increased LFA-1 dependency for T cell migration, indicating that lymphocyte density modulates the use of LFA-1. Therefore, our results indicate that LFA-1/ICAM-1–dependent interactions between T cells and DCs play a crucial role not only in supporting firm arrest during Ag recognition but also in facilitating the Ag scanning processes.

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.

Hippo-Foxa2 signaling pathway plays a role in peripheral lung maturation and surfactant homeostasis

Respiratory distress syndrome (RDS), which is induced by insufficient production of surfactant, is the leading cause of mortality in preterm babies. Although several transcription factors are known to be involved in surfactant protein expression, the molecular mechanisms and signaling pathways upstream of these transcription factors have remained elusive. Here, using mammalian Hippo kinases (Mst1/2, mammalian sterile 20-like kinase 1/2) conditional knockout mice, we demonstrate that Mst1/2 kinases are critical for orchestration of transcription factors involved in surfactant protein homeostasis and prevention of RDS. Mice lacking Mst1/2 in the respiratory epithelium exhibited perinatal mortality with respiratory failure and their lungs contained fewer type I pneumocytes and more immature type II pneumocytes lacking microvilli, lamellar bodies, and surfactant protein expression, pointing to peripheral lung immaturity and RDS. In contrast to previous findings of YAP (Yes-associated protein)-mediated canonical Hippo signaling in the liver and intestine, loss of Mst1/2 kinases induced the defects in pneumocyte differentiation independently of YAP hyperactivity. We instead found that Mst1/2 kinases stabilized and phosphorylated the transcription factor Foxa2 (forkhead box A2), which regulates pneumocyte maturation and surfactant protein expression. Taken together, our results suggest that the mammalian Hippo kinases play crucial roles in surfactant homeostasis and coordination of peripheral lung differentiation through regulation of Foxa2 rather than of YAP.

Rab13 acts downstream of the kinase Mst1 to deliver the integrin LFA-1 to the cell surface for lymphocyte trafficking

Chemokines promote the delivery and proper organization of an integrin at the leading edge of migrating lymphocytes. Integrin Delivery Service Optimal immune responses depend on lymphocytes getting to the right place at the right time. Chemokines are secreted proteins that bind to cell surface chemokine receptors and act as the tracking signals for lymphocytes to follow to sites of infection or injury. Integrins are another type of cell surface protein that are important for lymphocyte activation and migration. Nishikimi et al. identified a mechanism through which the kinase Mst1 and the vesicle-associated protein Rab13 respond to chemokine signals by transporting integrins containing LFA-1 from the inside of lymphocytes to the plasma membrane at the leading edge of migrating cells. Rab13-deficient lymphocytes exhibited impaired migration in culture, and Rab13-deficient mice had smaller lymphoid organs than those of wild-type mice because of defective lymphocyte trafficking. In lymphocytes, the kinase Mst1 is required for the proper organization of integrins in the plasma membrane at the leading edge of migrating cells, which is critical for lymphocyte trafficking. We found a functional link between the small G protein Rab13 and Mst1 in lymphocyte adhesion and migration. In response to stimulation of T lymphocytes with chemokine, Mst1 promoted phosphorylation of the guanine nucleotide exchange factor DENND1C (differentially expressed in normal and neoplastic cells domain 1C), which activated Rab13. Active Rab13 associated with Mst1 to facilitate the delivery of the integrin LFA-1 (lymphocyte function–associated antigen 1) to the leading edge of lymphocytes. Delivery of LFA-1 involved the recruitment of myosin Va along actin filaments, which extended as a result of the localization of the actin regulatory protein VASP to the cell periphery through phosphorylation of VASP at Ser157 by Mst1. Inhibition of Rab13 function reduced the adhesion and migration of lymphocytes on ICAM-1 (intercellular adhesion molecule–1), the ligand for LFA-1, and inhibited the formation of a ring-like arrangement of LFA-1 at the contact sites between T cells and antigen-presenting cells. The lymphoid tissues of Rab13-deficient mice had reduced numbers of lymphocytes because of the defective trafficking capability of these cells. These results suggest that Rab13 acts with Mst1 to regulate the spatial distribution of LFA-1 and the motility and trafficking of lymphocytes.

Antigen-Specific Suppression and Immunological Synapse Formation by Regulatory T Cells Require the Mst1 Kinase

Although the cell-to-cell contact between CD4+Foxp3+ regulatory T (Treg) and their target cells is important for the suppressor function of Treg cells, the regulation of this process is not well understood. Here we show that the Mst1 kinase plays a critical role in the suppressor function of Treg cells through regulation of cell contact dependent processes. Mst1 -/- Treg cells failed to prevent the development of experimental colitis and antigen-specific suppression of naïve T cells proliferation in vitro. Mst1 -/- Treg cells exhibited defective interactions with antigen-presenting dendritic cells (DCs), resulting in reduced down-regulation of costimulatory molecules. While wild-type CD4+ Foxp3+ Treg cells formed mobile immunological synapses on supported planar membrane, Mst1 -/- Treg cells did not exhibit ICAM-1 ring or central peptide-MHC clustering. Using two-photon imaging we showed that antigen-specific wild-type Treg cells exhibited dynamic mobile contacts with antigen-pulsed DCs bearing stably associated naïve T cells. In contrast, Mst1 -/- Treg had impairments in their interactions with DCs. Thus, Mst1 is required for Treg cells to mediate contact-dependent suppressor functions.

Surf4 modulates STIM1-dependent calcium entry

Store-operated Ca(2+) entry (SOCE) is crucial for various physiological responses in immune cells. Although it is known that STIM1 relocates into discrete puncta juxtaposed to the plasma membrane to initiate SOCE, the machinery modulating the function of STIM1 remains unclear. We explored to find its modulators using affinity purification for STIM1-binding proteins and identified surfeit locus protein 4 (Surf4). Surf4 associated with STIM1 in the endoplasmic reticulum. Deletion of Surf4 in DT40 B cells resulted in marked increase of SOCE and facilitation of STIM1 clustering upon store-depletion. These findings suggest the modulatory function of Surf4 for STIM1-mediated SOCE.

Longest neurite-specific activation of Rap1B in hippocampal neurons contributes to polarity formation through RalA and Nore1A in addition to PI3-kinase.

In a developing nervous system, axon‐dendrite formation is instructed by extrinsic cues, and the mechanism whereby a developing neuron interprets these cues using intracellular signaling is particularly important. Studies using dissociated hippocampal neurons have identified many signaling pathways underlying neuronal polarization. Among the components of these pathways, Rap1B is essential for axon specification in hippocampal cultures. However, spatiotemporal regulation of Rap1B activity in polarizing neurons and how it affects neuronal polarization remain unclear. Herein, we investigated spatiotemporal activity‐change of Rap1B and its target molecules in hippocampal neurons. FRET imaging showed that specific activation of Rap1B was observed at the tip of a future axon. To dissect downstream signaling, we used three effector mutants of Rap1B. Expression of Rap1B‐G12V/E37G and G12V/Y40C mutants resulted in supernumerary axons. The targets of Rap1B‐G12V/E37G were RalA and Nore1A, whereas Rap1B‐G12V/Y40C activated PI3‐kinase. RalA was activated in the tip of stage 3 axons, and RalA‐S28N expression reduced the fraction of neurons with supernumerary axons induced by Rap1B‐G12V/E37G. Furthermore, Nore1A depletion reduced the number of cells without axons. These results indicate that specific activation of Rap1B contributes to neuronal polarization via interaction with RalA and Nore1A in addition to PI3‐kinase.

Hypermethylation of MST1 in IgG4-related autoimmune pancreatitis and rheumatoid arthritis

The serine/threonine kinase Mst1 plays important roles in the control of immune cell trafficking, proliferation, and differentiation. Previously, we reported that Mst1 was required for thymocyte selection and regulatory T-cell functions, thereby the prevention of autoimmunity in mice. In humans, MST1 null mutations cause T-cell immunodeficiency and hypergammaglobulinemia with autoantibody production. RASSF5C(RAPL) is an activator of MST1 and it is frequently methylated in some tumors. Herein, we investigated methylation of the promoter regions of MST1 and RASSF5C(RAPL) in leukocytes from patients with IgG4-related autoimmune pancreatitis (AIP) and rheumatoid arthritis (RA). Increased number of CpG methylation in the 5 region of MST1 was detected in AIP patients with extrapancreatic lesions, whereas AIP patients without extrapancreatic lesions were similar to controls. In RA patients, we detected a slight increased CpG methylation in MST1, although the overall number of methylation sites was lower than that of AIP patients with extrapancreatic lesions. There were no significant changes of the methylation levels of the CpG islands in the 5 region of RASSF5C(RAPL) in leukocytes from AIP and RA patients. Consistently, we found a significantly down-regulated expression of MST1 in regulatory T cells of AIP patients. Our results suggest that the decreased expression of MST1 in regulatory T cells due to hypermethylation of the promoter contributes to the pathogenesis of IgG4-related AIP.