Min-Sung Kwon

Swiprosin-1 Is a Novel Actin Bundling Protein That Regulates Cell Spreading and Migration

Protein functions are often revealed by their localization to specialized cellular sites. Recent reports demonstrated that swiprosin-1 is found together with actin and actin-binding proteins in the cytoskeleton fraction of human mast cells and NK-like cells. However, direct evidence of whether swiprosin-1 regulates actin dynamics is currently lacking. We found that swiprosin-1 localizes to microvilli-like membrane protrusions and lamellipodia and exhibits actin-binding activity. Overexpression of swiprosin-1 enhanced lamellipodia formation and cell spreading. In contrast, swiprosin-1 knockdown showed reduced cell spreading and migration. Swiprosin-1 induced actin bundling in the presence of Ca2+, and deletion of the EF-hand motifs partially reduced bundling activity. Swiprosin-1 dimerized in the presence of Ca2+ via its coiled-coil domain, and a lysine (Lys)-rich region in the coiled-coil domain was essential for regulation of actin bundling. Consistent with these observations, mutations of the EF-hand motifs and coiled-coil region significantly reduced cell spreading and lamellipodia formation. We provide new evidence of how swiprosin-1 influences cytoskeleton reorganization and cell spreading.

TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse.

TAGLN2 stabilizes cortical F-actin and thereby maintains F-actin contents at the immunological synapse, which allows T cell activation following T cell receptor stimulation.

Swiprosin‐1 is expressed in mast cells and up‐regulated through the protein kinase CβI/η pathway

Swiprosin‐1 exhibits the highest expression in CD8+ T cells and immature B cells and has been thought to play a role in lymphocyte physiology. Here we report that swiprosin‐1 is also expressed in mast cells and up‐regulated in both in vitro cultured mast cells by phorbol ester and in vivo model tissues of passive cutaneous anaphylaxis and atopic dermatitis. Targeted inhibition of the specific protein kinase C (PKC) isotypes by siRNA revealed that PKC‐βI/η are involved in the expression of swiprosin‐1 in the human mast cell line HMC‐1. In contrast, down‐regulation of swiprosin‐1 by A23187 or ionomycin suggests that calcium‐signaling plays a negative role. The ectopic expression of swiprosin‐1 augmented PMA/A23187‐induced NF‐κB promoter activity, and resulted in increased expression of cytokines. Moreover, knock‐down of swiprosin‐1 attenuated PMA/A23187‐induced cytokine expression. Collectively, these results suggest that swiprosin‐1 is a PKC‐βI/η‐inducible gene and it modulates mast cell activation through NF‐κB‐dependent pathway. J. Cell. Biochem. 108: 705–715, 2009.

Swiprosin-1 Regulates Cytokine Expression of Human Mast Cell Line HMC-1 through Actin Remodeling.

Background Swiprosin-1 was identified in human CD8+ lymphocytes, mature B cells and non-lymphonoid tissue. We have recently reported that swiprosin-1 is expressed in mast cells and up-regulated in both in vitro and in vivo. Methods The expression of cytokines and swiprosin-1 were determined by by real time PCR and conventional PCR. Pharmacological inhibitors were treated to investigate potential mechanism of swiprosin-1 in mast cell activation. Actin content was evaluated by confocal microscopy and flow cytometry. Results The swiprosin-1 augmented PMA/A23187-induced expression of cytokines and release of histamine. However, knock-down of swiprosin-1 showed only a modest effect on PMA/A23187-induced cytokine expression, suggesting that swiprosin-1 has gain-of-function characteristics. Swiprosin-1 was found in microvilli-like membrane protrusions and highly co-localized with F-actin. Importantly, either disruption of actin by cytochalasin B or inhibition of PI3 kinase, an enzyme involved in actin remodeling, by wortmannin blocked cytokine expression only in swiprosin-1-overexpressing cells. Conclusion These results suggest that swiprosin-1 modulates mast cell activation potentially through actin regulation.

Weissella cibaria WIKIM28 ameliorates atopic dermatitis-like skin lesions by inducing tolerogenic dendritic cells and regulatory T cells in BALB/c mice.

The occurrence of atopic dermatitis (AD), a chronic inflammatory skin disease, has been increasing steadily in children and adults in recent decades. In this study, we evaluated the ability of the lactic acid bacterium Weissella cibaria WIKIM28 isolated from gatkimchi, a Korean fermented vegetable preparation made from mustard leaves, to suppress the development of AD induced by 2,4-dinitrochlorobenzene in a murine model. Oral administration of W. cibaria WIKIM28 reduced AD-like skin lesions, epidermal thickening, and serum immunoglobulin E levels. Furthermore, the production of type 2 helper T (Th2) cytokines such as interleukin (IL)-4, IL-5, and IL-13 decreased in peripheral lymph node cells. Moreover, the intake of W. cibaria WIKIM28 increased the proportion of CD4+CD25+Foxp3+ regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) and IL-10 levels in polyclonally stimulated MLN cells. In conclusion, the oral administration of W. cibaria WIKIM28 isolated from gatkimchi ameliorated AD-like symptoms by suppressing allergic Th2 responses and inducing Treg responses. These results suggest that W. cibaria WIKIM28 may be applicable as a probiotic for the prevention and amelioration of AD.

Recycling and LFA‐1‐dependent trafficking of ICAM‐1 to the immunological synapse

Little is known about how adhesion molecules on APCs accumulate at immunological synapses. We show here that ICAM‐1 on APCs is continuously internalized and rapidly recycled back to the interface after antigen‐priming T‐cell contact. The internalization rate is high in APCs, including Raji B cells and dendritic cells, but low in endothelial cells. Internalization is significantly reduced by inhibitors of Na+/H+ exchangers (NHEs), suggesting that members of the NHE‐family regulate this process. Once internalized, ICAM‐1 is co‐localized with MHC class II in the polarized recycling compartment. Surprisingly, not only ICAM‐1, but also MHC class II, is targeted to the immunological synapse through LFA‐1‐dependent adhesion. Cytosolic ICAM‐1 is highly mobile and forms a tubular structure. Inhibitors of microtubule or actin polymerization can reduce ICAM‐1 mobility, and thereby block accumulation at immunological synapses. Membrane ICAM‐1 also moves to the T‐cell contact zone, presumably through an active, cytoskeleton‐dependent mechanism. Collectively, these results demonstrate that ICAM‐1 can be transported to the immunological synapse through the recycling compartment. Furthermore, the high‐affinity state of LFA‐1 on T cells is critical to induce targeted movements of both ICAM‐1 and MHC class II to the immunological synapse on APCs. J. Cell. Biochem. 111: 1125–1137, 2010.

Aplotaxene blocks T cell activation by modulation of protein kinase C-θ-dependent pathway

Aplotaxene, (8Z, 11Z, 14Z)-heptadeca-1, 8, 11, 14-tetraene, is one of the major components of essential oil obtained from Inula helenium root, which is used in Oriental medicine. However, the effects of aplotaxene on immunity have not been investigated. Here, we show that aplotaxene inhibits T cell activation in terms of IL-2 and CD69 expression. Aplotaxene, at a concentration that optimally inhibits IL-2 production, has little effect on apoptotic or necrotic cell death, suggesting that apoptosis is not a mechanism for aplotaxene-mediated inhibition of T cell activation. Aplotaxene affects neither superantigeninduced conjugate formation between Jurkat T cells and Raji B cells nor clustering of CD3 and LFA-1 at the immunological synapse. Aplotaxene significantly inhibits PKC-θ phosphorylation and translocation to the immunological synapse, and blocks PMA-induced T-cell receptor internalization. Furthermore, aplotaxene leads to inhibition of mitogen-activated protein kinases (JNK, ERK and p38) phosphorylation and NF-κB, NF-AT, and AP-1 promoter activities in Jurkat T cells. Taken together, our findings provide evidence for the immunosuppressive effect of aplotaxene on activated T cells through the modulation of the PKC-θ and MAPK pathways, suggesting that aplotaxene may be a novel immunotherapeutic agent for immunological diseases related to the overactivation of T cells.

Phytocomponent 4-hydroxy-3-methoxycinnamaldehyde ablates T-cell activation by targeting protein kinase C-θ and its downstream pathways

Autoreactive T-cell responses have a crucial role in the pathology and clinical course of autoimmune diseases. Therefore, controlling the activation of these cells is an important strategy for developing therapies and therapeutics. Here, we identified that 4-hydroxy-3-methoxycinnamaldehyde (4H3MC) has a therapeutic potential for T-cell activation by modulating protein kinase C-θ (PKCθ) and its downstream pathways. Pre- and post-treatment with 4H3MC prevented IL-2 release from human transformed and untransformed T cells at the micromolar concentrations without any cytotoxic effects, in fact more efficiently than its structural analogue 4-hydroxycinnamic acid-a previously reported T-cell inhibitor. In silico analysis showed that 4H3MC is a potential inhibitor of PKC isotypes, including PKCθ-a crucial PKC isotype in T cells. Consistently, 4H3MC significantly blocked PKC activity in vitro and also inhibited the phosphorylation of PKCθ in T cells. 4H3MC had no effect on TCR-mediated membrane-proximal-signalling events such as phosphorylation of Zap70. Instead, it attenuated the phosphorylation of mitogen-activated protein kinases (ERK and p38) and promoter activities of NF-κB, AP-1 and NFAT. Taken together, our results provide the evidences that 4H3MC may have curative potential as a novel immune modulator in a broad range of immunopathological disorders by modulating PKCθ activity.

Intermediate monomer–dimer equilibrium structure of native ICAM-1: Implication for enhanced cell adhesion

Dimeric intercellular adhesion molecule-1 (ICAM-1) has been known to more efficiently mediate cell adhesion than monomeric ICAM-1. Here, we found that truncation of the intracellular domain of ICAM-1 significantly enhances surface dimerization based on the two criteria: 1) the binding degree of monomer-specific antibody CA-7 and 2) the ratio of dimer/monomer when a mutation (L42→C42) was introduced in the interface of domain 1. Mutation analysis revealed that the positively charged amino acids, including very membrane-proximal ⁵⁰⁵R, are essential for maintaining the structural transition between the monomer and dimer. Despite a strong dimer presentation, the ICAM-1 mutants lacking an intracellular domain (IC1ΔCTD) or containing R to A substitution in position 505 (⁵⁰⁵R/A) supported a lower degree of cell adhesion than did wild-type ICAM-1. Collectively, these results demonstrate that the native structure of surface ICAM-1 is not a dimer, but is an intermediate monomer-dimer equilibrium structure by which the effectiveness of ICAM-1 can be fully achieved.

Structural implications of Ca(2+)-dependent actin-bundling function of human EFhd2/Swiprosin-1.

EFhd2/Swiprosin-1 is a cytoskeletal Ca2+-binding protein implicated in Ca2+-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca2+ and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region. However, the molecular mechanism for the regulation of F-actin binding and bundling by EFhd2 is unknown. Here, the Ca2+-bound crystal structure of the EFhd2 core region is presented and structures of mutants defective for Ca2+-binding are also described. These structures and biochemical analyses reveal that the F-actin bundling activity of EFhd2 depends on the structural rigidity of F-actin binding sites conferred by binding of the EF-hands to Ca2+. In the absence of Ca2+, the EFhd2 core region exhibits local conformational flexibility around the EF-hand domain and C-terminal linker, which retains F-actin binding activity but loses the ability to bundle F-actin. In addition, we establish that dimerisation of EFhd2 via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction.