Yoe-Sik Bae

Proteomic analysis of tumor necrosis factor-alpha-induced secretome of human adipose tissue-derived mesenchymal stem cells.

Human adipose tissue-derived mesenchymal stem cells (hASCs) are useful for regeneration of inflamed or injured tissues. To identify secreted hASC proteins during inflammation, hASCs were exposed to tumor necrosis factor-alpha (TNF-alpha) and conditioned media derived from hASCs were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified 187 individual proteins as secreted proteins (secretome) in hASC-conditioned media; 118 proteins were secreted at higher levels upon TNF-alpha treatment. The TNF-alpha-induced secretome included a variety of cytokines and chemokines such as interleukin-6 (IL-6), IL-8, chemokine (C-X-C motif) ligand 6, and monocyte chemotactic protein-1 (MCP-1). TNF-alpha also increased expression of various proteases including cathepsin L, matrix metalloproteases and protease inhibitors, and induced secretion of long pentraxin 3, a key inflammatory mediator implicated in innate immunity. TNF-alpha-conditioned media stimulated migration of human monocytes, which play a key role in inflammatory responses. This migration was abrogated by pretreatment with neutralizing anti-IL-6, anti-IL-8, and anti-MCP-1 antibodies, suggesting that IL-6, IL-8, and MCP-1 are involved in migration of monocytes. Taken together, these results suggest that TNF-alpha-induced secretome may play a pivotal role in inflammatory responses and that shotgun proteomic analysis will be useful for elucidation of the paracrine functions of mesenchymal stem cells.

Serum amyloid A induces CCL2 production via formyl peptide receptor-like 1-mediated signaling in human monocytes.

Although the presence of an elevated level of serum amyloid A (SAA) has been regarded as a cardiovascular risk factor, the role of SAA on the progress of atherosclerosis has not been fully elucidated. In the present study, we investigated the effect of SAA on the production of CCL2, an important mediator of monocyte recruitment, and the mechanism underlying the action of SAA in human monocytes. The stimulation of human monocytes with SAA elicited CCL2 production in a concentration-dependent manner. The production of CCL2 by SAA was found to be mediated by the activation of NF-κB. Moreover, the signaling events induced by SAA included the activation of ERK and the induction of cyclooxygenase-2, which were required for the production of CCL2. Moreover, SAA-induced CCL2 induction was inhibited by a formyl peptide receptor-like 1 (FPRL1) antagonist. We also found that the stimulation of FPRL1-expressing RBL-2H3 cells induced CCL2 mRNA accumulation, but the vector-expressing RBL-2H3 cells combined with SAA did not. Taken together, our findings suggest that SAA stimulates CCL2 production and, thus, contributes to atherosclerosis. Moreover, FPRL1 was found to be engaged in SAA-induced CCL2 induction, and cyclooxygenase-2 induction was found to be essential for SAA-induced CCL2 expression. These results suggest that SAA and FPRL1 offer a developmental starting point for the treatment of atherosclerosis.

Proteoglycan isolated from Phellinus linteus inhibits tumor growth through mechanisms leading to an activation of CD11c+CD8+ DC and type I helper T cell-dominant immune state.

Dendritic cells (DC) are known to not only induce the activation of T cells, but are also associated with the polarization of T cells. This study investigated whether or not proteoglycan (PG) isolated from Phellinus linteus induces the phenotypic and functional maturation of CD11c+ DC in vitro and in vivo. PG was found to induce the phenotypic and functional maturation of bone marrow‐derived DC via Toll‐like receptors (TLR) 2 and 4 in vitro. Administration of PG in vivo strongly inhibited the MCA‐102 tumor growth and increase in vivo. The ratio of CD8+ DC to CD8− DC increased, and PG enhanced IL‐12 and IFN‐γ production, and expression of surface molecules including major histocompatibility complexes (MHC) classes I, MHC II, CD80, and CD86 in MCA‐102‐challenged mice. PG also caused a marked increase in the production of Th (helper T cells)‐1 cytokine (IFN‐γ) and a decrease in the production of Th‐2 cytokine (IL‐4) by splenic cells and inguinal lymph node cells in MCA‐102 tumor‐bearing mice. Furthermore, PG stimulated the proliferation of CD4+ and CD8+ T cells. In addition, a combination of PG and tumor lysate‐pulsed DC inhibited completely the growth of MCA‐102 cells in tumor‐bearing mice. These results indicate that the administration of PG inhibited the tumor growth through a mechanism leading to a Th‐1 dominant immune state and the activation of CD11c+CD8+ DC.

Toxoplasma gondii–derived heat shock protein 70 stimulates maturation of murine bone marrow–derived dendritic cells via Toll-like receptor 4

Abstract Toxoplasma gondii –derived heat shock protein 70 (T.g.HSP70) induced maturation of bone marrow–derived dendritic cells (DCs) of wild-type (WT) C57BL/6 mice as evidenced by an increase in surface expression of MHC class I and II molecules and costimulatory molecules such as CD40, CD80, and CD86. Functionally, decreased phagocytic ability and increased alloreactive T cell stimulatory ability were observed in T.g.HSP70-stimulated DCs. These phenotypic and functional changes of T.g.HSP70-stimulated DCs were demonstrated in Toll-like receptor (TLR) 2- and myeloid differentiation factor 88 (MyD88)-deficient but not TLR4-deficient C57BL/6 mice. DCs from WT and TLR2-deficient but not TLR4-deficient mice produced IL-12 after T.g.HSP70 stimulation. T.g.HSP70-stimulated DCs from WT, TLR2-deficient, and MyD88-deficient, but not TLR4-deficient mice expressed IFN-β mRNA. Thus, T.g.HSP70 stimulates murine DC maturation via TLR4 through the MyD88-independent signal transduction cascade.

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif.

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1α, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1α were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1α were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1α were specific targets for Chk1 and/or Chk2 at least in vitro.

A novel collagen-binding peptide promotes osteogenic differentiation via Ca2+/calmodulin-dependent protein kinase II/ERK/AP-1 signaling pathway in human bone marrow-derived mesenchymal stem cells.

The intracellular signaling events controlling human mesenchymal stem cell (hMSC) differentiation into osteoblasts are poorly understood. Collagen-binding domain is considered an essential component of bone mineralization. In the present study, we investigated the regulatory mechanism of osteoblastic differentiation of hMSC by the peptide with a novel collagen-binding motif derived from osteopontin. The peptide induced influx of extracellular Ca2+ via calcium channels and increased intracellular Ca2+ concentration ([Ca2+]i) independent of both pertussis toxin and phospholipase C, and activated ERK, which was inhibited by Ca2+/calmodulin-dependent protein kinase (CaMKII) antagonist, KN93. The peptide-induced increase of [Ca2+]i is correlated with ERK activation in a various cell types. The peptide stimulated the migration of hMSC but suppressed cell proliferation. Furthermore, the peptide increased the phosphorylation of cAMP-response element-binding protein, leading to a significant increase in the transactivation of cAMP-response element and serum response element. Ultimately, the peptide increased AP-1 transactivation, c-jun expression, and bone mineralization, which are suppressed by KN93. Taken together, these results indicate that the novel collagen-binding peptide promotes osteogenic differentiation via Ca2+/CaMKII/ERK/AP-1 signaling pathway in hMSC, suggesting the potential application in cell therapy for bone regeneration.

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor.

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK‐OV3 ovarian cancer cells and OVCAR‐3 ovarian cancer cells were responsive to LPE. LPE‐stimulated intracellular calcium concentration ([Ca2+]i) increase was inhibited by U‐73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca2+]i increase by LPE, indicating the involvement of PTX‐sensitive G‐proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK‐OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE‐stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B‐driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK‐OV3 ovarian cancer cells.

Glycogen synthase kinase 3β and β-catenin pathway is involved in toll-like receptor 4-mediated NADPH oxidase 1 expression in macrophages

Macrophage activation contributes to the pathogenesis of atherosclerosis. In the vascular system, the major source of reactive oxygen species is the NADPH oxidase (Nox) family. Nox1 is induced by lipopolysaccharide (LPS) in macrophages, but the expression mechanism is not fully understood. We found that LPS causes β‐catenin accumulation by glycogen synthase kinase 3β (GSK3β) inactivation, and that β‐catenin accumulation increases Nox1 expression. LPS induced Nox1 mRNA expression and reactive oxygen species generation in Raw264.7 cells. Using bone marrow‐derived macrophages from toll‐like receptor 4 mutant mice, we also tested whether LPS‐induced Nox1 expression is toll‐like receptor 4 dependent. LPS caused GSK3β phosphorylation, induced β‐catenin accumulation and increased nuclear translocation. The GSK3β inhibitor LiCl potentiated LPS‐induced Nox1 expression in accordance with β‐catenin accumulation and nuclear translocation. Conversely, ectopic expression of a constitutively active GSK3β mutant severely attenuated Nox1 expression. These findings identify a novel regulatory pathway controlling Nox1 expression by LPS‐stimulated macrophages.

The Agonists of Formyl Peptide Receptors Prevent Development of Severe Sepsis after Microbial Infection

Severe sepsis, a principal cause of death in intensive care units, occurs when host immune defenses fail to combat invading microbes. In this paper, we report that the administration of peptide agonists of formyl peptide receptors, including Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), protected against death by enhanced bactericidal activity and inhibition of vital organ inflammation and immune cell apoptosis in a cecal ligation and puncture (CLP) sepsis mouse model. The administration of WKYMVm also enhanced the production of type 1 (IFN-γ and IL-12) and type 17 (IL-17 and TGF-β) cytokines in CLP mice. In contrast, the administration of WKYMVm inhibited the production of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) in the CLP mice. The therapeutic and bactericidal effects of WKYMVm were partly reversed in IFN-γ–deficient mice, whereas target organ inflammation was not. Meanwhile, the therapeutic and anti-inflammatory effects of WKYMVm were partly reversed in IL-17–deficient mice. In addition, the administration of WKYMVm also enhanced type 1 and type 17 Th cell responses in mice sensitized with LPS plus Ags. These results suggest that the agonists of formyl peptide receptors effectively prevent development of severe sepsis following microbial infection partly via augmentation of type 1 and type 17 immune responses.

Nek6 is involved in G2/M phase cell cycle arrest through DNA damage-induced phosphorylation.

Nek6 is a recently identified NIMA-related kinase that is required for mitotic cell cycle progression. In the present study, we examined the role of Nek6 in the DNA damage response. We found that Nek6 is phosphorylated upon IR and UV irradiation through the DNA damage checkpoint in vivo. Nek6 is also directly phosphorylated by the checkpoint kinases Chk1 and Chk2 in vitro. Notably, Nek6 activation during mitosis is completely abolished by IR and UV irradiation. Moreover, the ectopic expression of Nek6 overrides DNA damage-induced G2/M arrest. These results suggest that Nek6 is a novel target of the DNA damage checkpoint and that the inhibition of Nek6 activity is required for proper cell cycle arrest in the G2/M phase upon DNA damage.