Seung Hoon Lee

Angiogenin Reduces Immune Inflammation via Inhibition of TANK-Binding Kinase 1 Expression in Human Corneal Fibroblast Cells

Angiogenin (ANG) is reportedly multifunctional, with roles in angiogenesis and autoimmune diseases. This protein is involved in the innate immune system and has been implicated in several inflammatory diseases. Although ANG may be involved in the anti-inflammatory response, there is no evidence that it has direct anti-inflammatory effects. In this study we sought to determine whether ANG has an anti-inflammatory effect in human corneal fibroblasts (HCFs) exposed to media containing tumor necrosis factor-alpha (TNF-α). We found that ANG reduced the mRNA expression of interleukin-1 beta (IL-1β), -6, -8 and TNF-α receptors (TNFR) 1 and 2. In contrast, ANG increased the mRNA expression of IL-4 and -10. Protein levels of TANK-binding kinase 1 (TBK1) were reduced by ANG in HCFs treated with TNF-α. Moreover, ANG diminished the expression of IL-6 and -8 and monocyte chemotactic protein- (MCP-) 1. The protein expression of nuclear factor-κB (NF-κB) was downregulated by ANG treatment. These findings suggest that ANG suppressed the TNF-α-induced inflammatory response in HCFs through inhibition of TBK1-mediated NF-κB nuclear translocation. These novel results are likely to play a significant role in the selection of immune-mediated inflammatory therapeutic targets and may shed light on the pathogenesis of immune-mediated inflammatory diseases.

Leucine-rich glioma inactivated 3 regulates adipogenesis through ADAM23.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein and a member of LGI/epitempin family. We previously showed that LGI3 was highly expressed in brain and played regulatory roles in neuronal exocytosis and differentiation. Besides the nervous system, LGI3 was shown to be expressed in diverse tissues. In this study, we found that LGI3 and its receptor candidate ADAM23 were expressed in adipose tissues and 3T3-L1 cells. 3T3-L1 preadipocytes secreted a 60-kDa protein, a major secreted form of LGI3, which declined with adipocyte differentiation. LGI3 was also expressed in adipose tissue macrophages in the ob/ob mice and in macrophage cell line. The 60-kDa LGI3 protein was selectively increased in the ob/ob adipose tissues comparing with the lean mice. Pull-down experiments, coimmunoprecipitation and immunocytochemistry indicated that LGI3 associated with ADAM23 in adipose tissues and 3T3-L1 cells. Knockdown of LGI3 or ADAM23 by siRNA increased adipogenesis in 3T3-L1 cells. Treatment with LGI3 protein did not affect preadipocyte proliferation but attenuated adipogenesis and this effect was reversed by siRNA-mediated knockdown of ADAM23. Taken together, we propose that LGI3 may be a candidate adipokine that is perturbed in obesity and suppresses adipogenesis through its receptor, ADAM23.

Upregulated Stromal Cell-Derived Factor 1 (SDF-1) Expression and its Interaction With CXCR4 Contribute to the Pathogenesis of Severe Pterygia

PURPOSE Stromal cell-derived factor 1 (SDF-1) and its interaction with chemokine receptor 4 (CXCR4) have been noted for participating in the wound healing process, and may paradoxically develop hypertrophic scarring. With viewing pterygia as a product of exaggerated wound formation, we evaluated the effects of SDF-1 and CXCR4 on determining the severity of pterygia. METHODS Human pterygial fibroblasts were cultured from excised tissues. Then, expression levels of SDF-1 and CXCR4 were assessed at both the mRNA and protein levels and analyzed with respect to the severity (grade T1 to T3) of pterygia. Expression patterns of SDF-1 and CXCR4 in pterygium tissues were evaluated by immunohistochemistry. Additionally, to investigate the SDF-1-induced myofibroblast transformation of pterygial fibroblasts, the correlation between SDF-1 and α-smooth muscle actin (α-SMA) expression levels was evaluated. Furthermore, α-SMA levels in pterygial fibroblasts were determined before and after knockdown of SDF-1 and blockade of CXCR4 by AMD3100. RESULTS Stromal cell-derived factor 1 and CXCR4 were expressed in identical areas in severe pterygium tissues (grade T3) and CXCR4-immunopositive cells were concentrated at perivascular regions. Stromal cell-derived factor 1 levels in cultured pterygial fibroblasts correlated positively with the severity of pterygia. Stromal cell-derived factor 1 levels had a significant, positive correlation with α-SMA levels in pterygial fibroblasts. Furthermore, each knockdown of SDF-1 expression and blockade of SDF-1/CXCR4 signaling in severe pterygia significantly reduced α-SMA levels. CONCLUSIONS Stromal cell-derived factor 1 expression is upregulated in severe pterygia, and SDF-1 and CXCR4 interaction may contribute to the myofibroblast transformation, which can be possibly restored through the downregulation of the SDF-1/CXCR4 axis.

Involvement of mTOR signaling in sphingosylphosphorylcholine-induced hypopigmentation effects

BackgroundSphingosylphosphorylcholine (SPC) acts as a potent lipid mediator and signaling molecule in various cell types. In the present study, we investigated the effects of SPC on melanogenesis and SPC-modulated signaling pathways related to melanin synthesis.MethodsMelanin production was measured in Mel-Ab cells. A luciferase assay was used to detect transcriptional activity of the MITF promoter. Western blot analysis was performed to examine SPC-induced signaling pathways.ResultsSPC produced significant hypopigmentation effects in a dose-dependent manner. It was found that SPC induced not only activation of Akt but also stimulation of mTOR, a downstream mediator of the Akt signaling pathway. Moreover, SPC decreased the levels of LC3 II, which is known to be regulated by mTOR. Treatment with the mTOR inhibitor rapamycin eliminated decreases in melanin and LC3 II levels by SPC. Furthermore, we found that the Akt inhibitor LY294002 restored SPC-mediated downregulation of LC3 II and inhibited the activation of mTOR by SPC.ConclusionsOur data suggest that the mTOR signaling pathway is involved in SPC-modulated melanin synthesis.

IL-1 Receptor Blockade Alleviates Graft-versus-Host Disease through Downregulation of an Interleukin-1β-Dependent Glycolytic Pathway in Th17 Cells

T helper (Th) 17 cells are a subset of Th cells expressing interleukin- (IL-) 17 and initiating an inflammatory response in autoimmune diseases. Graft-versus-host disease (GVHD) is an immune inflammatory disease caused by interactions between the adaptive immunity of donor and recipient. The Th17 lineage exhibits proinflammatory activity and is believed to be a central player in GVHD. IL-1 performs a key function in immune responses and induces development of Th17 cells. Here, we show that blockade of IL-1 signaling suppresses Th17 cell differentiation and alleviates GVHD severity. We hypothesized that the IL-1 receptor antagonist (IL-1Ra) would suppress Th17 cell differentiation in vitro via inhibition of glycolysis-related genes. Blockade of IL-1 using IL-1Ra downregulated Th17 cell differentiation, an alloreactive T cell response, and expression of genes of the glycolysis pathway. Severity of GVHD was reduced in mice with a transplant of IL-Ra-treated cells, in comparison with control mice. To clarify the mechanisms via which IL-1Ra exerts the therapeutic effect, we demonstrated in vivo that IL-1Ra decreased the proportion of Th17 cells, increased the proportion of FoxP3-expressing T regulatory (Treg) cells, and inhibited expression of glycolysis-related genes and suppressed Th17 cell development and B-cell activation. These results suggest that blockade of IL-1 signaling ameliorates GVHD via suppression of excessive T cell-related inflammation.

Ultraviolet B-induced LGI3 secretion protects human keratinocytes.

Leucine‐rich glioma inactivated 3 (LGI3) is known to be expressed mainly in the brain. However, the expression and physiological roles of LGI3 in skin cells remain unknown. In this study, it was found for the first time that LGI3 is expressed mostly by normal human keratinocytes. Furthermore, ELISA analysis showed that HaCaT human keratinocytes increased LGI3 secretion after exposure to ultraviolet B (UVB) in a time‐ and dose‐dependent manner. We next investigated the possible role of LGI3 in keratinocytes. LGI3 (50 ng/ml) increased survival of HaCaT cells by 20% after UVB irradiation (150 mJ/cm2). It was also found that LGI3 stimulates the phosphorylation of Akt, which is involved in the cell survival‐signalling cascade. Furthermore, LGI3 led to the phosphorylation of MDM2 and subsequent p53 degradation. Taken together, the data suggest that LGI3 may regulate p53 levels and that keratinocyte‐derived LGI3 may act as a novel cytokine for skin homoeostasis.

Angiogenin ameliorates corneal opacity and neovascularization via regulating immune response in corneal fibroblasts.

BackgroundAngiogenin (ANG), a component of tears, is involved in the innate immune system and is related with inflammatory disease. We investigated whether ANG has an immune modulatory function in human corneal fibroblasts (HCFs).MethodsHCFs were cultured from excised corneal tissues. The gene or protein expression levels of interleukin (IL)-1beta (β), IL-4, IL-6, IL-8, IL-10, complements, toll-like receptor (TLR)4, myeloid differentiation primary response gene (MYD)88, TANK-binding kinase (TBK)1, IkappaB kinase-epsilon (IKK-ε) and nuclear factor-kappaB (NF-κB) were analyzed with or without ANG treatment in tumor necrosis factor-alpha (TNF-α)- or lipopolysaccharide (LPS)-induced inflammatory HCFs by real-time polymerase chain reaction (PCR), Western blotting and immunocytochemistry. Inflammatory cytokine profiles with or without ANG were evaluated through immunodot blot analysis in inflammatory HCFs. Corneal neovascularization and opacity in a rat model of corneal alkali burn were evaluated after application of ANG eye drops.ResultsANG decreased the mRNA levels of IL-1β, IL-6, IL-8, TNF-α receptor (TNFR)1, 2, TLR4, MYD88, and complement components except for C1r and C1s and elevated the mRNA expression of IL-4 and IL-10. Increased signal intensity of IL-6, IL-8 and monocyte chemotactic protein (MCP)-1 and MCP-2 induced by TNF-α or LPS was weakened by ANG treatment. ANG reduced the protein levels of IKK-ε by either TNF-α and LPS, and decreased TBK1 production induced by TNF-α, but not induced by LPS. The expression of NF-κB in the nuclei was decreased after ANG treatment. ANG application lowered corneal neovascularization and opacity in rats compared to controls.ConclusionThese results demonstrate that ANG reduces the inflammatory response induced by TNF-α or LPS in HCFs through common suppression of IKK-ε-mediated activation of NF-κB. This may support the targeting of immune-mediated corneal inflammation by using ANG.

Scopoletin from Cirsium setidens Increases Melanin Synthesis via CREB Phosphorylation in B16F10 Cells.

In this study, we isolated scopoletin from Cirsium setidens Nakai (Compositae) and tested its effects on melanogenesis. Scopoletin was not toxic to cells at concentrations less than 50 µM and increased melanin synthesis in a dose-dependent manner. As melanin synthesis increased, scopoletin stimulated the total tyrosinase activity, the rate-limiting enzyme of melanogenesis. In a cell-free system, however, scopoletin did not increase tyrosinase activity, indicating that scopoletin is not a direct activator of tyrosinase. Furthermore, Western blot analysis showed that scopoletin stimulated the production of microphthalmia-associated transcription factor (MITF) and tyrosinase expression via cAMP response element-binding protein (CREB) phosphorylation in a dose-dependent manner. Based on these results, preclinical and clinical studies are needed to assess the use of scopoletin for the treatment of vitiligo.

Leucine-rich glioma inactivated 3 is a melanogenic cytokine in human skin.

Recently, we demonstrated that leucine‐rich glioma inactivated 3 (LGI3) is expressed in human skin. However, the effects of LGI3 on melanocytes remain unknown. The present study demonstrated that LGI3 can serve to stimulate melanogenesis without affecting cell viability. To determine the effects of LGI3 on melanin synthesis, normal human melanocytes and Mel‐Ab cells were treated with recombinant LGI3 and melanin content was measured. Our results showed that LGI3 promoted melanin synthesis in both cell types. Moreover, upregulation of microphthalmia‐associated transcription factor (MITF) and tyrosinase was observed at both the mRNA and protein levels via RT‐PCR and Western blotting, respectively. Furthermore, immunohistochemical staining showed that the expression of LGI3 increased in the basal layer of melasma skin samples, whereas it decreased slightly in vitiligo samples. These results suggest that LGI3 may play a role as a melanogenic cytokine in human skin.

Changes in Activation of Serratus Anterior, Trapezius and Latissimus Dorsi With Slouched Posture.

Objective To compare quantitative muscle activation between erect and slouched sitting postures in the muscles around the scapula, and to investigate the correlation between the angle of thoracic kyphosis and the alteration of muscle activity depending on two different sitting postures. Methods Ten healthy males participated in the study. Unilateral surface electromyography (SEMG) was performed for serratus anterior, middle trapezius (MT), and lower trapezius (LT), which are scapular stabilizer muscles, as well as latissimus dorsi. Participants elevated their shoulders for 3 seconds up to 90° abduction in the scapular plane, tilting 30° anterior in the coronal plane. They were told to hold the position for 10 seconds and voluntary isometric contractions were recorded by SEMG. These movement procedures were conducted for three times each for erect and slouched sitting postures and data were averaged. Results Activities of MT and LT increased significantly more in the slouched sitting posture than in the erect one. There was no significant correlation between kyphotic angle and the area under curve of each muscle. Conclusion Because MT and LT are known as prime movers of scapular rotation, the findings of this study support the notion that slouched sitting posture affects scapular movement. Such scapular dyskinesis during arm elevation leads to scapular stabilizers becoming overactive, and is relevant to muscle fatigue. Thus, slouched sitting posture could be one of the risk factors involved in musculoskeletal pain around scapulae.