• Journal of Life Science
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• v.32 no.11
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• pp.833-840
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• 2022

Chronic inflammation has been shown to be closely associated with tumor development and progression. Nuclear factor kappa B (NF-κB) is composed of a family of five transcription factors. NF-κB signaling plays a crucial role in the inflammatory response and is often found to be dysregulated in various types of cancer, making it an attractive target in cancer therapeutics. In this study, CDC-like kinase 3 (CLK3) was identified as a novel kinase that regulates the NF-κB signaling pathway. Our data demonstrate that CLK3 inhibits the canonical and non-canonical NF-κB pathways. Luciferase assays following the transient or stable expression of CLK3 indicated that this kinase inhibited NF-κB activation mediated by Tumor necrosis factor-alpha (TNFα) and Phorbol 12-myristate 13-acetate (PMA), which are known to activate NF-κB signaling via the canonical pathway. Consistent with data on the ectopic expression of CLK3, CLK3 knockdown using shRNA constructs increased NF-κB activity 1.5-fold upon stimulation with TNFα in HEK293 cells compared with the control cells. Additionally, overexpression of CLK3 suppressed the activation of this signaling pathway induced by NF-κB-inducing kinase (NIK) or CD40, which are well-established activators of the non-canonical pathway. To further examine the negative impact of CLK3 on NF-κB signaling, we performed Western blotting following the TNFα treatment to directly identify the molecular components of the NF-κB pathway that are affected by this kinase. Our results revealed that CLK3 mitigated the phosphorylation/activation of transforming growth factor-α-activated kinase 1 (TAK1), inhibitor of NF-κB kinase alpha/beta (IKKα/α), NF-κB p65 (RelA), NF-κB inhibitor alpha (IκBα), and Extracellular signal-regulated kinase 1/2-Mitogen-activated protein kinase (ERK1/2-MAPK), suggesting that CLK3 inhibits both the NF-κB and MAPK signaling activated by TNFα exposure. Further studies are required to elucidate the mechanism by which CLK3 inhibits the canonical and non-canonical NF-κB pathways. Collectively, these findings reveal CLK3 as a novel negative regulator of NF-κB signaling.

• Archives of Pharmacal Research
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• v.19 no.2
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• pp.116-121
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• 1996

Antisense oligonucleotides seem to provide a promising new tool for the therapy. Choi et al. (1995) reported antisense phosphorothioate oligonucleotides (PS-ODN, 25 mer) complementary to TGF-.betha. mRNA designed for scar formation inhibitor to eliminate scars, which was caused by undesired collagen deposition due to overexpression of TGF-.betha., in wounded skin. PS-ODN were evaluated in vitro for skin penetration using normal and tape-stripped damaged rat skin. The in vitro skin transports were carried out with partially modified PS-ODN (6S) and fully modified PS-ODN (25S). The cumulative amount of PS-ODN (6S) penetrated through normal rat skin was $0.234{\pm}0.041{\mu}g/cm^2$ and that of tape-stripped damaged rat skin was $1.077{\pm}0.301{\mu}g/cm^2$ over 8 hrs. PS-ODN (25S) can not be found in receptor medium through normal skin due to high molecular weight (Mol.Wt.=8,000) and polyanionic charge. However, the cumulative amount of PS-ODN (25S) penetrated across damaged rat skin in PBS was $0.340{\pm}0.296{\mu}g/cm^2$ over 8 hrs. The absense of dermis raised the cumulative amount of PS-ODN (6S) penetrated through rat skin. And the fluxes of PS-ODN (6S) and PSODN (25S) at 8hrs across damaged rat skin were $134.63{\pm}37.67{\mu}g/cm^2$ h, and $42.50{\pm}36.95ng/cm^2$ h, respectively. While PS-ODN (25S) was stable in 10% heat inactivated fetal bovine serum (FBS) during 24 hrs, PS-ODN (6S) was less stable than PS-ODN (25S), but was markedly stable than unmodified phosphodiester. It is suggested that the cumulative amount of PS-ODN (6S) penetrated through damaged rat skin is larger than that of PS-ODN (25S) since the former is easier to degrade by nuclease than the latter and then is apt to penetrate into skin. Thus, PS-ODN represents a logical candidate for further evaluation due to the potential for delivery into the wounded skin.

• Radiation Oncology Journal
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• v.18 no.4
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• pp.314-320
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• 2000

Purpose :To investigate whether changes in plasma concentrations of transforming growth factor-$\beta$1(TGF-$\beta$1), tumor necrosis factor-alpha (TNF-$\alpha$) and interleukin-6 (IL-6) could be used to identify the development of radiation-induced pneumonitis in the lung cancer patients. Methods and Materials :Seventeen patients with lung cancer (11 NSCLC, 6 SCLC) were enrolled in a prospective study designed to evaluate clinical and molecular biologic correlation of radiation-induced pneumonitis. The study began in May 1998 and completed in July 1999. All patients were treated with radiotherapy with curative intent : 1.8 Gy per day, 5 fractions per week. Serial measurements of plasma TGF-$\beta$1, TNF-$\alpha$ and IL-6 were obtained in all patients before, weekly during radiotherapy and at each follow-up visits after completion of treatment. These measurements were quantified using enzyme linked immunosorbent assay (ELISA). All patients were evaluated for signs and symptoms of pneumonitis at each follow-up visit after completion of radiotherapy. High resolution CT (HRCT) scans were obtained when signs and symptoms of pneumonitis were developed after completion of radiotherapy. Results : Thirteen patients eventually developed signs and symptoms of clinical pneumonitis 씬file four patients did not. TGF-$\beta$ 1 levels were elevated in all 13 patients with pneumonitis, which showed characteristic pattern of elevation (38.45 ng/ml at pretreatment, 13.66 ng/ml during radiotherapy, then 60.63 ng/ml at 2-4 weeks after completion of radiotherapy). The levels of TNF- $\alpha$ and IL-6 were also elevated In the group of patients who developed pneumonitis but the pattern was not characteristic. Conclusions : Changes in plasma TGF$\beta$-1 levels before, during and after radiotherapy appears to be a useful means by which to identify patients at risk for the development of symptomatic pneumonitis. Other cytokines like TNF- $\alpha$ and IL-6 shows no meaningful changes in association with radiation pneumonitis.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.457-473
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• 2002

The purposes of this study is to evaluate the combination effects of TGF-${\beta}_1$ and PDGF-BB on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were prepared from the first premolar tooth extracted for the orthodontic treatment and were cultured in DMEM/100% FBS at the $37^{\circ}C$, 5% $CO_2$ incubator. Authors measured the DNA synthesis, total protein, collagen and noncollagenous protein synthesis according to the concentration of TGF-${\beta}_1$,(1,5ng/ml) and PDGF-BB (1,10 ng/ml) in combination. To explore further this delayed effect of TGF-${\beta}_1$, we preincubated human periodontal ligament cells with TGF-${\beta}_1$ for 4 or 24 hours before PDGF-BB stimulation. The results were as follows: The DNA synthetic activity was increased dose dependently by TGF-${\beta}_1$, PDGF-BB. The combination of TGF-${\beta}_1$ and PDGF-BB consistently enhanced the DNA synthetic activity to PDGF-BB alone. The ability of TGF-${\beta}_1$ to enhance DNA synthetic activity in PDGF-BB treated periodontal ligament cells was dose dependent. The maximum mitogenic effect was at the 5ng/ml of TGF-${\beta}_1$ and l0ng/ml of PDGF-BB. Preincubation of cell with TGF-${\beta}_1$ resulted in significantly greater response to PDGF-BB at all TGF-${\beta}_1$ concentration studied, and may be useful for clinical application in periodontal regenerative procedures. The total protein, collagen and noncollagen synthesis was increased dose pendently by TGF-${\beta}_1$, PDGF-BB. The % of collagen was slightly decreased according to the concentration of TGF-${\beta}_1$, PDGF-BB. The effect of TGF-${\beta}_1$, PDGF-BB were not specific for collagen synthesis since it also increased noncollagenous protein synthesis. This study demonstrates that PDGF-BB is major mitogens for human periodontal ligament cells in vitro, and supports a role for TGF-${\beta}_1$ as a regulation of the mitogenic and total protein formation to PDGF-BB in these cells.

• Journal of Life Science
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• v.28 no.1
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• pp.17-25
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• 2018

In this study, we examined the efficacy of the immune regulation of ${\beta}$-1,3/1,6-glucan and Lactobacillus plantarum LM1004 on atopic dermatitis models. The oral administration of ${\beta}$-1,3/1,6-glucan and L. plantarum LM1004 on mice significantly decreased the amount of scratching, leakage to evans blue, and concentrations of serum immunoglobulin E (IgE) and histamine compared with the atopic dermatitis - induced group. When atopic dermatitis was induced, the transcription factors (GATA-3, retinoic acid-related orphan receptor ${\gamma}$ T [$ROR{\gamma}T$]) and cytokines (interleukin-4 [IL-4], IL-17) of Th2 and Th17 cells were overexpressed at the transcriptional level, and they significantly decreased with oral administration of ${\beta}$-1,3/1,6-glucan and L. plantarum LM1004. In addition, ${\beta}$-1,3/1,6-glucan and L. plantarum LM1004 were shown to modulate the immune balance by increasing the expression of Th1 and Treg transcription (T-bet, forkhead box p3 [Foxp3]) and cytokines (interferon-${\gamma}$ [IFN-${\gamma}$], transforming growth factor-${\beta}$ [TGF-${\beta}$]). Galectin-9 and filaggrin were significantly lower in the atopic dermatitis - induced group and significantly higher in the ${\beta}$-1,3/1,6-glucan-treated group. In contrast, thymic stromal lymphopoietin (TSLP) was highest in the atopic dermatitis-induced group, while mice that were orally administered ${\beta}$-1,3/1,6-glucan and L. plantarum LM1004 showed similar TSLP levels to the control group. These results indicate that ${\beta}$-1,3/1,6-glucan and L. plantarum LM1004 have immunomodulatory effects and atopic dermatitis improvement effects in an animal model of atopic dermatitis. Therefore, it is expected that ${\beta}$-1,3/1,6-glucan and L. plantarum LM1004 can be used as natural materials in the treatment of atopic dermatitis.

• Radiation Oncology Journal
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• v.23 no.3
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• pp.161-168
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• 2005

Purpose: The changed expressions of $TGF-{\beta}1$, as a key cytokine in the fibrotic process, due to melatonin with potent antioxidative effects, were investigated in the irradiated lung using fibrosis-sensitive C57BL/6 mice. Materials and Methods: Female C57BL/6 mice were divided into control irradiation-only, and melatonin (300 mg/kg i.p. 1 hr before irradiation) pretreatment groups. The thoraces of the mice were irradiated with a single dose of 12 Gy. The mRNA expressions of $TGF-{\beta}1$ in the lung tissue 2 and 4 weeks after irradiation were quantified using semiquantitive RT-PCR, and the cellular origin and expression levels of $TGF-{\beta}1$ protein were identified using immunohistochemical staining. Results: The relative mRNA expression levels in the irradiation-only and melatonin pretreatment groups 2 and 4 weeks after irradiation were 1.92- and 1.80-fold (p=0.064) and 2.38- and 1.94-fold (p=0.004) Increased, respectively compared to those in the control group. increased expressions of $TGF-{\beta}1$ protein were prominently detected in regions of histopathologicai radiation injury, with alveolar macrophages and septal epithelial cells serving as important sources of $TGF-{\beta}1$ expression. At 2 and 4 weeks after irradiation, the expression levels of protein were $15.8\%\;vs.\;16.9\%$ (p=0.565) and $36.1\%\;vs.\;25.7\%$ (p=0.009), respectively. Conclusion: The mRNA and protein expressions of $TGF-{\beta}1$ in the lung tissue following thoracic irradiation with 12 Gy were significantly decreased by melatonin pretreatment at 4 weeks. These results indicate that melatonin may have a possible application as an antifibrotic agent in radiation-induced lung injury.

• Journal of Life Science
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• v.34 no.7
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• pp.443-452
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• 2024

This study aimed to examine the influence of 3,6-anhydroxygalactose (L-AHG) on the pro-inflammatory M1 polarization and pro-inflammatory responses observed in the RAW264.7 mouse macrophage cell line following stimulation with lipopolysaccharides (LPS). L-AHG exhibited a significant and dose-dependent inhibition of inducible nitric oxide synthase (iNOS) expression, a hallmark of M1 polarization, and subsequent NO production in LPS-stimulated RAW264.7 cells. Furthermore, the LPS-induced upregulation of cyclooxygenase-2 (COX-2), which drives the production of prostaglandin E2, an inflammatory mediator, was also inhibited by L-AHG. L-AHG did not affect the LPS-triggered Toll-like receptor 4 (TLR4)-mediated pro-inflammatory signaling pathway, which culminated in the activation of transforming growth factor-β-activated kinase 1 (TAK1). However, it was observed to inhibit the generation of reactive oxugen species (ROS) in a dose-dependent manner, as well as the TAK1-driven activation of JNK and p38 MAPK. Given that the active p38 MAPK is known to contribute to the assembly of active nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which catalyzes the intracellular generation of pro-inflammatory ROS in LPS-stimulated macrophages, the dose-dependent reduction in the LPS-induced ROS generation by L-AHG may be mainly due to the prevention of TAK1-driven activation of p38 MAPK. Together, these results demonstrate that the L-AHG-mediated inhibition of the TAK1-JNK/p38 MAPK activation phase of the pro-inflammatory signaling pathway in LPS-stimulated RAW264.7 cells by L-AHG represents a promising mechanism for suppressing M1 polarization and pro-inflammatory responses in macrophages.

• Food Science of Animal Resources
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• v.32 no.3
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• pp.339-345
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• 2012

This experiment was carried out in order to separate bovine colostral whey protein from Imsil province and to test the effect of immunological activity on RAW 264.7 cells. The colostral whey protein contained TGF-${\beta}$ 7, 475 pg/g in total. We first tested the effect of the colostral whey protein on the proliferation of RAW 264.7 cells and it demonstrated cytotoxicity at concentrations greater than 20 mg/mL. Therefore, the immunological activities of colostral whey protein were investigated in maximum concentration of 10 mg/mL on LPS-induced RAW 264.7 cells. Results indicated that colostral whey protein inhibited the LPS-induced nitric oxide (NO) production in a dose-dependent manner. The colostral whey protein also suppressed the productions of proinflammatory cytokines (TNF-${\alpha}$, IL-$1{\beta}$, IL-6) in a dose-dependent manner. In addition to the immunological activity, colostral whey protein led to the expression of heme oxygenase-1 (HO-1) in RAW 264.7 cells. In conclusion, colostral whey protein containing TGF-${\beta}$ inhibited the production of NO, TNF-${\alpha}$, IL-$1{\beta}$, and IL-6 via expression of HO-1.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.2
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• pp.238-246
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• 2001

Bone morphogenetic proteins(BMPs), members of the transforming growth factor $\beta$(TGF-$\beta$) superfamily were first identified as the factors that induce ectopic bone formation in vivo, when implanted into muscular tissue. Especially BMP-2 inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. In the molecular mechanism of the signal transduction of TGF-$\beta$ and related factors, intracellular signaling proteins were identified as Smad. In previous study, it has been reported that Smad 1 and Smad 5, which belong to the R-Smad family mediate BMP signaling, were involved in the induction of osteoblast differentiation in C2C12 cells. To understnad the role of Smads involved in osteogenic transdifferentiation in C2C12 cell, in present study, after we stably transfected C2C12 cells with each. Smad(Smad 1,Smad 5) expression vector, cultured for 3 days and stained for alkaline phophatase activity. ALP activity positive cells appeared in the Smad 1, Smad 5 stably transfected cell even in the abscence of BMP. After transiently co-transfected C2C12 cells with each Smad expression vector and ALP promoter, it was examined that Smad 1 and Smad 5 expression vector had increased about 2 fold ALP promoter activity in the abscence of BMP. These result suggested that both Smad 1 and Smad 5 were involved in the intracellular BMP signals which induce osteoblast differentiation in C2C12 cells. The effect of BMP on C2C12 cells with Smad 1, Smad 5 transfected were studied by using northern blot analysis. the treatment of BMP upregulated ALP mRNA level in three groups, especially upregulation of ALP was larger in Smad 1, Smad 5 transfected cell than control group. Pretreatment with cycloheximide($10{\mu}g/ml$), a protein synthesis inhibitor resulted in blocking the ALP gene expression even in BMP(100ng/ml) treated cell. These results suggested that Smad increased the level of ALP mRNA via the synthesis of a certain transcriptional regulatory protein.

• Radiation Oncology Journal
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• v.28 no.1
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• pp.32-38
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• 2010

Purpose: Although radiation-induced fibrosis is one of the common sequelae occurring after irradiation of skin and soft tissues, the treatment methods are not well standardized. This study aimed to establish the skin fibrosis mouse model by fractionated radiation for the further mechanism studies or testing the efficacy of therapeutic candidates. Materials and Methods: The right hind limbs of BALB/c mice received two fractions of 20 Gy using a therapeutic linear accelerator. Early skin damages were scored and tissue fibrosis was assessed by the measurement of a leg extension. Morphological changes were assessed by H&E staining and by Masson's Trichrome staining. TGF-${\beta}1$ expression from soft tissues was also detected by immunohistochemistry and PCR. Results: Two fractions of 20 Gy irradiation were demonstrated as being enough to induce early skin damage effects such as erythema, mild skin dryness, dry and wet desquamation within several weeks of radiation. After 13 weeks of irradiation, the average radiation-induced leg contraction was $11.1{\pm}6.2mm$. Morphologic changes in irradiated skin biopsies exhibited disorganized collagen and extracellular matrix fibers, as well as the accumulation of myofibroblasts compared to the non-irradiated skin. Moreover, TGF-${\beta}1$ expression in tissue was increased by radiation. Conclusion: These results show that two fractions of 20 Gy irradiation can induce skin fibrosis in BALB/c mice accompanied by other common characteristics of skin damages. This animal model can be a useful tool for studying skin fibrosis induced by radiation.