• 생명과학회지
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• 제9권1호
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• pp.50-57
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• 1999

STAT들은 다양한 cytokine이나 성장호르몬 등에 의해 활성화되어 핵으로 빠르게 이동되어 유전자 발현을 조절한다. 우리는 D-raf 유전자의 5' 발현조절영역에서 STAT결합부위 염기서열을 발견하였다. 본 연구는 D-raf 유전자발현조절에 있어서 STAT결합부위의 역할을 형질전환 초파리를 사용하여 조사하였다. STAT결합부위에 염기치환돌연변이를 도입시킨 D-raf promoter부분을 P인자 벡터의 IacZ유전자에 융합시킴으로써 리포터 플라스미드 pDraf-STATmut-lacZ를 제작하였다. 이 리포터 플라스미드를 이용하여 얻은 Draf-STATmut-lacZ형질전환 초파리의 lacZ발현을 발생단계별, 조직별로 조사한 결과, 거의 모든 발생단계에서 정상형 Draf-lacZ 형질전환 초파리의 lacZ발현에 비해 크게 감소하였다. 본 연구결과들은 STAT결합배열 부위가 D-raf유전자발현조절에 있어서 중요한 역할을 함을 개체수준에서 증명하고 있다.

• International Journal of Oral Biology
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• 제44권4호
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• pp.160-164
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• 2019

Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), Tannerella forsythia (Tf), Prevotella intermedia (Pi), and Fusobacterium nucleatum (Fn) are major periodontal pathogens. Lipopolysaccharides (LPSs) from periodontal bacteria play an important role in periodontal pathogenesis by stimulating host cells to produce inflammatory cytokines. In this study, highly pure LPSs from the five major periodontopathogens were prepared, and their monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α)-inducing activities were compared in human umbilical vein endothelial cells (HUVECs) and THP-1 macrophagic cells, respectively. In HUVECs, LPSs from Aa and Fn were potent stimulators for MCP-1 induction; however, LPSs from Pg, Pi, and Tf were much weaker MCP-1 inducers. In THP-1 cells, LPSs from Pg, Aa, and Fn were relatively strong inducers of TNF-α, whereas LPSs from Pi and Tf produced little activity. The Toll-like receptor (TLR)2/TLR4 dependency of various LPSs was also determined by measuring NF-κB reporter activity in TLR2- or TLR4-expressing 293 cells. LPSs from Aa, Fn, and Tf stimulated only TLR4; however, LPSs from Pg and Pi stimulated both TLR2 and TLR4. These results suggest that LPSs from major periodontal bacteria differ considerably in their cell-stimulating activity.

• 농업과학연구
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• 제47권1호
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• pp.83-93
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• 2020

Lactoferrin (LF) is an iron-binding glycoprotein that is present in colostrum, milk, and other body secretions. The objective of this study was to investigate the effects of lactoferrin hydrolysates (LHs) on the production of immunomodulatory factors, including inflammatory related cytokines. The nuclear factor (NF)-κB reporter assay using human embryonic kidney 293 cells (HEK-293) revealed that NF-κB activity was significantly decreased by 1, 50, and 100 ㎍/mL of LH and the fractions above and below the 10 kDa LH. The mRNA expression of interferon (IFN)-γ in rat basophilic leukemia mast cells (RBL-2H3) treated with the fraction above the 10 kDa LH decreased in a dose-dependent manner, but the cells treated with LH and the fraction below the 10 kDa LH showed an increased expression of IFN-γ in a dose-dependent manner. The level of cyclooxygenase (COX)-2 expression decreased dose-dependently in RBL-2H3 cells treated with LH and the fraction above the 10 kDa LH, but the cells treated with the fraction below the 10 kDa LH showed an increased COX-2 expression in a dose-dependent manner. The mRNA expression of interleukin (IL)-4) was dose-dependently decreased by the fraction below the 10 kDa LH in human mast cells (HMC-1). The mRNA expressions of tumor necrosis factor (TNF)-α and IL-6 were significantly dose-dependently decreased by the fractions above and below the 10 kDa LH, but was dose-dependently increased by LH. The production of IL-4 was a little increased by the fraction above the 10 kDa LH compared to the positive control, but was decreased with LH and the fraction below the 10 kDa LH in HMC-1 cells. It was concluded that LF hydrolysates had an immunomodulating effect on anti-, pro-inflammatory and anti-allergic reactions.

• 대한한방내과학회지
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• 제25권4호
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• pp.260-273
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• 2004

Backgrounds : In recent years, asthma has become recognized as a chronic inflammatory disease associated pathologically with airway epithelial inflammation and airway remodeling. Objectives : To evaluate the different effects of Hirudo depending upon pharmaceutical manufactures on the expression and the activities of IL-6 and GM-CSF in airway epithelial cells, samples of Hirudo(水蛭), Hirudo toasted with Talcum(水蛭滑石炒) and Hirudo toasted with Ephedrae Herba(水蛭麻黃炒) were tested. Methods : After inducing enhanced messenger RNA(mRNA) expression and secretion of each cytokine by tumor necrosis factor-alpha(10 ng/ml) treatment, cultured human bronchial epithelial cell line BEAS-2B was added to each sample$(l,\;10,\;100\;&\;1000\;{\mu}g/ml)$. Subsequently, DNA activities were analyzed. Specifically mRNA expression and culture supernatants(protein levels) of IL-6 and GM-CSF from BEAS-2B cells, were analyzed using luciferase reporter gene assay, reverse transcription-polymerase chain reaction(RT-PCR) analysis and enzyme-linked immunosorbent assay. Results : Hirudo toasted with Ephedrae Herba(水蛭麻黃炒) and Hirudo(水蛭) inhibited IL-6 activities in BEAS-2B cells remarkably, and inhibited mRNA expression levels and protein levels in supernatant of IL-6 and GM-CSF at various concentrations, significantly(p<0.05). However, Hirudo toasted with Talcum(水蛭滑石炒) had no effect on mRNA expression levels and showed a slight inhibitory effect on GM-CSF protein levels in supernatant of culture medium. Conclusions : These results strongly suggest that Hirudo toasted with Ephedrae Herba(水蛭麻黃炒) and Hirudo(水蛭) would be serve as effective medicaments in the treatment of airway inflammation and remodeling of asthmatic patients.

• IMMUNE NETWORK
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• 제15권2호
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• pp.100-109
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• 2015

Controlling balance between T-helper type 1 (Th1) and T-helper type 2 (Th2) plays a pivotal role in maintaining the biological rhythm of Th1/Th2 and circumventing diseases caused by Th1/Th2 imbalance. Interleukin 4 (IL-4) is a Th2-type cytokine and often associated with hypersensitivity-related diseases such as atopic dermatitis and allergies when overexpressed. In this study, we have tried to elucidate the function of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (EC-18) as an essential modulator of Th1/Th2 balance. EC-18 has showed an inhibitory effect on the production of IL-4 in a dose-dependent manner. RT-PCR analysis has proved EC-18 affect the transcription of IL-4. By analyzing the phosphorylation status of Signal transducer and activator of transcription 6 (STAT6), which is a transcriptional activator of IL-4 expression, we discovered that EC-18 induced the decrease of STAT6 activity in several stimulated cell lines, which was also showed in STAT6 reporter analysis. Co-treatment of EC-18 significantly weakened atopy-like phenotypes in mice treated with an allergen. Collectively, our results suggest that EC-18 is a potent Th2 modulating factor by regulating the transcription of IL-4 via STAT6 modulation, and could be developed for immune-modulatory therapeutics.

• Molecules and Cells
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• 제23권1호
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• pp.23-29
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• 2007

Cyclic AMP-responsive element binding protein (CREB) is known to be associated with angiogenesis. In the present study we investigated the possible role of CREB in the expression of vascular endothelial growth factor (VEGF) by mouse macrophages. Over-expression of CREB increased VEGF secretion by cells of the RAW264.7 mouse macrophage cell line. It also increased the promoter activity of a mouse reporter driven by the VEGF promoter, while a dominant negative CREB (DN-CREB) abrogated the activity, suggesting that CREB mediates VEGF transcription. Forskolin, an adenylyl cyclase activator, stimulated VEGF transcription, and the PKA inhibitor H89 abolished this effect. IFN-${\gamma}$, a potent cytokine, stimulated VEGF expression only in part through the PKA-CREB pathway. These results indicate that PKA phosphorylates CREB and so induces VEGF gene expression. An analysis of mutant promoters revealed that one of the putative CREB responsive elements (CREs), at -399 ~ -388 in the promoter, is critical for CREB-mediated VEGF promoter activity, and the significance of this CRE was confirmed by chromatin immunoprecipitation assays.

• Asian Pacific Journal of Cancer Prevention
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• 제14권1호
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• pp.359-365
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• 2013

The high mobility group box 1 (HMGB1) protein is a multifunctional cytokine-like molecule that plays an important role in the pathogenesis of tumors. In this study, real-time polymerase chain reactions and Western blot assays indicated that HMGB1 transcriptional activity and protein level are increased in $Tax^+$-T cells (TaxP). To clarify the mechanisms, a series of HMGB1 deletion reporter plasmids (pHLuc1 to pHLuc6) were transfected into $Tax^-$-T cells (TaxN, Jurkat) and $Tax^+$-T cells (TaxP). We found that promoter activity in $Tax^+$-T cells to be higher than that in $Tax^-$-T cells, indicating a significant increase in pHLuc6. Bay11-7082 (NF-${\kappa}B$ inhibitor) treatment did not block the enhancing effect. Chromatin immunoprecipitation assays revealed that Tax was retained on a HMGB1 promoter fragment encompassing -1163 to -975. Bioinformatics analysis showed six characteristic cis-elements for CdxA, AP-1, AML-1a, USF, v-Myb, and C/EBP in the fragment in question. Mutation of cis-elements for C/EBP reduced significant HMGB1 promoter activity induced by Tax. These findings indicate that Tax enhances the expression of HMGB1 gene at the transcriptional level, possibly by interacting with C/EBP.

• BMB Reports
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• 제53권4호
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• pp.223-228
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• 2020

Dysregulation of histone deacetylase 6 (HDAC6) can lead to the pathologic states and result in the development of various diseases including cancers and inflammatory diseases. The objective of this study was to elucidate the regulatory role of microRNA-22 (miR-22) in HDAC6-mediated expression of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated macrophages. LPS stimulation induced HDAC6 expression, but suppressed miR-22 expression in macrophages, suggesting possible correlation between HDAC6 and miR-22. Luciferase reporter assays revealed that 3'UTR of HDAC6 was a bona fide target site of miR-22. Transfection of miR-22 mimic significantly inhibited LPS-induced HDAC6 expression, while miR-22 inhibitor further increased LPS-induced HDAC6 expression. LPS-induced activation of NF-κB and AP-1 was inhibited by miR-22 mimic, but further increased by miR-22 inhibitor. LPS-induced expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 was inhibited by miR-22 mimic, but further increased by miR-22 inhibitor. Taken together, these data provide evidence that miR-22 can downregulate LPS-induced expression of pro-inflammatory cytokines via suppression of NF-κB and AP-1 axis by targeting HDAC6 in macrophages.

• International Journal of Oral Biology
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• 제38권3호
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• pp.121-126
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• 2013

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional inflammatory cytokine that regulates various cellular and biological processes. Increased levels of $TNF{\alpha}$ have been implicated in a number of human diseases including diabetes and arthritis. Sympathetic nervous system stimulation via the beta2-adrenergic receptor (${\beta}2AR$) in osteoblasts suppresses osteogenic activity. We previously reported that $TNF{\alpha}$ upregulates ${\beta}2AR$ expression in murine osteoblastic cells and that this modulation is associated with $TNF{\alpha}$ inhibition of osteoblast differentiation. In our present study, we explored whether $TNF{\alpha}$ induces ${\beta}2AR$ expression in human osteoblasts and then identified the downstream signaling pathway. Our results indicated that ${\beta}2AR$ expression was increased in Saos-2 and C2C12 cells by $TNF{\alpha}$ treatment, and that this increase was blocked by the inhibition of NF-${\kappa}B$ activation. Chromatin immunoprecipitation and luciferase reporter assay results indicated that NF-${\kappa}B$ directly binds to its cognate elements on the ${\beta}2AR$ promoter and thereby stimulates ${\beta}2AR$ expression. These findings suggest that the activation of $TNF{\alpha}$ signaling in osteoblastic cells leads to an upregulation of ${\beta}2AR$ and also that $TNF{\alpha}$ induces ${\beta}2AR$ expression in an NF-${\kappa}B$-dependent manner.

• Radiation Oncology Journal
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• 제19권2호
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• pp.171-180
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• 2001

목적 : Tissue inhibitor of matrix metalloproteinase (TIMP)는 matrix metalloproteinase (MMP)에 작용하여 암세포의 침윤과 전이를 억제하고 염증, angiogenesis, fibrosis에 중요한 역할을 한다. TIMP 유전자는 여러 cytokine 및 signal molecule에 의하여 조절되는 유전자이므로 방사선에 의한 TIMP의 발현을 측정하고 전사 조절 기전을 연구하고자 하였다. 대상 및 방법 : 두경부암 환자의 병변에서 유도하여 확립한 두경부암 세포주를 이용하여 방사선에 의한 TIMP 유전자 발현을 측정하였다. 각 세포주의 방사선 민감도를 측정하고 transwell을 이용한 invasion assay로 전이성을 측정하였다. TIMP1, TIMP2 발현은 conditioned medium을 취해 ELISA assay로 측정하였다. 방사선조사는 2 Gy, 10 Gy 군으로 나누어 관찰했고 조사 후 시간 간격은 24, 48시간이었다. MTT assay로 생존세포 수를 측정하여 방사선 세포치사로 인한 발현 변화를 보정하였다. hTIMP1 promoter region을 PCR하여 pGL2-basic luciferase reporter vector에 cloning하여 인간 두경부암 세포주에 이입하여 functional TIMP1 발현이 증가하는지 확인하였고 protein kinase C (PKC) activator인 PMA (phorbol 12-myristate 13-acetate)와 Ras에 의한 TIMP1 발현이 유도되는지 확인하였다. 결과 : HN-1, HN-2, HN-3, HN-5, HN골 세포주의 $D_0$는 각각 1.55 Gy, 1.8 Gy, 1.5 Gy, 1.55 Gy, 2.45 Gy 이었다. 각 세포주의 방사선조사 후 MTT assay에 의한 cell viability는 24, 48시간에서 2 Gy인 경우 모두 $94\%$ 이상 그리고 10 Gy에서는 $73\%$ 이상의 생존 세포를 확인하였다. TIMP1, TIMP2 단백의 basal 농도는 24시간 48시간에서 점점 증가하여 세포에서 계속 합성되어 분비되고 있음을 확인하였다. 2 Gy 조사 후 24시간에서 TIMP2는 HN-1, HN-9 세포주에서 감소하였으나, 10 Gy 조사 후에는 두 세포주에서 모두 증가하여 방사선량에 따라 반응이 달랐고, 방사선조사 후 48시간에는 HN-9 세포주에서는 증가하나 HN-9 세포주에서는 감소하여 세포주에 따라 반응이 달랐다. 그러나 방사선에 의한 TIMP1 발현 변화는 미미하였다. TIMP1 reporter gene을 인간 두경부암 세포주에 transfection하고 PMA (100 ng/ml)을 가한 경우 HN-1세포주에서는 유의하게 증가하고 HN-9 세포주에서는 감소하였다. Ras 발현 벡터와 co-transfection한 경우 TIMP1 promoter가 활성화 되었다. 결론 : 모두 두경부 암에서 유래된 세포주 이지만 방사선에 의한 TIMP의 발현 및 전사조절 기전은 세포주 마다 차이가 있었고 이온화 방사선의 용량에 따라서, 방사선조사 후의 시간 경과에 따라서도 TIMP 발현에 차이가 있었다. 이 결과는 TIMP의 전사 및 발현이 여러 종류의 signal molecule에 의하여 영향을 받고, 이 signal molecule들이 각 세포주 마다 다르기 때문으로 사료된다.