• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제33권6호
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• pp.636-642
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• 2007

Background: CpG DNA plays an important role in immune cell function. This study examined whether the temporal control of toll-like receptor (TLR)9 by CpG DNA can regulate the expression of matrix metalloproteinase-9(MMP-9). Methods and materials: Macrophages were cultured in the presence of 10% FBS. For the various MMP genes analysis, RT-PCR and real-time PCR were performed. In addition, zymography assay performed for the MMP activity. The phosphorylation assay did for the ERK1/2 and NF${\kappa}B$ activation, and luciferase promoter assay was for the NF${\kappa}B$ activity. Results: CpG DNA induced the mRNA expression of MMP-2, MMP-9, and MMP-13, but not of MMP-7, MMP-8, and MMP-12, in a time-dependent manner. Especially, the mRNA expression of MMP-9 was strongly induced by CpG DNA using real-time RT-PCR. The TLR9 inhibitor, chloroquine, suppressed CpG DNA-induced MMP-9 expression and its activity. Moreover, CpG DNA induced the phosphorylation of ERK and the inhibition of ERK by U0126 suppressed CpG DNA-induced MMP-9 expression and its activity. CpG DNA stimulated $I{\kappa}B-{\alpha}$ degradation and luciferase activity. In addition, pretreatment of SN-50, the inhibitor of NF${\kappa}B$, strongly blocked the CpG DNA-induced MMP-9 expression and activity. Conclusion: These observations suggest that CpG DNA may play important roles in the activation of macrophages by regulating the production of MMP-9 via the sequential TLR9-ERK-NF${\kappa}B$ signaling pathway.

• Tuberculosis and Respiratory Diseases
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• 제44권6호
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• pp.1271-1284
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• 1997

연구배경 : 종양괴사인자(tumor necrosis factor ; TNF)는 다양한 생물학적 기능을 가지고 있는 바, 그 중 생체 외에서 증명된 뚜렷한 항암 효과로 말미암아 최근 항암 유전자요법의 중요한 대상으로 관심을 모으고 있다. 현재 유전자 이입의 기술적 문제로 생체 외에서 암세포에 유전자 이입을 시행한 후 이를 다시 환자의 생체내로 이식하는 방법이 연구의 주종을 이루고 있다. 그러나 저자들의 과거의 연구를 포함한 여러 연구에서 TNF가 이입된 암세포는 TNF에 대해 내성을 보이는 것으로 증명되었고 이에는 새로이 방어 단백질을 합성하는 것이 관여할 것이라는 시사가 있었다. 이 획득내성의 기전을 밝히는 것이 종양생물학의 이해를 넓히고 보다 효과적인 항암 유전자요법을 개발하기위한 매우 중요한 과제로 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제7권2호
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• pp.79-84
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• 2003

We have previously reported that expression of the somatostatin receptor subtypes, sst1-5, is differentially regulated by growth hormone (GH)-releasing hormone (GHRH) and forskolin (FSK), in vitro. GHRH binds to membrane receptors selectively located on pituitary somatotropes, activates adenylyl cyclase (AC) and increases sst1 and sst2 and decreases sst5 mRNA levels, without significantly altering the expression of sst3 and sst4. In contrast FSK directly activates AC in all pituitary cell types and increases sst1 and sst2 mRNA levels and decreases sst3, sst4 and sst5 expression. Two explanations could account for these differential effects: 1) GHRH inhibits sst3 and sst4 expression in somatotropes, but this inhibitory effect is masked by expression of these receptors in unresponsive pituitary cell types, and 2) FSK inhibits sst3 and sst4 expression levels in pituitary cell types other than somatotropes. To differentiate between these two possibilities, somatotropes were sequentially labeled with monkey anti-rat GH antiserum, biotinylated goat anti-human IgG, and streptavidin-PE and subsequently purified by fluorescent-activated cell sorting (FACS). The resultant cell population consisted of 95% somatotropes, as determined by GH immunohistochemistry using a primary GH antiserum different from that used for FACS sorting. Purified somatotropes were cultured for 3 days and treated for 4 h with vehicle, GHRH (10 nM) or FSK ($10{\mu}M$). Total RNA was isolated by column extraction and specific receptor mRNA levels were determined by semi-quantitative multiplex RT-PCR. Under basal conditions, the relative expression levels of the various somatostatin receptor subtypes were sst2＞sst5＞sst3=sst1＞ sst4. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst3, sst4 and sst5 mRNA levels in purified somatotropes, comparable to the effects of FSK on purified somatotropes and mixed pituitary cell cultures. Taken together, these results demonstrate that GHRH acutely modulates the expression of all somatostatin receptor subtypes within GH-producing cells and its actions are likely mediated by activation of AC.

• Nutritional Sciences
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• 제8권3호
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• pp.159-168
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• 2005

Phytoestrogens, especially Yak-kong or soybean-derived isoflavones have been traditionally used as a supplement of estrogen for preventing postmemopausal osteoporosis in oriental folk medicine. In our previous study, the treatment of Yak-kong and soybean increased estrogen receptor-a (ERa) expression and proliferation of MG-63 osteoblastic cells. In contrast, the increase of estrogen receptor-$\beta$ (ER$\beta$) expression in proliferating MG-63 cells with Yak-kong and soybean treatment was less pronounced, which suggested that ER$\beta$ may play a role rather in the regulation of bone cell differentiation To determine the role of ER$\beta$ in Yak-kong or soybean mediated regulation of bone cell differentiation, we established MG-63 cell lines stably expressing either ER$\beta$ or antisense ER$\beta$ RNAs. Increased expression of ER$\beta$ did not affect ERa expression and proliferation of MG-63 cells. However, increased expression of ER$\beta$ in MG-63 cells (ER$\beta$-MG63 cells) selectively enhanced Yak-kong or soybean induced expression of osteoprotegerin (OPG), a novel soluble glycoprotein which is secreted from osteoblasts and mediates the signal for osteoclast differentiation. Inhibition of ER$\beta$ expression by antisense ER$\beta$ RNAs (As-ER$\beta$-MG63) caused these cells to insensitize Yak-kong or soybean induced expression of OPG but increased MG-63 cell proliferation. Furthermore, the comparable effects between Yak-kong and the combined treatment of genistein and daidzein at $0.5{\times}l0^{-8}$ M, which is a concentration of these two isoflavones similar to Yak-kong at 0.001 mg/mL, on OPG expression in ER$\beta$-MG63 cell demonstrate that the enhanced expression of OPG with Yak-kong treatment is mediated by the synergistic effect of low leveled isoflavones in the extracts. Together, coupled with low level of ER expression in osteoclasts, our data demonstrate that ER$\beta$ in osteoblasts plays an important role in Yak-kong and soybean mediated inhibition of osteoclast differentiation indirectly by enhancing the expression of OPG.

• 대한예방한의학회지
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• 제14권2호
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• pp.77-89
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• 2010

Objectives : This study was performed to evaluate the effect of CJ(Cuscuta japonica Chois) on osteoclast differentiation and gene expression. Methods : The osteoclastogenesis and gene expression were determined in RANKL(receptor activator of nuclear factor kappa B ligand)－stimulated RAW 264.7. The results were summarized as followes. Results : CJ decreased the number of TRAP positive cell in RANKL-stimulated RAW264.7 cell. CJ decreased the expression of RANK(receptor activator of nuclear factor kappa B), $TNF{\alpha}$, and IL-6 in RANKL-stimulated RAW264.7 cell. CJ decreased the expression of iNOS and COX-2 in RANKL-stimulated RAW264.7 cell. CJ decreased the expression of Cathepsin K in RANKL-stimulated RAW264.7 cell. Conclusions : It is concluded that CJ might decrease the bone resorption resulted from decrease of osteoclast differentiation and it's related gene expression.

• International Journal of Oral Biology
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• 제46권1호
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• pp.39-44
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• 2021

This study aimed to investigate whether neurotransmitter receptors in the nervous system were also expressed in oral keratinocytes. Expressions of various neurotransmitter receptor genes in immortalized mouse oral keratinocyte (IMOK) cells were examined by reverse transcriptase polymerase chain reaction. IMOK cells expressed calcitonin gene-related peptide (CGRP) receptor subunit genes Ramp1 and Ramp3 and glutamate receptor subunit genes Grina, Gria3, Grin1, Grin2a, and Grin2d. Moreover, IMOK cells expressed Adrb2 and Chrna5 that encode beta 2 adrenergic receptor and cholinergic receptor nicotinic alpha 5 for sympathetic and parasympathetic neurotransmitters, respectively. The expression of Bdkrb1 and Ptger4, which encode receptors for bradykinin and prostaglandin E2 involved in inflammatory responses, was also observed at low levels. Expressions of Ramp1 and Grina in the mouse gingival epithelium were also confirmed by immunohistochemistry. When the function of neurotransmitter receptors expressed on IMOK cells was tested by intracellular calcium response, CGRP, glutamate, and cholinergic receptors did not respond to their agonists, but the bradykinin receptor responded to bradykinin. Collectively, oral keratinocytes express several neurotransmitter receptors, suggesting the potential regulation of oral epithelial homeostasis by the nervous system.

• 한국발생생물학회지:발생과생식
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• 제4권2호
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• pp.231-236
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• 2000

Gonadotropin-releasing hormone (GnRH)과 그 수용체가 흰쥐의 난소, 정소, 자궁, 태 반 그리고 유선 등의 생식기관에서 발현됨이 알려져 있다. 더욱이, 뇌하수체 전엽에 작용하는 GnRH의 표적 산물로 알려진 luteinizing hormone (LH)이 흰쥐 생식소에서도 발현됨이 알려졌는데, 이는 생식소 내에 GnRH-LH로 이루어진 국부 회로 (local circuit)가 존재함을 시사하는 것이다 본 연구는 LH와 그 수용체 유전자가 흰쥐 유선에서도 발현되는가를 규명한 것이다. 이를 위해 reverse transcription-polymerase chain reaction (RT-PCR)과 LH 방사면역측정법 (radioimmunoassay, RIA)을 사용하였다. RT-PCR을 시행한 결과 생식 주기중인 임신하지 않은 흰쥐 유선에서 뇌하수체 유형의 LH${\beta}$ 전사체 (exon 1-3)가 증폭되었으나 정소특이적 LH${\beta}$ exon 부분은 검출되지 않았다. 뇌하수체 glycoprotein hormone에서 공통적으로 존재하는 ${\alpha}$-subunit과 LH 수용체에 대한 전사체 역시 흰쥐 유선에 존재함이 확인되었다. 또한 기존의 보고에서 수유중인 흰쥐 유선에서만 발현된다고 알려진 GnRH가 임신하지 않은 흰쥐 유선에서도 발현됨을 확인하였다. LH 방사면역측정법을 시행한 결과 흰쥐 유선조직 추출물에서 immunoreactive LH분자들이 검출되었으며, LH standard curve와 parallelism을 보이므로 흰쥐 유선의 LH가 뇌하수체 형과 동일할 가능성을 확인하였다. 본 연구는 흰쥐 유선에서 LH subunit들과 수용체 유전자가 발현됨을 최초로 보고한 것으로서, 흰쥐 유선이 LH의 생성처이면서 동시에 작용처이며 유선에서 합성된 GnRH의 조절하에 국부적인 인자로 작용할 가능성을 시사한다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권6호
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• pp.481-489
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• 2010

Introduction: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. Materials and Methods: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. Results: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. Conclusion: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.

• Journal of Korean Neurosurgical Society
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• 제29권7호
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• pp.868-876
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• 2000

Objective : Parkinsonian rat models have generally been characterized by unilateral destruction of both the nigrostriatal pathway and the mesolimbic pathway using the neurotoxin 6-hydroxydopamine. The induction of contraversive turning by apomorphine in these models is thought to reflect the stimulation of supersensitive dopamine D2 receptor or receptor-mediated mechanisms in denervated neostriatum. The present study was undertaken to investigate the expression of dopamine D2 receptor in denervated striatum according to modalities of apomorphine(dopamine agonist) treatment after creating a hemiparkinsonian rat model in which there is 6-hydroxydopamine induced destruction of the unilateral dopaminergic nigrostriatal pathway. Methods : After making complete lesion in left side substantia nigra pars compacta(SNpc) by stereotactic injection of 6-hydroxydopamine into medial and lateral areas of SNpc, and confirming successful animal model by apomorphine induced contraversive turning behavior without recovery and complete destruction of ipsilateral SNpc with tyrosine hydroxylase immunostaining in 7th day after operation, 15 rats of parkinsonian model were studied with or without administration of apomorphine at varying doses and durations. According to the modalities of apomorphine treatment for 4 days, these rats were divided into 3 groups, as not-treated group, intermittently treated group and constantly treated group. For investigating the extent of the expression of dopamine D2 receptor in denervated striatum, immunohistochemical staining by dopamine D2 receptor antibody and Western blot were performed. Results : In the D2 receptor antibody immunohistochemical staining, the mean number of positive stained neurons was highest in not-treated group($20.5{\pm}1.14$) of 3 groups. In constantly treated group, the mean number of positive stained neurons was less($3.9{\pm}1.79$) than intermittently treated group(p<0.05). The Western blotting with the D2 receptor antibody revealed that expression of receptors was also highest in not-treated group and less in constantiy treated group than intermittently treated group. Conclusion : Dopamine D2 receptors in denervated striatum of parkinsonian rat models, which were not treated with apomorphine, revealed to be most highly expressed. And, according to doses and durations of apomorphine administration, desensitization of the receptor was more apt to develop with constant treatment than intermittent treatment. In clinical setting, the authors believe that, in long-term treated parkinsonian patients, desensitization of dopamine receptors due to chronic dopaminergic stimulation seems to be partially related to mechanisms of drug tolerance.

• Asian Pacific Journal of Cancer Prevention
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• 제15권2호
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• pp.989-997
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• 2014

AMFR, autocrine motility factor receptor, also called gp78, is a cell surface cytokine receptor which has a dual role as an E3 ubiquitin ligase in endoplasmic reticulum-associated degradation. AMFR expression is associated with tumor malignancy. We here investigated the clinical significance of AMFR and its role in metastasis and prognosis in gastric cancer. Expression of AMFR, E-cadherin and N-cadherin in cancer tissues and matched adjacent normal tissues from 122 gastric cancer (GC) patients undergoing surgical resection was assessed by immunohistochemistry. Levels of these molecules in 17 cases selected randomly were also analysed by Western blotting. AMFR expression was significantly increased in gastric cancer tissues, and associated with invasion depth and lymph node metastasis. Kaplan-Meier analysis showed AMFR expression correlated with poor overall survival and an increased risk of recurrence in the GC cases. Cox regression analysis suggested AMFR to be an independent predictor for overall and recurrence-free survival. E-cadherin expression was decreased in gastric cancer tissues; conversely, N-cadherin was increased. Expression of AMFR negatively correlated with E-cadherin expression, whereas N-cadherin expression showed a significant positive correlation with AMFR expression. AMFR might be involved in the regulation of epithelial-mesenchymal transition, with aberrant expression correlating with a poor prognosis and promoting invasion and metastasis in GCs.