• Biomolecules & Therapeutics
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• 제29권6호
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• pp.667-677
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• 2021

The elevated expression of the hyaluronan-mediated motility receptor (HMMR) is known to be highly associated with tumor progression in prostate cancer, but the molecular mechanisms underlying the regulation of HMMR expression remain unclear. Here, we report that mammalian target of rapamycin (mTOR) is a key regulator of HMMR expression, for which its kinase activity is required. Pharmacological inhibitors of mTOR, such as rapamycin and Torin2, markedly suppressed the mRNA level as well as the protein level of HMMR in LNCaP and PC-3 cells. Our data demonstrate that such regulation occurs at the transcription level. HMMR promoter reporter assays revealed that the transcription factor SRF is responsible for the mTOR-mediated transcriptional regulation of HMMR gene. Consistently, the suppression of HMMR expression by Torin2 was noticeably reversed by the overexpression of SRF. Moreover, our findings suggest that the SRF binding sites responsible for the transcriptional regulation of HMMR through the mTOR-SRF axis are located in HMMR promoter sequences carrying the first intron, downstream of the translational start site. Furthermore, the upregulation of HMMR by DHT was abolished by stimulation with rapamycin, prior to DHT treatment, suggesting that mTOR activity is required for the induction of HMMR expression by androgen. Collectively, our study provides new mechanistic insights into the role of mTOR/SRF/AR signaling in HMMR regulation in prostate cancer cells.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.71-71
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• 2002

Promoters for milk proteins have been used far producing transgenic animals due to their temporal and spatial expression patterns. ${\beta}$-casein, a calcium-sensitive casein, is a major milk protein that corresponds ca. 30 per cent of total milk protein. Expression of ${\beta}$-casein is controlled by lactogenic hormones such as prolactin (PRL), composite response elements (CoREs) and transcription factors. CoREs are clusters of transcription factor binding sites containing both positive and negative regulatory elements. ${\beta}$-casein gene promoter contains various regions (CoREs) for gene transcription. We analyzed the promoter region by mutagenesis using exonuclease III and linker-scanning. Transcription control elements usually are positioned in 5'-flanking region of the gene. However, in some cases, these elements are located in other regions such as intron 1. The nucleotide sequences of ${\beta}$-casein promote. region has been reported (E12614). However, the properties of the promoter is not yet clear. In this study, we plan to investigate the properties of cis-regulating elements of porcine ${\beta}$-casein by mutation analysis and expression analysis using dual-luciferase repoter assay system.

• Asian-Australasian Journal of Animal Sciences
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• 제31권3호
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• pp.309-315
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• 2018

Objective: This study aimed to test the expression patterns of ERBB receptor feedback inhibitor 1 (ERRFI1) before and after exercise and the association of non-synonymous single-nucleotide polymorphisms (nsSNPs) of horse ERRFI1 with racing traits in Thoroughbreds. Methods: We performed bioinformatics and gene expression analyses for horse ERRFI1. Transcription factor (TF) binding sites in the 5'-regulatory region of this gene were identified through a tool for prediction of TF-binding site (PROMO). A general linear model was used to detect the association between the nsSNP (LOC42830758 A to G) and race performance. Results: Quantitative polymerase chain reaction analysis showed that expression level of ERRFI1 after exercise was 1.6 times higher than that before exercise. Ten transcription factors were predicted from the ERRFI1 regulatory region. A novel nsSNP (LOC42830758 A to G) was found in ERRFI1, which was associated with three racing traits including average prize money, average racing index, and 3-year-old starts percentile ranking. Conclusion: Our analysis will be helpful as a basis for studying genes and SNPs that affect race performance in racehorses.

• 생명과학회지
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• 제17권2호통권82호
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• pp.167-173
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• 2007

생쥐 신경교세포 유래 신경영양인자 유도성 전사인자(mGIF)의 발현조절에 필요한 전사기작을 연구하기 위하여 mGIF cDNA를 탐침자로 이용하여 genomic clone을 분리하였다. 전체 유전자 13-kb 영역 중 전사개시점에서 4-kb 상류영역의 유전자 서열을 파악한 결과, 프로모터 영역에서 TATA box와 CAAT box는 발견할 수 없었으며 G+C content는 높은 것으로 나타났고 여러 개의 Sp1 전사인자 결합영역이 있었다. 또한 mGIF 유전자는 AP2 결합에 필요한 보존적 영역이 있었다. mGIF 유전자의 프로모터 영역의 단편들을 프로모터가 없는 pGL2-Basic 플라스미드의 luciferase 유전자의 상류에 연결하여 서로 다른 5종류의 결손 돌연변이체를 제조하고 NB41A3 세포주를 이용하여 전사활성을 측정하였다. Transient expression assays 결과, 모든 결손 돌연변이체에서 전사활성이 나타났으며 -213과 -129사이에 전사촉진 영역이 존재하며 -806과 -214사이에 전사억제 영역이 있는 것으로 나타났다. 신경세포주인 NB41A3과 신경교세포주인 C6 그리고 간세포주인 HepG2에서 mGIF 유전자 프로모터의 높은 활성이 관찰되었으며, 근육세포주인 C2C12에서는 낮은 활성이 관찰되었다. 따라서 mGIF 유전자는 조직특이적으로 발현하며 도파민 수용체 유전자와 구조적, 기능적 유사성이 있는 것을 알 수 있었다.

• BMB Reports
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• 제40권1호
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• pp.88-94
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• 2007

Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in the turnover of extracellular matrix (ECM) and in the migration of normal and tumor cells in response to normal physiologic and numerous pathologic conditions. Here, we show that the transcription of the MMP-9 gene is induced by lipopolysaccharide (LPS) stimulation in cells of a macrophage lineage (RAW 264.7 cells). We provide evidence that the NF-$\kappa$B binding site of the MMP-9 gene contributes to its expression in the LPS-signaling pathway, since mutation of NF-$\kappa$B binding site of MMP-9 promoter leads to a dramatic reduction in MMP-9 promoter activation. In addition, the degradation of l$\kappa$B$\alpha$;, and the presences of myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated kinase 6 (TRAF6) were found to be required for LPS-activated MMP-9 expression. Chromatin immunoprecipitation (ChIP) assays showed that functional interaction between NF-$\kappa$B and the MMP-9 promoter element is necessary for LPS-activated MMP-9 induction in RAW 264.7 cells. In conclusion, our observations demonstrate that NF-$\kappa$B contributes to LPS-induced MMP-9 gene expression in a mouse macrophage cell line.

• BMB Reports
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• 제55권6호
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• pp.287-292
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• 2022

The acute response to hypoxia is mainly driven by hypoxia-inducible factors, but their effects gradually subside with time. Hypoxia-specific histone modifications may be important for the stable maintenance of long-term adaptation to hypoxia. However, little is known about the molecular mechanisms underlying the dynamic alterations of histones under hypoxic conditions. We found that the phosphorylation of histone H3 at Ser-10 (H3S10) was noticeably attenuated after hypoxic challenge, which was mediated by the inhibition of aurora kinase B (AURKB). To understand the role of AURKB in epigenetic regulation, DNA microarray and transcription factor binding site analyses combined with proteomics analysis were performed. Under normoxia, phosphorylated AURKB, in concert with the repressor element-1 silencing transcription factor (REST), phosphorylates H3S10, which allows the AURKB-REST complex to access the MDM2 proto-oncogene. REST then acts as a transcriptional repressor of MDM2 and downregulates its expression. Under hypoxia, AURKB is dephosphorylated and the AURKB-REST complex fails to access MDM2, leading to the upregulation of its expression. In this study, we present a case of hypoxia-specific epigenetic regulation of the oxygen-sensitive AURKB signaling pathway. To better understand the cellular adaptation to hypoxia, it is worthwhile to further investigate the epigenetic regulation of genes under hypoxic conditions.

• International Journal of Oral Biology
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• 제35권2호
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• pp.69-74
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• 2010

The mu opioid receptor (MOR) has been regarded as the main site of interaction with analgesics in major clinical use, particularly morphine. The repressor element-1 silencing transcription factor (REST) functions as a transcriptional repressor of neuronal genes in non-neuronal cells. However, it is expressed in certain mature neurons, suggesting that it may have complex and novel roles. In addition, the interactions between MOR and REST and their functions remain unclear. In this study, we examined the effects of morphine on the expression of REST mRNA and protein in human neuroblastoma NMB cells to investigate the roles of REST induced by MOR activation in neuronal cells. To determine the effects of morphine on REST expression, we performed RT-PCR, real-time quantitative RT-PCR, western blot analysis and radioligand binding assays in NMB cells. By RTPCR and real-time quantitative RT-PCR, the expression of REST was found to be unchanged by either the MOR agonist morphine or the MOR specific antagonist CTOP. By western blot, morphine was shown to significantly inhibit the expression of REST, but this suppression was completely blocked by treatment with CTOP. In the radioligand binding assay, the overexpression of REST led to an increased opioid ligand binding activity of endogenous MOR in the NMB cells. These results together suggest that morphine inhibits the expression of REST in human neuroblastoma cells through a post-transcriptional regulatory mechanism mediated through MOR.

• Molecules and Cells
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• 제40권3호
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• pp.211-221
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• 2017

Transforming growth factor ${\beta}1$ $(TGF{\beta}1)/Smad4$ signaling plays a pivotal role in maintenance of the dynamic balance between bone formation and resorption. The microRNA miR-155 has been reported to exert a significant role in the differentiation of macrophage and dendritic cells. The goal of this study was to determine whether miR-155 regulates osteoclast differentiation through $TGF{\beta}1/Smad4$ signaling. Here, we present that $TGF{\beta}1$ elevated miR-155 levels during osteoclast differentiation through the stimulation of M-CSF and RANKL. Additionally, we found that silencing Smad4 attenuated the upregulation of miR-155 induced by $TGF{\beta}1$. The results of luciferase reporter experiments and ChIP assays demonstrated that $TGF{\beta}1$ promoted the binding of Smad4 to the miR-155 promoter at a site located in 454 bp from the transcription start site in vivo, further verifying that miR-155 is a transcriptional target of the $TGF{\beta}1/Smad4$ pathway. Subsequently, TRAP staining and qRT-PCR analysis revealed that silencing Smad4 impaired the $TGF{\beta}1$-mediated inhibition on osteoclast differentiation. Finally, we found that miR-155 may target SOCS1 and MITF to suppress osteoclast differentiation. Taken together, we provide the first evidence that $TGF{\beta}1/Smad4$ signaling affects osteoclast differentiation by regulation of miR-155 expression and the use of miR-155 as a potential therapeutic target for osteoclast-related diseases shows great promise.

• 생명과학회지
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• 제20권5호
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• pp.655-661
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• 2010

흑색종세포주 SK-MEL-2에서 레티노이드에 의한 GD3합성효소(hST8Sia I)의 발현억제기작을 규명하게 위하여 hST8Sia I의 프로모터 활성을 조사해 본 결과 -1146에서 -646영역에서 레티노이드에 의한 활성억제를 나타내었다. 또한 부위특이적 변이와 ChIP분석은 -731에서 -722영역에 위치한 전사인자NF-kB 결합부위가 hST8Sia I의 레티노이드에 의한 활성억제에 중요하게 관여하고 있음을 나타내었다. 이러한 발현 억제는 PKC/ERK 신호전달경로를 통하여 일어난다는 것을 신호전달경로 저해제를 이용한 RT-PCR과 프로모터 활성조사에 의해 규명하였다.

• Genomics & Informatics
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• 제8권4호
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• pp.201-205
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• 2010

The H1 histone family, member N, testis-specific (H1FNT) is exclusively expressed in the testis, and had its possible role for sperm chromatin formation. The purpose of this study is to investigate any genetic association of H1FNT gene with male infertility, especially at the promoter region. We examined the promoter single nucleotide polymorphisms (SNP) of H1FNT gene which is located within transcription factor binding site for its association with male infertility. The statistical analysis showed that the -1129A>T polymorphism was present at a statistically significance in male infertility (p=0.0059 and 0.0349 for hetero and risk type, respectively). The dual-luciferase promoter assay was performed to examine the polymorphic effect of this promoter SNP by the cloning of promoter region (1700bp fragment) into pGL3-basic vector. In our plasmid based reporter system, there is no big difference between wild and risk type. In conclusion, H1FNT -1129A>T promoter SNP is statistically significant with male infertility, especially with subfertile (non-azoospermia) group. Further analysis of its functional polymorphic effect in vivo may provide the biological significance of testis-specific histone with spermatogenesis.