The classical and a non-classical pathways associated with NF-κB are involved in estrogen-medicated regulation of Calbindin-D9k gene in rat pituitary cells

Lee, G.S.,Choi, K.C.,Han, H.J.,Jeung, E.B. North-Holland 2007 Molecular and cellular endocrinology Vol.277 No.1-2

Calbindin-D9k (CaBP-9k) is a high affinity calcium binding protein that is highly expressed in the duodenum, kidney, uterus, and pituitary glands. Previous studies have shown that CaBP-9k expression is regulated by several steroid hormones, such as 1,25-dihydroxyvitamin D3, glucocorticoids, 17β-estradiol (E2), and progesterone (P4), in a tissue-specific manner. However, the promoter elements that mediate transcriptional regulation by these steroid hormones are not clearly understood, mainly due to the lack of an appropriate cell line expressing CaBP-9k. Recently it was shown that CaBP-9k was constitutively expressed in the rat pituitary gland, and is expressed in an E2-dependent manner in a pituitary gland tumor-derived cell line, GH3. In the current study, we examined the activity of the estrogen responsive element (ERE) in rat CaBP-9k gene in GH3 cells, using a luciferase gene reporter assay, electrophoretic mobility shift assay, and mutagenesis. A nuclear factor-κB (NF-κB) binding site in the CaBP-9k promoter region was identified (nucleotides -848 to -834 from the transcriptional start site), and we demonstrated that addition of an NF-κB blocker to GH3 cells reduced E2-induced CaBP-9k transcription. In the present study, we further showed a previously reported imperfect ERE (nucleotides +51 to +65) between exon I and intron A of CaBP-9k, indicating that the interaction of estrogen receptor (ER) α with this region is involved in the regulation of CaBP-9k promoter activity and its expression. Taken together, these results suggest that in GH3 cells, both the classical ERα-ERE pathway and a non-classical pathway involving NF-κB are involved in E2-mediatd regulation of CaBP-9k expression in the pituitary gland.

Aerosol delivery of eukaryotic translation initiation factor 4E-binding protein 1 effectively suppresses lung tumorigenesis in K-ras^LA1 mice

Chang, S-H,Kim, J-E,Lee, J-H,Minai-Tehrani, A,Han, K,Chae, C,Cho, Y-H,Yun, J-H,Park, K,Kim, Y-S,Cho, M-H Nature America, Inc. 2013 Cancer gene therapy Vol.20 No.6

Conventional radiotherapy or chemotherapy for the long-term survival of patients with lung cancer is still difficult for treatment in metastatic and advanced tumors. Therefore, the safe and effective approaches to the treatment of lung cancer are needed. In this study, the effect of delivered eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) on lung cancer progression was evaluated. Recombinant adeno-associated virus (rAAV)-M3/4E-BP1 was delivered into 6-week-old K-ras<SUP>LA1</SUP> lung cancer model mice through a nose-only inhalation system twice a week for 4 weeks. Long-term repeated delivery of 4E-BP1 effectively reduced tumor progression in the lungs of K-ras<SUP>LA1</SUP> mice. Reduction of eIF4E by overexpression of 4E-BP1 resulted in suppression of cap-dependent protein expression of basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF). In addition, delivered 4E-BP1 inhibited the proliferation of lung cancer cells in K-ras<SUP>LA1</SUP> mice model. Our results suggest that long-term repeated viral delivery of 4E-BP1 may provide a useful tool for designing lung cancer treatment.

Bacteroides fragilis의 독소유전자 분석 및 재조합독소 발현에 관한 연구

오희복,정경태,이기은,성원근,이용진,이근영 국립보건연구원 1999 국립보건원보 Vol.36 No.-

Association of Extracellular Cleavage of E-Cadherin Mediated by MMP-7 with HGF-Induced in vitro Invasion in Human Stomach Cancer Cells

Lee, K.H.,Choi, E.Y.,Hyun, M.S.,Jang, B.I.,Kim, T.N.,Kim, S.W.,Song, S.K.,Kim, J.H.,Kim, J.-R. S. Karger AG 2007 European surgical research Vol.39 No.4

<P><I>Background:</I> Proteolytic shedding of the ectodomain of a variety of transmembrane proteins, including cell-to-cell adhesion molecules, has been observed in solid cancers. We have investigated whether extracellular cleavage of E-cadherin mediated by matrix metalloproteinase-7 (MMP-7) is involved in hepatocyte growth factor (HGF) induced in vitro invasion in stomach cancer cells. <I>Methods:</I> The effects of HGF on the expression of E-cadherin/β-catenin and MMP-7 at both the protein and mRNA levels were assessed in stomach cancer cells, NUGC-3 and MKN-28, and in cells in which the expression of MMP-7 was downregulated by transfection with a MMP-7 short hairpin RNA plasmid. <I>Results:</I> Treatment with HGF increased the extracellular cleavage of E-cadherin and the release of MMP-7 and reduced the level of E-cadherin in a dose- and time-dependent manner. HGF treatment repressed the phosphorylation of β-catenin in a Triton-soluble fraction, but enhanced this phosphorylation in a Triton-insoluble fraction. The association of E-cadherin with β-catenin was decreased by HGF treatment in the Triton-soluble fraction. In addition, treatment of MMP-7 short hairpin RNA transfected NUGC-3 cells with HGF resulted in no extracellular cleavage of E-cadherin and also decreased the in vitro cell invasion. <I>Conclusions:</I> These results suggest that incubation with HGF mediated the release of MMP-7, resulting in extracellular cleavage of E-cadherin from stomach cancer cells. This might be a key mechanism in HGF-induced in vitro invasion and metastasis.</P><P>Copyright © 2007 S. Karger AG, Basel</P>

한국인 Long Term Non Progressors로부터 분리된 HIV-1 env 및 nef 유전자와 이들의 면역 유전학적 특성 분석에 의한 질병진전 요인 규명

이주실,강춘,김성순,박근용,고운영,기미경,최병선,남정구,서순덕,이성래,구본기,박혜경,도경미,김태규,최희백,조현일,김요숙,김지영,이해정,안수진 국립보건연구원 1999 국립보건원보 Vol.36 No.-

EZH2 Generates a Methyl Degron that Is Recognized by the DCAF1/DDB1/CUL4 E3 Ubiquitin Ligase Complex

Lee, J.,Lee, Jason S.,Kim, H.,Kim, K.,Park, H.,Kim, J.Y.,Lee, S.,Kim, I.,Kim, J.,Lee, M.,Chung, C.,Seo, S.B.,Yoon, J.B.,Ko, E.,Noh, D.Y.,Kim, K.,Kim, K.,Baek, S. Cell Press 2012 Molecular cell Vol.48 No.4

Ubiquitination plays a major role in protein degradation. Although phosphorylation-dependent ubiquitination is well known for the regulation of protein stability, methylation-dependent ubiquitination machinery has not been characterized. Here, we provide evidence that methylation-dependent ubiquitination is carried out by damage-specific DNA binding protein 1 (DDB1)/cullin4 (CUL4) E3 ubiquitin ligase complex and a DDB1-CUL4-associated factor 1 (DCAF1) adaptor, which recognizes monomethylated substrates. Molecular modeling and binding affinity studies reveal that the putative chromo domain of DCAF1 directly recognizes monomethylated substrates, whereas critical binding pocket mutations of the DCAF1 chromo domain ablated the binding from the monomethylated substrates. Further, we discovered that enhancer of zeste homolog 2 (EZH2) methyltransferase has distinct substrate specificities for histone H3K27 and nonhistones exemplified by an orphan nuclear receptor, RORα. We propose that EZH2-DCAF1/DDB1/CUL4 represents a previously unrecognized methylation-dependent ubiquitination machinery specifically recognizing ''methyl degron''; through this, nonhistone protein stability can be dynamically regulated in a methylation-dependent manner.

A novel chimeric promoter that is highly responsive to hypoxia and metals

Lee, J-Y,Lee, Y-S,Kim, J-M,Kim, K L,Lee, J-S,Jang, H-S,Shin, I-S,Suh, W,Jeon, E-S,Byun, J,Kim, D-K Nature Publishing Group 2006 Gene Therapy Vol.13 No.10

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 × HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS–MRE-3 × HRE (E–M–H) gave a hypoxia induction ratio of 69. The expression induced from E–M–H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E–M–H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1α, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E–M–H chimeric promoter. E–M–H was also induced by hypoxia mimetics such as Co<SUP>2+</SUP> and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E–M–H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E–M–H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E–M–H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.Gene Therapy (2006) 13, 857–868. doi:10.1038/sj.gt.3302728; published online 9 February 2006

Magnetic resonance imaging of macrophage activity in atherosclerotic plaques of apolipoprotein E-deficient mice by using polyethylene glycolated magnetic fluorescent silica-coated nanoparticles

Lee, K.,Moon, H.Y.,Park, C.,Kim, O.R.,Ahn, E.,Lee, S.Y.,Park, H.E.,Ihm, S.H.,Seung, K.B.,Chang, K.,Yoon, T.J.,Lee, C.,Cheong, C.,Hong, K.S. Elsevier 2009 Current Applied Physics Vol.9 No.1

We have recently synthesized organic dye-incorporated silica-coated core-shell magnetic nanoparticles (MFSNs) that enable the detection of both fluorescence and magnetic properties in cells and tissues by using magnetic resonance imaging (MRI). Furthermore, polyethylene glycolation of the surface of these MFSNs would render them more stable and biocompatible, and thus allow their in vivo use as a circulating agent with a long half-life. Atherosclerotic vascular diseases are the leading cause of death worldwide. A noninvasive diagnostic imaging method is required to identify vulnerable plaques prior to the occurrence of a clinical event. Macrophages are the key cellular mediators in the pathogenesis of plaque inflammation and vulnerability. We evaluated whether the use of polyethylene glycolated (PEGylated) MFSNs could help in effectively detecting the macrophage activity in the aorta of apolipoprotein E (apoE)-deficient mice. PEGylated MFSNs (Fe, 30mg/kg) were injected via the tail vein in 1.2% cholesterol-fed 30-week-old apoE-deficient mice. After 24h, ex vivo MRI was carried out. The atheromas were then observed by confocal laser scanning microscopy (CLSM), and immunohistochemical staining targeted toward the macrophages was performed. Ex vivo MRI demonstrated robust enhancement of the atherosclerotic plaques along the aortic wall. CLSM images showed accumulation of PEGylated MFSNs in the atherosclerotic plaques, and histological examination revealed the localization of MFSNs in the macrophages present in the lesion. Therefore, PEGylated MFSNs could function as an effective multimodal imaging agent in the identification of macrophage activity in atherosclerotic plaques.

Relationship Between K_trans and K₁ with Simultaneous Versus Separate MR/PET in Rabbits with VX2 Tumors

Lee, K. H.,Kang, S. K.,Goo, J. M.,Lee, J. S.,Cheon, G. J.,Seo, S.,Hwang, E. J. INTERNATIONAL INSTITUTE OF ANTICANCER RESEARCH 2017 Anticancer research Vol.37 No.3

<P>Background/Aim: To compare the relationship between Ktrans from DCE-MRI and K1 from dynamic (NNH3)-N-13- PET, with simultaneous and separate MR/PET in the VX-2 rabbit carcinoma model. Materials and Methods: MR/PET was performed simultaneously and separately, 14 and 15 days after VX-2 tumor implantation at the paravertebral muscle. The Ktrans and K-1 values were estimated using an in-house software program. The relationships between Ktrans and K-1 were analyzed using Pearson's correlation coefficients and linear/non-linear regression function. Results: Assuming a linear relationship, Ktrans and K-1 exhibited a moderate positive correlations with both simultaneous ( r=0.54-0.57) and separate ( r=0.53-0.69) imaging. However, while the Ktrans and K-1 from separate imaging were linearly correlated, those from simultaneous imaging exhibited a non-linear relationship. The amount of change in K-1 associated with a unit increase in Ktrans varied depending on Ktrans values. Conclusion: The relationship between K-trans and K-1 may be mis-interpreted with separate MR and PET acquisition.</P>

Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II

Lee, K‐,E,Kang, H‐,Y,Lee, S‐,K,Yoo, S‐,H,Lee, J‐,C,Hwang, Y‐,H,Nam, KH,Kim, J‐,S,Park, J‐,C,Kim, J‐,W Blackwell Publishing Ltd 2011 Clinical genetics Vol.79 No.4

<P>Lee K‐E, Kang H‐Y, Lee S‐K, Yoo S‐H, Lee J‐C, Hwang Y‐H, Nam KH, Kim J‐S, Park J‐C, Kim J‐W. Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II.</P><P>The dentin sialophosphoprotein (<I>DSPP</I>) gene encodes the most abundant non‐collagenous protein in tooth dentin and DSPP protein is cleaved into several segments including the highly phosphorylated dentin phosphoprotein (DPP). Mutations in the <I>DSPP</I> gene have been solely related to non‐syndromic form of hereditary dentin defects. We recruited three Korean families with dentinogenesis imperfecta (DGI) type II and sequenced the exons and exon–intron boundaries of the <I>DSPP</I> gene based on the candidate gene approach. Direct sequencing of PCR products and allele‐specific cloning of the highly repetitive exon 5 revealed novel single base pair (bp) deletional mutations (c.2688delT and c.3560delG) introducing hydrophobic amino acids in the hydrophilic repeat domain of the DPP coding region. All affected members of the three families showed exceptionally rapid pulp chambers obliteration, even before tooth eruption. Individuals with the c.3560delG mutation showed only mild, yellowish tooth discoloration, in contrast to the affected individuals from two families with c.2688delT mutation. We believe that these results will help us to understand the molecular pathogenesis of DGI type II as well as the normal process of dentin biomineralization.</P>