👤 Soon Won Choi

Liver X receptor (LXR) is a ligand-activated transcription factor that plays important roles in cholesterol and lipid homeostasis. However, ligand-induced posttranslational modification of LXR is largely unknown. Here, we show that ligand-free LXRalpha is rapidly degraded by ubiquitination. Without ligand, LXRalpha interacts with an ubiquitin E3-ligase protein complex containing breast and ovarian cancer susceptibility 1 (BRCA1)-associated RING domain 1 (BARD1). Interestingly, LXR ligand represses ubiquitination and degradation of LXRalpha, and the interaction between LXRalpha and BARD1 is inhibited by LXR ligand. Consistently, T0901317, a synthetic LXR ligand, increased the level of LXRalpha protein in liver. Moreover, overexpression of BARD1/BRCA1 promoted the ubiquitination of LXRalpha and reduced the recruitment of LXRalpha to the target gene promoters, whereas BARD1 knockdown reversed such effects. Taken together, these data suggest that LXR ligand prevents LXRalpha from ubiquitination and degradation by detaching BARD1/BRCA1, which might be critical for the early step of transcriptional activation of ligand-stimulated LXRalpha through a stable binding of LXRalpha to the promoters of target genes. Show less

Although hepatitis B virus X protein (HBx) has been implicated in abnormal lipid metabolism in hepatitis B virus (HBV)-associated hepatic steatosis, its underlying molecular mechanism remains unclear. Liver X receptor (LXR) plays an important role in regulating the expression of genes involved in hepatic lipogenesis. Here we demonstrate that LXRalpha and LXRbeta mediate HBV-associated hepatic steatosis. We have found that HBx induces the expression of LXR and its lipogenic target genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and peroxisome proliferator-activated receptor, and this is accompanied by the accumulation of lipid droplets. RNA interference with LXR expression decreases the amount of lipid droplets as well as the expression of the lipogenic genes, and this indicates that HBx-induced lipogenesis is LXR-dependent. LXRalpha and HBx colocalize in the nucleus and are physically associated. HBx induces the transactivation function of LXRalpha by recruiting CREB binding protein to the promoter of the target gene. Furthermore, we have observed that expression of LXR is increased in the livers of HBx-transgenic mice. Finally, there is a significant increase in the expression of LXRbeta (P = 0.036), SREBP-1c (P = 0.008), FAS, and stearoyl-coenyzme A desaturase-1 (P = 0.001) in hepatocellular carcinoma (HCC) in comparison with adjacent nontumorous nodules in human HBV-associated HCC specimens. Our results suggest a novel association between HBx and LXR that may represent an important mechanism explaining HBx-induced hepatic lipogenesis during HBV-associated hepatic carcinogenesis. Show less

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Recently, abnormal activation of the Wnt pathway has been found to be involved in the carcinogenesis of HCC. However, the relationship between genetic changes in the Wnt pathway-associated genes and its protein expression has not been studied in patients with HCC and cirrhotic nodules. The purpose of this study is to explore the contribution of inappropriate activation of the Wnt pathway in liver carcinogenesis. Somatic mutation in exons 3-5 of AXIN1 and exon 3 of beta-catenin were analyzed by direct sequencing and expression of axin and beta-catenin proteins by immunohistochemistry in a series of 36 patients with HCC and cirrhosis. The AXIN1 and beta-catenin gene mutations were observed in 25% (9/36) and 2.8% (1/36) of HCCs, respectively. All mutations detected in AXIN1 and beta-catenin genes were missense point mutations. Abnormal nuclear expression of beta-catenin was observed in 11 of 36 cases of HCCs (30.6%), but not in cirrhotic nodules. Reduced or absent expression of axin was seen in 24 of 36 HCCs (66.7%). The abnormal expression of beta-catenin and axin proteins was closely correlated with mutations of AXIN1 and beta-catenin (P < 0.0001 and P = 0.008, respectively). These data suggest that mutation of AXIN1 gene is a frequent and late event for HCC associated with cirrhosis, and is correlated significantly with abnormal expression of axin and beta-catenin. Therefore, activation of Wnt signaling through AXIN1 rather than beta-catenin mutation might play an important role in liver carcinogenesis. Show less

The cholesteryl ester transfer protein (CETP), a key player in cholesterol metabolism, has been shown to promote the transfer of triglycerides from very low density lipoprotein (VLDL) and low density lipoprotein (LDL) to high density lipoprotein (HDL) in exchange for cholesterol ester. Here we demonstrate that farnesoid X receptor alpha (FXRalpha; NR1H4) down-regulates CETP expression in HepG2 cells. A FXRalpha ligand, chenodeoxycholic acid (CDCA), suppressed basal mRNA levels of the CETP gene in HepG2 cells in a dose-dependent manner. Using gel shift and chromatin immunoprecipitation (ChIP) assays, we found that FXRalpha could bind to the liver X receptor alpha (LXRalpha; NR1H3) binding site (LXRE; DR4RE) located within the CETP 5' promoter region. FXRalpha suppressed LXRalpha-induced DR4RE-luciferase activity and this effect was mediated by a binding competition between FXRalpha and LXRalpha for DR4RE. Furthermore, the addition of CDCA together with a LXRalpha ligand, GW3965, to HepG2 cells was shown to substantially decrease mRNA levels of hepatic CETP gene, which is typically induced by GW3965. Together, our data demonstrate that FXRalpha down-regulates CETP gene expression via binding to the DR4RE sequence within the CETP 5' promoter and this FXRalpha binding is essential for FXRalpha inhibition of LXRalpha-induced CETP expression. Show less

Recent studies using human and mice reported that apolipoprotein A-V (APOA5) gene plays an important role in controlling triglyceride (TG) concentrations. The purpose of the present study was to investigate the correlation between single nucleotide polymorphisms (SNPs) and haplotypes in the APOA5 gene and TG in subjects and to search for possible associations of the APOA5 gene variants and common haplotypes with hypertriglyceridemia (HTG). We examined the case-control subjects including 100 HTG patients and 243 unrelated healthy control. The genes were screened for SNPs by direct sequencing in 48 genetically unrelated individuals. Six SNPs (-1390C>T, -1020G>A, -3A>G, V150M, G182C and 1259T>C) were genotyped in case and control populations. In this study, our results indicated a strong association between APOA5 SNP -3A>G and G182C and elevated TG levels (p<0.001). Analysis of the SNPs from APOA5 gene has identified major haplotype showing very strong association with HTG, CGGGTT (p<0.001). Likelihood ratio test (LRT) of these six SNPs revealed that haplotypes were strong independent predictors of HTG (p<0.001). Haplotype-trend logistic regression (HTR) analysis revealed a significant association between the CGGGGC (haplotype 2) and CGGGTT (haplotype 4) and HTG (OR=2.48, 95% CI=1.06-5.76 and OR=8.54, 95% CI=2.66-27.42, respectively). We confirm that the APOA5 variants are associated with triglyceride levels and the haplotype may be strong independent predictors of HTG among Koreans. Show less

Calsenilin/DREAM/KChIP3, a neuronal Ca(2+)-binding protein, has multifunctions in nucleus and cytosol. Here, we identified CLN3 as a calsenilin-binding partner whose mutation or deletion is observed in Batten disease. In vitro binding and immunoprecipitation assays show that calsenilin interacts with the C-terminal region of CLN3 and the increase of Ca(2+) concentration in vitro and in cells causes significant dissociation of calsenilin from CLN3. Ectopic expression of CLN3 or its deletion mutant containing only the C-terminus (153-438) and capable of binding to calsenilin suppresses thapsigargin or A23187-induced death of neuronal cells. In contrast, CLN3 deletion mutant containing the N-terminus (1-153) or (1-263), which is frequently found in Batten disease, induces the perturbation of Ca(2+) transient and fails to inhibit the cell death. In addition, the expression of calsenilin is increased in the brain tissues of CLN3 knock-out mice and SH-SY5Y/CLN3 knock-down cells. Down-regulation of CLN3 expression sensitizes SH-SY5Y cells to thapsigargin or A23187. However, additional decrease of calsenilin expression rescues the sensitivity of SH-SY5Y/CLN3 knock-down cells to Ca(2+)-mediated cell death. These results suggest that the vulnerability of CLN3 knock-out or CLN3 deletion (1-153)-expressing neuronal cells to Ca(2+)-induced cell death may be mediated by calsenilin. Show less

The objective of this study was to investigate the molecular mechanisms underlying the attenuating effect of (-)-epigallocatechin-3-gallate (EGCG) on proliferation and lipid accumulation of 3T3-L1 cells, with a focus on the duration of EGCG treatment. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and diamidino-2-phenylindole staining. The anti-adipogenic effect of EGCG on 3T3-L1 cells was analyzed by glycerol-3-phosphate dehydrogenase activity and Oil red O staining. Western blot analysis was used to detect adenosine monophosphate-activated protein kinase (AMPK) activation and phosphorylation of its substrate, acetyl-CoA carboxylase (ACC), and expression of insulin (INS) receptor, INS receptor substrate-1 (IRS-1), and adipocyte marker proteins. Exposure to EGCG during the early period of adipogenesis (7 days) was sufficient to prevent lipid accumulation. During this period, EGCG greatly decreased expression of the adipocyte marker proteins peroxisome proliferator-activated receptor gamma2 (PPARgamma2) and liver X receptor (LXR)-alpha. Furthermore, EGCG significantly induced generation of reactive oxygen species (ROS), which led to AMPK activation, and these effects were eliminated by N-acetylcysteine (NAC) treatment. Also, EGCG increased the tyrosine phosphorylation of INS receptor and INS-1 with increasing incubation time. In contrast, EGCG treatment did not alter glycerol release in the presence or absence of 2',5'-dideoxyadenosine (DDA), indicating that EGCG had no effect on lipolysis. Our data demonstrate that EGCG decreased cell viability and inhibited differentiation of 3T3-L1 cells in a manner dependent on the duration of treatment. Also, we showed that inhibition of adipocyte differentiation by EGCG was associated with decreased glycerol-3-phosphate dehydrogenase (GPDH) activity accompanied by a strong inhibition of PPARgamma2-induced transcriptional activity. Furthermore, the inhibition of adipocyte differentiation by EGCG involved generation of ROS and activation of AMPK. Show less

The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Dpatj (Drosophila homolog of mammalian Patj) is a multi-PDZ domain protein that localizes to the apical cell membrane and forms a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (Sdt). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the dpatj gene. Our genetic analysis of hypomorph, null, and RNA interference reveals distinct dual functions of Dpatj in developing and mature photoreceptors. The C-terminal region (PDZ domains 2-4) of Dpatj is not essential for development of the animal but is required to prevent late-onset photoreceptor degeneration. In contrast, the N-terminal region of Dpatj is essential for animal viability and photoreceptor morphogenesis during development. The localization and maintenance of Crb and Sdt in the apical photoreceptor membrane are strongly affected by reduced levels of Dpatj. Dpatj is necessary for proper positioning of AJs and the integrity of photoreceptors in the developing retina as well as for the maintenance of adult photoreceptors. Our study provides evidence that Dpatj has domain-specific early and late functions in regulating the localization and stability of the Crb-Sdt complex in photoreceptor cells. Show less

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling. Show less

Glycogen synthase kinase 3beta (GSK3 beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and insulin response. GSK3 beta has now been shown to induce activation of the mitogen-activated protein kinase kinase kinase MEKK1 and thereby to promote signaling by the stress-activated protein kinase pathway. GSK3 beta-binding protein blocked the activation of MEKK1 by GSK3 beta in human embryonic kidney 293 (HEK293) cells. Furthermore, co-immunoprecipitation analysis revealed a physical association between endogenous GSK3 beta and MEKK1 in HEK293 cells. Overexpression of axin1, a GSK3 beta-regulated scaffolding protein, did not affect the physical interaction between GSK3 beta and MEKK1 in transfected HEK293 cells. Exposure of cells to insulin inhibited the activation of MEKK1 by GSK3 beta, and this inhibitory effect of insulin was abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1 activity under either basal or UV- or tumor necrosis factor alpha-stimulated conditions was reduced in embryonic fibroblasts derived from GSK3 beta knockout mice compared with that in such cells from wild-type mice. Ectopic expression of GSK3 beta increased both basal and tumor necrosis factor alpha-stimulated activities of MEKK1 in GSK3 beta(-/-) cells. Together, these observations suggest that GSK3 beta functions as a natural activator of MEKK1. Show less

The pathology of diabetic retinopathy includes dilatation and beading of retinal vessels, and vascular sheathing. To gain a better understanding of the molecular events leading to diabetic retinopathy, we investigated disease-specific gene responses by screening differential expression using cDNA microarray. Male Sprague-Dawley rats were intraperitoneally injected with streptozotocin (STZ, 50 mg/kg) or the control buffer and were maintained for 6 weeks. Total RNA extracted from the retinas of both groups was used for cDNA microarray analysis. Signals from all the spots representing hybridized DNA were quantified and compared between the normal and diabetic rat retinas. Among 1176 genes analyzed, the retinal expression of glucose-dependent insulinotropic polypeptide (GIP) was found to increase in STZ-induced diabetic rats compared to controls. GIP is a secreted protein, known to be released from the small intestine, which potentiates glucose-induced insulin secretion from the pancreas. However, the expression of GIP and its receptor (GIPR) has not been previously noted in the rat retina. To further validate the expression of GIP in the rat retina and to determine its possible role in the development of early diabetic retinopathy, we investigated its expression by RT-PCR, Northern blotting, and immunohistochemistry in normal and diabetic rat retinas. GIP mRNA and protein are not only expressed in the rat retina, but their levels are greater in the diabetic rat as compared to controls. And GIPR expression was also upregulated in the retinas of STZ-induced diabetic rats. We here demonstrate for the first time the expression of GIP and GIPR in the rat retina. And we also revealed some genetic events in the early stage of diabetic retinopathy including the de novo increment of GIP and GIPR expression in the retina. Show less

A partial C-terminal cDNA sequence of a novel Drosophila mitogen-activated protein kinase phosphatase (MKP), designated DMKP-3, was identified from an epitope expressed sequence tag database, and the missing N-terminal cDNA fragment was cloned from a Drosophila cDNA library. DMKP-3 is a protein of 411 amino acids, with a calculated molecular mass of 45.8 kDa; the deduced amino acid sequence is most similar to that of mammalian MKP-3. Recombinant DMKP-3 produced in Escherichia coli retained intrinsic tyrosine phosphatase activity. In addition, DMKP-3 specifically inhibited extracellular-signal-regulated kinase (ERK) activity, but was without a significant affect on c-Jun N-terminal kinase (JNK) and p38 activities, when it was overexpressed in Schneider cells. DMKP-3 interacted specifically with Drosophila ERK (DERK) via its N-terminal domain. In addition, DMKP-3 specifically inhibited Elk-1-dependent trans-reporter gene expression in mammalian CV1 cells, and dephosphorylated activated mammalian ERK in vitro. DMKP-3 is uniquely localized in the cytoplasm within Schneider cells, and gene expression is tightly regulated during development. Thus DMKP-3 is a Drosophila homologue of mammalian MKP-3, and may play important roles in the regulation of various developmental processes. Show less

Oculodentodigital dysplasia (ODDD) is an autosomal dominant condition with congenital anomalies of the craniofacial and limb regions and neurodegeneration. Genetic anticipation for the dysmorphic and neurologic features has been inferred in a few families. Our previous linkage studies have refined the ODDD candidate region to chromosome 6q22-->q23. In an attempt to clone the ODDD gene, we created a yeast artificial chromosome contig with 31 redundant clones spanning the region and identified and ordered candidate genes and markers. Fluorescent IN SITU hybridization mapped two of these YAC clones to chromosome 6q22.2 telomeric to a known 6q21 fragile site, excluding it as a possible cause of the suggested anticipation. We performed mutation analysis on thirteen candidate genes - GRIK2, HDAC2, COL10A1, PTD013, KPNA5, PIST, ROS1, BRD7, PLN, HSF2, PKIB, FABP7, and HEY2. Although no mutations were found, we identified 44 polymorphisms, including 28 single nucleotide polymorphisms. Direct cDNA selection was performed and fifty-five clones were found to contain sequences that were not previously reported as known genes or ESTs. These clones and polymorphisms will assist in the further characterization of this region and identification of disease genes. Show less

Many patients with chronic renal failure (CRF) requiring hemodialysis present with hypertriglyceridemia (HTG). But the exact cause of HTG in CRF is still unknown. Genetic variation of the apo AI-CIII-AIV gene cluster was reported to be associated with primary HTG, atherosclerosis and coronary artery disease. This study was designed to evaluate the association between the restriction fragment length polymorphism (RFLP) of the apo AI-CIII-AIV gene cluster and HTG in patients with CRF undergoing hemodialysis. Genetic variations of the apo AI-CIII-AIV gene cluster were analysed in peripheral leukocyte samples from 59 patients with CRF undergoing hemodialysis: 17 patients with HTG (CRF-HTG) and 42 patients without HTG (CRF-NTG). The RFLP was achieved through the digestion of PCR products by two restriction enzymes, SstI and MspI. The frequency of SstI minor allele (S2) in CRF-HTG was 0.44, which was significantly higher than that in CRF-NTG (0.17). Frequencies of MspI minor allele (M2) in CRF-HTG and CRF-NTG were not significantly different (0.5 vs 0.32) (p=0.07). Frequencies of S2-M2 genotype were 0.65 in CRF-HTG, and 0.27 in CRF-NTG (p<0.005). These data indicate that genetic variation of the apo AI-CIII-AIV gene cluster may serve as one of the causes of HTG in CRF. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominantly inherited disease characterized by the formation of cartilage-capped prominences (exostoses) that develop from the juxtaepiphyseal regions of the long bones. Recently, EXT1 and EXT2 genes were cloned and germline mutations of EXT1 and EXT2 were identified in EXT families. In this study, we performed a mutational analysis of EXT1 and EXT2 genes in eight unrelated Korean EXT families by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing. As a result, we were able to identify one family (SNU-OC3) with the EXT1 mutation and another family (SNU-OC15) with the EXT2 mutation. The EXT1 mutation was a 10-bp deletion at the 3' end of exon 5 (CTAATTTAGg) including the splice site of this exon. The EXT2 mutation identified in the SNU-OC15 family was a missense mutation at codon 85 of exon 2 (TGC-->CGC), resulting in an amino acid change from cysteine to arginine. This missense mutation cosegregated with the disease phenotype in this family, suggesting that it is the disease-causing mutation. These two mutations identified in EXT1 and EXT2 are novel ones. Show less

The mutation responsible for the juvenile form of Batten disease was mapped to a single gene, Cln3 (T. J. Lerner et al. (1995) Cell 82:949-957). Yeast Saccharomyces cerevisiae has an open reading frame, BTN1 (YHC3), that encodes the putative homologue of Cln3p. Primary structure comparison indicates that the human Cln3p and yeast Btn1p are 59% similar and 39% identical and they have similar hydropathy profiles. Gene disruption of BTN1 in yeast has no apparent effect on growth or viability of the cells under a variety of conditions. Gene fusion protein constructs of green fluorescent protein (GFP) and Btn1p, with GFP at the amino and carboxyl ends of Btn1p, localize to the vacuole in yeast. These data indicate that BTN1 is a nonessential gene under most growth conditions which functions in the vacuole in yeast Saccharomyces cerevisiae. Show less