Epithelial-Mesenchymal Transition in Kidney Tubular Epithelial Cells Induced by Globotriaosylsphingosine and Globotriaosylceramide.

Fabry disease is a lysosomal storage disorder caused by deficiency of alpha-galactosidase A (α-gal A), which results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. Globotriaosylsphingosine (lyso-Gb3), a deacylated Gb3, is also increased in the plasma of patients with Fabry disease. Renal fibrosis is a key feature of advanced Fabry disease patients. Therefore, we evaluated the association of Gb3 and lyso-Gb3 accumulation and the epithelial-mesenchymal transition (EMT) on tubular epithelial cells of the kidney. In HK2 cells, exogenous treatments of Gb3 and lyso-Gb3 increased the expression of TGF-ß, EMT markers (N-cadherin and α-SMA), and phosphorylation of PI3K/AKT, and decreased the expression of E-cadherin. Lyso-Gb3, rather than Gb3, strongly induced EMT in HK2 cells. In the mouse renal mesangial cell line, SV40 MES 13 cells, Gb3 strongly induced phenotype changes. The EMT induced by Gb3 was inhibited by enzyme α-gal A treatment, but EMT induced by lyso-Gb3 was not abrogated by enzyme treatment. However, TGF-ß receptor inhibitor (TRI, SB525334) inhibited the activation of TGF-ß and EMT markers in HK2 cells with Gb3 and lyso-Gb3 treatments. This study suggested that increased plasma lyso-Gb3 has a crucial role in the development of renal fibrosis through the cell-specific induction of the EMT in Fabry disease, and that TRI treatment, alongside enzyme replacement therapy, could be a potential therapeutic option for patients with Fabry disease.

Substrate-specific gene expression profiles in different kidney cell types are associated with Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene encoding the α-galactosidase A (α-Gal A) lysosomal enzyme, which results in globotriaosylceramide (Gb3) storage in vascular endothelial cells and different cell types throughout the body. Involvement of the kidney and heart is life threatening, and fibrosis of these organs is considered to be involved in the pathogenesis of Fabry disease. An increased concentration of deacylated Gb3 (lyso­Gb3) in the plasma of symptomatic patients has also been suggested as a causative molecular event. To elucidate the molecular mechanisms involved in renal fibrosis in Fabry disease, the present analyzed the changes in global gene expression prior to and following Gb3 or lyso­Gb3 treatment in two types of kidney cell lines, human proximal renal tubular epithelial (HK­2) and mouse renal glomerular mesangial (SV40 MES 13) cells. Gb3 and lyso­Gb3 treatment regulated the expression of 199 and 328 genes in each cell type, demonstrating a >2.0­fold change. The majority of the biological functions of the regulated genes were associated with fibrogenesis or epithelial­mesenchymal transition (EMT). The gene expression patterns of sphingolipid­treated HK­2 cells were distinguishable from the patterns in the SV40 MES 13 cells. Several genes associated with the EMT were selected and evaluated further in kidney cells and in Fabry mouse kidney tissues. In the SV40 MES 13 cells, the DLL1, F8, and HOXA11 genes were downregulated, and FOXP2 was upregulated by treatment with Gb3 or lyso­Gb3. In the HK­2 cells, the ADAMTS6, BEST1, IL4, and MYH11 genes were upregulated. Upregulation of the FOXP2, COL15A1, IL4, and MYH11 genes was also observed in the Fabry mouse kidney tissues. The gene expression profiles in kidney cells following the addition of Gb3 or lyso­Gb3 revealed substrate­specific and cell­specific patterns. These findings suggested that Gb3 and lyso­Gb3 lead to renal fibrosis in Fabry disease through different biochemical modulations.

Possible role of transforming growth factor-ß1 and vascular endothelial growth factor in Fabry disease nephropathy.

Fabry disease is a lysosomal storage disorder (LSD) caused by deficiency of α-galactosidase A (α-gal A), resulting in deposition of globotriaosylceramide (Gb3; also known as ceramide trihexoside) in the vascular endothelium of many organs. A gradual accumulation of Gb3 leads to cardiovascular, cerebrovascular and renal dysfunction. Endothelial cell dysfunction leads to renal complications, one of the main symptoms of Fabry disease. However, the pathological mechanisms by which endothelial dysfunction occurs in Fabry disease are poorly characterized. The purpose of this study was to investigate whether the expression of transforming growth factor-ß1 (TGF-ß1) and vascular endothelial growth factor (VEGF) is associated with the renal pathogenesis of Fabry disease. We found that the protein expression levels of renal thrombospondin-1 (TSP-1), TGF-ß1 and VEGF were higher in the kidneys from Fabry mice compared to wild-type mice. The expression levels of VEGF receptor 2 (VEGFR2), fibroblast growth factor-2 (FGF-2) and phospho-p38 (P-p38) were also higher in the kidneys from Fabry mice compared with wild-type mice. Activities of cysteine aspartic acid protease (caspase)-6 and caspase-9 were higher in kidneys from Fabry than from the wild-type mice. These results suggest that overexpression of TGF-ß1 and VEGF in the Fabry mouse kidney might contribute to Fabry disease nephropathy by inducing apoptosis. To test whether Gb3 accumulation can induce apoptosis, we incubated bovine aortic endothelial cells with Gb3 and found increased expression of TGF-ß1, VEGFR2, VEGF, FGF-2 and P-p38. The combination of increased expression of TGF-ß1 and VEGF caused by Gb3 accumulation may allow upregulation of FGF-2, VEGFR2 and P-p38 expression, and these changes may be associated with Fabry disease nephropathy by inducing apoptosis.

Genome-wide identification of chemosensitive single nucleotide polymorphism markers in gastric cancer.

A chemosensitive single nucleotide polymorphism (SNP) discovery schema is presented that utilizes (i) genome-wide SNP screening, with a human SNP array and an in vitro chemosensitivity assay, in 93 patients with gastric cancer (GC), and (ii) biological utility assessment using cell viability assays of transfected GC cells. Cytotoxicity analysis showed that most of the MKN1 and SNU638 clones transfected with the G allele of Deoxyribonuclease II beta (DNASE2B) rs3738573 were more sensitive to docetaxel than those with the C allele (p≤0.001-0.029) and most of the AGS and SNU638 clones transfected with the T allele of 5-hydroxytryptamine receptor IE (HTRIE) rs3828741 were more sensitive to paclitaxel than those with the C allele (p≤0.001-0.019). Our findings show that the two novel markers, DNASE2B rs3738573 and HTR1E rs3828741, have potential for improving the prediction of chemosensitivity of GC patients.

Genome-wide association of serum bilirubin levels in Korean population.

A large-scale, genome-wide association study was performed to identify genetic variations influencing serum bilirubin levels using 8841 Korean individuals. Significant associations were observed at UGT1A1 (rs11891311, P = 4.78 x 10(-148)) and SLCO1B3 (rs2417940, P = 1.03 x 10(-17)), which are two previously identified loci. The two single-nucleotide polymorphisms (SNPs) were replicated (rs11891311, P = 3.18 x 10(-15)) or marginally significant (rs2417940, P = 8.56 x 10(-4)) in an independent cohort of 1096 individuals. In a conditional analysis adjusted for the top UGT1A1 variant (rs11891311), another variant in UGT1A1 (rs4148323, P = 1.22 x 10(-121)) remained significant; this suggests that in UGT1A1 at least two independent genetic variations influence the bilirubin levels in the Korean population. The protein coding variant rs4148323, which is monomorphic in European-derived populations, may be specifically associated with serum bilirubin levels in Asians (P = 2.56 x 10(-70)). The SLCO1B3 variant (rs2417940, P = 1.67 x 10(-18)) remained significant in a conditional analysis for the top UGT1A1 variant. Interestingly, there were significant differences in the associated variations of SLCO1B3 between Koreans and European-derived populations. While the variant rs2417940 at intron 7 of SLCO1B3 was more significantly associated in Koreans, variants rs17680137 (P = 0.584) and rs2117032 (P = 2.76 x 10(-5)), two of the top-ranked SNPs in European-derived populations, did not reach the genome-wide significance level. Also, variants in SLCO1B1 did not reach genome-wide significance in Koreans. Our result supports the idea that there are considerable ethnic differences in genetic association of bilirubin levels between Koreans and European-derived populations.

Placental gene expression is related to glucose metabolism and fetal cord blood levels of insulin and insulin-like growth factors in intrauterine growth restriction.

Despite the importance of glucose for fetal growth, placental gene expression related to the glycolytic pathway has not been studied. Insulin-like growth factors (IGFs) and insulin are known to play a critical role in fetal growth. In our study, we identified differentially expressed genes related to the glycolytic pathway using oligonucleotide microarray analysis and confirmed these genes with quantitative real-time PCR between uncomplicated pregnancies and pregnancies with intrauterine growth restriction (IUGR). We also compared the concentrations of insulin and IGFs in cord blood between the two groups. Microarray experiments identified increased expression of glycolytic enzyme-related genes, including lactate dehydrogenase C (LDHC), dihydrolipoamide S-acetyltransferase (DLAT), 6 phosphofructo-2-kinase/fructose-2, 6-biphosphatase 2 (PFKFB2), oxoglutarate dehydrogenase, phosphorylase, and insulin-like growth factor (IGF)-II and decreased expression of IGF-I in placentas from pregnancies with IUGR (p<0.05). There were significantly lower concentrations of glucose, insulin, IGF-1, and IGF-II in the fetal cord blood of pregnancies with IUGR (p<0.05). Microarray analysis revealed increased expression of enzyme genes related to the tricarboxylic acid cycle pathway in placentas from pregnancies with IUGR; the cause of hypoglycaemia in IUGR is attributed to increased glycolytic pathway activity in placentas from pregnancies with IUGR.

Generation of expression clone set for functional proteomics of human gastric and liver cancers.

Two thousand sixty-eight multi-purpose expression clones for the 326 candidate genes related to gastric or liver cancers were constructed using the Gateway system. These clones can be expressed as His, Glutathione-S-transferase (GST) or Enhanced version of the green fluorescent protein (EGFP) fusion proteins in E. coli, insect cells or mammalian cells. For the 246 E. coli expression clones, the GST fusion proteins had greater expression efficiency and solubility than the His fusion proteins. Approximately 20% of the expressed proteins had unexpected molecular weights. A detailed sequence analysis of these clones revealed frameshift mutations resulting from insertion, deletion or substitution of nucleotides. The results indicate that these changes in the candidate genes may affect the occurrence of gastric or liver cancers. In addition, when 105 proteins, which were expressed in E. coli at very low or undetectable levels, were expressed in insect cells, 76% of the proteins were expressed very well and most were soluble. We also found that most of the 30 proteins prepared using EGFP mammalian expression clones were localized to cellular compartments expected by Gene ontology (GO) and this localization was unaffected if the EGFP-fusion was at the N-terminal or C-terminal region of the protein. Antibody production and subcellular localization analysis of the candidate genes as well as a screen of genes involved in carcinogenesis pathways are currently in progress using these expression clones. These studies provide a valuable resource for developing a better understanding of the molecular mechanism of carcinogenesis in both gastric and liver cancer and would be very helpful in diagnosis and therapeutic predictions.

Copy number variations (CNVs) identified in Korean individuals.

BACKGROUND: Copy number variations (CNVs) are deletions, insertions, duplications, and more complex variations ranging from 1 kb to sub-microscopic sizes. Recent advances in array technologies have enabled researchers to identify a number of CNVs from normal individuals. However, the identification of new CNVs has not yet reached saturation, and more CNVs from diverse populations remain to be discovered. RESULTS: We identified 65 copy number variation regions (CNVRs) in 116 normal Korean individuals by analyzing Affymetrix 250 K Nsp whole-genome SNP data. Ten of these CNVRs were novel and not present in the Database of Genomic Variants (DGV). To increase the specificity of CNV detection, three algorithms, CNAG, dChip and GEMCA, were applied to the data set, and only those regions recognized at least by two algorithms were identified as CNVs. Most CNVRs identified in the Korean population were rare (<1%), occurring just once among the 116 individuals. When CNVs from the Korean population were compared with CNVs from the three HapMap ethnic groups, African, European, and Asian; our Korean population showed the highest degree of overlap with the Asian population, as expected. However, the overlap was less than 40%, implying that more CNVs remain to be discovered from the Asian population as well as from other populations. Genes in the novel CNVRs from the Korean population were enriched for genes involved in regulation and development processes. CONCLUSION: CNVs are recently-recognized structural variations among individuals, and more CNVs need to be identified from diverse populations. Until now, CNVs from Asian populations have been studied less than those from European or American populations. In this regard, our study of CNVs from the Korean population will contribute to the full cataloguing of structural variation among diverse human populations.

Prenatal increased asymmetric dimethylarginine is associated with placental heat-shock protein 70 and lectin-like oxidized low-density lipoprotein receptor-1 expression.

BACKGROUND: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, induces endothelial dysfunction by reversibly blocking NO production from L-arginine. To elucidate the association of prenatal status of ADMA with placental heat-shock protein 70 (HSP70) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in normal full-term pregnancies, we evaluated the expression of placental HSP70 and LOX-1. METHODS: Tissue samples of placentas obtained from 60 normal-term pregnancies were categorized into 30 cases with a lower level of prenatal ADMA (0.28 +/- 0.07 microM) and 30 cases with a higher level of prenatal ADMA (1.31 +/- 0.44 microM). We evaluated the placental expression of HSP70 and LOX-1 with Western blot analysis and immunohistochemistry and determined the correlation between HSP70 and LOX-1. RESULTS: We found that prenatal increased ADMA is associated with increased placental HSP70 and LOX-1 expression. CONCLUSIONS: We postulate that higher ADMA levels are associated with excessive levels of oxidative damage and stress markers (HSP, LOX-1) in placental tissues.

Identification of novel regulators of apoptosis using a high-throughput cell-based screen.

High-throughput subcellular imaging is a powerful tool for investigating the function of genes. In order to identify novel regulators of apoptosis we transiently transfected HeLa cells with 938 hypothetical genes of unknown function, and captured their nuclear images with an automated fluorescence microscope. We selected genes that induced greater than 3-fold increase in the percentage of apoptotic nuclei compared with vector-transfected cells. The full-length genes C10orf61, MGC 26717, and FLJ13855 were identified as candidate proapoptotic genes, and their apoptotic effects were confirmed by DNA fragmentation ELISAs and Western blotting for caspase-7 and PARP. We conclude that a subcellular image-based apoptotic screen is useful for identifying genes with proapoptotic activity.

Gene expression profiling of human HBV- and/or HCV-associated hepatocellular carcinoma cells using expressed sequence tags.

Liver cancer is one of the leading causes of cancer death worldwide. To identify novel target genes that are related to liver carcinogenesis, we examined new genes that are differentially expressed in human hepatocellular carcinoma (HCC) cell lines and tissues based on the expressed sequence tag (EST) frequency. Eleven libraries were constructed from seven HCC cell lines and three normal liver tissue samples obtained from Korean patients. An analysis of gene expression profiles for HCC was performed using the frequency of ESTs obtained from these cDNA libraries. Genes were identified (n=120) as being either up- or down-regulated in human liver cancer cells. Among these, 14 genes (FTL, K-ALPHA1, LDHA, RPL4, ENO1, ANXA2, RPL9, RPL10, RPL13A, GNB2L1, AMBP, GC, A1BG, and SERPINC1), in addition to previously well-known liver cancer related genes, were confirmed to be differentially expressed in seven liver cancer cell lines and 17 HCC tissues by semi-quantitative RT-PCR. In addition, 73 genes, in which there was a significant difference (P>0.99) between HBV- and HCV-associated HCC cells, were selected. Of these, expression patterns of 14 (RPLP0, AKR1C, KRT8, GPX4, RPS15, ID1, RPS21, VIM, EEF1G, EIF4A1, HLA-C, FN1, CD44, and RPS10) were confirmed by semi-quantitative RT-PCR in four of HBV- and three of HCV-associated HCC cell lines. Among those genes, an immunohistochemical analysis for ANXA2 showed that it is expressed at high levels in HCC. Using an analysis of EST frequency, the newly identified genes, especially ANXA2, represent potential biomarkers for HCC and useful targets for elucidating the molecular mechanisms associated with HCC involving virological etiology.

Identification of intrahepatic cholangiocarcinoma related genes by comparison with normal liver tissues using expressed sequence tags.

Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the bile duct epithelium, is one of the leading causes of death from cancer, worldwide. However, the mechanisms related to it remain largely unknown. In this study, an analysis of the gene expression profiles for ICC was done using the frequency of the ESTs obtained from nine cDNA libraries that constructed from 4 ICC cell lines and 4 normal liver tissues. One hundred and thirty-seven genes were identified as being either up- or down-regulated in human ICC cells. Thirty genes were randomly selected to confirm their differential expression in 4 human ICC cell lines and 5 ICC tissues compared to normal liver tissues by semi-quantitative RT-PCR. Among these genes, ANXA1, ANXA2, AMBP, and SERPINC1 were further verified by immunohistochemical analyses. In conclusion, these identified genes represent potential biomarkers for ICC and represent potential targets for elucidating the molecular mechanisms that are associated with ICC.

Gene expression profiling between embryonic and larval stages of the silkworm, Bombyx mori.

To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the different developmental stages, specifically the embryonic (ES) and larval (LS) stages of B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (P>0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of B. mori.

Identification of genes related to Parkinson's disease using expressed sequence tags.

In a search for novel target genes related to Parkinson's disease (PD), two full-length cDNA libraries were constructed from a human normal substantia nigra (SN) and a PD patient's SN. An analysis of the gene expression profiles between them was done using the expressed sequence tags (ESTs) frequency. Data for the differently expressed genes were verified by quantitative real-time RT-PCR, immunohistochemical analysis and a cell death assay. Among the 76 genes identified with a significant difference (P > 0.9), 21 upregulated genes and 13 downregulated genes were confirmed to be differentially expressed in human PD tissues and/or in an MPTP-treated mice model by quantitative real-time RT-PCR. Among those genes, an immunohistochemical analysis using an MPTP mice model for alpha-tubulin including TUBA3 and TUBA6 showed that the protein levels are downregulated, as well as the RNA levels. In addition, MBP, PBP and GNAS were confirmed to accelerate cell death activity, whereas SPP1 and TUBA3 to retard this process. Using an analysis of ESTs frequency, it was possible to identify a large number of genes related to human PD. These new genes, MBP, PBP, GNAS, SPP1 and TUBA3 in particular, represent potential biomarkers for PD and could serve as useful targets for elucidating the molecular mechanisms associated with PD.

Transcriptome analysis of human gastric cancer.

To elucidate the genetic events associated with gastric cancer, 124,704 cDNA clones were collected from 37 human gastric cDNA libraries, including 20 full-length enriched cDNA libraries of gastric cancer cell lines and tissues from Korean patients. An analysis of the collected ESTs revealed that 97,930 high-quality ESTs coalesced into 13,001 clusters, of which 11,135 clusters (85.6%) were annotated to known ESTs. The analysis of the full-length cDNAs also revealed that 4862 clusters (51.7%) contained at least one putative full-length cDNA clone with an initiation codon, with the average length of the 5' UTR of 140 bp. A large number appear to have a diverse transcription start site (TSS). An examination of the TSS of some genes, such as TEGT and GAPD, using 5' RACE revealed that the predicted TSSs are actually found in human gastric cancer cells and that several TSSs differ depending on the specific gastric cell line. Furthermore, of the human gastric ESTs, 766 genes (9.5%) were present as putative alternatively spliced variants. Confirmation of the predicted spliced isoforms using RT-PCR showed that the predicted isoforms exist in gastric cancer cells and some isoforms coexist in gastric cell lines. These results provide potentially useful information for elucidating the molecular mechanisms associated with gastric oncogenesis.

HESAS: HERVs Expression and Structure Analysis System.

SUMMARY: HESAS (HERVs Expression and Structure Analysis System) database was developed to understand the human endogenous retroviruses (HERVs) that have an effect on the expression of human functional genes. The database products are generated by the exon-based expressed sequence tag clustering and reconstructing of partial HERV structures that result from various mutations during primate evolution. The expression types were classified according to the existence of splicing, transcriptional start and polyadenylation signal sites. The database currently contains HERV information on 26,981 human genes of exon-intron structure. The HERV elements were inserted into 17,317 of these genes and linked to expression with 898 genes. AVAILABILITY: http://www.primate.or.kr/HESAS CONTACT: khs307@pusan.ac.kr.

The distribution and expression of HERV families in the human genome.

The families of human endogenous retroviruses (HERVs) are widely distributed in the human genome. Here we examined their distribution and expression. Approximately forty thousand HERV elements including truncated and solitary long terminal repeats (LTRs) were identified. These elements were most dense on chromosomes 4, 20, X, and Y. From an analysis of genomic stability during primate evolution, the 5 cent -LTR of the HERV genome (5 cent LTR - internal HERV - 3 cent LTR) appeared to be more often truncated than the 3 cent -LTR. ESTs derived from normal placenta, skeletal muscle, hypothalamus, and testis gave frequent matches to HERV elements. We present a classification of genes associated with HERV elements according to the hierarchical structure of gene ontology.