Young Mee Kim

Functional regulation of Slug / Snail2 is dependent on GSK‐3β‐mediated phosphorylation

Snail family proteins regulate transcription of molecules for cell–cell adhesion during epithelial–mesenchymal transition (EMT). Based on putative glycogen synthase kinase 3β (GSK‐3β) phosphorylation sites within the Slug/Snail2, we explored the significance of GSK‐3β‐mediated phosphorylation in Slug/Snail2 expression during EMT. Mutation of the putative GSK‐3β phosphorylation sites (S92/96A or S100/104A) enhanced the Slug/Snail2‐mediated EMT properties of E‐cadherin repression and vimentin induction, compared with wild‐type Slug/Snail2. S92/96A mutation inhibited degradation of Slug/Snail2 and S100/104A mutation extended nuclear stabilization. Inhibition of GSK‐3β activity caused similar effects, as did the phosphorylation mutations. Thus, our study suggests that GSK‐3β‐mediated phosphorylation of Slug/Snail2 controls its turnover and localization during EMT.

Activation of NADPH oxidase subunit NCF4 induces ROS-mediated EMT signaling in HeLa cells

The epithelial-mesenchymal transition (EMT) is a critical biological process characterized by morphological and behavioral changes in cells. The regulatory and signaling mechanisms of both developmental and pathological EMT have been investigated. Reactive oxygen species (ROS) play a role in early EMT, but the exact mechanism by which ROS are involved is unclear. We investigated ROS-mediated EMT in human HeLa cells. Transforming growth factor beta (TGF-β) treatments lead to dramatic NADPH oxidase 2 (NOX2) inductions in HeLa cells; antioxidant treatment prevented TGF-β-driven EMT. Over-expression of the p40phox subunit (NCF4) led to activation of the NOX2 complex and ROS production. We showed that NOX2 and NOX5 mRNA was increased, along with increased expression of several matrix metalloproteinases (MMPs) in response to NCF4 expression. Moreover, these changes were reversible upon ROS scavenging. Down-regulation of E-cadherin and up-regulation of Snail, Slug and vimentin occurred at the transcriptional level. We also showed that new EMT regulator, YB-1 is a downstream target in ROS-induced EMT. Together, these data suggest that ROS switching is necessary for increased EMT but is not required for the morphological changes that accompany EMT.

Genetic Analysis of 10 Unrelated Korean Families with p22-phox-deficient Chronic Granulomatous Disease: An Unusually Identical Mutation of the CYBA Gene on Jeju Island, Korea

Chronic granulomatous disease (CGD) is a rare hereditary disorder characterized by recurrent life-threatening bacterial and fungal infections. The underlying defect in CGD is an inability of phagocytes to produce reactive oxygen species as a result of defects in NADPH oxidase. Considering that CGD generally affects about 3-4 in 1,000,000 individuals, it is surprising that the prevalence of CGD on Jeju Island is 20.7 in 1,000,000 individuals. We performed genetic analysis on 12 patients from 10 unrelated families and found that all patients had an identical homozygous single-base substitution of C to T in exon 1 (c.7C>T) of the CYBA gene, which was expected to result in a nonsense mutation (p.Q3X). Because Jeju Island has long been a geologically isolated region, the high prevalence of CGD on Jeju Island is presumably associated with an identical mutation inherited from a common ancestor or proband.

Effects of the Novel Compound DK223 ((1E,2E-1,2-Bis(6-methoxy-2H-chromen-3-yl)methylene)hydrazine) on Migration and Proliferation of Human Keratinocytes and Primary Dermal Fibroblasts

Wound healing plays an important role in protecting the human body from external infection. Cell migration and proliferation of keratinocytes and dermal fibroblasts are essential for proper wound healing. Recently, several studies have demonstrated that secondary compounds produced in plants could affect skin cells migration and proliferation. In this study, we identified a novel compound DK223 ([1E,2E-1,2-bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) that concomitantly induced human keratinocyte migration and dermal fibroblast proliferation. We evaluated the regulation of epithelial and mesenchymal protein markers, such as E-cadherin and Vimentin, in human keratinocytes, as well as extracellular matrix (ECM) secretion and metalloproteinase families in dermal fibroblasts. DK223 upregulated keratinocyte migration and significantly increased the epithelial marker E-cadherin in a time-dependent manner. We also found that reactive oxygen species (ROS) increased significantly in keratinocytes after 2 h of DK223 exposure, returning to normal levels after 24 h, which indicated that DK223 had an early shock effect on ROS production. DK223 also stimulated fibroblast proliferation, and induced significant secretion of ECM proteins, such as collagen I, III, and fibronectin. In dermal fibroblasts, DK223 treatment induced TGF-β1, which is involved in a signaling pathway that mediates proliferation. In conclusion, DK223 simultaneously induced both keratinocyte migration via ROS production and fibroblast proliferation via TGF-β1 induction.

Soy Isoflavone Glycitin (4'-Hydroxy-6-Methoxyisoflavone-7-D-Glucoside) Promotes Human Dermal Fibroblast Cell Proliferation and Migration via TGF-β Signaling

Glycitin is a soy isoflavone that exhibits antioxidant, antiallergic, and anti‐osteoporosis activities. We investigated the effects of glycitin on dermal fibroblast proliferation and migration. Treatment of primary dermal fibroblasts with glycitin increased cell proliferation and migration. In addition, treatment with 20 μM glycitin for 24 h induced the synthesis of collagen type I and type III at both the mRNA and protein levels. Fibronectin was also increased by 20% after treatment. Matrix metalloproteinase‐1 collagenase was decreased in the media after 24‐h incubation with glycitin, and the synthesis of transforming growth factor‐beta (TGF‐β) mRNA increased approximately twofold in cells following glycitin treatment. Phosphorylation of Smad2 and Smad3 increased after 1 h of glycitin treatment, and phosphorylation continued for 24 h. Furthermore, the phosphorylated form of AKT was increased in glycitin‐treated cells after 3 h and remained higher for 24 h. Thus, glycitin treatment produces anti‐aging effects including increased total collagen in the culture media, decreased elastase, and decreased β‐galactosidase. Together, these results indicate that glycitin stimulates TGF‐β secretion, and the subsequent autocrine actions of TGF‐β induce proliferation of fibroblasts, ultimately protecting skin cells from aging and wrinkling. Copyright

New method for detection of p22-phox-deficient chronic granulomatous disease heterozygote carriers in Jeju

Chronic granulomatous disease (CGD) is a genetically heterogeneous disease caused by defects in the genes encoding any one of the NADPH oxidase components. The estimated prevalence of CGD is between 1 in 200,000 and 1 in 250,000 individuals, with variable rates in different countries. According to a compilation by the Korean College of Pediatric Clinical Immunology, the prevalence of CGD in Korea is 0.9 in 1,000,000 individuals. Surprisingly, the prevalence of CGD in Jeju Island is 20.7 in 1,000,000 individuals. We reported an identical homozygous single-base substitution of C to T in exon 1 (c.7C > T) of the CYBA gene from 12 CGD patients in Jeju Island. We hypothesized that the high prevalence of CGD in Jeju Island is associated with an identical mutation inherited from a common ancestor or proband. The aim of this study was to develop an assay to detect heterozygote carriers of the genotype specific to Jeju Island. We developed three specific primers, and nested polymerase chain reaction was employed using whole blood samples as a source of genomic DNA. Using the new detection method, 704 individuals were tested, 9 of which were detected as carriers, and the expected number of carriers is 1.3 in 100 individuals.

TGF-β secreted from activated hepatic stellate cells may induce the transdifferentiation of hepatocytes into hepatocarcinoma in HBx-expressing livers

Hepatic stellate cells (HSCs) are the main extra cellular matrix-producing cells in the liver. Several reports have indicated that activated HSCs are involved in hepatic carcinogenesis by way of transforming growth factor β (TGF-β) secretion. This study aimed to investigate the effects of TGF-β, derived from HSCs activated by the chronic hepatitis B virus x protein (HBx), on the transdifferentiation of hepatocytes into hepatocarcinoma cells. Normal hepatocytes (the Chang liver cell line) were treated with a low concentration of TGF-β for 2 weeks, after which cell cycle- and cell signaling- related protein expression was analyzed. Lon-term treatment of TGF-β clearly induced the proliferation and the expression of cancer signaling proteins in the Chang cell line. TGF-β treatment also increased the expression of c-Jun N-terminal kinase (JNK) and c-Myc, indicating that induction of the JNK/pSmad3/c-Myc oncogenic signaling pathway is involved in hepatocyte transformation. Similar results were observed after culturing Chang cells with conditioned media derived from the activated LX-2 hepatic stellate cell line, suggesting that TGF-β paracrine effects are involved in the transformation of hepatocyte cells into hepatocarcinoma cells. Immunohistochemical results showed that the livers from HBx transgenic mice were composed of more activated HSCs and produced more TGF-β compared with those from normal mice. The TGF-β secreted from HBx-infected HSCs might induce transdifferentiation of hepatocytes into hepatocarcinoma, which is the fact that suggested a potential knowledge on liver cancer inhibition.

Proteasome Down-regulation is Partly Mediated by Slug/Snail2 in Hepatocarcinoma Cells

Snail family proteins (Snail1 and Slug/Snail2) are transcription factors that regulate transcription of molecules during epithelial-mesenchymal transition (EMT). Snail1/2 is known to bind to the E-box motif (CANNTG). The proteasome activity is decreased in EMT (Kim et al., 2011), and several E-box motifs are found in the promoters of genes coding for proteasome subunits. We used a new protein-binding microarray to specify the Slug/Snail2 binding sequence. Among 563 9-mer clusters, the motif CACCTGC yielded the highest P-value in the Wilcoxon-Mann-Whitney test. Within this motif, the A and T were absolutely required, and CC was preferred, but could be replaced by GG with little effect. In hepatocytes overexpressing Slug/Snail2, the 20S proteasome expression and proteasome activity were decreased partly due to the down-regulation of proteasome subunit beta type 2 (PSMB2) and PSMB3 transcription.

Erratum to: Development of a lentiviral vector and an efficient infection method for gene therapy for p22phoxdefective chronic granulomatous disease

Chronic granulomatous disease (CGD) is caused by impaired antimicrobial activity in phagocytes due to the absence or malfunction of the respiratory burst NADPH oxidase. In a previous study, we found that 12 patients from 10 unrelated families on Jeju Island had an identical homozygous single-base C-to-T substitution in exon 1 (c.7C > T) of CYBA, which encodes p22phox. Autosomal recessive p22phox-defective CGD carrierderived white blood cells were efficiently transduced by the elongation factor 1-alpha lentivirus constructs, as up to 90% of cells were green fluorescent protein (eGFP)-positive at 3 days post-transduction. pLL3.7-driven eGFP expression was stable for at least 4 weeks after transduction and persisted after CGD carrierderived cells were immortalized by human telomerase reverse transcriptase (hTERT) and B lymphoma Mo-MLV insertion region 1 (Bmi-1). Upon macrophage-like differentiation of the transduced HL-60 cells by dimethyl sulfoxide, up to 28% of the cells had higher mean levels of superoxide production than undifferentiated cells, and lentivirus efficiently transduced cells and induced the expression of genes for extended periods.