• Molecules and Cells
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• v.40 no.2
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• pp.123-132
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• 2017

Insulin signaling is coordinated by insulin receptor substrates (IRSs). Many insulin responses, especially for blood glucose metabolism, are mediated primarily through Irs-1 and Irs-2. Irs-1 knockout mice show growth retardation and insulin signaling defects, which can be compensated by other IRSs in vivo; however, the underlying mechanism is not clear. Here, we presented an Irs-1 truncated mutated mouse ($Irs-1^{-/-}$) with growth retardation and subcutaneous adipocyte atrophy. $Irs-1^{-/-}$ mice exhibited mild insulin resistance, as demonstrated by the insulin tolerance test. Phosphatidylinositol 3-kinase (PI3K) activity and phosphorylated Protein Kinase B (PKB/AKT) expression were elevated in liver, skeletal muscle, and subcutaneous adipocytes in Irs-1 deficiency. In addition, the expression of IRS-2 and its phosphorylated version were clearly elevated in liver and skeletal muscle. With miRNA microarray analysis, we found miR-33 was down-regulated in bone marrow stromal cells (BMSCs) of $Irs-1^{-/-}$ mice, while its target gene Irs-2 was up-regulated in vitro studies. In addition, miR-33 was down-regulated in the presence of Irs-1 and which was up-regulated in fasting status. What's more, miR-33 restored its expression in re-feeding status. Meanwhile, miR-33 levels decreased and Irs-2 levels increased in liver, skeletal muscle, and subcutaneous adipocytes of $Irs-1^{-/-}$ mice. In primary cultured liver cells transfected with an miR-33 inhibitor, the expression of IRS-2, PI3K, and phosphorylated-AKT (p-AKT) increased while the opposite results were observed in the presence of an miR-33 mimic. Therefore, decreased miR-33 levels can up-regulate IRS-2 expression, which appears to compensate for the defects of the insulin signaling pathway in Irs-1 deficient mice.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.57-64
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• 2009

This study was carried out to investigate expression of apoptosis-related differentially expressed gene (DEG) in ovaries of Korean cattle with follicular and luteal cysts and to identify the relationship between cyst and apoptosis using microarray, real-time PCR, TUNEL staining, and Western blot analysis. Microarray data showed that PIK3R2 and AKT1 were significantly up-regulated in follicular cyst, and TNF-RAF2, PRLR, FOXL2, STK4, and COL4A3 were up-regulated whereas INHA, CIDEB, BCL10, and FASLG were down-regulated in luteal cyst. Real-time PCR was performed to validate DEGs altered in luteal cyst. Of nine DEGs, four DEGs down-regulated in luteal cyst showed a positive corelation between microarray data and real-time PCR data. In this study, we focused on INHA, among many DEGs, which was highly down-regulated in both follicular and luteal cysts. Real-time PCR and micro array data showed that INHA was down-regulated by 12.3-fold and by 1.4-fold, respectively, in the bovine follicular cyst. TUNEL assay and Western blot analysis for ERK, JNK, p38, PI3K, and Akt, which were used to detect whether apoptosis is occurred, showed no significant changes in cystic ovaries (p>0.05). In the expression and activity of caspase-3, Bax, Bel-2, and Bel-xL, there was no significant changes between follicular cystic ovary and normal ovary. Rather, the expression levels of PI3K and p-Akt were decreased in follicular cystic ovary. These results suggest that deficiency of apoptosis in cystic ovary is associated with decreased expression of apoptotic effectors.

• Animal cells and systems
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• v.9 no.3
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• pp.161-171
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• 2005

Integrin-linked kinase 1 (ILK1) regulates several protein kinases, including PKB/Akt kinase and glycogen synthase kinase ${\beta}$. ILK1 is also involved distinctively in the cell morphological and structural functions by interacting with the components of the extracellular matrix or integrin. According to the information of serum- and glucocorticoid-induced kinase 1 (SGK1) substrate specificity (R-X-R-X-X(S/T)-${\phi};{\phi}$ indicates a hydrophobic amino acid), two putative phosphorylation sites, $Thr^{181}\;and\;Ser^{246}$, were found in ILK1. We showed that ILK1 fusion protein and two fluorescein-labeled ILK1 peptides, $FITC-^{174}RTRPRNGTLN^{183}$ and $FITC-^{239}CPRLRIFSHP^{248}$, were phosphorylated by SGK1 in vitro. We also identified that 14-3-3 ${\theta}\;{\varepsilon}\;and\;{\xi}$, among several 143-3 isotypes $({\beta},\;{\gamma},\;{\varepsilon},\;{\eta},\;{\sigma},\;{\theta},\;{\tau}\;and\;{\xi})$ formed protein complex with ILK1 in COS-1 cells. Furthermore, the phosphorylation of $Ser^{246}$ by SGK1 induced the binding with 14-3-3. It was also demonstrated that 14-3-3-bound ILK1 has reduced kinase activity. Thus, these data suggest that SGK1 phosphorylates $Thr^{181}\;and\;Ser^{246}$ of ILK1 and the phosphorylation of its $Ser^{246}$ makes ILK1 bind to 14-3-3, resulting in the inhibition of ILK1 kinase activity.

• Clinical and Experimental Pediatrics
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• v.52 no.2
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• pp.213-219
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• 2009

Purpose:Iron is a critical nutritional element that is essential for a variety of important biological processes, including cell growth and differentiation, electron transfer reactions, and oxygen transport, activation, and detoxification. Iron is also required for neoplastic cell growth due to its catalytic effects on the formation of hydroxyl radicals, suppression of host defense cell activities, and promotion of cancer cell multiplication. Chronic transfusion-dependent patients receiving chemotherapy may have iron overload, which requires iron-chelating therapy. We performed this study to demonstrate whether the iron chelating agent deferoxamine induces apoptosis in Saos-2 osteosarcoma cells, and to investigate the underlying apoptotic mechanism. Methods:To analyze the apoptotic effects of an iron chelator, cultured Saos-2 cells were treated with deferoxamine. We analyzed cell survival by trypan blue and crystal violet analysis, apoptosis by nuclear condensation, DNA fragmentation, and cell cycle analysis, and the expression of apoptotic related proteins by Western immunoblot analysis. Results:Deferoxamine inhibited the growth of Saos-2 cell in a time- and dose-dependent manner. The major mechanism for growth inhibition with the deferoxamine treatment was by the induction of apoptosis, which was supported by nuclear staining, DNA fragmentation analysis, and flow cytometric analysis. Furthermore, bcl-2 expression decreased, while bax, caspase-3, caspase-9, and PARP expression increased in Saos-2 cells treated with deferoxamine. Conclusion:These results demonstrated that the iron chelating agent deferoxamine induced growth inhibition and mitochondrial-dependent apoptosis in osteosarcoma Saos-2 cells, suggesting that iron chelating agents used in controlling neoplastic cell fate can be potentially developed as an adjuvant agent enhancing the anti-tumor effect for the treatment of osteosarcoma.