• Molecules and Cells
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• v.38 no.6
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• pp.528-534
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• 2015

Hypoxia-inducible factor (HIF) is a key regulator of tumor growth and angiogenesis. Recent studies have shown that, BIX01294, a G9a histone methyltransferase (HMT)-specific inhibitor, induces apoptosis and inhibits the proliferation, migration, and invasion of cancer cells. However, not many studies have investigated whether inhibition of G9a HMT can modulate HIF-$1{\alpha}$ stability and angiogenesis. Here, we show that BIX01294 dose-dependently decreases levels of HIF-$1{\alpha}$ in HepG2 human hepatocellular carcinoma cells. The half-life of HIF-$1{\alpha}$, expression of proline hydroxylase 2 (PHD2), hydroxylated HIF-$1{\alpha}$ and von Hippel-Lindau protein (pVHL) under hypoxic conditions were decreased by BIX01294. The mRNA expression and secretion of vascular endothelial growth factor (VEGF) were also significantly reduced by BIX01294 under hypoxic conditions in HepG2 cells. BIX01294 remarkably decreased angiogenic activity induced by VEGF in vitro, ex vivo, and in vivo, as demonstrated by assays using human umbilical vein endothelial cells (HUVECs), mouse aortic rings, and chick chorioallantoic membranes (CAMs), respectively. Furthermore, BIX01294 suppressed VEGF-induced matrix metalloproteinase 2 (MMP2) activity and inhibited VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR-2), focal adhesion kinase (FAK), and paxillin in HUVECs. In addition, BIX01294 inhibited VEGF-induced formation of actin cytoskeletal stress fibers. In conclusion, we demonstrated that BIX01294 inhibits HIF-$1{\alpha}$ stability and VEGF-induced angiogenesis through the VEGFR-2 signaling pathway and actin cytoskeletal remodeling, indicating a promising approach for developing novel therapeutics to stop tumor progression.

• International Journal of Oral Biology
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• v.32 no.1
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• pp.35-43
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• 2007

Tooth loss in elderly is mainly caused by alveolar bone loss via severe periodontitis. Although the severity of periodontitis is known to be affected by age, the aging process or the genetic changes during the aging of periodontal tissue cells are not well characterized. In this study, we investigated the effect of in vitro aging on the change of gene expression pattern in periodontal fibroblasts. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDL) were obtained from two young patients and replicative senescence was induced by sequential subcultivation. When more than 90% cells were positively stained with senescence-associated ${\beta},-galactosidase$, those cells were regarded as aged cells. In aged GF and PDL, the level of phosphorylated retinoblastoma (RB) and $p16^{INK4A}$ protein was significantly decreased and increased, respectively. However, the protein level of p53 and p21, well known senescence-inducing genes, did not increase in aged GF and PDL. Although $p27^{Kip1}$ and $p15^{INK4B}$, another cyclin-dependent kinase inhibitors, were reported to be involved in replicative senescence of human cells, they were decreased in aged GF and PDL. Because senescent cells showed flattened and enlarged cell shape and are known to have increased focal adhesion, we examined the protein level of several integrins. Aged GF and PDL showed increased protein level of integrin ${\alpha}2$, ${\alpha}v$, and ${\beta}1$. When the gene expression profiles of actively proliferating young cells and aged cells were compared by cDNA microarray of 3,063 genes and were confirmed by reverse transcription-polymerase chain reaction, 7 genes and 15 genes were significantly and commonly increased and decreased, respectively, in aged GF and PDL. Among them, included are the genes that were known to be involved in the regulation of cell cycle, gene transcription, or integrin signaling. The change of gene expression pattern in GF and PDL was minimally similar to that of oral keratinocyte. These results suggest that $p16^{INK4A}/RB$ might be involved in replicative senescence of periodontal fibroblasts and the change of gene expression profile during aging process is cell type specific.

• Journal of Periodontal and Implant Science
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• v.32 no.1
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• pp.143-160
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• 2002

타이태늄은 뛰어난 생체적합성과 적절한 물리적 성질을 바탕으로 치과 및 정형외과 영역의 매식재로 널리사용되어져 왔으며, 골과 매식재 사이의 골 융합 정도를 증가시킬 목적으로 물리, 화학적인 방법을 이용한 타이태늄의 표면처리에 관한 많은 연구들이 진행되어 왔다. 최근에는 부착단백질 또는 성장인자를 이용한 생체재료의 표면개질을 통하여 조직적합성 및 치유 능의 개선을 위한 시도들이 있어왔다. Fibronectin(FN)은 주요 세포외기질중의 하나로 생체 내 널리 분포하여 세포의 부착, 이동 및 증식에 관여하는 거대 당단백으로, RGD및 PHSRN 펩타이드 서열이 세포의 인테그린과 결합하여 세포의 활성을 조절하는 것으로 알려져 있다. 이 연구에서는 FN으로 처리된 타이태늄이 조골세포의 부착, 증식 및 분화에 미치는 영향과 이에 따른 석회화 정도에 미치는 영향을 관찰하여 부착분자를 이용한 타이태늄 표면개질의 효과를 규명하고자 하였다. 상업용 순수 타이태늄을 gold thiol법을 이용하여 표면처리 후, 혈장 FN(plasma FN, pFN)과 유전자재조합법을 이용하여 얻은 FN조각(FN type III 7-10, FNIII 7-10)을 피복한 시편을 실험군으로, 아무런 처리를 하지 않은것(smooth surface, SS)과 산 부식(Sandblasted and acid etched, SLA)처리된것을 대조군으로 이용하였다. 배양된 조골세포주(MC3T3-E1)를 사용하여 타이태늄 표면 처리에 따른 세포의 증식, 형태변화, 알칼리성 인산분해효소(ALPase) 생산 및 세포면역형광법을 이용한 분화정도를 시간 경과에 따라 관찰하였다. 조골세포증식의 경우 FNIII 7-10 처리군에서 pFN 처리군 및 대조군에 비해 시간경과에 따라 유의성있는 세포수의 증식이 관찰되었으며(p<0.05), ALPase 생성의 경우에도 FNIII 7-10 처리 군에서 아무 처리도 하지 않은 군에 비해 유의성 있게 높은 효소의 생성이 관찰되었다(p<0.05). 주사전자현미경을 이용한 세포의 형태관찰결과 아무 처리도 하지 않은 군에서는 마름모형태를 나타내었으며, 산 부식 처리된 군에서는 세포가 가시모양의 형태를 보인 반면 FN으로 처리된 두 군에서는 세포의 부착 및 펴짐이 매우 발달되어 있는 모습이 관찰되었다. 세포의 분화정도를 관찰하기 위하여 국소부착키나제(focal adhesion kinase, FAK), 및 actin stress fiber의 분포양상을 세포면역형광법을 이용하여 관찰한 결과 FN으로 표면처리된 두 군에서 아무런 처리도 하지않은 군 및 산 부식처리 한 군에 비해 프라크의 발현이 높게 나타났으며 잘 발달된 actin stress fiber의 소견을 나타내었다. 이 실험의 결과들은 gold thiol 법을 이용한 표면처리 후 FN부착을 통한 타이태늄의 표면개질이 조골세포의 부착, 증식 및 분화에 중요한 역할을 담당하여 석회화 정도를 촉진시키는 것을 보여주었으며, 이런 결과들은 더 짧은 FN조각을 이용한 다른 생체재료의 표면개질에 폭 넓게 응용할 수 있으리라 생각된다.

• Journal of Biomedical and Translational Research
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• v.19 no.4
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• pp.116-124
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• 2018

Korean Native Pig (KNP) has a uniform black coat color, excellent meat quality, white colored fat, solid fat structure and good marbling. However, its growth performance is low, while the western origin Yorkshire pig has high growth performance. To take advantage of the unique performance of the two pig breeds, we raised crossbreeds (KNP ${\times}$ Yorkshire to make use of the heterotic effect. We then analyzed the liver transcriptome as it plays an important role in fat metabolism. We sampled at two stages: 10 weeks and at 26 weeks. The stages were chosen to correspond to the change in feeding system. A total of 16 pigs (8 from each stage) were sampled and RNA sequencing was performed. The reads were mapped to the reference genome and differential expression analysis was performed with edgeR package. A total of 324 genes were found to be significantly differentially expressed (${\left|log2FC\right|}$ > 1 & q < 0.01), out of which 180 genes were up-regulated and 144 genes were down-regulated. Principal Component Analysis (PCA) showed that the samples clustered according to stages. Functional annotation of significant DEGs (differentially expressed genes) showed that GO terms such as DNA replication, cell division, protein phosphorylation, regulation of signal transduction by p53 class mediator, ribosome, focal adhesion, DNA helicase activity, protein kinase activity etc. were enriched. KEGG pathway analysis showed that the DEGs functioned in cell cycle, Ras signaling pathway, p53 signaling pathway, MAPK signaling pathway etc. Twenty-nine transcripts were also part of the DEGs, these were predominantly Cys2His2-like fold group (C2H2) family of zinc fingers. A protein-protein interaction (PPI) network analysis showed that there were three highly interconnected clusters, suggesting an enrichment of genes with similar biological function. This study presents the first report of liver tissue specific gene regulation in a cross-bred Korean pig.