Mi-Ock Lee

Genistein enhances expression of genes involved in fatty acid catabolism through activation of PPARα

Although evidences are emerging that dietary isoflavones have beneficial effects in treatment of hyperlipidemia and cardiovascular diseases, the underlying molecular mechanism has not yet been extensively characterized. In this report, we showed that genistein, one of the major isoflavones, increased expression of genes involved in lipid catabolism such as carnitine palmitoyltransferase 1, liver form (CPT1L) in HepG2 cells, when assayed by real-time reverse-transcriptase polymerase chain reactions as well as Western blotting analysis. The increase in mRNA-level of CPT1L after genistein treatment was not changed in the presence of ICI182780, a potent inhibitor of estrogen receptor, suggesting that this effect of genistein was estrogen receptor-independent. Since these genes involved in fatty acid catabolism are considered putative downstream target genes of peroxisome proliferators-activated receptor alpha (PPARalpha), we examined whether expression of PPARalpha was modulated by genistein treatment. Interestingly, genistein induced expression of PPARalpha at both mRNA- and protein-level. Further, genistein activated transcriptional activity of PPARalpha, when determined by reporter gene analysis, suggesting genistein as a potential ligand for PPARalpha. Taken together, this study provides a picture of the regulatory action of genistein, as an activator of PPARalpha in fatty acid catabolism and potential use of genistein as lipid-lowering agent.

Analysis of ceramic/metal armour systems

A combined numerical and experimental study for the analysis of ceramic/metal composite armour system against 40.7 g steel projectiles has been performed. The ballistic performance of the add-on lightweight armours was examined by varying the thickness of tiles, while maintaining equal areal density of the system. A numerical study using smoothed particle hydrodynamics scheme is promising since the major distinguishing features of composite armour systems such as, projectile erosion, crack propagation, ceramic conoid formation and failure of backing plate, are successfully captured. Simulation results for ballistic limits appear to match fairly well with the test values and reveal an optimum value of the front plate to back plate thickness ratio.

The carboxy-terminus of the hepatitis B virus X protein is necessary and sufficient for the activation of hypoxia-inducible factor-1α

Hepatitis B virus X protein (HBx) of the hepatitis B virus is strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Previously, we reported that HBx enhances activity of hypoxia‐inducible factor‐1α (HIF‐1α), a potent transactivator that induces angiogenic factors. Here, we delineate the structural region of HBx that potentiates HIF‐1α. The carboxy‐terminus of HBx increased the stability of HIF‐1α protein, probably through inhibiting interaction with von Hippel‐Lindau protein. Further, the carboxy‐terminus of HBx enhanced the transactivation function of HIF‐1α by enhancing its association with CREB binding protein (CBP). Finally, we demonstrated the physical association of HBx with the basic helix–loop–helix/PER–ARNT–SIM domain, the inhibitory domain, and the carboxy‐terminal transactivation domain of HIF‐1α in vivo.

Hepatitis B virus X protein induced expression of interleukin 18 (IL-18): a potential mechanism for liver injury caused by hepatitis B virus (HBV) infection.

BACKGROUND/AIMS The hepatitis B virus X protein (HBx), a major viral transactivator, is implicated in hepatic inflammation, since it induces many pro-inflammatory cytokines at transcriptional level. The aim of this study was to investigate role of HBx in expression of interleukin 18 (IL-18), a newly identified cytokine that up-regulates Fas ligand (FasL) expression. METHODS Chang X-34 that expressing HBx under the control of a doxycycline-inducible promoter, and hepatitis B virus (HBV)-integrated hepatoma cell lines were examined for IL-18 expression by Northern and Western blotting analysis. To test the role of IL-18 produced by hepatoma cells, FasL expression was examined by flow cytometry after treatment with neutralizing anti-IL-18 antibodies. Further, IL-18 expression was examined in the liver tissues of HBx-transgenic mice. RESULTS Induction of IL-18 following HBx expression in Chang X-34 and the pattern of IL-18 expression in HBV-integrated cell lines, implicated that HBx transcriptionally induces IL-18 expression. Neutralizing anti-IL-18 antibodies blocked the expression of FasL, suggesting that IL-18 plays a critical role in FasL expression. Further, IL-18 expression in the HBx-transgenic liver, was correlated with the degree of hepatitis. CONCLUSIONS Our results demonstrated that HBx induces IL-18 expression in liver, which may be associated with hepatic injury by amplifying FasL expression during HBV infection.

α-Lipoic Acid Prevents Neointimal Hyperplasia Via Induction of p38 Mitogen-Activated Protein Kinase/Nur77-Mediated Apoptosis of Vascular Smooth Muscle Cells and Accelerates Postinjury Reendothelialization

Objective—To explore whether &agr;-lipoic acid (ALA), a naturally occurring antioxidant, inhibits neointimal hyperplasia by inducing apoptosis of vascular smooth muscle cells and to examine its potential effects on reendothelialization and platelet aggregation. Methods and Results—Restenosis and late stent thrombosis, caused by neointimal hyperplasia and delayed reendothelialization, are significant clinical problems of balloon angioplasty and drug-eluting stents. ALA treatment strongly induced apoptosis of vascular smooth muscle cells and enhanced the expression and cytoplasmic localization of Nur77, which triggers intrinsic apoptotic events. Small interfering RNA–mediated downregulation of Nur77 diminished this proapoptotic effect of ALA. Moreover, ALA increased p38 mitogen-activated protein kinase phosphorylation, and inhibition of p38 mitogen-activated protein kinase completely blocked ALA-induced vascular smooth muscle cell apoptosis and Nur77 induction and cytoplasmic localization. In balloon-injured rat carotid arteries, ALA enhanced Nur77 expression and increased TUNEL-positive apoptotic cells in the neointima, leading to inhibition of neointimal hyperplasia. This preventive effect of ALA was significantly reduced by infection of an adenovirus encoding Nur77 small hairpin (sh)RNA. Furthermore, ALA reduced basal apoptosis of human aortic endothelial cells and accelerated reendothelialization after balloon injury. ALA also suppressed arachidonic acid–induced platelet aggregation. Conclusion—ALA could be a promising therapeutic agent to prevent restenosis and late stent thrombosis after angioplasty and drug-eluting stent implantation.

Radicicol represses the transcriptional function of the estrogen receptor by suppressing the stabilization of the receptor by heat shock protein 90

The estrogen receptor (ER) is a hormone-dependent transcription factor that belongs to the steroid/thyroid hormone receptor superfamily. Since the ER contributes to development and progression in human breast cancer, a number of studies have explored ways to inactivate this receptor. Previous studies have suggested that the 90-kDa heat shock protein (Hsp90) interacts with the ER, thus stabilizing the receptor in an inactive state. Here, we report that radicicol, an Hsp90-specific inhibitor, repressed estrogen-dependent transactivation of the ER as measured by pS2 gene transcription and a reporter gene encoding an estrogen-responsive element. Furthermore, we showed that radicicol induced rapid degradation of ERalpha, while the amount of ubiquitinated ERalpha was increased. A proteasome inhibitor, LLnL, almost completely abrogated the radicicol-induced decrease in expression level, as well as in transcriptional activity of ERalpha. These results suggest that radicicol disrupts the ER-Hsp90 heterodimeric complex, thereby generating ERalpha that is susceptible to ubiquitin/proteasome-induced degradation.

Identification of genes that are induced after cadmium exposure by suppression subtractive hybridization.

The heavy metal cadmium is a xenobiotic toxicant of environmental and occupational concern and it has been classified as a human carcinogen. Inhalation of cadmium has been implicated in the development of emphysema and pulmonary fibrosis, but, the detailed mechanism by which cadmium induces adverse biological effects is not yet known. Therefore, we undertook the investigation of genes that are induced after cadmium exposure to illustrate the mechanism of cadmium toxicity. For this purpose, we employed the polymerase chain reaction (PCR)-based suppression subtractive hybridization (SSH) technique. We identified 29 different cadmium-inducible genes in human peripheral blood mononuclear cells (PBMCs), such as macrophage migration inhibitory factor (MIF), lysophosphatidic acid acyltransferase-alpha, enolase-1alpha, VEGF, Bax, and neuron-derived orphan receptor-1 (Nor-1), which are known to be associated with inflammation, cell survival, and apoptosis. Induction of these genes by cadmium treatment was further confirmed by semi-quantitative reverse-transcription PCR. Further, we found that these genes were also induced after cadmium exposure in normal human lung fibroblast cell line, WI-38, suggesting potential use of this induction profile to monitor cadmium toxicity in the lung.

Hepatitis B virus X protein modulates peroxisome proliferator-activated receptor γ through protein–protein interaction

Ligand activation of peroxisome proliferator‐activated receptor γ (PPARγ) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARγ activation has not been characterized in hepatitis B virus (HBV)‐associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARγ ligand in HBx‐associated HCC cells. We found that HBx bound to DNA binding domain of PPARγ and HBx/PPARγ interaction blocked nuclear localization and binding to recognition site of PPARγ. HBx significantly suppressed a PPARγ‐mediated transactivation. These results suggest that HBx modulates PPARγ function through protein–protein interaction.

Positive Cross-Talk Between Hypoxia Inducible Factor-1α and Liver X Receptor α Induces Formation of Triglyceride-Loaded Foam Cells

Objective—Atherosclerosis is a chronic and progressive inflammatory disease of the arteries that is characterized by subendothelial accumulation of lipid-rich macrophages, called foam cells. We sought to identify the molecular details of cross-talk between liver X receptor &agr; (LXR&agr;) and hypoxia-inducible factor 1&agr; (HIF-1&agr;) for the formation of triglyceride-rich foam cells under hypoxic conditions. Methods and Results—We first observed that expression of LXR&agr; and its target lipogenic genes was time-dependently induced in human primary macrophages and RAW 264.7 cells under hypoxia. Similarly, TO901317, an activator of LXR&agr;, enhanced the expression level and the transcriptional activity of HIF-1&agr;. Second, we demonstrated that LXR&agr; increased HIF-1&agr; protein stability through a physical interaction between the ligand binding domain of LXR&agr; and the oxygen-dependent degradation domain of HIF-1&agr;. Third, we found that the activation of HIF-1&agr; or LXR&agr; synergistically induced triglyceride accumulation in macrophages. Finally, we showed that LXR&agr; and HIF-1&agr; were codistributed in the macrophages of atherosclerotic lesions of patients. Conclusion—These results suggest that the positive feed-forward regulation of transcriptional activity and protein stability of LXR&agr; and HIF-1&agr; has an important impact in foam cell formation.

Transcriptional profiling in human HaCaT keratinocytes in response to kaempferol and identification of potential transcription factors for regulating differential gene expression

Kaempferol is the major flavonol in green tea and exhibits many biomedically useful properties such as antioxidative, cytoprotective and anti-apoptotic activities. To elucidate its effects on the skin, we investigated the transcriptional profiles of kaempferol-treated HaCaT cells using cDNA microarray analysis and identified 147 transcripts that exhibited significant changes in expression. Of these, 18 were up-regulated and 129 were down-regulated. These transcripts were then classified into 12 categories according to their functional roles: cell adhesion/cytoskeleton, cell cycle, redox homeostasis, immune/defense responses, metabolism, protein biosynthesis/modification, intracellular transport, RNA processing, DNA modification/ replication, regulation of transcription, signal transduction and transport. We then analyzed the promoter sequences of differentially-regulated genes and identified over-represented regulatory sites and candidate transcription factors (TFs) for gene regulation by kaempferol. These included c-REL, SAP-1, Ahr-ARNT, Nrf-2, Elk-1, SPI-B, NF-κB and p65. In addition, we validated the microarray results and promoter analyses using conventional methods such as real-time PCR and ELISA-based transcription factor assay. Our microarray analysis has provided useful information for determining the genetic regulatory network affected by kaempferol, and this approach will be useful for elucidating gene-phytochemical interactions.