Establishment of Nrf2-deficient HaCaT and immortalized primary human foreskin keratinocytes and characterization of their responses to ROS-induced cytotoxicity.

Nuclear factor erythroid 2-like 2 (Nrf2) is a key transcription factor responsible for the induction of cytoprotective genes when a cell is exposed to reactive oxygen species (ROS). Insufficient ROS neutralization has been associated with undesirable changes in the skin caused by age and disease. In order to mimic the pathological conditions of these oxidative stress-induced skin disorders, we established Nrf2-deficient HaCaT and immortalized human foreskin keratinocyte (iHFK) cell lines via lentiviral transduction of Nrf2-targeting short-hairpin RNAs. Their transcriptional, as well as translational blockage of Nrf2 expression, was verified by using a proteasomal inhibitor (MG132) and well-known Nrf2 activator (α-lipoic acid (ALA)). Reduced expression of NADPH dehydrogenase quinone 1 (NQO-1) and heme oxygenase 1 (HO-1) genes, which are well-characterized downstream targets of Nrf2-mediated transactivation, was also confirmed by using ALA and another Nrf2 activator, marliolide. In general, iHFK cells displayed more enhanced cytotoxicity to menadione, a ROS-generating reference compound, than HaCaT cells. In addition, the Nrf2 deficiency highly potentiated the cytotoxic effects of menadione in both HaCaT and iHFK cells. Interestingly, pretreatment of either ALA or marliolide conferred protection against the ROS induction and the subsequent development of cytotoxicity by menadione in both HaCaT and iHFK cells regardless of the Nrf2 status. These data suggest a possibility for activation of Nrf2-independent ROS detoxification pathways by either ALA or marliolide. These newly established Nrf2-deficient HaCaT and iHFK cell lines should be useful as a highly ROS-sensitive damaged skin model for the study of age-dependent cellular changes in an in vitro setting.

Suppression of Hepatitis C Virus Genome Replication and Particle Production by a Novel Diacylglycerol Acyltransferases Inhibitor.

Diacylglycerol acyltransferases (DGATs) play a critical role in the biosynthesis of endogenous triglycerides (TGs) and formation of lipid droplets (LDs) in the liver. In particular, one member of DGATs, DGAT-1 was reported to be an essential host factor for the efficient production of hepatitis C virus (HCV) particles. By utilizing our previously characterized three different groups of twelve DGAT inhibitors, we found that one of the DGAT inhibitors, a 2-((4-adamantylphenoxy) methyl)-N-(furan-2-ylmethyl)-1H-benzo[d]imidazole-5-carboxam (10j) is a potent suppressor of both HCV genome replication and particle production. 10j was able to induce inhibition of these two critical viral functions in a mutually separate manner. Abrogation of the viral genome replication by 10j led to a significant reduction in the viral protein expression as well. Interestingly, we found that its antiviral effect did not depend on the reduction of TG biosynthesis by 10j. This suggests that the inhibitory activity of 10j against DGATs may not be directly related with its antiviral action.

Establishment of Immortalized Primary Human Foreskin Keratinocytes and Their Application to Toxicity Assessment and Three Dimensional Skin Culture Construction.

In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced blockage of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals.

Immortalization of Primary Keratinocytes and Its Application to Skin Research.

As a major component of the epidermal tissue, a primary keratinocyte has served as an essential tool not only for the study of pathogenesis of skin-related diseases but also for the assessment of potential toxicities of various chemicals used in cosmetics. However, its short lifespan in ex vivo setting has been a great hurdle for many practical applications. Therefore, a number of immortalization attempts have been made with success to overcome this limitation. In order to understand the immortalization process of a primary keratinocyte, several key biological phenomena governing its lifespan will be reviewed first. Then, various immortalization methods for the establishment of stable keratinocyte cell lines will be explained. Finally, its application to a three-dimensional skin culture system will be described.

Mangosteen xanthones suppress hepatitis C virus genome replication.

Hepatitis C virus (HCV) is a hepatotropic single-stranded RNA virus. HCV infection is causally linked with development of liver cirrhosis and hepatocellular carcinoma. Enhanced production of reactive oxygen species by HCV has been implicated to play an important role in HCV-induced pathogenesis. Mangosteen has been widely used as a traditional medicine as well as a dietary supplement ,thanks to its powerful anti-oxidant effect. In the present study, we demonstrated that the ethanol extract from mangosteen fruit peels (MG-EtOH) is able to block HCV genome replication using HCV genotype 1b Bart79I subgenomic (EC50 5.1 µg/mL) and genotype 2a J6/JFH-1 infectious replicon systems (EC50 3.8 µg/mL). We found that inhibition of HCV replication by MG-EtOH led to subsequent down-regulation of expression of HCV proteins. Interestingly, MG-EtOH exhibited a modest inhibitory effect on in vitro RNA polymerase activity of NS5B. Among a number of xanthones compounds identified within this MG-EtOH, we discovered α-MG (EC50 6.3 µM) and γ-MG (EC50 2.7 µM) as two major single molecules responsible for suppression of HCV replication. This finding will provide a valuable molecular basis to further develop mangosteen as an important dietary supplement to combat HCV-induced liver diseases.

Chemical genetics-based discovery of indole derivatives as HCV NS5B polymerase inhibitors.

In order to identify the inhibitors of hepatitis C virus (HCV) replication with a novel scaffold via a mechanistically unbiased approach, we screened our in-house library composed of ∼6000 compounds with various chemical structures by using the renilla luciferase-linked genotype 2a reporter virus, and we identified a series of compounds containing an indole moiety that were active against HCV replication. Based on this result, we further synthesized three groups of indole derivatives and evaluated their inhibitory effects on HCV replication. In the present structure-activity relationship study of these indole derivatives, we discovered that compound 12e was the most potent inhibitor of HCV replication with minimal cytotoxicity (EC50 = 1.1 µM, EC90 = 2.1 µM, and CC50 = 61.8 µM). We also confirmed that compound 12e caused a dose- and time-dependent reduction of viral RNA as well as viral protein levels in both genotype 2a J6/JFH1 RNA-transfected cells and genotype 1b Bart79I subgenomic replicon cells. Finally, a genetic mapping study of mutant viruses resistant to compound 12e revealed that NS5B RNA polymerase was the potential target. This finding was further validated by demonstration of inhibition of NS5B RNA polymerase in vitro by compound 12e (IC50 = 292 nM). Compound 12e may serve as a valuable candidate for the development of a new class of HCV NS5B RNA polymerase inhibitors in the future.

Roles of the PDZ domain-binding motif of the human papillomavirus type 16 E6 on the immortalization and differentiation of primary human foreskin keratinocytes.

A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI1, MAGI2, and MAGI3, MUPP1, 14-3-3zeta, Na/H exchange regulatory factor 1, PTPN13, TIP-2/GIPC, Tip-1, and PATJ. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. However, contribution of degradation of PDZ proteins by E6 to HPV-induced oncogenesis is still controversial. In order to clarify potential roles of molecular interactions between high-risk HPV E6 and one of best characterized PDZ proteins, hDlg in HPV-induced transformation, we used a retroviral infection system to overexpress HPV16 E7 gene alone or together with either HPV16 E6 wild type or E6 mutant gene lacking the PDZ domain-binding motif and investigated the effect of mutating the PDZ domain-binding motif of E6 on the immortalization and differentiation of human foreskin keratinocytes (HFKs) by the high-risk type HPV E6 and E7. Although the PDZ domain-binding motif of E6 was found to be required for the efficient growth of HFKs, it was not necessary for the E6 and E7-induced immortalization of HFKs. Furthermore, the overexpression of E6 and E7 neither induced degradation nor altered cellular localization of hDlg in undifferentiated or differentiated HFKs. These data indicate that the PDZ domain-binding motif of E6 contributes to the efficient cellular growth through mechanisms other than degradation and changes in the subcellular localizations of hDlg.

A hepatitis C virus NS4B inhibitor suppresses viral genome replication by disrupting NS4B's dimerization/multimerization as well as its interaction with NS5A.

Chronic hepatitis C virus (HCV) infection is responsible for severe liver diseases including liver cirrhosis and hepatocellular carcinoma. An HCV non-structural protein 4B (NS4B) plays an essential role in viral RNA genome replication by building multi-vesicular structures around endoplasmic reticulum membranes. Especially, the second amphipathic helix of NS4B (NS4B-AH2) was shown to be essential for this process. By screening compounds against a membrane-aggregating activity of NS4B-AH2, several anti-HCV replication small molecules targeting NS4B-AH2 were discovered. However, little is known about detailed molecular mechanism of action for these NS4B-AH2 inhibitors. In this report, we provide evidences that NS4B-AH2 is required for NS4B's dimerization/multimerization, its proper subcellular localization, as well as its interaction with NS5A. More importantly, one of NS4B-AH2 inhibitors called "anguizole" was found to be able to disrupt all of these NS4B-AH2-mediated biological functions of NS4B. This newly elucidated mechanism of action will enable us not only to better understand a central role of NS4B-AH2 in HCV life cycle but also to develop a more safe and effective new class of NS4B-AH2 inhibitors of HCV replication in the future.