Kyoung Sang Cho

Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production

The Akt/protein kinase B (PKB) serine/ threonine kinase is well known as an important mediator of many cell survival signaling pathways. Here, we demonstrate for the first time a major role of Akt/PKB in the cell invasion properties of the highly metastatic cell line HT1080. Using confocal microscopic analyses of live samples, we found Akt/PKB to be localized in the leading edge membrane area of migrating HT1080 cells. This localization was dependent on phosphoino‐sitide 3‐kinase and required the lipid binding ability of the phosphoinositide binding pleckstrin homology domain of Akt/PKB. We examined the possible function of Akt/PKB in HT1080 invasion. Surprisingly, Akt/ PKB potently promoted HT1080 invasion, by increasing cell motility and matrix metalloproteinase‐9 (MMP‐9) production, in a manner highly dependent on its kinase activity and membrane‐translocating ability. The increase in MMP‐9 production was mediated by activation of nuclear factor‐κB transcriptional activity by Akt/PKB. However, Akt/PKB did not affect the cell‐cell or cell‐matrix adhesion properties of HT1080. Our findings thus establish Akt/PKB as a major factor in the invasive abilities of cancer cells.

Discrete Functions of TRAF1 and TRAF2 in Drosophila melanogaster Mediated by c-Jun N-Terminal Kinase and NF-κB-Dependent Signaling Pathways

ABSTRACT Two Drosophila tumor necrosis factor receptor-associated factors (TRAF), DTRAF1 and DTRAF2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, their in vivo functions and related signaling pathways are not fully understood yet. Here, we show that DTRAF1 is an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila melanogaster. Ectopic expression of DTRAF1 in the developing eye induced apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses revealed that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by DTRAF1 are dependent on JNK and its upstream kinases, Hep and DTAK1. In support of these results, DTRAF1-null mutant showed a remarkable reduction in JNK activity with an impaired development of imaginal discs and a defective formation of photosensory neuron arrays. In contrast, DTRAF2 was demonstrated as an upstream activator of nuclear factor-κB (NF-κB). Ectopic expression of DTRAF2 induced nuclear translocation of two Drosophila NF-κBs, DIF and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and drosomycin. Consistently, the null mutant of DTRAF2 showed immune deficiencies in which NF-κB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, our findings demonstrate that DTRAF1 and DTRAF2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-κB-dependent signaling pathway, respectively.

Drosophila phosphoinositide-dependent kinase-1 regulates apoptosis and growth via the phosphoinositide 3-kinase-dependent signaling pathway

Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Here, we have isolated Drosophila PDK-1 (dPDK-1) mutants and characterized the in vivo roles of the kinase. Drosophila deficient in the dPDK-1 gene exhibited lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increased cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only could activate Drosophila Akt/PKB (Dakt1), but also substitute the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila. On the other hand, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1, did not interact with dPDK-1. In conclusion, our findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate our Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1.

blistery encodes Drosophila tensin protein and interacts with integrin and the JNK signaling pathway during wing development.

Tensin is an actin-binding protein that is localized in focal adhesions. At focal adhesion sites, tensin participates in the protein complex that establishes transmembrane linkage between the extracellular matrix and cytoskeletal actin filaments. Even though there have been many studies on tensin as an adaptor protein, the role of tensin during development has not yet been clearly elucidated. Thus, this study was designed to dissect the developmental role of tensin by isolating Drosophila tensin mutants and characterizing its role in wing development. The Drosophila tensin loss-of-function mutations resulted in the formation of blisters in the wings, which was due to a defective wing unfolding process. Interestingly, by1-the mutant allele of the gene blistery (by)-also showed a blistered wing phenotype, but failed to complement the wing blister phenotype of the Drosophila tensin mutants, and contains Y62N/T163R point mutations in Drosophila tensin coding sequences. These results demonstrate that by encodes Drosophila tensin protein and that the Drosophila tensin mutants are alleles of by. Using a genetic approach, we have demonstrated that tensin interacts with integrin and also with the components of the JNK signaling pathway during wing development; overexpression of by in wing imaginal discs significantly increased JNK activity and induced apoptotic cell death. Collectively, our data suggest that tensin relays signals from the extracellular matrix to the cytoskeleton through interaction with integrin, and through the modulation of the JNK signal transduction pathway during Drosophila wing development.

Drosophila DJ-1 decreases neural sensitivity to stress by negatively regulating Daxx-like protein through dFOXO.

DJ-1, a Parkinsons disease (PD)–associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1β, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1β mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1β mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1β mutants and that dFOXO activity was increased in the heads of DJ-1β mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1β mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1β under oxidative stress conditions; and, furthermore, targeted expression of DJ-1β to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1β protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD.

Inhibition of JNK/dFOXO pathway and caspases rescues neurological impairments in Drosophila Alzheimer’s disease model

Amyloid-β-42 (Aβ42) has been implicated in the pathogenesis of Alzheimers disease (AD). Neuronal Aβ42 expression induces apoptosis and decreases survival and locomotive activity in Drosophila. However, the mechanism by which Aβ42 induces these neuronal impairments is unclear. In this study, we investigated the underlying pathway in theses impairments. JNK activity was increased in Aβ42-expressing brains, and the Aβ42-induced defects were rescued by reducing JNK or caspase activity through genetic modification or pharmacological treatment. In addition, these impairments were restored by Drosophila forkhead box subgroup O (dFOXO) deficiency. These results suggest that the JNK/dFOXO pathway confers a therapeutic potential for AD.

Drosophila PDZ-GEF, a Guanine Nucleotide Exchange Factor for Rap1 GTPase, Reveals a Novel Upstream Regulatory Mechanism in the Mitogen-Activated Protein Kinase Signaling Pathway

ABSTRACT PDZ-GEF is a novel guanine nucleotide exchange factor for Rap1 GTPase. Here we isolated Drosophila melanogaster PDZ-GEF (dPDZ-GEF), which contains the all-conserved domains of mammalian and nematode PDZ-GEF including cyclic nucleotide monophosphate-binding, Ras exchange motif, PDZ, RA, and GEF domains. dPDZ-GEF loss-of-function mutants were defective in the development of various organs including eye, wing, and ovary. Many of these phenotypes are strikingly similar to the phenotype of the rolled mutant, implying that dPDZ-GEF functions upstream of the mitogen-activated protein (MAP) kinase pathway. Indeed, we found that dPDZ-GEF is specifically involved in photoreceptor cell differentiation, facilitating its neuronal fate via activation of the MAP kinase pathway. Rap1 was found to link dPDZ-GEF to the MAP kinase pathway; however, Ras was not involved in the regulation of the MAP kinase pathway by dPDZ-GEF and actually had an inhibitory function. The analyses of ovary development in dPDZ-GEF-deficient mutants also demonstrated another role of dPDZ-GEF independent of the MAP kinase signaling pathway. Collectively, our findings identify dPDZ-GEF as a novel upstream regulator of various morphogenetic pathways and demonstrate the presence of a novel, Ras-independent mechanism for activating the MAP kinase signaling pathway.

A modified formulation of Chinese traditional medicine improves memory impairment and reduces Aβ level in the Tg-APPswe/PS1dE9 mouse model of Alzheimer's disease

ETHNOPHARMACOLOGICAL RELEVANCE SuHeXiang Wan (SHXW), a Chinese traditional medicine has been used orally for the treatment of seizures, infantile convulsion, stroke and so forth. Previously, we reported the effects of modified SHXW essential oil mixture of the fragrance containing herbs on the sedative effect, anticonvulsant property and antioxidative activity after fragrance inhalation. MATERIALS AND METHODS This study was undertaken to evaluate beneficial effects of a modified recipe of SHXW (termed as KSOP1009) consisting of a ethanol extract of 8 herbs including resin of Liquidambar orientalis Miller, seed of Myristica fragrans Houtt., rhizome of Cnidium officinale Makino, lumber of Santalum album L., fructus of Piper longum L., flower buds of Eugenia caryophyllata Merrill et Perry, pollen of Typha orientalis Presl., and root of Salvia miltiorrhiza Bunge in the neurodegenerative diseases such as Alzheimers disease (AD). The transgenic mice of AD, Tg-APPswe/PS1dE9, were fed KSOP1009 or as a positive control, donepezil for 3 months from 4.5 months of age. Behavioral, immunological and ELISA analyses were used to assess memory impairment, Aβ accumulation and plaque deposition in the brain. Other in vitro works were performed to examine whether KSOP1009 inhibits the Aβ(1-42)-induced neurotoxicity in human neuroblastoma cell line, SH-SY5Y cells. RESULTS Intake of KSOP1009 improved the Aβ-induced memory impairment and suppressed Aβ levels and plaque deposition in the brain of Tg-APPswe/PS1dE9 mice as much as that of donepezil treatment. KSOP1009 prevented the down-regulation of phospho-CREB and increased AKT phosphorylation in the AD-like brains. Moreover, KSOP1009 suppresses Aβ-induced apoptosis and ROS production in SH-SY5Y cells. CONCLUSION The present study suggests that KSOP1009 may develop as a therapeutic drug for treatment of AD patients.

Neuroprotective effect of SuHeXiang Wan in Drosophila models of Alzheimer's disease.

AIM OF THE STUDY SuHeXiang Wan (SHXW) is a Chinese traditional medicinal prescription that consists of 15 crude herbs. SHXW has been used to treat central nervous depression, seizures, infantile convulsion and stroke, and its essential oil has been shown to have anticonvulsant and antioxidative activity. The goal of this study was to investigate the beneficial effects of SHXW on the neurological phenotypes of Drosophila AD models. MATERIALS AND METHODS We evaluated the effects of a modified SHXW (called KSOP1009) intake on the AD-like phenotypes of Drosophila AD models, which express human Aβ42 in their developing eyes or neurons. RESULTS When the flies were kept on the media containing 5 μg/ml of KSOP1009 extract, Aβ42-induced eye degeneration, apoptosis, and the locomotive dysfunctions were strongly suppressed. However, Aβ42 fibril deposits in the Aβ42 overexpressing model were not affected by treatment with KSOP1009 extract. Conversely, KSOP1009 extract intake significantly suppressed the constitutive active form of hemipterous, a JNK activator, while it induced eye degeneration and JNK activation, which has been recognized as an important mediator of Aβ42-associated neuro-cytotoxicity. CONCLUSIONS In conclusion, the results of this study suggest that KSOP1009 confers a therapeutic potential to AD-like pathology of Aβ42 overexpressing Drosophila model via suppression of the hyperactivation of JNK activity and apoptosis.

Parkin Suppresses c-Jun N-terminal kinase-induced cell death via transriptional regulation in Drosophila

Parkin is the most prevalent genetic factor in the onset of autosomal recessive juvenile parkinsonism (AR-JP), and mutations in parkin has been reported to cause motor defects, which result from dopamine deficiency caused by dopaminergic neuronal cell death. Activation of c-Jun N-terminal kinase (JNK) has also been implicated in neuronal cell death in Parkinson’s disease (PD). Moreover, Drosophila models for AR-JP, loss of function mutants of Drosophila parkin, also show dopaminergic neural degeneration associated with hyperactivation of JNK, increased apoptosis, and mitochondrial defects. However, the molecular mechanism by which Parkin protects cells from apoptosis remains unclear. In the present study, we tested whether Drosophila Parkin suppressed the JNK signaling pathway in developing tissues. Ectopically expressed parkin strongly suppressed the constitutively active form of Hemipterous (HepCA), a Drosophila JNK kinase that induces an eye degeneration phenotype and apoptosis in the eye imaginal disc. Moreover, parkin also suppressed extra vein formation induced by Basket (Bsk), a Drosophila JNK. Interestingly, the bsk mRNA level was markedly reduced by parkin over-expression, suggesting that the effect of parkin on the phenotype induced by activation of JNK signaling was achieved by transcriptional regulation. Furthermore, we found that the expression level of JNK target genes was reduced by parkin over-expression. Taken together, these results suggest that Drosophila Parkin suppresses JNK signaling by reducing bsk transcription.