Su Ryeon Seo

Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells.

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia‐ and seizure‐induced neuronal death. However, the intracellular signaling pathways involved in Zn2+‐induced cell death are largely unknown. In the present study, we investigated the roles of mitogen‐activated protein kinases (MAPKs), such as c‐Jun N‐terminal kinase (JNK), p38 MAPK and extracellular signal‐regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+‐induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 µm), a Zn2+ ionophore, and Zn2+ (10 µm) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+‐induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol‐3 kinase had little effect on Zn2+‐induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+‐induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.

NF-κB-inducing Kinase Phosphorylates and Blocks the Degradation of Down Syndrome Candidate Region 1

Down syndrome, the most frequent genetic disorder, is characterized by an extra copy of all or part of chromosome 21. Down syndrome candidate region 1 (DSCR1) gene, which is located on chromosome 21, is highly expressed in the brain of Down syndrome patients. Although its cellular function remains unknown, DSCR1 expression is linked to inflammation, angiogenesis, and cardiac development. To explore the functional role of DSCR1 and the regulation of its expression, we searched for novel DSCR1-interacting proteins using a yeast two-hybrid assay. Using a human fetal brain library, we found that DSCR1 interacts with NF-κB-inducing kinase (NIK). Furthermore, we demonstrate that NIK specifically interacts with and phosphorylates the C-terminal region of DSCR1 in immortalized hippocampal cells as well as in primary cortical neurons. This NIK-mediated phosphorylation of DSCR1 increases its protein stability and blocks its proteasomal degradation, the effects of which lead to an increase in soluble and insoluble DSCR1 levels. We show that an increase in insoluble DSCR1 levels results in the formation of cytosolic aggregates. Interestingly, we found that whereas the formation of these inclusions does not significantly alter the viability of neuronal cells, the overexpression of DSCR1 without the formation of aggregates is cytotoxic.

Neuroprotective roles of pituitary adenylate cyclase-activating polypeptide in neurodegenerative diseases

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic bioactive peptide that was first isolated from an ovine hypothalamus in 1989. PACAP belongs to the secretin/glucagon/vasoactive intestinal polypeptide (VIP) superfamily. PACAP is widely distributed in the central and peripheral nervous systems and acts as a neurotransmitter, neuromodulator, and neurotrophic factor via three major receptors (PAC1, VPAC1, and VPAC2). Recent studies have shown a neuroprotective role of PACAP using in vitro and in vivo models. In this review, we briefly summarize the current findings on the neurotrophic and neuroprotective effects of PACAP in different brain injury models, such as cerebral ischemia, Parkinson’s disease (PD), and Alzheimer’s disease (AD). This review will provide information for the future development of therapeutic strategies in treatment of these neurodegenerative diseases. [BMB Reports 2014; 47(7): 369-375]

Xanthorrhizol induces apoptosis through ROS-mediated MAPK activation in human oral squamous cell carcinoma cells and inhibits DMBA-induced oral carcinogenesis in hamsters

Xanthorrhizol, a natural sesquiterpenoid compound isolated from Curcuma xanthorrhiza Roxb, has been known to inhibit the growth of human colon, breast, liver and cervical cancer cells. In this study, xanthorrhizol decreased cell viability, induced apoptosis and decreased the level of full‐length PARP in SCC‐15 oral squamous cell carcinoma (OSCC) cells. A decrease in cell viability and PARP degradation was not prevented by treatment with the caspase inhibitor Z‐VAD‐fmk in xanthorrhizol‐treated cells. Xanthorrhizol treatment elevated intracellular Ca2+ and ROS levels in SCC‐15 cells. Treatment with a Ca2+ chelator, EGTA/AM, did not affect xanthorrhizol‐ induced cytotoxicity, but cell viability was partly recovered by treatment with endogenous antioxidant, GSH, or hydroxy radical trapper, MCI‐186. Furthermore, the viability of xanthorrhizol‐treated SCC‐15 cells was significantly restored by treatment with SB203580 and/or SP600125 but not significantly by PD98059 treatment. Xanthorrhizol‐induced activation of p38 MAPK and JNK was blocked by MCI‐186. Finally, xanthorrhizol suppressed the number of tumors in buccal pouches and increased the survival rate in hamsters treated with 7,12‐dimethylbenz[a]anthracene. In conclusion, xanthorrhizol may induce caspase‐independent apoptosis through ROS‐mediated p38 MAPK and JNK activation in SCC‐15 OSCC cells and prevent chemical‐induced oral carcinogenesis. Therefore, xanthorrhizol seems to be a promising chemopreventive agent. Copyright

CREB activates proteasomal degradation of DSCR1/RCAN1.

The cyclic AMP response element‐binding protein (CREB) is involved in the development and function of the nervous system. Here, we find that CREB decreases the protein level of Regulator of Calcineurin Activity 1 (RCAN1/DSCR1/MCIP1), which is overexpressed in the brain of Down Syndrome (DS) patients. Decrease of RCAN1 by CREB was blocked by proteasome inhibitors, indicating that this decrease is mediated by the ubiquitin‐proteasome pathway. Furthermore, we found that the ability of CREB to activate the degradation of RCAN1 depends on its transcriptional activation. Consistently, CREB‐enhanced the ubiquitination and turnover rate of RCAN1. Our results reveal a new regulatory role for CREB in DS pathology through the proteasomal degradation of RCAN1.

Overexpression of DSCR1 blocks zinc-induced neuronal cell death through the formation of nuclear aggregates.

The Down syndrome (DS) candidate region gene 1 (DSCR1) is localized near DS critical region on chromosome 21 and is overexpressed in the brains of DS patients. Although DSCR1 was known for a modulator of calcineurin, the overexpression of DSCR1 is thought to play a role in neuronal cell death. Zinc, one of the most abundant transition metals in the brain, may also contribute to selective neuronal cell death when present in excessive amounts. In the present study, we investigated the effect of DSCR1 overexpression on zinc-induced cell death in hippocampal neuroprogenitor cells. The overexpression of DSCR1 caused apoptotic cell death without an apparent formation of intracellular protein inclusions. Upon exposure to zinc, soluble DSCR1 levels were significantly decreased and insoluble levels were enhanced to a similar extent, which were partially caused by the zinc-induced inhibition of proteasomal activity and a consequently diminished degradation of DSCR1. Furthermore, zinc treatment induced the formation of nuclear DSCR1 aggregates, which blocked zinc-induced cell death. These findings indicate that, although the up-regulation of DSCR1 levels exerts a cytotoxic effect, the addition of zinc leads to the formation of cytoprotective nuclear aggregates in neuronal cells.

Protein kinase A phosphorylates Down syndrome critical region 1 (RCAN1)

The Down syndrome critical region 1 (DSCR1) gene encodes a regulator of the calcineurin 1 (RCAN1) protein, and the elevated levels of RCAN1 are associated with Alzheimers disease (AD) and Down syndrome (DS). In this report, we found that protein kinase A (PKA) was able to phosphorylate RCAN1 in vitro and in vivo. In addition, we found that the phosphorylation of RCAN1 by PKA caused an increase of RCAN1 expression by increasing of the half-life of the protein. Consistently, the pharmacological inhibition of intracellular PKA using H-89 and the knockdown of the endogenous PKA catalytic subunit with siRNA decreased the expression of RCAN1. Furthermore, the phosphorylation of RCAN1 by PKA enhanced the inhibitory function of RCAN1 on calcineurin-mediated gene transcription. Our data provide the first evidence that PKA acts as an important regulatory component in the control of RCAN1 function through phosphorylation.

The Regulator of Calcineurin 1 (RCAN1/DSCR1) Activates the cAMP Response Element-binding Protein (CREB) Pathway

Background: The regulator of the calcineurin 1 (RCAN1) gene is located on human chromosome 21, the trisomy of which causes Down syndrome (DS). Results: RCAN1 increases the phosphorylation of transcription factor CREB. Conclusion: RCAN1 activates CREB signaling by inhibition of calcineurin activity. Significance: Identification of RCAN1 as a CREB signaling regulator could contribute to the understanding of DS pathogenesis. cAMP response element-binding protein (CREB) is one of the best known transcription factors in the development and function of the nervous system. In this report, we found that the regulator of calcineurin 1 (RCAN1), which is overexpressed in the brain of patients with Down syndrome, increased the phosphorylation of CREB and cAMP response element-mediated gene transcription in response to the activation of the intracellular cAMP pathway. Furthermore, we found that the increased activation of CREB signaling by RCAN1 depended on the ability of RCAN1 to inhibit calcineurin activity. Our data provide the first evidence that RCAN1 acts as an important regulatory component in the control of CREB signaling.

Sirtuin 3 (SIRT3) Regulates α-Smooth Muscle Actin (α-SMA) Production through the Succinate Dehydrogenase-G Protein-coupled Receptor 91 (GPR91) Pathway in Hepatic Stellate Cells.

Sirtuin 3 (SIRT3) is an NAD+-dependent protein deacetylase. Recent studies have shown that SIRT3 expression is decreased in nonalcoholic fatty liver disease (NAFLD). Moreover, SIRT3 is a key regulator of succinate dehydrogenase (SDH), which catalyzes the oxidation of succinate to fumarate. Increased succinate concentrations and the specific G protein-coupled receptor 91 (GPR91) are involved in the activation of hepatic stellate cells (HSCs). In this study, we aimed to establish whether SIRT3 regulated the SDH activity, succinate, and GPR91 expression in HSCs and an animal model of NAFLD. Our goal was also to determine whether succinate released from hepatocytes regulated HSC activation. Inhibiting SIRT3 using SIRT3 siRNA exacerbated HSC activation via the SDH-succinate-GPR91 pathway, and SIRT3 overexpression or honokiol treatment attenuated HSC activation in vitro. In isolated liver and HSCs from methionine- and choline-deficient (MCD) diet-induced NAFLD, the expression of SIRT3 and SDH activity was decreased, and the succinate concentrations and GPR91 expression were increased. Moreover, we found that GPR91 knockdown or resveratrol treatment improved the steatosis in MCD diet-fed mice. This investigation revealed a novel mechanism of the SIRT3-SDH-GPR91 cascade in MCD diet-induced HSC activation in NAFLD. These findings highlight the biological significance of novel strategies aimed at targeting SIRT3 and GPR91 in HSCs with the goal of improving NAFLD treatment.

Carmustine induces ERK- and JNK-dependent cell death of neuronally-differentiated PC12 cells via generation of reactive oxygen species

Accumulation of reactive oxygen species (ROS) caused by the inhibition of glutathione reductase (GR) has been proposed as one of the mechanisms responsible for carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU)-induced cytotoxicity. Since mitogen-activated protein kinases (MAPKs) are known to mediate ROS-dependent cell death in multiple cell types, we examined whether redox-sensitive MAPK activation mediated the carmustine-induced cell death of neuronally differentiated PC12 cells. Carmustine induced a concentration- and time-dependent cell death, which was associated with increased caspase-3 activation, a reduction in GR activity accompanied by a concomitant decrease in reduced glutathione levels, and accumulation of ROS. Carmustine-induced caspase-3 activation and cell death were prevented by pretreatment with anti-oxidants or a reducing agent, indicating that carmustine-induced caspase-3 activation and cell death occur via redox-dependent processes. Carmustine induced phosphorylation of the MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. The activation of these kinases was inhibited by pretreatment with N-acetyl-L-cysteine (NAC). Although all the MAPKs were activated by carmustine, only the inhibitors of JNK and ERK prevented carmustine-induced cell death and caspase-3 activation. Our data suggest that carmustine-induced neurotoxicity is, at least in part, due to the activation of ROS-dependent JNK and ERK signaling.