Jeong Taeg 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.

Na+-dependent transporters mediate HCO3– salvage across the luminal membrane of the main pancreatic duct

To study the roles of Na(+)-dependent H(+) transporters, we characterized H(+) efflux mechanisms in the pancreatic duct in wild-type, NHE2(-/-), and NHE3(-/-) mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na(+)-dependent H(+) efflux in the microperfused duct was inhibited by 1.5 microM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 microM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na(+)-dependent H(+) efflux by approximately 45%. Notably, the remaining luminal Na(+)-dependent H(+) efflux in ducts from NHE3(-/-) mice was inhibited by 50 microM HOE 694. Hence, approximately 55% of luminal H(+) efflux (or HCO(3)(-) influx) in the pancreatic duct is mediated by a novel, HOE 694-sensitive, Na(+)-dependent mechanism. H(+) transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na(+)-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na(+) and HCO(3)(-) to produce a pancreatic juice that is poor in HCO(3)(-) and rich in Cl(-) during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO(3)(-)-rich pancreatic juice.

Oxidative Stress Induced Cytokine Production in Isolated Rat Pancreatic Acinar Cells: Effects of Small-Molecule Antioxidants

Reactive oxygen species are considered important regulators in the pathogenesis and in the development of pancreatitis. The transcription factor nuclear factor ĸB (NF-ĸB) is activated by reactive oxygen species and regulates the gene expressions of inflammatory cytokines. The present study investigates (1) the susceptibility of isolated rat pancreatic acinar cells to oxidant attacks produced by adenosine diphosphate/ferrous iron, hypoxanthine/xanthine oxidase, and neutrophils primed with 4β-phorbol 12β-myristate 13α-acetate (PMA) and (2) the potential of small-molecule antioxidants (N-acetylcysteine, β-carotene, rebamipide, allopurinol) and superoxide dismutase (SOD) to prevent such injury and oxidant-mediated NF-ĸB activation and inflammatory cytokine production in the cells. As a result, oxidative stress resulted in a time-dependent increase in lipid peroxide production in pancreatic acinar cells which was inhibited by small-molecule antioxidants and SOD. PMA-primed neutrophils induced NF-ĸB activation and increased the production of cytokines (IL-6, TNF-α) in the cells. This was in parallel with lipid peroxide production. Small-molecule antioxidants and SOD inhibited NF-ĸB activation and cytokine production in acinar cells caused by PMA-primed neutrophils. In conclusion, oxidative stress activates NF-ĸB, resulting in upregulation of inflammatory cytokines in pancreatic acinar cells. Small-molecule antioxidants might be clinically useful anti-inflammatory agents by inhibiting oxidant-induced cytokine production.

Critical role of phospholipase Cγ1 in the generation of H2O2-evoked [Ca2+]i oscillations in cultured rat cortical astrocytes

Reactive oxygen species, such as the superoxide anion, H2O2, and the hydroxyl radical, have been considered as cytotoxic by-products of cellular metabolism. However, recent studies have provided evidence that H2O2 serves as a signaling molecule modulating various physiological functions. Here we investigated the effect of H2O2 on the regulation of intracellular Ca2+ signaling in rat cortical astrocytes. H2O2 triggered the generation of oscillations of intracellular Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner over the range 10–100μm. The H2O2-induced [Ca2+]i oscillations persisted in the absence of extracellular Ca2+ and were prevented by depletion of intracellular Ca2+ stores with thapsigargin. The H2O2-induced [Ca2+]i oscillations were not inhibited by pretreatment with ryanodine but were prevented by 2-aminoethoxydiphenyl borate and caffeine, known antagonists of inositol 1,4,5-trisphosphate receptors. H2O2 activated phospholipase C (PLC) γ1 in a dose-dependent manner, and U73122, an inhibitor of PLC, completely abolished the H2O2-induced [Ca2+]i oscillations. In addition, RNA interference against PLCγ1 and the expression of the inositol 1,4,5-trisphosphate-sequestering “sponge” prevented the generation of [Ca2+]i oscillations. H2O2-induced [Ca2+]i oscillations and PLCγ1 phosphorylation were inhibited by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Finally, epidermal growth factor induced H2O2 production, PLCγ1 activation, and [Ca2+]i increases, which were attenuated by N-acetylcysteine and diphenyleneiodonium and by the overexpression of peroxiredoxin type II. Therefore, we conclude that low concentrations of exogenously applied H2O2 generate [Ca2+]i oscillations by activating PLCγ1 through sulfhydryl oxidation-dependent mechanisms. Furthermore, we show that this mechanism underlies the modulatory effect of endogenously produced H2O2 on epidermal growth factor-induced Ca2+ signaling in rat cortical astrocytes.

Chitinase Activates Protease-Activated Receptor-2 in Human Airway Epithelial Cells

Mammalian chitinase released by airway epithelia is thought to be an important mediator of disease manifestation in an experimental model of asthma. However, the intracellular signaling mechanisms engaged by exogenous chitinase in human airway epithelial cells are unknown. Here, we investigated the direct effects of exogenous chitinase from Streptomyces griseus on Ca(2+) signaling in human airway epithelial cells. Spectrofluorometry was used to measure intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-AM-loaded cells. S. griseus chitinase induced dose-dependent [Ca(2+)](i) increases in normal human bronchial epithelial cells and promoted [Ca(2+)](i) oscillations in H292 cells. Chitinase-induced [Ca(2+)](i) oscillations were independent of extracellular Ca(2+), suggesting that the observed [Ca(2+)](i) increases were due to Ca(2+) release from intracellular stores. Accordingly, after depleting endoplasmic reticulum (ER) Ca(2+) with the ER Ca(2+) ATPase inhibitor, thapsigargin, chitinase-mediated [Ca(2+)](i) increases were abolished. Treatment with the phospholipase C (PLC) inhibitor U73122 or the 1, 4, 5-trisinositolphosphate (IP(3)) receptor inhibitor 2-APB attenuated chitinase-induced [Ca(2+)](i) increases. Desensitization of protease-activated receptor-2 (PAR-2) by repetitive agonist stimulation or siRNA-mediated PAR-2 knock-down revealed that chitinase-mediated [Ca(2+)](i) increases were exclusively mediated by PAR-2 activation. Finally, chitinase was found to cleave a model peptide representing the cleavage site of PAR-2 and enhanced IL-8 production. These results indicate that exogenous chitinase is a potent proteolytic activator of PAR-2 that can directly induce PLC/IP(3)-dependent Ca(2+) signaling in human airway epithelial cells.

Biphasic effects of dithiocarbamates on the activity of nuclear factor-κB

Abstract Dithiocarbamates are well-known antioxidants and nuclear factor-κB (NF-κB) inhibitors. Recently, they have been characterized as zinc ionophores. Concentration-dependent biphasic effects of dithiocarbamates on NF-κB activity have been widely reported. We studied the mechanism of this phenomenon in relation to Zn 2+ influx. Two dithiocarbamates, pyrrolidine dithiocarbamate and diethyldithiocarbamate, showed concentration-dependent biphasic effects in inhibiting NF-κB activation in cerebral endothelial cells. These unique effects of dithiocarbamates on NF-κB were tightly linked to their ability to elevate intracellular Zn 2+ levels. At high concentrations (>500 μM), dithiocarbamates started to lose their ability to promote Zn 2+ influx and to inhibit NF-κB activation. These results might provide insight into the appropriate use of dithiocarbamates in various disorders.

Pyrrolidine dithiocarbamate‐induced neuronal cell death is mediated by Akt, casein kinase 2, c‐Jun N‐terminal kinase, and IκB kinase in embryonic hippocampal progenitor cells

Pyrrolidine dithiocarbamate (PDTC) is known to induce cell death by the stimulation of intracellular zinc transport and subsequent modulation of nuclear factor‐κB (NF‐κB) activity. Zinc is a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes to severe neuronal cell death. In the present study, we explored how PDTC modulates intracellular signal transduction pathways, leading to neuronal cell death. The exposure of immortalized embryonic hippocampal cells (H19‐7) to PDTC within the range of 1–100 μM caused cell death in a dose‐dependent manner. During the cell death, NF‐κB activity increased in response to PDTC, and this activity corresponded well with the increase of intracellular free zinc levels, implying that the activation of NF‐κB transmits the cell death signals of PDTC. Furthermore, PDTC caused the activation of IκB kinase (IKK), casein kinase 2 (CK2), phosphatidylinositol 3‐kinase (PI‐3K), and Akt, as well as mitogen‐activated protein kinases (MAPKs), such as extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK), but not p38 kinase. The blockade of PI‐3K, JNK, and CK2 pathways resulted in a remarkable suppression of PDTC‐induced cell death and also the activation of IKK, which subsequently led to a decrease of IκB phosphorylation. Although the overexpression of dominant‐negative SEK in a transient manner did not inhibit the activation of Akt by PDTC, the transfection of kinase‐inactive Akt mutants did cause a remarkable blockade of JNK activation, implying that Akt is present upstream of JNK in the PDTC‐signaling pathways. Moreover, whereas selective CK2 inhibitors suppressed PDTC‐induced JNK activation, the inhibition of JNK did not affect CK2 activity, suggesting that CK2 is directly related to the regulation of cell viability by PDTC and that the CK2‐JNK pathway could be a downstream target of PDTC. Taken together, our results suggest that PDTC‐mediated accumulation of intracellular zinc ions may affect cell viability by modulating several intracellular signaling pathways in neuronal hippocampal progenitor cells.

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

Alteration of RANKL-induced osteoclastogenesis in primary cultured osteoclasts from SERCA2+/- mice.

RANKL is essential for the terminal differentiation of monocytes/marcrophages into osteoclasts. RANKL induces long‐lasting oscillations in the intracellular concentration of Ca2+ ([Ca2+]i) only after 24 h of stimulation. These Ca2+ oscillations play a switch‐on role in NFATc1 expression and osteoclast differentiation. Which Ca2+ transporting pathway is induced by RANKL to evoke the Ca2+ oscillations and its specific role in RANKL‐mediated osteoclast differentiation is not known. This study examined the effect of a partial loss of sarco/endoplasmic reticulum Ca2+ ATPase type2 (SERCA2) on osteoclast differentiation in SERCA2 heterozygote mice (SERCA2+/−). The BMD in the tibias of SERCA2+/− mice increased >1.5‐fold compared with wildtype mice (WT). RANKL‐induced [Ca2+]i oscillations were generated 48 h after RANKL treatment in the WT mice but not in the SERCA2+/− bone marrow–derived macrophages (BMMs). Forty‐eight hours after RANKL treatment, there was a lower level of NFATc1 protein expression and markedly reduced translocation of NFATc1 into the nucleus during osteoclastogenesis of the SERCA2+/− BMMs. In addition, RANKL treatment of SERCA2+/− BMMs incompletely induced formation of multinucleated cells, leading to reduced bone resorption activity. These results suggest that RANKL‐mediated induction of SERCA2 plays a critical role in the RANKL‐induced [Ca2+]i oscillations that are essential for osteoclastogenesis.

Ciliary Phosphoinositide Regulates Ciliary Protein Trafficking in Drosophila

Cilia are highly specialized antennae-like cellular organelles. Inositol polyphosphate 5-phosphatase E (INPP5E) converts PI(4,5)P2 into PI4P and is required for proper ciliary function. Although Inpp5e mutations are associated with ciliopathies in humans and mice, the precise molecular role INPP5E plays in cilia remains unclear. Here, we report that Drosophila INPP5E (dINPP5E) regulates ciliary protein trafficking by controlling the phosphoinositide composition of ciliary membranes. Mutations in dInpp5e lead to hearing deficits due to the mislocalization of dTULP and mechanotransduction channels, Inactive and NOMPC, in chordotonal cilia. Both loss of dINPP5E and ectopic expression of the phosphatidylinositol-4-phosphate 5-kinase Skittles increase PI(4,5)P2 levels in the ciliary base. The fact that Skittles expression phenocopies the dInpp5e mutants confirms a central role for PI(4,5)P2 in the regulation of dTULP, Inactive, and NOMPC localization. These data suggest that the spatial localization and levels of PI(4,5)P2 in ciliary membranes are important regulators of ciliary trafficking and function.