Kyong Hoon Ahn

Hypoxia-induced neuronal apoptosis is mediated by de novo synthesis of ceramide through activation of serine palmitoyltransferase

Cellular hypoxia can lead to cell death or adaptation and has important effects on development, physiology, and pathology. Here, we investigated the role and regulation of ceramide in hypoxia-induced apoptosis of SH-SY5Y neuroblastoma cells. Hypoxia increased the ceramide concentration; subsequently, we observed biochemical changes indicative of apoptosis, such as DNA fragmentation, nuclear staining, and poly ADP-ribose polymerase (PARP) cleavage. The hypoxic cell death was potently inhibited by a caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone). l-Cycloserine, a serine palmitoyltransferase (SPT) inhibitor, and fumonisin B(1) (FB(1)), a ceramide synthase inhibitor, inhibited the hypoxia-induced increase in ceramide, indicating that the increase occurred via the de novo pathway. Hypoxia increased the activity and protein levels of SPT2, suggesting that the hypoxia-induced increase in ceramide is due to the transcriptional up-regulation of SPT2. Specific siRNA of SPT2 prevented hypoxia-induced cell death and ceramide production. However, hypoxia also increased the cellular level of glucosylceramide, which was inhibited by a glucosylceramide synthase (GCS) inhibitor and specific siRNA, but not a ceramidase inhibitor. The increase in glucosylceramide was accompanied by increases in both PARP cleavage and DNA fragmentation. Together, the current results suggest that both SPT and GCS may regulate the cellular level of ceramide, and thus may be critical enzymes for deciding the fate of the cells exposed to hypoxia.

Age-related effects of sodium arsenite on splenocyte proliferation and Th1/Th2 cytokine production

Aging is associated with immune dysfunction and conditions such as inflamm-aging and immunosuppression. Arsenic, an environmental contaminant distributed worldwide, affects the immune system. This study tested the hypothesis that arsenic has distinct effects on T cell proliferation and the production of cytokines by activated T cells. Murine splenocytes from young (2 months) and aged (24–26 months) C57BL/6 mice were exposed to arsenite (As3+), the most toxic form of inorganic arsenic, and stimulated with concanavalin A (Con A) or anti-CD3 antibody. T cell proliferation decreased significantly in response to Con A and anti-CD3 at subtoxic doses of arsenite in splenocytes from both young and aged mice. Arsenite, added concurrently with Con A or anti-CD3, significantly inhibited the production of interleukin-2 (IL-2), interferon-γ (IFN-γ), and interleukin-4 (IL-4) by splenocytes from young mice and significantly reduced the production of IL-10 by splenocytes from aged mice. In contrast, the production of IL-2 and IL-4 by splenocytes from aged mice was only slightly affected by arsenite. The results show that arsenic exposure reduces the immune response in splenocytes. Moreover, this effect may be influenced by aging.

Neutral sphingomyelinase 2 induces dopamine uptake through regulation of intracellular calcium

Ceramide serves as a second messenger produced from sphingomyelin by the activation of sphingomyelinase (SMase). Here, we suggest that neutral SMase 2 (nSMase2) may regulate dopamine (DA) uptake. nSMase2 siRNA-transfected PC12 cells showed lower levels of nSMase activity and ceramide than scramble siRNA-transfected and control cells. Interestingly, transfection of nSMase2 siRNA or pretreatment with the nSMase2-specific inhibitor GW4869 resulted in decreased DA uptake. Reciprocally, exposure of PC12 cells to cell-permeable C(6)-ceramide induced a concentration-dependent increase in DA uptake. Removal of extracellular calcium by EGTA increased DA uptake in scramble-transfected and control cells, but not in nSMase2 siRNA-transfected or GW4869-pretreated cells. Moreover, siRNA-transfected cells showed higher levels of intracellular calcium than scramble cells, while C(6)-ceramide treatment resulted in decreased intracellular calcium compared to vehicle treatment alone. Taken together, these data suggest that nSMase2 may increase DA uptake through inducing ceramide production and thereby decreasing intracellular calcium levels.

Identification and Evaluation of Neutral Sphingomyelinase 2 Inhibitors

Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to generate ceramide, an important molecule involved in the regulation of various cellular responses. In this study, we partially purified the neutral sphingomyelinase2 (nSMase2) and identified the inhibitors, d-lyxophytosphingosine and d-arabino-phytosphingosine, which have an inhibitory effect on nSMase2 in a concentration-dependent manner. A Dixon plot of each phytosphingosines revealed their probable inhibitory pattern, i.e., apparent competitive inhibition. These compounds did not inhibit the Mg2+-independent neutral SMase activity, although the known nSMase2 inhibitor, GW4869, showed inhibitory effects on Mg2+-independent neutral SMase activity. Further, the two phytosphingosines specifically inhibited the ceramide generation regulated by nSMase2.

Subcutaneous Four-Week Repeated Dose Toxicity Studies of Rice Cell-Derived Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor in Rats

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a glycoprotein and hematopoietic growth factors that regulates the proliferation of myeloid precursor cells and activates mature granulocytes and macrophages. In a previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study, we examined the repeated dose toxicity of rhGM-CSF in SD rats. The repeated dose toxicity study was performed at each dose of 50 and 200 µg/kg subcutaneous administration of rhGM-CSF everyday for 28-days period. The results did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the tested groups. The hematological and blood biochemical parameters were statistically not different in all groups. These results suggest that rhGM-CSF may show no repeated dose toxicity in SD rats under the conditions.

DA-9801 Promotes Neurite Outgrowth via ERK1/2-CREB Pathway in PC12 Cells

In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2.

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).

C6-ceramide enhances phagocytic activity of Kupffer cells through the production of endogenous ceramides

Ceramide has been suggested to be not only a tumorsuppressive lipid but also a regulator of phagocytosis. We examined whether exogenous cell-permeable C6-ceramide enhances the phagocytic activity of Kupffer cells (KCs) and affects the level of cellular ceramides. Rat KCs were isolated by collagenase digestion and differential centrifugation, using Percoll system. Phagocytic activity was measured by FACS analysis after incubating KCs with fluorescence-conjugated latex beads, and the level of cellular ceramide was analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). In this study we found that permeable C6-ceramide increases the cellular levels of endogenous ceramides via a sphingosine-recycling pathway leading to enhanced phagocytosis by KCs.

Purification of neutral sphingomyelinase 2 from bovine brain and its calcium-dependent activation

J. Neurochem. (2010) 112, 1088–1097.

Identification of three competitive inhibitors for membrane-associated, Mg2+-dependent and neutral 60 kDa sphingomyelinase activity.

Methanol extracts of domestic plants of Korea were evaluated as a potential inhibitor of neutral pH optimum and membrane-associated 60 kDa sphingomyelinase (N-SMase) activity. In this study, we partially purifiedN-SMase from bovine brain membranes using ammonium sulfate. It was purified approximately 163-fold by the sequential use of DE52, Butyl-Toyopearl, DEAE-Cellulose, and Phenyl-5PW column chromatographies. The purifiedN-SMase activity was assayed in the presence of the plant extracts of three hundreds species. Based on thein vitro assay, three plant extracts significantly inhibited theN-SMase activity in a time- and concentration-dependent manner. To further examine the inhibitory pattern, a Dixon plot was constructed for each of the plant extracts. The extracts ofAbies nephrolepis, Acer tegmentosum, andGinkgo biloba revealed a competitive inhibition with the inhibition constant (Ki) of 11.9 μg/mL, 9.4 μg/mL, and 12.9 μg/mL, respectively. These extracts also inhibited in a dose-dependent manner the production of ceramide induced by serum deprivation in human neuroblastoma cell line SH-SY5Y.