P2Y6 Nucleotide Receptor Activates PKC to Protect 1321N1 Astrocytoma Cells Against Tumor Necrosis Factor-Induced Apoptosis

Share Embed


Descrição do Produto

NIH Public Access Author Manuscript Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

NIH-PA Author Manuscript

Published in final edited form as: Cell Mol Neurobiol. 2003 June ; 23(3): 401–418.

P2Y6 Nucleotide Receptor Activates PKC to Protect 1321N1 Astrocytoma Cells Against Tumor Necrosis Factor-Induced Apoptosis Seong G. Kim1, Zhan-Guo Gao1, Kelly A. Soltysiak1, Tong-Shin Chang2, Chaya Brodie3, and Kenneth A. Jacobson1,4 1Molecular

NIH-PA Author Manuscript

Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 2Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 3Department of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

SUMMARY

NIH-PA Author Manuscript

1.

We recently reported that the activation by UDP of rat P2Y6 nucleotide receptors expressed in 1321N1 astrocytoma cells protected them from TNFα-induced apoptosis by suppressing activation of caspase 3 and 8. This study aims to characterize the involvement of intracellular signaling pathways, including kinases, involved in the antiapoptotic effect of UDP.

2.

Cell death was induced in 1321N1 astrocytoma cells permanently expressing the rat P2Y6 receptor by exposure to TNFα in the presence of cycloheximide. The apoptotic fraction was analyzed using flow cytometry.

3.

The activation of P2Y6 receptors by UDP both protected the astrocytes from TNF-α induced apoptosis and activated protein kinase C (PKC) isotypes. The phorbol ester PMA also activated PKC and protected the cells from TNFα-induced cell death. The α- and εisotypes of PKC were both activated in a persistent fashion upon 5-min exposure to either UDP (10 µM) or the phorbol ester PMA (100 nM). The PKCζ isotype was markedly activated upon UDP treatment.

4.

The addition of PKC inhibitors, GF109203X or Gö6976, partially antagonized the protective effect of UDP and reduced the UDP-induced phosphorylation of extracellular signal-regulated protein kinases (Erk). The inhibitors of Erk, PD98,059 or U0126, antagonized UDP-induced protection.

5.

The antiapoptotic protein, Akt, was not affected by P2Y6 receptor activation. Incubation of the astrocytes with calcium modifiers, BAPTA-AM or dantrolene, did not affect the UDP-induced protection from apoptosis.

6.

The addition of phospholipase C (PLC) inhibitors, D609 or U73122, partially antagonized both UDP-induced protection and PKC activation.

© 2003 Plenum Publishing Corporation 4 To whom correspondence should be addressed at Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, NIH, Building 8A, Room. B1A-19, Bethesda, Maryland 20892-0810; [email protected]

Kim et al.

Page 2

7.

NIH-PA Author Manuscript

Therefore, it is suggested that P2Y6 receptors in 1321N1 cells, through coupling to PCPLC and PI-PLC, activate PKC to protect against TNFα-induced apoptosis, in which the activation of Erk is involved in part.

Keywords nucleotide; pyrimidines; agonist; cell death; protein kinase; phospholipase C

INTRODUCTION P2Y6 nucleotide receptors are activated by UDP, but not by UTP or by adenine nucleotides (Communi et al., 1996; Nicholas et al., 1996). Like most other P2Y receptors, the P2Y6 receptor is coupled through Gq to the activation of PLCβ. Activation by the stable agonist analogue UDP-β-S of a P2Y6 receptor in the rat mesenteric artery results in vasoconstriction (Malmsjö et al., 2000).

NIH-PA Author Manuscript

We found that the activation of the rat P2Y6 nucleotide receptor expressed in 1321N1 human astrocytes was able to prevent TNFα-induced apoptosis (Kim et al., 2002). Interestingly, UDP did not protect the P2Y6-1321N1 cells from cell death induced by other methods, i.e., oxidative stress (H2O2) or chemically induced ischemia. In parallel experiments, activation of the human P2Y4 receptor expressed in the astrocytes did not elicit a protective effect. The interaction of P2Y6-receptor-induced mechanisms with the TNFαrelated intracellular signals to prevent apoptotic cell death appeared to occur rapidly and early in the process.

NIH-PA Author Manuscript

Among the several mechanisms of apoptosis, which rapidly and selectively eliminates cells that are no longer needed, is the fine-tuned interaction of cell surface death receptors with their cognate ligands. Currently, five such death receptors are known to regulate cell survival, including tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNFreceptor-related apoptosis-mediated protein (TRAMP), and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2. The signaling pathways by which these receptors induce apoptosis are rather similar, i.e., association with TRADD or FADD followed by caspase activation (Aggarwal, 2000; Schulze-Osthoff et al., 1998). The recruitment of these proteins affects intracellular signaling markers, including mitogen-activated protein kinases (MAPKs), such as p44/42 MAPK (Erk1/2), p38 MAPK, and SAPK/JNK. The MAPK inases are implicated in the apoptotic process initiated through death receptors. The kinase JNK has been shown to be involved in the cellular mechanism of apoptotic cell death resulting from certain stimuli, including death receptor activation (Aggarwal, 2000; Ip and Davis, 1998; Javelaud and Besancon, 2001; Minden and Karin, 1997; Tang et al., 2001). The role of p38 MAP kinase, however, is controversial. The kinase Erk also is believed to control the death receptor signal (Nosaka et al., 2001; Tran et al., 2001; Yazlovitskaya et al., 1999). It has been reported that P2Y receptors affect MAPKs under several conditions. When P2Y receptors were activated, JNK was either stimulated (Huwiler et al., 1997; Paul et al., 2000) or inhibited (Paul et al., 2000), suggesting the effect mediated by P2Y receptors might be cell-specific. Erk can be activated by P2Y receptors (King et al., 1996; Lenz et al., 2000). It was suggested that Erk activity mediated the mitogenic properties of P2Y receptors (Neary et al., 1996a,b, 2001; Tu et al., 2000). Hou et al. (2002) proposed that the P2Y6 receptor acts as a growth signal in vascular smooth muscle cells. Erk MAP kinase is activated by growth factors playing a crucial role in cellular proliferation and differentiation (Neary et al., 2001), and can also negatively regulate JNK and p38 MAP kinase (Cheng and Feldman, 1998; Gardner and Johnson, 1996; Xia et al., 1995). It is also considered to regulate the death

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 3

signal from the TNF receptor (Nosaka et al., 2001; Tran et al., 2001; Yazlovitskaya et al., 1999).

NIH-PA Author Manuscript

The activation of PKC by various P2Y receptors was reported to mediate their biological effects. Signaling from P2Y receptors to Erk in astrocytes was PKC-dependent (Neary, 1996; Neary et al., 2001). UTP-mediated MAPK activation was also modulated by Ca2+, PKC, and tyrosine kinase in cultured C6 glioma cells, which were associated with cell proliferation (Tu et al., 2000). The activation of endogenous P2Y2 receptors in CHO cells caused the PKC-dependent phosphorylation and subsequent inactivation of PLC-β3 (Strassheim and Williams, 2000). PKC is also known to be involved in death receptormediated TNFα-induced apoptosis (Laouar et al., 1999; Li et al., 1999). Phosphorylation of Akt, dependent on phosphatidylinositol 3-kinase (PI3K), is known to prevent death-receptor-induced apoptosis (Gibson et al., 2002; Hatano and Brenner, 2001; Sautin et al., 2001). In some cases, ATP or adenosine induced Akt activation or enhanced Akt activation by growth factors such as insulin (She et al., 2000; Takasuga et al., 1999). In case of the adenosine A3 receptor, Akt was reported to be involved in the protective effect of the nucleoside against UV-induced apoptosis (Gao et al., 2001).

NIH-PA Author Manuscript

Therefore JNK, Erk, Akt, and PKC are hypothesized to have important roles in the biological events resulting from P2Y receptor activation. To probe the mechanism of cellular protection by P2Y6 receptors in 1321N1 astrocytes, we investigated each of the proposed intracellular markers in relation to the initial steps of receptor activation such as phospholipase C activities and intracellular calcium increase. We found that PKC and Erk1/2 play significant roles in the antiapoptotic effect of the P2Y6 receptor, and proposed the importance of the receptor in cellular homeostasis and proliferation.

METHODS Materials

NIH-PA Author Manuscript

1321N1 astrocytoma cells stably transfected with rat P2Y6 (rP2Y6) receptors were kindly provided by Dr Robert Nicholas (University of North Carolina, Chapel Hill). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Life Technologies, Inc. (Rockville, MD). HRP-linked anti-mouse IgG and HRP-linked antirabbit IgG antibodies, p-p38, and p-JNK were purchased from Cell Signaling Technology (Beverly, MA). Tumor necrosis factor α (TNFα) was purchased from Biosource International (Camarillo, CA). Antibodies for p-Erk, p-Akt, actin, Erk, p-PKCζ (Thr410), pPKCε (ser729), and p-PKCα (ser657) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).Gö6976, U73122, U0126, and D609 were purchased from Biomol (Plymouth, PA). BAPTA-AM was obtained from Molecular Probes (Eugene, OR). LY294002 was purchased from Upstate (Waltham, MA). All other reagents were purchased from Sigma (St. Louis, MO). Cell Culture and Preparation 1321N1 cells stably transfected with the rP2Y6 receptors were grown at 37°C in a humidified incubator with 5% CO2/95% air in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine. The cells were grown to ~60% confluence for the experiments. Induction of Apoptosis TNFα was used to induce apoptosis. Medium containing 5 µg/mL cycloheximide was added to the cells grown to ~60% confluence. In all the experiments concerning TNFα-induced cell

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 4

NIH-PA Author Manuscript

apoptosis cycloheximide was included. The cells were treated with appropriate concentrations of TNFα for 4 or 16 h. UDP was added as indicated in each figure. Cell death was observed 16 h later. Western Blots The cells grew to 60% confluence when the assays were performed. For JNK and p38 MAPK activities, media was changed to fresh media 2 h before each experiment. UDP was added 10 min prior to TNFα addition. After the appropriate treatment, the cells were lysed with lysis buffer (2 mM EGTA, 25 mM β-glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM Na3VO4, 5 mM NaF, 10 µg/µL leupeptin, 10 µg/µL aprotinin, 1 mM PMSF, 20 mM HEPES (pH 7.4)) and the lysates were subjected to Western blotting using appropriate antibodies. For Erk and Akt activities, cells were cultured overnight in 0.5% serum-containing DMEM, and then further incubated in serum-free media for 4 h before the experiments. The treatment of chemicals and extraction of proteins for Western blotting were same as above. For the measuring of phosphorylation of each isotype of PKCs, the cells were treated with UDP or PMA as indicated in each figure, and the lysate was immunoblotted using anti-phospho-PKCα (Ser657), PKCε (Ser729), and PKCζ (Thr410). Cell Death Analysis

NIH-PA Author Manuscript

After the proper treatment, the cells in the supernatant and the cells detached by trypsinEDTA were combined and centrifuged. The cells were washed with PBS once and resuspended to 2~5 × 105/mL in PBS. The cells were treated with propidium iodide solution (final; 2 µg/mL). After 10-min incubation at room temperature in the dark, the cell death ratio was analyzed by flow cytometry (FACSCaliber™, Becton Dickinson).

RESULTS Prevention of Cell Death by UDP and PMA In preliminary experiments, we observed that cotreatment of a PKC inhibitor, GF109203X (Toullec et al., 1991), at 2 or 4 µM for 16 h increased the TNFα-induced cell death in 1321N1 astrocytoma cells stably expressing the rP2Y6-receptor (data not shown). Therefore, activities of PKC appear to be involved in controlling TNFα receptor signals. Indeed, the cells were protected from TNFα-induced cell death upon a 10-min pretreatment with the PKC activator PMA (100 nM), followed by its removal from the media by washing (Fig. 1). This implied a role for PKC in regulating cell death signals originating through TNFα receptors.

NIH-PA Author Manuscript

To understand the role of UDP in activation of PKC, inhibition of PKC in UDP protected status was investigated. A nonselective PKC inhibitor, GF109203X (2µM), was added 30 min prior to the addition of TNFα and remained only for the additional 4 h in the presence of TNFα and/or UDP to inhibit the PKC activities without producing its own cytotoxicity; more than a 16-h incubation with 2 µM GF109203X caused cell death (data not shown). In this condition, GF109203X was able to antagonize the protective effect of UDP against TNFα-induced apoptosis in P2Y6-1321N1 cells (Fig. 2). At the concentration tested, 2 µM, GF109203X did not display its own toxicity and increase the toxicity of TNFα. The effect of GF109203X was usually more pronounced at a lower UDP concentration. The inhibitor of calcium-dependent PKC, Gö6976 (Martiny-Baron et al., 1993), which inhibits PKCα and β, but does not affect δ-, ε-, or ζ-isotypes, was less effective than GF109203X in suppressing the protection by UDP.

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 5

BAPTA-AM and Dantrolene Did Not Antagonize the Protective Effect of UDP

NIH-PA Author Manuscript

The production of inositol phosphates and the concentration of intracellular calcium have been shown to increase upon the activation of P2Y6 receptors (Chang et al., 1995; Nicholas et al., 1996). We have introduced the calcium chelator, BAPTA-AM (12.5 µM), and intracellular calcium depleter, dantrolene (25 µM), to see if calcium-dependent PKC is responsible for the P2Y6-receptor-induced protective effect. As shown in Fig. 3, there were no changes in the cell death ratio in the UDP-treated group with either of the calcium modifiers. With higher than 25 µM of BAPTA-AM, cell detachment occurred under the experimental conditions (4-h incubation with cells), and 12.5 µM of BAPTA-AM was the maximal concentration usable without producing cell detachment. Activation of PKC Isotypes by UDP The human astrocytoma cell line 1321N1 has been shown to express only α-, ε-, and ζisozymes of PKC (Post et al., 1996). As shown in Fig. 4, PKCα, which is dependent on both Ca2+ and diacylglycerol (DAG), was phosphorylated after 5-min exposure of the cells to UDP (10 µM) or PMA (100 nM). This activation persisted through all the time-points tested.

NIH-PA Author Manuscript

The addition of UDP also increased the phosphorylation of the DAG-dependent isotype, PKCε, which was evident after only 5 min. This phosphorylation persisted for 30 min and was reduced at 60 min. Upon treatment with PMA, however, the phosphorylation of PKCε reached a peak at 30 min and persisted for 60 min. The novel isotype PKCζ was phosphorylated in the presence of UDP within 5 min (Fig. 4), and this phosphorylation persisted for 60 min. Thus, UDP strongly activated this isotype, which is known to be antiapoptotic. Consistent with the inability of PMA to activate PKCζ, our results showed that PMA did not affect the phosphorylation of this isotype. PKCδ, which was not investigated in the experiment shown in Fig. 4, was also present in the astrocytoma cells (data not shown). P2Y6-Receptor-Activated Erk1/2, but Not Akt

NIH-PA Author Manuscript

Erk has been considered to be involved in cell growth and/or in prevention of cell death (Sautin et al., 2001; Tran et al., 2001; Yazlovitskaya et al., 1999; Zhu et al., 1999). We have observed that the phosphorylation of Erk increased within 5 min after the addition of UDP to P2Y6-1321N1 cells, and this effect persisted for 1 h (Fig. 5). Akt is well documented as an antiapoptotic protein, and its activation has been linked to many G-protein-coupled receptors and growth factor receptors (Gibson et al., 2002; Hatano and Brenner, 2001; Kulik et al., 1997; Murga et al., 1998; Sautin et al., 2001). In our case, the addition of UDP did not increase Akt phosphorylation (Fig. 5), suggesting that the protective effect of UDP in P2Y6-1321N1 cells may not occur through the PI3K and Akt pathway. In fact, the addition of LY294002 (100 µM), a PI3K inhibitor, did not affect the protection by UDP against TNFα-induced cell death (data not shown) as well as the UDP-activated Erk1/2 phosphorylation (Fig. 6). The addition of GF109203X 10 min prior to the activation of P2Y6 significantly inhibited the phosphorylation of Erk (Fig. 6). The phosphorylation was affected to a lesser extent by BAPTA-AM (Fig. 6). The addition of Gö6976 (2 µM) or dantrolene (25 µM) also significantly suppressed the UDP-induced Erk phosphorylation (data not shown). Erk Inhibitors PD98,059 and U0126 Reversed the Effect of UDP As shown in Fig. 7, Erk inhibitors, PD98,059 and U0126, were able to antagonize the protective effect of UDP to roughly the same degree as PKC inhibitors did. U0126 was more potent than PD98,059. In fact, we observed precipitation of PD98,059 in the aqueous media Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 6

NIH-PA Author Manuscript

at the concentrations tested (25 and 50 µM). This insolubility may explain the lower effectiveness of PD98,059. Therefore, PKC appeared to affect the TNFα death pathway through Erk activation. As has been reported (Gardner and Johnson, 1996; Tran et al., 2001), Erk activation seems to be important in the regulation of TNFα cytotoxicity. Involvement of PC-PLC as Well as PI-PLC in PKC Activation The activation of PKC may be controlled by the activation of phosphatidyl inositol-specific PLC (PI-PLC), which has been known to couple to Gq-linked P2Y6 receptors (Chang et al., 1995; Nicholas et al., 1996). The PI-PLC-specific inhibitor, U73122 (2µM), partially antagonized the effect of UDP, as indicated by the cell death ratio (Fig. 8), implying that PIPLC is upstream of PKC in P2Y6 receptor signaling. Interestingly, D609 (100 µg/mL), an inhibitor of phosphatidyl-choline-specific PLC (PC-PLC), antagonized the protective effect of UDP against TNFα-induced cell death to a greater extent than U73122 did. As shown in Fig. 9(A), UDP-induced phosphorylation of each PKC isotype (α, ε, and ζ) was significantly inhibited by D609 as well as by U73122. U73122 showed stronger inhibition of PKC phosphorylation than D609. These two PLC inhibitors, however, did not affect the UDPinduced Erk phosphorylation (Fig. 9(B)).

DISCUSSION NIH-PA Author Manuscript

Our results demonstrated that the activation of P2Y6 receptors in 1321N1 astrocytes activated PKC, which appeared to control Erk phosphorylation and to be responsible for the antiapoptotic effect of this receptor. It is interesting that PI-PLC and PC-PLC also seemed to play an important role in the antiapoptotic effect, but they did not affect Erk. Although the importance of Erk for cellular proliferation and survival has been suggested (Leirdal and Sioud, 2000), its regulation appears to be dependent on the cell system being examined.

NIH-PA Author Manuscript

In U937 monocytic cells the P2Y2 receptor agonist UTP induced the phosphorylation of the MAP kinases MEK1/2 and Erk1/2 in a sequential manner, and this process was coupled to PI3K and c-src, not to PKC (Santiago-Perez et al., 2001). In that report, the phosphorylation of Erk1/2 was independent of the ability of P2Y2 receptors to increase the concentration of intracellular-free calcium, since chelation of intracellular calcium by BAPTA-AM did not diminish the phosphorylation of Erk1/2 induced by UTP. However, in P2Y6-1321N1 astrocytes GF109203X affected the UDP-induced phosphorylation of Erk as well as cell death ratio (Figs. 2 and 6). BAPTA-AM and dantrolene also diminished the Erk phosphorylation. When Erk phosphorylation was activated through muscarinic receptors in 1321N1 astrocytes, the addition of BAPTA-AM (2, 10, or 50 µM) was reported to significantly inhibit the carbachol-induced phosphorylation of Erk (Yagle et al., 2001). This suggested that muscarinic receptors could increase the proliferation of 1321N1 cells through Erk activation. In rat aorta smooth muscle cells, UDP induced thymidine incorporation, which was significantly attenuated by 1 µM of U73122 (Hou et al., 2002). Therefore, PI-PLC was suggested to be important in the action of UDP as growth factor. But Neary et al. (1999) observed the inhibition of ATP-stimulated Erk neither by 10 µM U73122 nor by 30 µM BAPTA-AM, although they found that D609 inhibited ATP-stimulated Erk. In the present study, two forms of PLC, both PI-PLC and PC-PLC, appear to be involved in the protective effect of the P2Y6 receptor in 1321N1 astrocytes. Nucleotides, including UDP, have been demonstrated to act as growth factors in vascular smooth muscle cells (Erlinge et al., 1998; Harper et al., 1998; Hou et al., 2002). P2Y receptors in thyroid FRTL-5 cell, the Erk activation following agonist treatment increased thymidine incorporation and DNA synthesis, demonstrating growth control effects of the receptors (Ekokoski et al., 2001). Since many growth factor receptors induce Erk activation and the activated Erk has been Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 7

NIH-PA Author Manuscript

shown to be essential to cell proliferation, differentiation, or survival, the rapid increase of Erk phosphorylation by UDP by UDP in P2Y6-1321N1 astrocytes might contribute to growth factor-like effects. The effects of P2Y6 receptor activation on proliferation have not yet been reported. Both DNA synthesis and phosphorylation of MAPK in response to UTP were attenuated by tyrosine kinase inhibitors, protein kinase C (PKC) inhibitors, or removal of calcium in C6 glioma cells (Tu et al., 2000). The P2Y2 receptors in U937 cells activated Erk via PI3K and c-src, not by PKC (Santiago-Perez et al., 2001). The activation of Erk by P2Y1 was partially inhibited by LY294002 (Sellers et al., 2001). In 1321N1 cells, however, PI3K did not seem to couple to P2Y6 (Fig. 6). Accordingly, with the treatment of UDP in P2Y6-1321N1 cells, there was no change in the phosphorylation of Akt (Fig. 4), a serine–threonine kinase that controls an intracellular pathway to prevent cell death (Dudek et al., 1997; Gao et al., 2001; Kulik et al., 1997; Marte and Downward, 1997; Murga et al., 1998; She et al., 2000). ATP had similar effects in uterine artery endothelial cells from pregnant ewes, i.e., no stimulation of Akt phosphorylation but rapid Erk activation (Di et al., 2001).

NIH-PA Author Manuscript

The cytotoxic effect of TNF is pronounced when the cell is activated by cycloheximide (Higuchi et al., 1995; Li et al., 2001; Woods and Chapes, 1993), which is probably due to the inhibition of transcription of survival factors following NF-κB activation (De Smaele et al., 2001; Tang et al., 2001). The proapoptotic JNK signaling was downregulated by the induction of recently identified genes, gadd35β and xiap, which are expressed in response to TNF in an NF-κB-dependent way (Kyriakis, 2001). The protective effect of UDP may not be related to those anti-JNK proteins, because in our study the media always contained cycloheximide. Paul et al. (2000) have reported that P2Y4 receptors in EAhy926 endothelial cells inhibited TNFα-induced SAPK inase activities in a cell-type-specific manner. It is interesting that UDP increased the phosphorylation of JNK in P2Y6-1321N1 astrocytes (data not shown). Since no cell death occurred upon exposure of the astrocytes to UDP, JNK activation by UDP did not seem to be proapoptotic. The physiological role of the JNK activation remains unknown. Recombinant P2Y1 receptors expressed in 1321N1 cells activated SAPK and ERK, and induced apoptosis (Sellers et al., 2001). In rat mesangial cells, the activation of P2Y receptors by ATP and UTP stimulated JNK by a pathway independent of PKC but requiring a pertussis-toxin-sensitive G-protein and tyrosine kinase activation (Huwiler et al., 1997).

NIH-PA Author Manuscript

In smooth muscle cells (Hou et al., 2002), the signal from P2Y6 receptors was transmitted via PKCδ, on the basis of several PKC inhibitor studies. In P2Y6-1321N1 cells, however, we have found that at least three PKC isotypes (α, ε, and ζ) were activated by UDP, and PKC inhibitors, Gö6976 and GF109203X, antagonized the protective effect of UDP (Figs. 2 and 4). In the case of primary astrocytes obtained from neonatal rat cerebral cortices (Neary et al., 1999), ATP activated the PKCδ isotype, which was suggested to be responsible for the activation of Erk. Yagle et al. (2001) reported that the ε isozyme of PKC was primarily involved in MAPK activation by carbachol in 1321N1 cells. UDP increased [3H]thymidine incorporation in vascular smooth muscle cells through the activation of PKCδ isotype following P2Y6 receptor activation (Hou et al., 2002). Leirdal and Sioud (2000) suggested that PKCα regulated the activation of Erk1/2 in human glioma cells involving cell survival and gene expression. PKCζ was also proposed to be important to cell proliferation and survival (Smith et al., 2000). In P2Y6-1321N1 cells, UDP activated all three types of PKC: Ca2+/DAG-dependent, DAG-dependent, and novel PKC. Since the calcium modifiers did not alter the protective effects of UDP, PKCε appeared to be more important in cellular protection, while Erk phosphorylation by UDP may in principle be controlled by other

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 8

NIH-PA Author Manuscript

isotypes. In primary cultured rat astrocytes, the activated PKC isotype was involved in the activation of Erk (Neary et al., 1999). Tran et al. (2001) reported that the inhibition of deathreceptor-mediated Erk1/2 activation was sufficient to sensitize the cells to apoptotic signals. Erk1/2 had a dominant protecting effect over apoptotic signaling from the death receptor. Therefore, in P2Y6-1321N1 cells, activation of Erk by UDP appeared to play an important role in cell protection against TNFα-induced cell death when it was not controlled by PLC. In summary, it is suggested that P2Y6 receptors in 1321N1 cells are coupled to both PCPLC and PI-PLC, which protect cells against TNFα-induced apoptosis through the activation of PKC isotypes. Activation of the P2Y6 receptors also stimulates Erk, which is controlled by PKC, and is a partial factor in the cell protection by UDP against TNFα-induced cell death.

LIST OF ABBREVIATIONS

NIH-PA Author Manuscript NIH-PA Author Manuscript

BAPTA-AM

1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)

D609

Tricyclodecan-9-yl-xanthate, potassium salt

Erk1/2

Extracellular signal-regulated protein kinases 1 and 2

GPCR

G protein-coupled receptor

GF109203X

[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide

Gö6976

12-(2-Cyanoethyl)-6,7,12,13,-tetrahydro-13-methyl-5-oxo-5H-indolo[2m, 3-a]pyrrolo[3,4-c]carbazole

LY294002

2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one

MAPK

Mitogen-activated protein kinase

PC-PLC

Phosphatidyl-choline-specific phospholipase C

PD98,059

2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one

PI-PLC

Phosphatidyl inositol specific phospholipase C

PMA

Phorbol 12-myristate 13-acetate

PKC

Protein kinase C

SAPK/JNK

Stress-activated protein kinase/c-Jun N-terminal kinase

TNF

Tumor necrosis factor

U0126

1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene

U73122

1-[6-((17β-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1Hpyrrole-2,5-dione

Acknowledgments ZGG thanks Gilead Sciences, Foster City, CA, for financial support. We thank Dr Robert Nicholas (University of North Carolina, Chapel Hill) for the gift of astrocytoma cells stably expressing P2Y6 receptors. We thank Dr Jane Treppel for use of instrumentation.

REFERENCES Aggarwal BB. Tumor necrosis factors receptor associated signaling molecules and their role in activation of apoptosis, JNK, and NF-κB. Ann. Rheum. Dis. 2000; 59 Suppl. 1:i6–i16. [PubMed: 11053079]

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 9

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Chang K, Hanaoka K, Kumada M, Takuwa Y. Molecular cloning and functional analysis of a novel P2 nucleotide receptor. J. Biol. Chem. 1995; 270:26152–26158. [PubMed: 7592819] Cheng H-L, Feldman EL. Bidirectional regulation of p38 kinase and c-Jun N-terminal protein kinase by insulin-like growth factor-I. J. Biol. Chem. 1998; 273:14560–14565. [PubMed: 9603971] Communi D, Parmentier M, Boeynaems JM. Cloning, functional expression and tissue distribution of the human P2Y6 receptor. Biochem. Biophys. Res. Commun. 1996; 222:303–308. [PubMed: 8670200] De Smaele E, Zazzeroni F, Papa S, Nguyen DU, Jin R, Jones J, Cong R, Franzoso G. Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature. 2001; 414:308– 313. [PubMed: 11713530] Di T, Sullivan JA, Magness RR, Zhang L, Bird IM. Pregnancy-specific enhancement of agoniststimulated ERK-1/2 signaling in uterine artery endothelial cells increases Ca2+ sensitivity of endothelial nitric oxide synthase as well as cytosolic phospholipase A2. Endocrinology. 2001; 142:3014–3026. [PubMed: 11416023] Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR, Greenberg ME. Regulation of neuronal survival by the serine–threonine protein kinase Akt. Science. 1997; 275:661–665. [PubMed: 9005851] Ekokoski E, Webb TE, Simon J, Tornquist K. Mechanisms of P2 receptor-evoked DNA synthesis in thyroid FRTL-5 cells. J. Cell. Physiol. 2001; 187:166–175. [PubMed: 11267996] Erlinge D, Hou M, Webb TE, Barnard EA, Möller S. Phenotype changes of the vascular smooth muscle cell regulate P2 receptor expression as measured by quantitative RT-PCR. Biochem. Biophys. Res. Commun. 1998; 248:864–870. [PubMed: 9704019] Gao Z, Li BS, Day YJ, Linden J. A3 adenosine receptor activation triggers phosphorylation of protein kinase B and protects rat basophilic leukemia 2H3 mast cells from apoptosis. Mol. Pharmacol. 2001; 59:76–82. [PubMed: 11125027] Gardner AM, Johnson GL. Fibroblast growth factor-2 suppression of tumor necrosis factor α-mediated apoptosis requires Ras and the activation of mitogen-activated protein kinase. J. Biol. Chem. 1996; 271:14560–14566. [PubMed: 8662985] Gibson EM, Henson ES, Haney N, Villanueva J, Gibson SB. Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by inhibiting cytochrome c release. Cancer Res. 2002; 62:488–496. [PubMed: 11809700] Harper S, Webb TE, Charlton SJ, Ng LL, Boarder MR. Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP. Br. J. Pharmacol. 1998; 124:703–710. [PubMed: 9690862] Hatano E, Brenner DA. Akt protects mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis through NK-kappa B activation. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 281:G1357– G1368. [PubMed: 11705740] Higuchi M, Singh S, Chan H, Aggarwal BB. Protease inhibitors differentially regulate tumor necrosis factor-induced apoptosis, nuclear factor-kappa B activation, cytotoxicity, and differentiation. Blood. 1995; 86:2248–2256. [PubMed: 7662972] Hou M, Harden TK, Kuhn CM, Baldetorp B, Lazarowski E, Pendergast W, Möller T, Edvinsson L, Erlinge D. UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y6 receptors. Am. J. Physiol. Heart Circ. Physiol. 2002; 282:H784–H792. [PubMed: 11788430] Huwiler A, van Rossum G, Wartmann M, Pfeilschifter J. Stimulation by extracellular ATP and UTP of the stress-activated protein kinase cascade in rat renal mesangial cells. Br. J. Pharmacol. 1997; 120:807–812. [PubMed: 9138685] Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)—From inflammation to development. Curr. Opin. Cell Biol. 1998; 10:205–219. [PubMed: 9561845] Javelaud D, Besancon F. NF-kappa B activation results in rapid inactivation of JNK in TNFα-treated Ewing sarcoma cells: A mechanism for the anti-apoptotic effect of NF-kappa B. Oncogene. 2001; 20:4365–4372. [PubMed: 11466617]

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 10

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Kim SG, Soltysiak KA, Gao ZG, Chang TS, Chung E, Jacobson KA. Tumor necrosis factor α-induced apoptosis in astrocytes is prevented by the activation of P2Y6, but not P2Y4 nucleotide receptors. Biochem. Pharmacol. 2003; 65:923–931. [PubMed: 12623123] King BF, Neary JT, Zhu Q, Wang S, Norenberg MD, Burnstock G. P2 purinoceptors in rat cortical astrocytes: Expression, calcium-imaging and signalling studies. Neuroscience. 1996; 74:1187– 1196. [PubMed: 8895885] Kulik G, Klippel A, Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol. Cell. Biol. 1997; 17:1595–1606. [PubMed: 9032287] Kyriakis JM. Life-or-death decision. Nature. 2001; 414:265–266. [PubMed: 11713514] Laouar A, Glesne D, Huberman E. Involvement of protein kinase C-β and ceramide in tumor necrosis factor α-induced but not Fas-induced apoptosis of human myeloid leukemia cells. J. Biol. Chem. 1999; 274:23526–23534. [PubMed: 10438532] Leirdal M, Sioud M. Protein kinase Cα isotype regulates the activation of the MAP kinase ERK1/2 in human glioma cells: Involvement in cell survival and gene expression. Mol. Cell Biol. Res. Commun. 2000; 4:106–110. [PubMed: 11170840] Lenz G, Gottfried C, Luo Z, Avruch J, Rodnight R, Nie WJ, Kang Y, Neary JT. P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes. Br. J. Pharmacol. 2000; 129:927–936. [PubMed: 10696092] Li D, Yang B, Mehta JL. Tumor necrosis factor α enhances hypoxia-reoxygenation-mediated apoptosis in cultured human coronary artery endothelial cells: Critical role of protein kinase C. Cardiovasc. Res. 1999; 42:805–813. [PubMed: 10533621] Li J, Zheng R, Li J, Wang Z. Mechanisms of the induction of apoptosis in human hepatoma cells by tumour necrosis factor-alpha. Cell Biol. Int. 2001; 25:1213–1219. [PubMed: 11748914] Malmsjö M, Adner M, Harden TK, Pendergast W, Edvinsson L, Erlinge D. The stable pyrimidines UDPβS and UTPγS discriminate between the P2 receptors that mediate vascular contraction and relaxation of the rat mesenteric artery. Br. J. Pharmacol. 2000; 131:51–56. [PubMed: 10960068] Marte BM, Downward J. PKB/Akt: Connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem. Sci. 1997; 22:355–358. [PubMed: 9301337] Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, Schachtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J. Biol. Chem. 1993; 268:9194–9197. [PubMed: 8486620] Minden A, Karin M. Regulation and function of the JNK subgroup of MAP kinases. Biochim. Biophys. Acta. 1997; 1333:F85–F104. [PubMed: 9395283] Murga C, Laguinge L, Wetzker R, Cuadrado A, Gutkind JS. Activation of Akt/protein kinase B by G protein-coupled receptors. A role for alpha and beta gamma subunits of heterotrimeric G proteins acting through phosphatidylinositol-3-OH kinasegamma. J. Biol. Chem. 1998; 273:19080–19085. [PubMed: 9668091] Neary JT, Zhu Q, Kang Y, Dash PK. Extracellular ATP induces formation of AP-1 complexes in astrocytes via P2 purinoceptors. Neuro report. 1996a; 7:2893–2896. Neary JT, Rathbone MP, Cattabeni F, Abbracchio MP, Burnstock G. Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells. Trends Neurosci. 1996b; 19:13–18. [PubMed: 8787135] Neary JT. Trophic actions of extracellular ATP on astrocytes, synergistic interactions with fibroblast growth factors, and underlying signal transduction mechanisms. Ciba Found. Symp. 1996; 198:130–141. [PubMed: 8879822] Neary JT, Kang Y, Bu Y, Yu E, Akong K, Peters CM. Mitogenic signaling by ATP/P2Y purinergic receptors in astrocytes: Involvement of a calcium-independent protein kinase C, extracellular signal-regulated protein kinase pathway distinct from the phosphatidylinositol-specific phospholipase C/calcium pathway. J. Neurosci. 1999; 19:4211–4220. [PubMed: 10341225] Neary JT, Lenz G, Kang Y, Rodnight R, Avruch J. Role of mitogen-activated protein kinase cascades in P2Y receptor-mediated trophic activation of astroglial cells. Drug Dev. Res. 2001; 53:158–165. Nicholas RA, Watt WC, Lazarowski ER, Li Q, Harden TK. Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: Identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Mol. Pharmacol. 1996; 50:224–229. [PubMed: 8700127]

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 11

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Nosaka Y, Arai A, Kanda E, Akasaki T, Sumimoto H, Miyasaka N, Miura O. Rac is activated by tumor necrosis factor α and is involved in activation of Erk. Biochem. Biophys. Res. Commun. 2001; 285:675–679. [PubMed: 11453646] Paul A, Torrie LJ, McLaren GJ, Kennedy C, Gould GW, Plevin R. P2Y receptor-mediated inhibition of tumor necrosis factor α-stimulated stress-activated protein kinase activity in EAhy926 endothelial cells. J. Biol. Chem. 2000; 275:13243–13249. [PubMed: 10788429] Post GR, Collins LR, Kennedy ED, Moskowitz SA, Aragay AM, Goldstein D, Brown JH. Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells. Mol. Biol. Cell. 1996; 7:1679–1690. [PubMed: 8930892] Santiago-Perez LI, Flores RV, Santos-Berrios C, Chorna NE, Krugh B, Garrad RC, Erb L, Weisman GA, Gonzalez FA. P2Y2 nucleotide receptor signaling in human monocytic cells: Activation, desensitization, and coupling to mitogen-activated protein kinases. J. Cell. Physiol. 2001; 187:196–208. [PubMed: 11267999] Sautin YY, Crawford JM, Svetlov SI. Enhancement of survival by LPA via Erk1/Erk2 and PI 3-kinase/ Akt pathways in a murine hepatocyte cell line. Am. J. Physiol. Cell Physiol. 2001; 281:C2010– C2019. [PubMed: 11698260] Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME. Apoptosis signaling by death receptors. Eur. J. Biochem. 1998; 254:439–459. [PubMed: 9688254] Sellers LA, Simon J, Lundahl TS, Cousens DJ, Humphrey PPA, Barnard EA. Adenosine nucleotides acting at the human P2Y1 receptor stimulate mitogen-activated protein kinases and induce apoptosis. J. Biol. Chem. 2001; 276:16379–16390. [PubMed: 11278310] She QB, Mukherjee JJ, Chung T, Kiss Z. Placental alkaline phosphatase, insulin, and adenine nucleotides or adenosine synergistically promote long-term survival of serum-starved mouse embryo and human fetus fibroblasts. Cell. Signal. 2000; 12:659–665. [PubMed: 11080618] Smith L, Chen L, Reyland ME, DeVries TA, Talanian RV, Omura S, Smith JB. Activation of atypical protein kinase C zeta by caspase processing and degradation by the ubiquitin-proteasome system. J. Biol. Chem. 2000; 275:40620–40627. [PubMed: 11016947] Strassheim D, Williams CL. P2Y2 purinergic and M3 muscarinic acetylcholine receptors activate different phospholipase C-beta isotypes that are uniquely susceptible to protein kinase Cdependent phosphorylation and inactivation. J. Biol. Chem. 2000; 275:39767–39772. [PubMed: 10995776] Takasuga S, Katada T, Ui M, Hazeki O. Enhancement by adenosine of insulin-induced activation of phosphoinositide 3-kinase and protein kinaseBin rat adipocytes. J. Biol. Chem. 1999; 274:19545– 19550. [PubMed: 10391887] Tang G, Minemoto Y, Dibling B, Purcell NH, Li Z, Karin M, Lin A. Inhibition of JNK activation through NF-kB target genes. Nature. 2001; 414:313–317. [PubMed: 11713531] Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 1991; 266:15771–15781. [PubMed: 1874734] Tran SE, Holmstrom TH, Ahonen M, Kahari VM, Eriksson JE. MAPK/ERK overrides the apoptotic signaling from Fas, TNF, and TRAIL receptors. J. Biol. Chem. 2001; 276:16484–16490. [PubMed: 11278665] Tu MT, Luo SF, Wang CC, Chien CS, Chiu CT, Lin CC, Yang CM. P2Y2 receptor-mediated proliferation of C6 glioma cells via activation of Ras/Raf/MEK/MAPK pathway. Br. J. Pharmacol. 2000; 129:1481–1489. [PubMed: 10742305] Woods KM, Chapes SK. Three distinct cell phenotypes of induced-TNF cytotoxicity and their relationship to apoptosis. J. Leukoc. Biol. 1993; 53:37–44. [PubMed: 8426090] Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis. Science. 1995; 270:1326–1331. [PubMed: 7481820] Yagle K, Lu H, Guizzetti M, Möller T, Costa LG. Activation of mitogen-activated protein kinase by muscarinic receptors in astroglial cells: Role in DNA synthesis and effect of ethanol. Glia. 2001; 35:111–120. [PubMed: 11460267]

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 12

NIH-PA Author Manuscript

Yazlovitskaya EM, Pelling JC, Persons DL. Association of apoptosis with the inhibition of extracellular signal-regulated protein kinase activity in the tumor necrosis factor α-resistant ovarian carcinoma cell line UCI 101. Mol. Carcinog. 1999; 25:14–20. [PubMed: 10331740] Zhu W, Zou Y, Aikawa R, Harada K, Kudoh S, Uozumi H, Hayashi D, Gu Y, Yamazaki T, Nagai R, Yazaki Y, Komuro I. MAPK superfamily plays an important role in daunomycin-induced apoptosis of cardiac myocytes. Circulation. 1999; 100:2100–2107. [PubMed: 10562267]

NIH-PA Author Manuscript NIH-PA Author Manuscript Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 13

NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 1.

Effects of UDP and PMA on TNFα-induced cell death in P2Y6-1321N1 cells. The cells were pretreated with UDP (0.1, 1, 10 µM) and PMA (50, 100 nM) for 10 min, and further treated with TNFα for 4 h. Then, old media was replaced with fresh media following washing with PBS. Cell death was observed on the following day (total of 16-h incubation). The media always contained 5 µg/mL cycloheximide. Concentration of TNFα was 20 ng/ mL. The degree of cell death was analyzed using a FacsCalibur instrument (Becton Dickinson). Data shown are mean ± SD from two independent experiments.

NIH-PA Author Manuscript Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 14

NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 2.

NIH-PA Author Manuscript

Effect of PKC inhibitors on protection by UDP against TNFα-induced cell death in P2Y6-1321N1 cells. The cells were pretreated with GF109203X or Gö6976 for 10 min, and treated further with TNFα for 4 h in the absence or presence of UDP. Then, old media was replaced with fresh media following washing with PBS. Cell death was observed on the following day (total of 16-h incubation). The media always contained 5 µg/mL cycloheximide. Concentrations of GF109203X, Gö6976, and TNFα were 2 µM, 2 µM and 20 ng/mL, respectively. The degree of cell death was analyzed using a FacsCalibur instrument (Becton Dickinson). Data shown are mean ± SD from a representative result in triplicate, out of three independent experiments. *Statistically significant (p < 0.01) by Student t test; compared with control of each UDP concentration.

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 15

NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 3.

NIH-PA Author Manuscript

Effect of calcium modifiers on protection by UDP against TNFα-induced cell death in P2Y6-1321N1 cells. The cells were pretreated with BAPTA-AM or dantrolene for 10 min, and treated further with TNFα for 4 h in the absence or presence of UDP. Then, old media was replaced with fresh media following washing with PBS. Cell death was observed on the following day (total of 16-h incubation). The media always contained 5 µg/mL cycloheximide. Concentrations of BAPTA-AM, dantrolene, and TNFα were 12.5 µM, 25 µM and 20 ng/mL, respectively. The degree of cell death was analyzed using a FacsCalibur instrument (Becton Dickinson). Data shown are mean ± SD from a representative result in triplicate out of three independent experiments.

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 16

NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 4.

PKC subtype phosphorylation by PMA and UDP in P2Y6-1321N1 cells. The cells were treated with PMA (100 nM) or UDP (10 µM) for 0, 5, 10, 30, and 60 min. Proteins were extracted and applied to immunoblotting as described in experimental procedures. A total of 45 µg of protein was applied to each lane.

NIH-PA Author Manuscript Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 17

NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 5.

NIH-PA Author Manuscript

Effects of UDP on the phosphorylation of Akt and Erk1/2 in P2Y6-1321N1 cells. The cells were treated with 10 µM of UDP for 0, 5, 10, 30, and 60 min. Proteins were extracted and applied to immunoblotting as described in experimental procedures. A total of 30 µg of protein was applied to each lane.

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 18

NIH-PA Author Manuscript Fig. 6.

Effects of GF109203X, BAPTA-AM, and LY294002 on UDP-induced Erk phosphorylation in P2Y6-1321N1 cells, compared to PBS control. The cells were pretreated for 30 min with 2 µM GF109203X, 12.5 µM BAPTA-AM, and 100 µM LY294002. UDP(10 µM) was added for 0 and 5 min, and then proteins were extracted and applied to immunoblotting as described in experimental procedures. A total of 30 µg of protein was applied to each lane.

NIH-PA Author Manuscript NIH-PA Author Manuscript Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 19

NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 7.

NIH-PA Author Manuscript

Effect of Erk inhibitors on protection by UDP against TNFα-induced cell death in P2Y6-1321N1 cells. The cells were pretreated with U0126 or PD98,059 for 10 min, and further treated with TNFα for 4 h in the absence or presence of UDP. Then, old media was replaced with fresh media following washing with PBS. Cell death was observed on the following day (total of 16-h incubation). The media always contained 5 µg/mL cycloheximide. Concentrations of U0126, PD98,059, and TNFα were 25 µM, 25 µM and 20 ng/mL, respectively. U0126 or PD98,059 alone inhibited the phosphorylation of Erk (data not shown). The degree of cell death was analyzed using a FacsCalibur instrument (Becton Dickinson). Data shown are mean ± SD from a representative result in triplicate out of three independent experiments. *Statistically significant (p < 0.01) by Student’s t test; compared with control of each UDP concentration.

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 20

NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 8.

NIH-PA Author Manuscript

Effects of PLC inhibitors on protection by UDP against TNFα-induced cell death in P2Y6-1321N1 cells. The cells were pretreated U73122 or D609 for 10 min, and treated further with TNFα for 4 h in the absence or presence of UDP. Then, media was replaced with fresh media following washing with PBS. Cell death was observed after a 16-h incubation. The media always contained 5 µg/mL cycloheximide. Concentrations of U73122, D609, and TNFα were 2 µM, 100 µg/mL and 20 ng/mL, respectively. The degree of cell death was analyzed using a FacsCalibur instrument (Becton Dickinson). Data shown are mean values ± SD from a representative result in triplicate out of three independent experiments. *Statistically significant (p < 0.01) by Student’s t test; compared with control of each UDP concentration.

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Kim et al.

Page 21

NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 9.

NIH-PA Author Manuscript

Effect of PLC inhibitors on phosphorylation of PKC subtypes (A) and Erk1/2 (B). The cells were pretreated for 30 min with 2 µM U73122 or 100 µg/mL D609 GF109203X, and then UDP was added for the indicated time. Proteins were extracted and applied to immunoblotting as described in experimental procedures. A total of 45 µg (A) or 30 µg (B) of protein was applied to each lane. The PLC-inhibitors alone in non-UDP-treated cells had no effect (data not shown).

Cell Mol Neurobiol. Author manuscript; available in PMC 2011 July 21.

Lihat lebih banyak...

Comentários

Copyright © 2017 DADOSPDF Inc.