The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by O’Brien, J. B.
Right arrow Articles by Laster, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O’Brien, J. B.
Right arrow Articles by Laster, S. M.
The Journal of Immunology, 1998, 161: 1525-1532.
Copyright © 1998 by The American Association of Immunologists

Sustained Phosphorylation of Cytosolic Phospholipase A2 Accompanies Cycloheximide- and Adenovirus-Induced Susceptibility to TNF1

Jennifer B. O’Brien*, Debra L. Piddington*, Christina Voelkel-Johnson{dagger}, Debra J. Richards*, Leslie A. Hadley* and Scott M. Laster2,*

* Department of Microbiology, North Carolina State University, Raleigh, NC 27695; and {dagger} Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this report we examine the phosphorylation state of cytosolic phospholipase A2 (cPLA2) in C3HA fibroblasts that have been treated with TNF, cycloheximide (CHI), or a combination of both compounds. Our experiments show that TNF and CHI, when used independently, caused the rapid phosphorylation of cPLA2 (within 10 min). In both cases, cPLA2 was subsequently dephosphorylated to pretreatment levels by 40 min. In addition, under these conditions [3H]arachidonic acid was not released, and we could not detect a change in the activity of cPLA2 in vitro. In contrast, in cells treated with a combination of TNF and CHI, we found that the dephosphorylation of cPLA2 was inhibited, and cPLA2 remained phosphorylated for up to 2 h. In vitro we found that sustained phosphorylation of cPLA2 was accompanied by a 60 to 80% increase in the activity of cPLA2. The sustained phosphorylation of cPLA2 also occurred in cells infected with the adenovirus mutant dl309, suggesting that sustained phosphorylation may be a general requirement for the activation of cPLA2 in apoptotic cells. We also found that sustained phosphorylation of phosphoproteins is not a general consequence of apoptotic death, since the phosphorylation of p42 mitogen-activated protein kinase was not sustained. Finally, we show that the phosphatase inhibitor orthovanadate acts as does CHI to render cells susceptible to TNF, suggesting that resistance to TNF may depend on TNF’s ability to induce the expression of tyrosine or dual specificity phosphatase(s).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor {alpha} is an inflammatory cytokine that can mediate a variety of different immune and inflammatory reactions. In this report we focus on the apoptosis-inducing activity of TNF. Most normal and transformed cells are resistant to TNF (1, 2), although their resistance can be overcome with inhibitors of transcription or translation, such as cycloheximide (CHI)3 (3) or actinomycin D (4). Resistance to TNF is, therefore, dependent on the ability of TNF itself to induce the expression of one or more resistance gene products. A number of gene products have been implicated in resistance to TNF. For example, manganous superoxide dismutase (5) has been shown to provide resistance to TNF in certain cells, such as 293 human embryonic kidney cells. The expression of manganous superoxide dismutase does not, however, predict resistance in SV40-transformed fibroblasts (6), suggesting that it may not play a general role in resistance to TNF. Plasminogen activator inhibitor type 2 (7, 8) has also been shown to mediate protection against TNF-induced cytotoxicity in certain cells, although its general role has not been tested. Recently, NF-{kappa}B has been shown to be responsible for the expression of resistance gene products (9, 10). At this time it is not known whether NF-{kappa}B is responsible for the TNF-induced expression of manganous superoxide dismutase or plasminogen activator inhibitor type 2.

Susceptibility to TNF can also be induced by infection with viruses, such as adenovirus (11, 12), hepatitis B virus (13), Newcastle’s disease virus (14), HIV (15), and herpes virus (16). The molecular mechanism of virus-induced susceptibility is not known, but it has been suggested that viruses may act by inhibiting the expression of the above-mentioned TNF-induced resistance gene products (17). The mechanism of virus-induced susceptibility to TNF has been studied most extensively in the adenovirus model system, where it has been shown that expression of the adenovirus E1A protein by infection (12) or transfection (11) causes susceptibility to TNF. E1A encodes two major proteins, 289R and 243R, that contain two conserved regions, CR1 and CR2, that are required for transformation (18). These same regions influence transcription of cellular genes by associating with cellular proteins, including p300 (19) and p105-Rb (20). By using deletion mutants (21) it has been shown that the binding of E1A via CR1 to either p300 or p105-Rb is sufficient to induce susceptibility to TNF. To overcome this effect, viruses have also evolved proteins that protect the infected cell against TNF-induced apoptosis. The adenovirus, for example, encodes four proteins, the E1B 19K protein (22), the E3 14.7K protein (23, 24), and the E3 10.4K/14.5K dimer (25), that function independently to inhibit TNF-induced apoptosis in specific cell types. Therefore, to cause susceptibility to TNF with adenovirus, infections must be performed with deletion mutants lacking one or more of these protective proteins (21, 22, 23, 24, 25). It should be noted that in addition to inducing susceptibility to TNF, expression of E1A has been found to induce susceptibility to lysis by NK cells and activated macrophages (26, 27).

The enzyme cytosolic phospholipase A2 (cPLA2) is an 85-kDa, Ca2+-sensitive phospholipase that selectively catalyzes the hydrolysis of arachidonic acid from the sn-2 position of membrane phospholipids. Activation and translocation of cPLA2 have been shown to require both phosphorylation by a kinase (28, 29, 30) and an increase in intracellular Ca2+ (29, 31, 32, 33). We have shown previously that the activity of cPLA2 is necessary for the killing of 3T3-like fibroblasts and melanoma-derived tumor cells sensitized to TNF by inhibitors of transcription and translation (34). The activity of cPLA2 has also been shown to be necessary for the killing of cells infected by human adenoviruses lacking the E3 14.7K gene product (35) and in the death of the TNF-sensitive cell line L929 (36). Current evidence suggests that the release of arachidonic acid by cPLA2 is required for sphingomyelinase activation (37). In the L929 cell line, it was shown that cPLA2 activation and the generation of arachidonic acid were necessary for the accumulation of ceramide in response to TNF (37). Finally, in the TNF-sensitive cell lines MCF-7S1 and WEHI-S, cPLA2 activation was shown to be caspase dependent, indicating that cPLA2 is downstream of caspase-3 in these cells (38).

As mentioned above, we have shown previously that the activity of cPLA2 is required for the lysis of cells rendered sensitive to TNF by CHI. We found that the release of arachidonic acid occurred only from cells treated with TNF and CHI, not in those treated with TNF alone. We concluded, therefore, that CHI is inhibiting the expression of a negative regulatory factor upstream from cPLA2 that normally prevents the apoptotic signal from reaching cPLA2. In an attempt to gain some insight into the identity of this product, we have examined the phosphorylation state of cPLA2 in cells treated with TNF, CHI, or a combination of both compounds. Our results show that the normal pattern of transient phosphorylation that is induced by TNF changes in the presence of CHI. Instead, in cells treated with TNF and CHI, cPLA2 remains phosphorylated for an extended period of time (up to 2 h), suggesting that CHI may be inhibiting the expression of a phosphatase. Our experiments with phosphatase inhibitors suggest that a tyrosine phosphatase or dual specificity phosphatase may be necessary for resistance to TNF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

C3HA and NIH-3T3 cell lines are murine 3T3-like cell lines that were cultured in DMEM supplemented with 10% FCS and maintained at 37°C in 8% CO2. Both cell lines were supplied by L. Gooding, Emory University (Atlanta, GA). L929 is a TNF-sensitive murine fibrosarcoma cell line that was obtained from the American Type Culture Collection (Rockville, MD) and was cultured in DMEM supplemented with 10% FCS and maintained at 37°C and 8% CO2. WEHI 164 is a TNF-sensitive murine cell line that was obtained from the American Type Culture Collection and was cultured in RPMI 1640 medium supplemented with 10% FCS and maintained at 37°C and 8% CO2.

Virus and infections

Construction of the mutant adenoviruses dl309 (39) and dl758 (40) have been described previously. The mutant dl309 is derived from an Ad5 virus and lacks the righthand region of the E3 transcription unit, including the 10.4K, 14.5K, and 14.7K protein genes. The mutant dl758 is derived from an Ad5-Ad2-Ad5 recombinant and lacks the E3 14.7K protein gene. C3HA were plated overnight, washed, and incubated with 20 to 50 plaque-forming units/cell for 1.5 to 2 h in serum-free medium. Cells were then returned to medium with serum and incubated 24 h before treatment with TNF in 51Cr release and [3H]arachidonic acid release assays. The dl309 was provided by L. Gooding (Emory University, Atlanta, GA), and dl758 was provided by W. S. M. Wold (St. Louis University, St. Louis, MO).

Reagents

Media and chemicals were purchased from Sigma (St. Louis, MO). TNF was obtained from Quality Control Biochemicals (Hopkinton, MA). SB 20358 was provided by Dr. J. Lee of SmithKline Beecham Pharmaceuticals (King of Prussia, PA). The radiolabeled compounds were purchased from DuPont-New England Nuclear (Boston, MA).

51Cr release assays

Cells were labeled with 100 mCi of Na251CrO4 overnight and harvested by trypsinization. Cells were then plated at 104 cells/well into 96-well flat-bottom dishes that contained the appropriate concentrations of reagents for a total volume of 200 µl. At 16 h, 100 µl of the supernatant was counted with an autogamma counter (Packard, Downers Grove, IL). Maximum release was determined by adding 100 µl of 1 N HCl to untreated cells. The percent specific 51Cr release was calculated by the following formula: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. All treatments were performed in triplicate.

[3H]Arachidonic acid release assays

Cells were plated in 12-well tissue culture plates and labeled overnight with 0.1 µCi of [3H]arachidonic acid/ml. The following morning, cells were washed twice with HBSS, incubated for 2 h in medium, and washed two more times with HBSS before adding TNF and/or CHI for a final volume of 600 µl. Radiolabel release in supernatants was determined by scintillation counting 200 µl of the supernatant and multiplying by a factor of 3 (Beckman, Fullerton, CA). All points were performed in triplicate, and maximal incorporation was determined by lysing untreated cells in 1% SDS.

Cell lysates

Whole cells were washed twice with cold PBS, solubilized in lysis buffer (50 mM HEPES (pH 7.4), 1 mM EGTA, 1 mM EDTA, 0.2 mM sodium orthovanadate (OV), 1 mM PMSF, 0.2 mM leupeptin, and 0.5% SDS), and collected by scraping. The protein concentration for each sample lysate was determined using the Bio-Rad Dc Protein Assay kit (Bio-Rad, Hercules, CA), and volumes equivalent to 20 to 60 µg of protein were lyophilized. For cPLA2 gel shift or enzyme assays, cells were incubated in serum-free medium overnight before addition of cytokines or reagents.

SDS-PAGE and immunoblotting

The lyophilized protein was resuspended in sample buffer and subjected to electrophoresis using the NOVEX system (San Diego, CA) with 10% Tris-glycine gels at 30 mA for 3 h for cPLA2 gel-shift detection and 1 h for visualization of p38 and p42 mitogen-activated protein (MAP) kinases. Following transfer and blocking, the nitrocellulose was probed with either rabbit polyclonal antiserum raised against human cPLA2, phospho-specific p44/42 MAP kinase Ab, or phospho-specific p38 MAP kinase Ab. cPLA2 Ab was provided by Jim Clark of Genetics Institute (Cambridge, MA), and p44/42 and p38 MAP kinase Abs were obtained from New England BioLabs (Beverly, MA). Horseradish peroxidase-conjugated goat anti-rabbit secondary Ab was obtained from Sigma. Bands were visualized using the SuperSignal Chemiluminescent system (Pierce, Rockford, IL).

Cytosolic PLA2 enzyme assay

For lysate preparation cells were washed with PBS, scraped into homogenization buffer (20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, and 0.2 mM Na3VO4), and disrupted by sonication. The lysate was then cleared by ultracentrifugation (100,000 x g for 1 h at 4°C), and the supernatant was used as the source of cytosolic protein. The activity of cPLA2 was measured in vitro in substrate vesicles, as previously described (41). To prepare the substrate vesicles, [14C]arachidonyl phosphatidylethanolamine and dioleoylglycerol were mixed, dried under nitrogen gas, and suspended in 50 mM HEPES, pH 7.4. The suspension was then sonicated for 10 s, frozen in liquid nitrogen, and sonicated again for 1 min. To initiate enzymatic reactions, 10 µl of substrate was combined with 90 µl of buffer (50 mM HEPES, 150 mM sodium chloride, 2 mM 2-ME, and 1 mM calcium chloride, pH 7.4, with 1 mg/ml BSA) and cytosolic protein. Reaction tubes were incubated for 1 h at 37°C, and the reaction was terminated by the addition of 50 µl of 100 mM EGTA. To extract free fatty acids, each reaction mixture was combined with 2 ml of Dole’s reagent and vortexed, 750 µl of ddH2O and 1.2 ml heptane were added, and the tubes were vortexed again. The tubes were then allowed to sit on the bench for 5 min until the aqueous phase and heptane phase were completely separated, and then 1 ml of the heptane phase was transferred to another tube containing 1 ml of heptane and 100 mg of silicic acid. After vortexing, the tubes were allowed to sit for 1 h, and 1 ml of heptane was removed for scintillation counting. All points were performed in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The phenotype of C3HA fibroblasts

C3HA cells are 3T3-like fibroblasts that are resistant to TNF except when treated with CHI or infected with the adenovirus E3 deletion mutant dl309 (Fig. 1GoA). Figure 1GoB shows that the death of these sensitized cell populations is accompanied by the release of [3H]arachidonic acid, which begins typically 2 to 4 h after treatment with TNF is initiated. We have shown previously that cPLA2 is responsible for the release of [3H]arachidonic acid from these cell populations (34, 35).



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 1. TNF-induced release of 51Cr and [3H]arachidonic acid from sensitized C3HA fibroblasts. C3HA fibroblasts were labeled overnight with 51Cr (A) and treated with increasing concentrations of TNF. Susceptibility was induced by either adenovirus infection (20 plaque-forming units/cell of dl309) or treatment with CHI (10 µg/ml). In B, C3HA cells were labeled with [3H]arachidonic acid and treated with 20 ng/ml TNF. Susceptibility to TNF was induced as described above. The release of 51Cr (A) was measured at 16 h, and the release of [3H]arachidonic acid (B) was measured at the indicated times. The experiments shown are representative and have been repeated many times.

 
TNF-induced phosphorylation of cPLA2 in sensitized cells

The requirement for CHI for the activation of cPLA2 suggests that CHI is inhibiting the expression of a negative regulatory protein upstream from cPLA2. To determine whether this protein controls the phosphorylation state of cPLA2, we used an electrophoretic mobility gel-shift assay (28). This assay has been used repeatedly to study phosphorylation of cPLA2 induced by a variety of agents, such as thrombin (28, 42), phorbol ester (28), and fibroblast growth factor (43). As shown in Figure 2GoA, cPLA2 in resting C3HA fibroblasts displays two electrophoretic mobilities, and treatment with TNF produced a very consistent shift to the slower migrating form. The shift upward to the slower migrating form was complete by 10 min and was sustained for up to 20 min (Fig. 2GoA). Also very consistent was the subsequent shift downward to the faster migrating form, which was complete by 40 min (Fig. 2GoA). We found that CHI itself caused some phosphorylation of cPLA2 (Fig. 2GoB), although to a lesser extent than did TNF. Treatment with both CHI and TNF, however, changed this sequence of events. Treatment with both TNF and CHI (Fig. 2GoC) produced a shift upward by 10 min; however, dephosphorylation was inhibited, and the mobility of cPLA2 failed to return to pretreatment levels. As shown in Figure 2GoD, the dephosphorylation cPLA2 was inhibited as long as 120 min in cells treated with TNF and CHI.



View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 2. Phosphorylation of cPLA2 in C3HA fibroblasts. C3HA fibroblasts were treated with TNF (20 ng/ml), CHI (10 µg/ml), or both TNF and CHI (A–D). In E, C3HA cells were first infected with dl309 for 24 h, then treated with TNF (20 ng/ml) alone. At the indicated times, the cells were lysed as described in Materials and Methods, and the proteins were examined by SDS-PAGE and immunoblotting. Each experiment was repeated three times. The experiments shown are representative.

 
To determine whether this effect was unique to treatment with CHI, we also examined C3HA fibroblasts that had been rendered sensitive to TNF by infection with dl309. As shown in Figure 2GoE, although some dephosphorylation did occur, we also found that phosphorylation of cPLA2 was sustained for up to 2 h.

Enzyme activity in TNF- and CHI-treated cells

Next, an in vitro enzyme assay was used to establish whether the change in mobility of cPLA2 to the slower migrating form was associated with a change in enzyme activity. We have shown previously (34) that this vesicle-based assay measures selectively the activity of cPLA2 in C3HA fibroblasts. As shown in Figure 3Go, we did not find any increase in enzyme activity in C3HA cells treated with either TNF or CHI alone. In contrast, we did find an increase in enzyme activity in cells treated with both TNF and CHI. Altered enzyme activity was evident as soon as 15 min post-treatment and was sustained for up to 1 h.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 3. The activity of cPLA2 in C3HA fibroblasts. C3HA cells were treated with TNF (20 ng/ml), CHI (10 µg/ml), or both TNF and CHI in combination. At the indicated times, the cells were lysed, and the enzyme activity (picomoles per minute per milligram) was determined as described in Materials and Methods. The data are shown as the mean results from three experiments.

 
Phosphorylation of MAP kinases in cells treated with TNF and CHI

Does treatment with CHI or infection by adenovirus alter the TNF-dependent phosphorylation of all cellular proteins? To test this hypothesis, we examined the phosphorylation state of the p42 MAP kinase (p42) and the p38 MAP kinase (p38) in C3HA treated with TNF, CHI, and a combination of TNF and CHI. Both p42 (44) and p38 (45) are activated by dual phosphorylation of threonine and tyrosine residues, and Abs that recognize the phosphorylated form of p42 and p38 are available commercially. As shown in Figure 4GoA, under all three treatment conditions, p42 is phosphorylated rapidly (within 5 min) and is dephosphorylated by 20 min, indicating that the sustained phosphorylation of phosphoproteins is not an ubiquitous effect of CHI. p38 is also rapidly phosphorylated by 5 min and then dephosphorylated by 20 min in response to either TNF or CHI (Fig. 4GoB). Unlike p42, however, treatment with both TNF and CHI produced some enhanced phosphorylation of p38 that did not return to pretreatment levels by 40 min (Fig. 4GoB), indicating that sustained phosphorylation is not restricted entirely to cPLA2.



View larger version (70K):
[in this window]
[in a new window]
  		FIGURE 4. TNF-induced phosphorylation of p42 MAP kinase and p38 MAP kinase. C3HA cells were treated with TNF (20 ng/ml), CHI (10 µg/ml), or both for the indicated times and then lysed as described in Materials and Methods. The proteins were examined by SDS-PAGE and immunoblotting to visualize phosphorylated p42 MAP kinase (A) and phosphorylated p38 MAP kinase. Experiments were repeated three times; the experiments shown are representative.

 
Phosphatase inhibitors and sensitization to TNF

The sustained phosphorylation of cPLA2 in cells treated with CHI suggests that CHI is inhibiting the TNF-induced expression of a phosphatase. We would predict, therefore, that treatment with a phosphatase inhibitor should produce susceptibility to TNF, similar to the effect of CHI. Two phosphatase inhibitors were examined, the serine/threonine phosphatase inhibitor okadaic acid (OA) and the tyrosine phosphatase inhibitor OV. As shown in Figure 5GoA, OA failed to cause susceptibility in C3HA fibroblasts. It also failed to cause any enhanced susceptibility in L929 and WEHI 164, two cell lines that are spontaneously susceptible to TNF (Fig. 5GoA). In contrast, as shown in Figure 5GoB, susceptibility to TNF in C3HA cells was enhanced significantly by the addition of OV at both 0.5 and 1.0 mM. OV also produced susceptibility in NIH-3T3 cells and enhanced the susceptibility of L929 and WEHI 164 cell lines (Fig. 5GoC).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 5. The effects of phosphatase inhibitors on susceptibility to TNF. C3HA cells were labeled overnight with 51Cr and treated with OA at the indicated concentrations alone or in combination with 20 ng/ml TNF (A), with the indicated concentrations of sodium OV alone or in combination with 20 ng/ml TNF (B), or with 0.5 mM OV alone or in combination with 20 ng/ml TNF (C). The release of 51Cr was measured at 16 h. The experiments were repeated four to six times. The experiments shown are representative.

 
To determine whether OV is affecting the activity of cPLA2, the influence of OV on the TNF-induced release of [3H]arachidonic acid in C3HA fibroblasts was also examined. We found that treatment with OV itself was sufficient to stimulate the release of some [3H]arachidonic acid (Fig. 6GoA) and that OV acted synergistically with TNF (Fig. 6GoA) to enhance the release of [3H]arachidonic acid. As shown in Figure 6GoB, the effects of OV, although occurring somewhat sooner, parallel those of CHI. Finally, as shown in Figure 6GoC, we found that OV alone or in combination with TNF caused the rapid and sustained phosphorylation of cPLA2.



View larger version (66K):
[in this window]
[in a new window]
  		FIGURE 6. The effects of OV on the TNF-induced phosphorylation of cPLA2. C3HA were labeled overnight with [3H]arachidonic acid and treated with 0.5 mM OV alone or in combination with 20 ng/ml TNF (A). The release of [3H]arachidonic acid (A) was measured at the indicated times. For immunoblotting (B), C3HA were treated with 0.5 mM OV alone or with 20 ng/ml TNF, and the cells were lysed as described in Materials and Methods at the indicated times. The proteins were examined by SDS-PAGE and immunoblotting. The experiments were repeated three times. The experiments shown are representative.

 
Ca2+ and its role in enzyme activation

As shown above, although phosphorylation of cPLA2 is seen as early as 10 min in dying cells (Fig. 2GoC), the release of [3H]arachidonic acid is not detected until 2 to 4 h, suggesting that a second signal is also required for enzyme activation in situ (Fig. 1GoB). Ca2+ has been reported to be necessary for the translocation of cPLA2 (46), and recently, Kong et al. (47) have shown that Ca2+ is required for the lysis of L929 fibroblasts. These authors used verapamil to inhibit the import of extracellular Ca2+ and prevent cell death (47). As shown in Figure 7GoA, in agreement with their findings, we found that the death of L929 fibroblasts can be inhibited by pretreatment with 100 µM verapamil. We also found that the death of C3HA cells sensitized by CHI was inhibited by verapamil, although not to the same extent as with L929 fibroblasts. We could not, however, use verapamil to block the death of cells infected with dl758, an adenovirus mutant lacking only the E3 14.7K gene (Fig. 7GoA). Similarly, we could not block the TNF-induced release of [3H]arachidonic acid from cells infected with dl758 (Fig. 7GoB), while verapamil prevented the release of [3H]arachidonic acid from L929 fibroblasts treated with TNF or from C3HA cells treated with TNF and CHI.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 7. The effects of verapamil on the TNF-induced release of 51Cr and [3H]arachidonic acid release. Cells were either infected or uninfected, radiolabeled overnight, and treated with 20 ng/ml TNF, 10 µg/ml CHI, and 100 mM verapamil where indicated. The release of 51Cr and [3H]arachidonic acid were measured at 16 h. The release of [3H]arachidonic acid was measured at 10 h for L929 and 6 h for C3HA cells. The experiments shown are representative and were repeated four to six times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we have examined the phosphorylation state of cPLA2. We found that treatment of C3HA cells with either TNF or CHI produced a pattern of transient phosphorylation. Peak phosphorylation occurred at 10 min, and dephosphorylation to pretreatment levels was complete by 40 min. In contrast, treatment of C3HA cells with a combination of TNF and CHI caused sustained phosphorylation of cPLA2 for up to 120 min. We also determined that the combined treatment of TNF and CHI enhanced the activity of cPLA2 in vitro, suggesting a direct link between phosphorylation and enzyme activity. Finally, we observed a similar pattern of sustained phosphorylation of cPLA2 in C3HA cells infected with the adenovirus deletion mutant dl309, which suggests that this may be an effect common to agents that induce susceptibility to TNF.

Taken together, these results can be used to construct a model for the TNF-dependent regulation of cPLA2 in C3HA fibroblasts. The data suggest that TNF initiates a signal leading to the activation of a kinase that phosphorylates cPLA2 within 10 to 15 min. This signal is attenuated quickly (by 30–40 min), suggesting that TNF is also inducing (via NF-{kappa}B) the activity of a phosphatase. In the presence of CHI, however, the signal is not attenuated, and the phosphorylation of cPLA2 is sustained. It is likely that CHI is exerting this effect by blocking expression of the aforementioned TNF-induced phosphatase; however, other effects of CHI on phosphatase activity cannot be ruled out. In addition, since TNF and CHI did not cause the release of [3H]arachidonic acid for 1 to 2 h after the enzyme itself was modified, a second signal must be required for the activation of cPLA2 in situ.

To test the role of phosphatases in this system, we examined the effects of both a serine/threonine phosphatase inhibitor and a tyrosine phosphatase inhibitor. Serine phosphatases could be important because cPLA2 can be activated by phosphorylation on a number serine residues (30, 48), and it may be the target of a variety of serine kinases, such as p44/42 MAP kinase (28), p38 MAP kinase (49), protein kinase C (50), or protein kinase A (30). Tyrosine phosphatases may also be important in the regulation of cPLA2, because both the p44/42 MAP kinase (44) and the p38 MAP kinase (45) are themselves regulated by dual specificity tyrosine phosphatases (51, 52, 53, 54). Tyrosine phosphorylation of cPLA2 itself has not been reported (28, 30). Consequently, inhibition of either serine/threonine phosphatases (by OA) or tyrosine phosphatases (by sodium OV) could shift the balance in a cell, leading to the sustained phosphorylation of cPLA2. We found that the tyrosine phosphatase inhibitor, sodium OV, functioned as did CHI in its ability to render cells susceptible to TNF and also enhance the killing of cells spontaneously susceptible to TNF. Several differences were noted, however, between OV and CHI. OV itself caused sustained phosphorylation of cPLA2 and the rapid, sustained release of [3H]arachidonic acid, minimizing its synergistic action with TNF (Fig. 6GoA). It is possible, therefore, that OV may indeed be inhibiting the activity of a dual specificity phosphatase, which by chain reaction sustains the phosphorylation of cPLA2. Alternatively, OV could be acting to inhibit a tyrosine phosphatase that dephosphorylates an unrecognized site on cPLA2. Finally, OV could be acting like pervanadate, another inhibitor of tyrosine phosphatases, which has been shown to block the TNF-induced activation of NF-{kappa}B (55, 56), which could, in turn, prevent the induction of resistance gene products.

Surprisingly, the serine/threonine phosphatase inhibitor, OA, had no effect on TNF-induced killing. These results contrast with those previously reported by Wright et al. (57). These authors found that the serine/threonine phosphatase inhibitors, OA and calyculin A, synergized with TNF in the killing of several TNF-sensitive tumor cell lines, including the U937 histiocytic lymphoma, the BT-20 mammary carcinoma, and the LNCap prostatic tumor cell line, as well as the TNF-resistant cell line U9-TR, derived from U937. The difference between our two studies may arise from biochemical differences in the apoptotic pathways of the cell lines tested. For example, we have found that TNF-induced apoptosis in U937 cells is cPLA2 independent (data not shown), indicating that C3HA and U937 cell lines use different apoptotic pathways. In addition, Wright et al. (57) reported that OA was toxic to U937 cells at doses >25 nM, while we did not observe any toxicity toward any of the cells tested here at concentrations as high as 200 nM.

To determine whether the sustained phosphorylation of phosphoproteins is a general consequence of apoptotic death we also examined the phosphorylation state of p42 MAP kinase and p38 MAP kinase. We found that p42 MAP kinase was rapidly dephosphorylated, even in the presence of CHI, indicating that sustained phosphorylation of phosphoproteins is not an obligate consequence of apoptosis. We did find some enhanced phosphorylation of p38 MAP kinase in CHI-treated cells, suggesting that p38 might be the kinase responsible for the activation of cPLA2. Since p38 MAP kinase has been implicated in the activation of cPLA2 (49, 58), we tested the compound SB 203580, which has been shown to be an effective inhibitor of p38 (58, 59). We found that SB 203580 did not inhibit cell death or the release of [3H]arachidonic acid (data not shown). In fact, it enhanced the release of [3H]arachidonic acid, suggesting that p38 is not the kinase responsible for phosphorylation of cPLA2. Another kinase that has been shown to become activated in response to TNF through the noncytotoxic pathway is the Jun N-terminal kinase (60, 61). The Jun N-terminal kinase, however, has not been implicated in the activation of cPLA2 (30).

We also investigated that nature of the second signal that is necessary for the activation of cPLA2 in the apoptotic cell. In certain situations Ca2+ has been shown to be necessary for the translocation of cPLA2 to its membrane phospholipid substrate (32, 33, 46, 62). It has been demonstrated previously by Kong et al. (47) that extracellular Ca2+ is necessary for the TNF-induced death of L929 cells. These authors showed a slow, steady rise in intracellular Ca2+ levels that could be blocked by verapamil. Our experiments with verapamil suggest that extracellular Ca2+ is also required for the activation of cPLA2 in CHI-sensitized C3HA fibroblasts. Verapamil was not effective in adenovirus-infected cells, suggesting that these cells may rely entirely on internal stores of Ca2+. Alternatively, virus infection itself (via E1A) may elevate basal levels of intracellular Ca2+, so that a ligand-dependent rise in intracellular Ca2+ is not necessary. It is also possible that a signal other than Ca2+ acts as the second signal in virus-infected cells. For example, Wissing et al. (16) have shown that a specific tetrapeptide inhibitor of caspase-3, Ac-DEVD-CHO, can inhibit the activation of cPLA2, suggesting that at least one caspase is upstream of cPLA2. In fact, a sequence on cPLA2 has been identified (38) that is similar to the caspase-3 recognition sequence on poly(A)DP ribose polymerase, and it has been suggested that cleavage of cPLA2 itself may be an activating signal. This hypothesis remains unproven, however, since enhanced cPLA2 activity following cleavage was not demonstrated (16). In fact, we have shown (63) that cleaved cPLA2, which does appear in C3HA fibroblasts at later time points, is less active than the intact molecule. Alternatively, exteriorization of annexin V, which has been shown to be an inhibitor of cPLA2 (64), could be the second signal for cPLA2 activation in adenovirus-infected cells. These hypotheses are under investigation at the present time.


   Acknowledgments
 
We thank L. R. Gooding and W. S. M. Wold for the supply of cell lines and viruses. We thank Dr. J. Lee (SmithKline Beecham Pharmaceuticals, King of Prussia, PA) for the supply of SB 203580.


   Footnotes
 
1 This work was supported by Grant CA-59032 (to S.M.L.) from the National Institutes of Health and a Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship (to J.B.O.). Back

2 Address correspondence and reprint requests to Dr. Scott M. Laster, Department of Microbiology, North Carolina State University, Box 7615, Raleigh, NC 27695. Back

3 Abbreviations used in this paper: CHI, cycloheximide; cPLA2, phospholipase A2; OV, orthovanadate; MAP, mitogen-activated protein; OA, okadaic acid. Back

Received for publication January 14, 1998. Accepted for publication April 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sugarman, B. J., B. B. Aggarwal, P. E. Hass, I. S. Figari, Jr M. A. Palladino, H. M. Shepard. 1985. Recombinant human TNF-{alpha}: effects on proliferation of normal and transformed cells in vitro. Science 230:943.[Abstract/Free Full Text]
  2. Fransen, L., J. Van Der Heyden, R. Ruysschaert, W. Fiers. 1986. Recombinant TNF: its effect and its synergism with IFN-{gamma} on a variety of normal and transformed cell lines. Eur. J. Cancer Clin. Oncol. 22:419.[Medline]
  3. Kull, F. C., P. Cuatrecasas. 1981. Possible requirement of internalization in the mechanism of in vitro cytotoxicity in tumor necrosis serum. Cancer Res. 41:4885.[Abstract/Free Full Text]
  4. Ostrove, J. M., G. E. Gifford. 1979. Stimulation of RNA synthesis in L929 cells by rabbit TNF. Proc. Soc. Exp. Biol. Med. 160:354.[Medline]
  5. Wong, G. H. W., J. H. Elwell, L. W. Oberley, D. V. Goeddel. 1989. Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of TNF. Cell 58:923.[Medline]
  6. Boss, J. M., S. M. Laster, L. R. Gooding. 1991. Sensitivity to TNF-mediated cytolysis is unrelated to manganous superoxide dismutase messenger RNA levels among transformed mouse fibroblasts. Immunology 73:309.[Medline]
  7. Kumar, S., C. Baglioni. 1991. Protection from TNF-mediated cytolysis by overexpression of plasminogen activator inhibitor type-2. J. Biol. Chem. 266:20960.[Abstract/Free Full Text]
  8. Pytel, B. A., K. Peppel, C. Baglioni. 1990. Plasminogen activator inhibitor type-2 is a major protein induced in human fibroblasts and SK-MEL-109 melanoma cells by TNF. J. Cell. Physiol. 144:416.[Medline]
  9. Beg, A. A., D. Baltimore. 1996. An essential role for NF-{kappa}B in preventing TNF-{alpha}-induced cell death. Science 274:782.[Abstract/Free Full Text]
  10. Van Antwerp, D. J., S. J. Martin, T. Kafri, D. R. Green, I. M. Verma. 1996. Suppression of TNF-{alpha}-induced apoptosis by NF-{kappa}B. Science 274:787.[Abstract/Free Full Text]
  11. Chen, M. J., B. Holskin, J. Strickler, J. Gorniak, M. A. Clark, P. J. Johnson, M. Mitcho, D. Shalloway. 1987. Induction by E1A oncogene expression of cellular susceptibility to lysis by TNF. Nature 330:581.[Medline]
  12. Duerksen-Hughes, P., W. S. M. Wold, L. R. Gooding. 1989. Adenovirus E1A renders infected cells sensitive to cytolysis by TNF. J. Immunol. 143:4193.[Abstract]
  13. Su, F., R. J. Schneider. 1997. Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by TNF. Proc. Natl. Acad. Aci USA 94:8744.[Abstract/Free Full Text]
  14. Lorence, R. M., P. A. Rood, K. W. Kelley. 1988. Newcastle disease virus as an antineoplastic agent: induction of TNF-{alpha} and augmentation of its cytotoxicity. J. Natl. Cancer Inst. 80:1305.[Abstract/Free Full Text]
  15. Matsuyama, T., Y. Hamamoto, G.-I. Soma, D. Mizuno, N. Yamamoto, N. Kobyashi. 1989. Cytocidal effect of TNF on cells chronically infected with human immunodeficiency virus (HIV): enhancement of HIV replication. J. Virol. 63:2504.[Abstract/Free Full Text]
  16. Koff, W. C., A. V. Fann. 1986. Human TNF-{alpha} kills herpes virus-infected cells but not normal cells. Lymphokine Res. 5:215.[Medline]
  17. Gooding, L. R., W. S. M. Wold. 1990. Molecular mechanisms by which adenoviruses counteract antiviral immune defenses. Crit. Rev. Immunol. 10:53.[Medline]
  18. Svensson, C., M. Bindesson, E. Nyberg, S. Linder, N. Jones, G. Akusjarvi. 1991. Independent transformation activity by adenovirus-5 E1A-conserved regions 1 or 2 mutants. Virology 182:553.[Medline]
  19. Arany, Z., W. R. Sellers, D. M. Livingston, R. Eckner. 1994. E1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators. Cell 77:799.[Medline]
  20. Bandara, L. R., N. B. La Thangue. 1991. Adenovirus E1A prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature 351:494.[Medline]
  21. Shisler, J., P. Duerksen-Hughes, T. M. Hermiston, W. S. M. Wold, L. R. Gooding. 1996. Induction of susceptibility to TNF by E1A is dependent of binding to either p300 or p105-Rb and induction of DNA synthesis. J. Virol. 70:68.[Abstract]
  22. White, E., P. Sabbatini, M. Debbas, W. S. M. Wold, D. I. Kusher, L. R. Gooding. 1992. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by TNF-{alpha}. Mol. Cell. Biol. 12:2570.[Abstract/Free Full Text]
  23. Gooding, L. R., L. W. Elmore, A. E. Tollefson, H. A. Brady, W. S. M. Wold. 1988. A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by TNF. Cell 53:341.[Medline]
  24. Gooding, L. R., I. O. Sofola, A. E. Tollefson, P. Duerksen-Hughes, W. S. M. Wold. 1990. The adenovirus E3–14.7K protein is a general inhibitor of TNF-mediated cytolysis. J. Immunol. 145:3080.[Abstract]
  25. Gooding, L. R., T. S. Ranheim, A. E. Tollefson, L. Aquino, P. Duerksen-Hughers, T. M. Horton, W. S. M. Wold. 1991. The 10,4000- and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by TNF. J. Virol. 65:4114.[Abstract/Free Full Text]
  26. Cook, J. L., D. L. May, B. A. Wilson, B. Holskin, M. Chen, D. Shalloway, T. A. Walker. 1989. Role of TNF-{alpha} in E1A oncogene-induced susceptibility of neoplastic cells to lysis by NK cells and activated macrophages. J. Immunol. 142:4527.[Abstract]
  27. Walker, T. A., B. A. Wilson, Jr A. M. Lewis, J. L. Cook. 1991. E1A oncogene induction of cytolytic susceptibility eliminates sarcoma cell tumorigenicity. Proc. Natl. Acad. Sci. USA 88:6491.[Abstract/Free Full Text]
  28. Lin, L.-L., M. Wartmann, A. Y. Lin, J. L. Knopf, A. Seth, R. J. Davis. 1993. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72:269.[Medline]
  29. Schalkwijk, C. G., M. A. G. van der Heijden, G. Bunt, R. Maas, L. G. J. Tertoolen, P. M. P. van Bergen en Henegouwen, A. J. Verkleij, H. van den Bosch, J. Boonstra. 1996. Maximal epidermal growth-factor-induced cytosolic phospholipase A2 activation in vivo requires phosphorylation followed by an increased intracellular calcium concentration. Biochem. J. 313:91.
  30. de Carvalho, M. G. S., A. L. McCormack, E. Olson, F. Ghomashchi, M. H. Gelb, III J. R. Yates, C. C. Leslie. 1996. Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes. J. Biol. Chem. 271:6987.[Abstract/Free Full Text]
  31. Fernandez, B., J. Basinde. 1991. Receptor-mediated activation of arachidonic acid release in mouse peritoneal-macrophages is linked to extracellular calcium influx. Biochem. Biophys. Res. Commun. 180:1036.[Medline]
  32. Clark, J. D., L.-L. Lin, R. W. Kriz, C. S. Ramesha, L. A. Sultzman, A. Y. Lin, N. Milona, J. L. Knopf. 1991. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell 65:1043.[Medline]
  33. Schievella, A. R., M. K. Regier, W. L. Smith, L.-L. Lin. 1995. Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J. Biol. Chem. 270:30749.[Abstract/Free Full Text]
  34. Voelkel-Johnson, C., T. E. Thorne, S. M. Laster. 1996. Susceptibility to TNF in the presence of inhibitors of transcription or translation is dependent on the activity of cytosolic phospholipase A2 in human melanoma tumor cells. J. Immunol. 156:201.[Abstract]
  35. Thorne, T. E., C. Voelkel-Johnson, W. M. Casey, L. W. Parks, S. M. Laster. 1996. The activity of cytosolic phospholipase A2 is required for the lysis of adenovirus-infected cells by TNF. J. Virol. 70:8502.[Abstract]
  36. Hayakawa, M., N. Ishida, K. Takeuchi, S. Shibamoto, T. Hori, N. Oku, F. Ito, M. Tsujimoto. 1993. Arachidonic acid-selective cytosolic phospholipase A2 is crucial in the cytotoxic action of TNF. J. Biol. Chem. 268:11290.[Abstract/Free Full Text]
  37. Jayadev, S., H. L. Hayter, N. Andrieu, C. J. Gamard, B. Liu, R. Balu, M. Hayakawa, F. Ito, Y. A. Hannun. 1997. Phospholipase A2 is necessary for TNF {alpha}-induced ceramide generation in L929 cells. J. Biol. Chem. 272:17196.[Abstract/Free Full Text]
  38. Wissing, D., H. Mouritzen, M. Egeblad, G. G. Poirier, M. Jaattela. 1997. Involvement of caspase-dependent activation of cytosolic phospholipase A2 in TNF-induced apoptosis. Proc. Natl. Acad. Sci. USA 94:5073.[Abstract/Free Full Text]
  39. Jones, N., T. Shenk. 1979. Isolation of adenovirus type 5 host range deletion mutants defective for transformation in rat embryo cells. Cell 17:683.[Medline]
  40. Brady, H. A., A. Scaria, W. S. M. Wold. 1992. Map of cis-acting sequences that determine alternative transcription unit of adenovirus. J. Virol. 66:5914.[Abstract/Free Full Text]
  41. Leslie, C. C., D. R. Voelker, J. Y. Channon, M. M. Wall, P. T. Zelarney. 1988. Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7. Biochim. Biophys. Acta 963:476.[Medline]
  42. Kramer, R. M., E. F. Roberts, P. A. Hyslop, B. G. Utterback, K. Y. Hui, J. A. Jokubowski. 1995. Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. J. Biol. Chem. 270:14816.[Abstract/Free Full Text]
  43. Sa, G., G. Murugesan, M. Jaye, Y. Ivashchenko, P. L. Fox. 1995. Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. J. Biol. Chem. 270:2360.[Abstract/Free Full Text]
  44. Payne, D. M., A. J. Rossamando, P. Martino, A. K. Erickson, J.-H. Her, J. Shabanowitz, D. F. Hunt, M. J. Weber, T. W. Sturgill. 1991. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 10:885.[Medline]
  45. Raingeaud, J., S. Gupta, J. Rogers, M. Dickens, J. Han, R. J. Ulevitch, R. J. Davis. 1995. Pro-inflammatory cytokines and environmental stress cause p38 MAP kinase activation by dual phosphorylation on tyrosine and threonine. J. Biol. Chem. 270:7420.[Abstract/Free Full Text]
  46. Channon, J. Y., C. C. Leslie. 1990. A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7. J. Biol. Chem. 265:5409.[Abstract/Free Full Text]
  47. Kong, S. K., K. P. Fung, Y. M. Choy, C. Y. Lee. 1997. Slow increase in intranuclear and cytosolic free calcium concentrations in L929 cells is important in tumour necrosis factor-{alpha}-mediated cell death. Oncology 54:55.[Medline]
  48. Lin, L.-L., A. Y. Lin, J. L. Knopf. 1992. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc. Natl. Acad. Sci. USA 89:6147.[Abstract/Free Full Text]
  49. Kramer, R. M., E. F. Roberts, S. L. Um, A. G. Borsch-Huabold, S. P. Watson, M. J. Fisher, J. A. Jakubowski. 1996. p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. J. Biol. Chem. 271:27723.[Abstract/Free Full Text]
  50. Nemenoff, R. A., S. Winitz, N.-X. Qian, V. Van Putten, G. L. Johnson, L. E. Heasley. 1993. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J. Biol. Chem. 268:1960.[Abstract/Free Full Text]
  51. Brondello, J.-M., A. Brunet, J. Pouyssegur, F. R. McKenzie. 1997. The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44MAPK cascade. J. Biol. Chem. 272:1368.[Abstract/Free Full Text]
  52. Franklin, C. C., A. S. Kraft. 1997. Conditional expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress-activated protein kinase in U937 cells. J. Biol. Chem. 272:16917.[Abstract/Free Full Text]
  53. Bokemeyer, D., A. Sorokin, M. Yan, N. G. Ahn, D. J. Templeton, M. J. Dunn. 1996. Induction of mitogen-activated protein kinase phosphatase 1 by the stress-activated protein kinase signaling pathway but not by extracellular signal-regulated kinase in fibroblasts. J. Biol. Chem. 271:639.[Abstract/Free Full Text]
  54. Sun, H., C. H. Charles, L. F. Lau, N. K. Tonks. 1993. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75:487.[Medline]
  55. Darnay, B. G., B. B. Aggarwal. 1997. Inhibition of protein tyrosine phosphatases causes phosphorylation of tyrosine-331 in the p60 TNF receptor and inactivates the receptor-associated kinase. FEBS Lett. 410:361.[Medline]
  56. Dhawan, S., S. Singh, B. B. Aggarwal. 1997. Induction of endothelial cell surface adhesion molecules by TNF is blocked by protein tyrosine phosphatase inhibitors: role of the nuclear transcription factor NF-{kappa}B. Eur. J. Immunol. 27:2172.[Medline]
  57. Wright, S. C., H. Zheng, J. Zhong, F. M. Torti, J. W. Larrick. 1993. Role of protein phosphorylation in TNF-induced apoptosis: phosphatase inhibitors synergize with TNF to activate DNA fragmentation in normal as well as TNF-resistant U937 variants. J. Cell. Biochem. 53:222.[Medline]
  58. Waterman, W. H., T. F. P. Molski, C.-K. Huang, J. L. Adams, R. I. Sha’afi. 1996. Tumour necrosis factor-{alpha}-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils. Biochem. J. 319:17.
  59. Cuenda, A., J. Rouse, Y. N. Doza, R. Meier, P. Cohen, T. F. Gallagher, P. R. Young, J. C. Lee. 1995. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364:229.[Medline]
  60. Liu, Z.-G., H. Hsu, D. V. Goeddel, M. Karin. 1996. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-{kappa}B activation prevents cell death. Cell 87:565.[Medline]
  61. Natoli, G., A. Costanzo, A. Ianni, D. J. Templeton, J. R. Woodgett, C. Balsano, M. Levrero. 1997. Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2-dependent pathway. Science 275:200.[Abstract/Free Full Text]
  62. Leslie, C. C., D. R. Voelker, J. Y. Channon, M. M. Wall, P. T. Zelarney. 1988. Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7. Biochim. Biophys. Acta 963:476.
  63. Voelkel-Johnson, C., A. J. Entingh, W. S. M. Wold, L. R. Gooding, S. M. Laster. 1995. Activation of intracellular proteases is an early event in TNF-induced apoptosis. J. Immunol. 154:1707.[Abstract]
  64. Mira, J.-P., T. Dubois, J.-P. Oudine, S. Lukowski, F. Russo-Marie, B. Geny. 1997. Inhibition of cytosolic phospholipase A2 by annexin V in differentiated permeabilized HL-60 cells. J. Biol. Chem. 272:10474.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
C. A. Culver and S. M. Laster
Adenovirus Type 5 Exerts Multiple Effects on the Expression and Activity of Cytosolic Phospholipase A2, Cyclooxygenase-2, and Prostaglandin Synthesis
J. Immunol., September 15, 2007; 179(6): 4170 - 4179.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Dong, M. Johnson, A. Rezaie, J. N.M. Ilsley, M. Nakanishi, M. M. Sanders, F. Forouhar, J. Levine, D. C. Montrose, C. Giardina, et al.
Cytoplasmic Phospholipase A2 Levels Correlate with Apoptosis in Human Colon Tumorigenesis
Clin. Cancer Res., March 15, 2005; 11(6): 2265 - 2271.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. W. Draper, V. G. Harris, C. A. Culver, and S. M. Laster
Calcium and Its Role in the Nuclear Translocation and Activation of Cytosolic Phospholipase A2 in Cells Rendered Sensitive to TNF-Induced Apoptosis by Cycloheximide
J. Immunol., February 15, 2004; 172(4): 2416 - 2423.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Liu, Y. Ma, L. J. Pagliari, H. Perlman, C. Yu, A. Lin, and R. M. Pope
TNF-{alpha}-Induced Apoptosis of Macrophages Following Inhibition of NF-{kappa}B: A Central Role for Disruption of Mitochondria
J. Immunol., February 1, 2004; 172(3): 1907 - 1915.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
M. Dong, K. Guda, P. R. Nambiar, A. Rezaie, G. S. Belinsky, G. Lambeau, C. Giardina, and D. W. Rosenberg
Inverse association between phospholipase A2 and COX-2 expression during mouse colon tumorigenesis
Carcinogenesis, February 1, 2003; 24(2): 307 - 315.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Duan, H. Gan, J. Arm, and H. G. Remold
Cytosolic Phospholipase A2 Participates with TNF-{{alpha}} in the Induction of Apoptosis of Human Macrophages Infected with Mycobacterium tuberculosis H37Ra
J. Immunol., June 15, 2001; 166(12): 7469 - 7476.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by O’Brien, J. B.
Right arrow Articles by Laster, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O’Brien, J. B.
Right arrow Articles by Laster, S. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS