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J. Biol. Chem., Vol. 276, Issue 30, 28364-28371, July 27, 2001

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Oxidative Stress-induced Phospholipase C-1 Activation Enhances Cell Survival^*

Xian-Tao Wang^§, Karen D. McCullough^§, Xue-Jie Wang^¶, Graham Carpenter^¶, and Nikki J. Holbrook^**

From the  Cell Stress and Aging Section, Laboratory of Cellular and Molecular Biology, NIA, National Institutes of Health, Baltimore, Maryland 21224-6825 and the ^¶ Departments of Biochemistry and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Received for publication, March 26, 2001, and in revised form, May 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phospholipase C-1 (PLC-1) is rapidly activated in response to growth factor stimulation and plays an important role in regulating cell proliferation and differentiation through the generation of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C (PKC) and increased levels of intracellular calcium, respectively. Given the existing overlap between signaling pathways that are activated in response to oxidant injury and those involved in responding to proliferative stimuli, we investigated the role of PLC-1 during the cellular response to oxidative stress. Treatment of normal mouse embryonic fibroblasts (MEF) with H₂O₂ resulted in time- and concentration-dependent tyrosine phosphorylation of PLC-1. Phosphorylation could be blocked by pharmacological inhibitors of Src family tyrosine kinases or the epidermal growth factor receptor tyrosine kinase, but not by inhibitors of the platelet-derived growth factor receptor or phosphatidylinositol 3-kinase. To investigate the physiologic relevance of H₂O₂-induced tyrosine phosphorylation of PLC-1, we compared survival of normal MEF and PLC-1-deficient MEF following exposure to H₂O₂. Treatment of PLC-1-deficient MEF with H₂O₂ resulted in rapid cell death, whereas normal MEF were resistant to the stress. Pretreatment of normal MEF with a selective pharmacological inhibitor of PLC-1, or inhibitors of inositol trisphosphate receptors and PKC, increased their sensitivity to H₂O₂, whereas treatment of PLC-1-deficient MEF with agents capable of directly activating PKC and enhancing calcium mobilization significantly improved their survival. Finally, reconstitution of PLC-1 protein expression in PLC-1-deficient MEF restored cell survival following H₂O₂ treatment. These findings suggest an important protective function for PLC-1 activation during the cellular response to oxidative stress.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As a consequence of our aerobic environment, we are continually exposed to reactive oxygen species. The deleterious and cumulative effects of oxidant injury to macromolecules appear to contribute to the development of a wide variety of disease processes, including diabetes, cancer, and Alzheimer's disease, and are believed to be a major factor in aging (1). For these reasons our laboratory has long been interested in the signaling pathways that mediate the effects of oxidative stress on cellular physiology.

Oxidants activate many signaling pathways, and we and others have demonstrated that growth factor receptors play an important role in initiating cellular responses to oxidative stress (2-6). Following exposure to H₂O₂, both the epidermal growth factor receptor (EGFR)¹ and the platelet-derived growth factor receptor (PDGFR) are activated, leading to their dimerization and autophosphorylation. Furthermore, we have demonstrated that certain proliferation-associated signaling pathways, including those leading to activation of extracellular signal-regulated kinase (ERK) and Akt, are activated by oxidative stress in a growth factor receptor-dependent manner and in such instances promote cell survival (2). Thus, it appears that at least some signaling pathways involved in regulating proliferation also participate in the cellular response to oxidative stress.

Phospholipase C 1 (PLC-1) is an enzyme that is recruited to the membrane following activation of growth factor receptor tyrosine kinases (7-10). As a result of its interaction with these signaling molecules, PLC-1 is activated by a mechanism that relies on tyrosine phosphorylation. Activated PLC-1 cleaves the membrane phospholipid phosphatidylinositol 4,5-bisphosphate generating two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). The former functions to activate protein kinase C (PKC); the latter stimulates the release of Ca²⁺ from internal stores. Through the pleiotropic actions of IP₃ and DAG, PLC-1 participates in the regulation of cellular proliferation and differentiation (8).

Several laboratories have demonstrated that PLC-1 undergoes tyrosine phosphorylation in response to oxidant exposure, but the mechanisms leading to this activation are not well understood (11-13). In addition, the physiologic consequences of PLC-1 activation by stress are unclear. Based on our prior studies demonstrating the importance of certain proliferation-associated signaling pathways in protecting the cell against oxidative stress (2, 14, 15), we hypothesized that PLC-1 might also be important for transducing survival signals resulting from oxidant exposure. To investigate this possibility, we have employed normal mouse embryo fibroblasts (wt MEF), MEF that have been rendered deficient for PLC-1 by targeted disruption of both plc-1 alleles (Plcg1 null), and Plcg1 null MEF in which PLC-1 function has been reconstituted through stable ectopic expression of PLC-1 (Plcg1 null+). Our findings reported herein demonstrate that H₂O₂ is a strong and specific activator of PLC-1, and further implicate PLC-1 and its downstream effectors in conferring protection against oxidative stress.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Hydrogen peroxide (H₂O₂) was purchased from Sigma. Thapsigargin, Go6983, xestospongin C, TPA, ionomycin, PP2, PP3, wortmannin, U73122, AG1478, compound 56, AG1296, and N-acetylcysteine were all obtained from Calbiochem (San Diego, CA). The JNK1 and PLC-1 polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, Inc. (Lake Placid, NY). The phospho-specific ERK rabbit polyclonal antibody was from Promega (Madison, WI). The phospho-specific p38 and phosphospecific Akt polyclonal antibodies were purchased from New England Biolabs, Inc. (Beverly, MA).

Cell Culture, Treatment, and Survival Assays-- Establishment of spontaneously immortalized wt MEF and Plcg1 null, derived from targeting vector I (TV-I), has been described previously (16). PLC-1 was stably re-expressed in plcg1 null MEF using a retroviral expression vector to produce plcg1 null+ MEF (17). Although plcg1 null do not mobilize calcium in response to growth factors, plcg1 null+ do. HeLa and T98G cells were obtained from American Type Culture Collection (Manassas, VA). All cell lines were grown in Dulbecco's modified Eagle's medium (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin, and were maintained in 5% CO₂.

For treatments with H₂O₂, an 8 M stock solution was diluted to a working concentration in deionized water and was added immediately to the culture medium (containing serum). For cell survival assays, cells were plated in 60-mm dishes and cultured overnight prior to treatment with H₂O₂ or other agents. Following treatment, cells were harvested and stained with trypan blue, and live cells were counted using a hemocytometer. The percentage of viable cells in the treatment groups was determined from cell counts in treatment groups divided by cell counts of untreated controls. Thus, a reduction in the number of viable cells in treatment groups reflects cell death and/or inhibition of proliferation.

DAPI Staining-- DAPI staining was performed as described previously (18). In brief, prior to staining, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then washed with phosphate-buffered saline. DAPI was added to the fixed cells for 1 h, after which they were examined by fluorescence microscopy. Apoptotic cells were identified by condensation and fragmentation of nuclei.

Immunoprecipitation and Immunoblot Analysis-- After stimulation, cells were washed in ice-cold phosphate-buffered saline, then harvested in 1 ml of lysis buffer (20 mM Hepes, pH 7.4, 2 mM EGTA, 50 mM -glycerophosphate, 1 mM Na₃VO₄, 5 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin). Equal amounts of protein were incubated with 5 µg of PLC-1 antibody and 35 µl of 50% slurry of protein A-Sepharose for 4 h at 4 °C. Immune complexes were washed four times with the same lysis buffer and were resuspended in 2× sample buffer. For immunoblot analysis, the precipitated proteins were resolved on 4-12% NuPAGE BisTris gels (Novex, San Diego, CA) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20, containing 5% milk, and were then probed with different antibodies. Proteins were detected by using enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech).

JNK Kinase Assays-- JNK activity was measured by an immunocomplex kinase assay as described previously (15). In brief, the cells were lysed in 1 ml of lysis buffer (20 mM Hepes, pH 7.4, 2 mM EGTA, 50 mM -glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 5 mM NaF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Equal amounts of protein samples were precipitated at 4 °C for 4 h with 5 µl of a JNK1 antibody with the addition of 35 µl of 50% slurry of protein A-Sepharose (Amersham Pharmacia Biotech). The protein A-Sepharose beads were washed three times each in lysis buffer and kinase assay buffer (20 mM MOPS, pH 7.2, 2 mM EGTA, 10 mM MgCl₂, 1 mM dithiothreitol, and 0.1% Triton X-100). JNK kinase assays were performed using GST-c-Jun-(1-135) as a substrate. Samples were separated on a 12% SDS-polyacrylamide gel and, after drying, were subjected to autoradiography.

Statistical Analysis-- An unpaired Student's t test was used to assess statistical significance of differences between normal MEF and Plcg1 null MEF. Differences were considered significant for p values of 0.05 or less.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

H₂O₂ Stimulates Tyrosine Phosphorylation of PLC-1-- PLC-1 is a tyrosine kinase substrate, and it is well established that tyrosine phosphorylation of PLC-1 is necessary for its activation (9, 10). To examine the effect of H₂O₂ treatment on PLC-1 phosphorylation, wt MEF were exposed to various doses of the oxidant for 15 min and tyrosine phosphorylation of PLC-1 was examined by precipitating PLC-1 with a PLC-1 antibody, followed by Western blotting analysis with a phosphotyrosine-specific antibody. Western blot analysis with a PLC-1 antibody was used to verify that equal amounts of PLC-1 protein were present in the immunoprecipitates. As shown in Fig. 1A, H₂O₂ treatment resulted in PLC-1 tyrosine phosphorylation in a dose- and time-dependent manner. A significant increase in PLC-1 phosphorylation was detected with H₂O₂ concentrations as low as 200 µM and activation was rapid, occurring within 5 min of treatment. Maximum levels of PLC-1 phosphorylation were recorded 10-20 min after the addition of H₂O₂. It is important to note that, in these experiments, H₂O₂ was added directly to the serum-containing culture medium, as opposed to cells being treated with H₂O₂ while in phosphate-buffered saline. The fact that such low doses of H₂O₂ increase PLC-1 tyrosine phosphorylation, despite the presence of anti-oxidants in the supplemented medium, supports the physiologic relevance of our observations. This response was not unique to MEF as H₂O₂ treatment also led to phosphorylation of PLC-1 in two human cell lines, HeLa and T98G glioblastoma cells (Fig. 1C). To confirm the involvement of reactive oxygen species in PLC-1 phosphorylation, the ability of N-acetylcysteine (NAC), a scavenger of reactive oxygen species and precursor of glutathione (19), to block phosphorylation was assessed. As shown, NAC acted in a dose-dependent manner to inhibit PLC-1 phosphorylation in wt MEF (Fig. 1D).

View larger version (48K): [in this window] [in a new window]   Fig. 1.   H2O2 treatment induces tyrosine phosphorylation of PLC-1. A, phosphorylation of PLC-1 by H2O2 is concentration-dependent. wt MEF were exposed to the indicated concentrations of H2O2 for 15 min. Cell lysates were generated, and PLC-1 was immunoprecipitated from ~500 µg of lysate using a polyclonal PLC-1 antibody. Western blotting was employed to assess the tyrosine phosphorylation of PLC-1 using a phosphotyrosine-specific antibody (top panel). The same membrane was then stripped and re-blotted with a PLC-1 antibody to demonstrate equal loading of protein in each lane (bottom panel). B, phosphorylation of PLC-1 by H2O2 is rapid and sustained. wt MEF were treated with 600 µM H2O2 for indicated times, and PLC-1 phosphorylation was assessed using immunoprecipitation and Western blotting techniques as described above. C, activation of PLC-1 by H2O2 is not specific to MEFs. HeLa and T98G cells were treated with H2O2 for 15 min, and PLC-1 phosphorylation was assayed using immunoprecipitation and Western blotting techniques. D, phosphorylation of PLC-1 by H2O2 is inhibited by the antioxidant NAC. Cells were pretreated with NAC (1 mM or 2 mM) for 30 min prior to the addition of H2O2 (600 µM) to the culture medium. Cells were harvested 15 min later for assessment of PLC-1 phosphorylation. All experiments were repeated three times, and data from one representative experiment are shown.

Upstream Mediators of PLC-1 Phosphorylation by H₂O₂ Treatment-- We and others have previously provided evidence that growth factor receptors, including EGFR and PDGFR, play an important role in mediating the activation of ERK and Akt in response to oxidant injury (2, 3, 5). Since PLC-1 is also strongly activated by growth factor stimulation of these receptors, we investigated their involvement in mediating PLC-1 activation following H₂O₂ treatment. As shown in Fig. 2A, AG1478 and compound 56, selective inhibitors of EGFR tyrosine kinase activity (20, 21), prevented PLC-1 tyrosine phosphorylation in response to H₂O₂ treatment. In contrast, AG1296, a specific inhibitor of PDGFR tyrosine kinase activity, had no effect on PLC-1 phosphorylation, although these cells have been shown to express functional PDGFRs (17). These findings suggest a role for EGFR, but not PDGFR in mediating PLC-1 phosphorylation by oxidative stress. Previous studies have shown that Src family tyrosine kinases are involved in signaling events stimulated by reactive oxygen species (22-24) and Src kinase family members have also been demonstrated to phosphorylate PLC-1 and PLC-2 in vitro (25, 26). To address the role of Src family kinases in H₂O₂-mediated phosphorylation of PLC-1, we utilized the selective Src family tyrosine kinase inhibitor PP2. Phosphorylation of PLC-1 by H₂O₂ was completely blocked by the presence of 10 µM PP2. Similar treatment with the same concentration of the inactive analog PP3 had no effect on PLC-1 phosphorylation by H₂O₂, indicating that the effect of PP2 was selective.

View larger version (58K): [in this window] [in a new window]   Fig. 2.   Upstream mediators of PLC-1 phosphorylation by H2O2 treatment. A, inhibition of H2O2-induced PLC-1 tyrosine phosphorylation by pretreatment with EGFR inhibitors. Cells were pretreated with various inhibitors or a solvent control for 30 min prior to treatment with H2O2 (600 µM, 15 min). PLC-1 phosphorylation was assayed by Western blotting. B, wt MEF were pretreated with the indicated doses of PP2 or its inactive analog PP3 for 30 min prior to H2O2 (600 µM) treatment. Lysates were prepared from cells 15 min after treatment with H2O2 and were subjected to immunoprecipitation (IP) with a PLC-1 antibody. Precipitates were then analyzed by immunoblotting (IB) using antibodies specific for either phosphotyrosine or PLC-1. C, inhibition of PI3-K has no effect on tyrosine phosphorylation of PLC-1 by H2O2. wt MEF were treated with the PI3-K inhibitor wortmannin (200 nM) for 30 min prior to treatment with H2O2 (600 µM), and cell lysates were prepared 15 min later. Phosphorylation of PLC-1 was assayed using immunoprecipitation and immunoblotting techniques, as described above. All experiments were repeated three times, and data from one representative experiment are shown.

Several studies have implicated PI3-K in the activation of PLC-1 (27-29), but others have not (30). As we have demonstrated previously that the PI3-K/Akt signaling pathway is activated in response to H₂O₂ treatment (2), we used the PI3-K inhibitor wortmannin to test the possibility that PI3-K is involved in mediating PLC-1 phosphorylation in response to H₂O₂. As shown in Fig. 2C, inhibition of PI3-K had no effect on the tyrosine phosphorylation of PLC-1 by H₂O₂. Consistent with this, wortmannin does not affect calcium mobilization in response to H₂O₂ (data not shown).

Influence of PLC-1 Status on Cell Survival following Oxidant Challenge-- H₂O₂ acts in a concentration-dependent manner to induce apoptosis in a variety of cell types (15, 31-33), and a number of different signaling pathways have been shown to be involved in controlling cellular sensitivity to oxidant injury. To explore the role of PLC-1 in influencing cell survival following H₂O₂ treatment, we compared the sensitivity of wt MEF and Plcg1 null MEF to various concentrations of the oxidant. Trypan blue exclusion was employed to assess viability 24 h after treatment. As shown in Fig. 3, treatment with 300 µM H₂O₂ inhibited the growth of both wt and Plcg1 null MEF, resulting in a reduction in the number of viable cells relative to untreated (control) cultures. At higher concentrations (450-600 µM), H₂O₂ became markedly cytotoxic for Plcg1 null MEF, with less than 1% of cells surviving treatment with 600 µM H₂O₂. However, little evidence of cytotoxicity was observed in wt MEF (Fig. 3, bottom panel; see also Fig. 4B and Fig. 7C). This differential sensitivity of wt and Plcg1 null MEF to H₂O₂ appears to be specific for oxidative stress as wt and Plcg1 null MEF did not differ in their susceptibility to another stressor, thapsigargin. Thapsigargin disrupts calcium homeostasis by inhibiting calcium ATPases on the membrane of the endoplasmic reticulum, which triggers a unique signaling cascade referred to as the unfolded protein response (34). Depending on the cell type, thapsigargin induces growth arrest and/or cell death. Thapsigargin had the same effect on wt and Plcg1 null MEF; although some growth arrest was observed, the treatment was toxic and resulted in significant cell death in both cell lines (Fig. 3, bottom panel). Consistent with the lack of a role for PLC-1 in influencing the responsiveness of MEF to thapsigargin treatment, no PLC-1 phosphorylation was evident in wt MEF following treatment with the agent (Fig. 3, top panel).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   PLC-1 protects cells from H2O2-induced death. Top panel, wt MEF and Plcg1 null MEF were treated with H2O2 (600 µM) and thapsigargin (500 nM) and cell lysates were prepared 15 min later. Western blotting techniques were then used to assess the tyrosine phosphorylation status of PLC-1. Bottom panel, cells were treated with indicated doses of H2O2 or thapsigargin for 24 h and surviving cells (those able to exclude trypan blue) were counted. Data are reported as percentage of survival relative to control (untreated) cells. Black bars correspond to wt MEF, and white bars correspond to Plcg1 null MEF. Data are means of at least three independent experiments, and error bars correspond to standard errors of the mean. Asterisk (*) denotes significant differences in values of wt MEF and Plcg1 null MEF determined using an unpaired Student's t test.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   H2O2 rapidly induces death in Plcg1 null MEF. A, wt MEF and Plcg1 null MEF were treated with 600 µM H2O2 for the indicated times, and surviving cells (those able to exclude trypan blue) were counted. Dramatic sensitivity of Plcg1 null MEF was observed as early as 4 h after addition of H2O2 to culture medium. Data are expressed as percentage of survival relative to control (untreated) cells, and values are means of three or more independent experiments. Error bars represent standard errors of the mean. B, representative DAPI staining of wt and Plcg1 null MEF, subjected to no treatment or treated with 600 µM H2O2 for 8 h. Nuclei of apoptotic cells are fragmented and condensed.

Examination of the kinetics of the response to 600 µM H₂O₂ treatment revealed that the onset of death occurred rapidly in Plcg1 null MEF (Fig. 4A). Within 4 h, these cells began to shrink, round up, and detach from the plate, and by 8 h less than 1% of the cells remained viable. The wt MEF, on the other hand, remained attached to the plate, but took on a more flattened, spread-out appearance. Thus, the growth inhibitory effects of H₂O₂ were already apparent at early time points. Plcg1 null MEF stained with DAPI displayed features typical of apoptosis including condensation and fragmentation of nuclei (Fig. 4B). In contrast, nuclei from H₂O₂-treated wt MEF remained homogeneously stained with little evidence of fragmentation (Fig. 4B).

H₂O₂-induced Activation of Mitogen-activated Protein Kinases (MAPK) and Akt Do Not Rely on PLC-1-- We have previously demonstrated that both the MAPK (including ERK, JNK, and p38 members) and PI3-K/Akt signaling pathways are activated in response to H₂O₂ treatment and influence cell survival (2, 15). To rule out the possibility that the differential sensitivity of wt MEF and Plcg1 null MEF to H₂O₂ might be attributed to effects of PLC-1 on one or more of these pathways, we examined whether the PLC-1 status affected activation of these kinases in response to oxidant treatment. wt MEF and Plcg1 null MEF were harvested at various times after exposure to 600 µM H₂O₂, and examined for ERK, p38, and Akt activation by Western blot analysis using phosphospecific ERK, p38, and Akt antibodies. JNK activity was assessed by an immunocomplex kinase assay using GST-c-Jun as a substrate. As shown in Fig. 5, no significant differences in the levels of ERK, JNK, or Akt phosphorylation were noted between wt MEF and Plcg1 null MEF. Although p38 activation was actually higher in the Plcg1 null MEF relative to wt MEF, neither SB202190 nor SB203580 (pharmacologic inhibitors of p38) affected MEF cell survival in response to H₂O₂ treatment (data not shown). Thus, the influence of PLC-1 on cell survival appears to be independent of MAPK and PI3-K/Akt signaling pathways.

View larger version (53K): [in this window] [in a new window]   Fig. 5.   H2O2-induced activation of MAPK and Akt does not rely on PLC-1. Cell lysates were prepared from wt and Plcg1 null MEF treated with 600 µM H2O2 for the indicated times. JNK1 activity was determined by an immune complex kinase assay using GST-c-Jun-(1-135) as substrate. Phosphorylation of ERK, p38, and Akt was assessed using immunoblotting techniques with phospho-ERK, phospho-p38, and phospho-Akt specific polyclonal antibodies. All assays were repeated three times, and data from one representative experiment are shown.

PLC-1 Function Is Required for Its Pro-survival Role in Response to H₂O₂-- Our results thus far suggest that PLC-1 serves a pro-survival function during H₂O₂ treatment. If so, then inhibition of PLC-1 activity would be expected to increase the sensitivity of wt MEF cells to H₂O₂. To test this possibility, wt MEF were treated with U73122, a selective inhibitor of phosphoinositide-specific PLC function, prior to challenge with H₂O₂. As shown in Fig. 6A, 30 min of pretreatment with 5 µM U73122 did significantly enhance the sensitivity of wt MEF to H₂O₂ (Fig. 6A). U73122 treatment in the absence of H₂O₂ had no effect on the cells (data not shown). The inactive analog of U73122, known as U73433, could not be included in these survival assays because it was toxic to both the wt and Plcg1 null MEF.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Pharmacologic inhibition of PLC-1 increases susceptibility wt MEF to H2O2-induced death. A, wt MEF were pretreated with vehicle control or the PLC-1 inhibitor U73122 (5 µM, 30 min) followed by H2O2 (600 µM, 24 h) and were compared with Plcg1 null MEF treated with H2O2 (600 µM, 24 h). Cells were harvested and counted, and survival was calculated relative to untreated cells. Survival of wt MEF was significantly reduced as a result of U73122 pretreatment. B, inhibitors of calcium release from the endoplasmic reticulum and PKC activity increase susceptibility of wt MEF to H2O2. wt MEF were treated with indicated doses of H2O2 (24 h) following pretreatment with vehicle control or a combination of XeC (1 µM) and Go6983 (5 µM). C, pharmacologic stimulation of calcium release and PKC activity enhance survival of H2O2-treated Plcg1 null MEF. Plcg1 null MEF were treated with indicated doses of H2O2 (24 h) in the absence or presence of pretreatment with TPA (100 ng/ml, 30 min) and ionomycin (2 µM, 30 min). In all experiments cells were harvested, stained with trypan blue, and counted. Percentage of survival is reported as the number of viable cells relative to control groups. All data are averages of at least three independent experiments and are reported along with standard errors of the mean. Asterisk (*) denotes significant difference in the value of wt MEF with and without pretreatment with pharmacologic inhibitors of PLC-1 or Plcg1 null with and without pretreatment with pharmacologic agents that mimic PLC-1 function.

PLC-1 catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate the second messenger molecules IP₃ and DAG. The former provokes a transient increase in intracellular free Ca (2+), whereas the latter serves as a direct activator of PKC. It is through these second messengers that PLC-1 exerts its influence on proliferation. To examine their importance in influencing cell survival in response to oxidative stress, two complementary approaches were employed. First, we pretreated wt MEF with a combination of XeC (an IP₃ receptor blocker) and Go6983 (a PKC inhibitor) to abrogate the PLC-1-mediated increases in intracellular Ca²⁺ and PKC activity. Cell survival was assessed 24 h later by trypan blue dye exclusion. As expected, the addition of XeC and Go6983 partially reduced the survival of wt MEF following exposure to H₂O₂ (Fig. 6B), although treatment with XeC and Go6983 in the absence of H₂O₂ had no effect on the cells (data not shown). In the second approach, Plcg1 null MEF were treated with pharmacologic agents that act downstream of PLC-1 to increase intracellular Ca²⁺ (ionomycin) and activate PKC (TPA). Consistent with the hypothesis that these PLC-1 effector functions are important in promoting cell survival during oxidant injury, the combination of ionomycin and TPA markedly improved survival of H₂O₂-treated Plcg1 null MEF.

Taken together, the experiments described above argue strongly that PLC-1 activation exerts a pro-survival influence during the cellular response to oxidant injury. As a final test of this hypothesis, we examined how reconstitution of PLC-1 expression in Plcg1 null MEF would affect their response to H₂O₂ treatment. Plcg1 null cells with restored PLC-1 expression (Plcg1 null+) were generated previously and have been shown to exhibit normal PLC-1 function in response to proliferative signals (17, 30). As shown in Fig. 7A, the ectopically expressed PLC-1 in these cells undergoes tyrosine phosphorylation in response to H₂O₂ treatment similar to that seen in wt MEF. Next, we compared the sensitivity of wt MEF, Plcg1 null, and Plcg1 null+ MEF to various doses of H₂O₂. As depicted in Fig. 7B, reconstitution of PLC-1 expression in the Plcg1 null MEF rescued them from H₂O₂-induced cytoxicity, resulting in cell viability essentially identical to that seen in wt MEF. As seen in Fig. 7C, morphologic examination of H₂O₂-treated wt MEF, Plcg1 null, and Plcg1 null+ MEF confirmed the viability measurements obtained by trypan blue dye exclusion in Fig. 7B, indicating that PLC-1 function is important in mediating resistance to harmful effects of H₂O₂.

View larger version (52K): [in this window] [in a new window]   Fig. 7.   Restoration of PLC-1 expression in Plcg1 null MEF protects against H2O2. A, PLC-1 protein is phosphorylated by H2O2 in Plcg1 null+ MEF. Cells were treated with H2O2 (600 µM, 15 min), and PLC-1 phosphorylation was assayed by Western blot analysis. B, wt, Plcg1 null, and Plcg1 null+ MEF were treated with the indicated doses of H2O2 for 24 h, and surviving cells (those able to exclude trypan blue) were counted. Percentage of survival is reported relative to untreated controls. Data represent averages from at least three independent experiments. Error bars correspond to standard errors of the mean. Asterisk (*) denotes significant difference in the value of Plcg1 null compared with wt MEF. Differences between survival rates of Plcg1 null+ and wt MEF are not statistically significant (p > 0.5). C, representative phase contrast micrographs of wt, Plcg1 null, and Plcg1 null+ MEF that were either left untreated or were treated with 600 µM H2O2 for 24 h. Nuclei of apoptotic cells are fragmented and condensed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PLC-1 is known to play an important role in regulating cell proliferation through its interactions with both receptor and non-receptor tyrosine kinases. However, despite the existence of significant overlap between proliferative and stress signaling pathways, a role for PLC-1 in mediating cellular responses to stress has not been appreciated. Here we have demonstrated that PLC-1 is tyrosine-phosphorylated in response to H₂O₂ treatment through Src family kinases and/or an EGFR-dependent mechanism. In addition, using Plcg1 null MEF and Plcg1 null MEF in which PLC-1 function is restored, we have provided evidence that PLC-1 expression supports cell survival following acute oxidant injury. Taken together, these findings implicate PLC-1 as an important regulatory molecule in the cellular response to oxidative stress.

That PLC-1 undergoes phosphorylation on tyrosine residues in response to H₂O₂ treatment has been reported by others (11-13). However, most of the previous studies have employed high doses of H₂O₂ (in excess of 1 mM, and usually between 5 and 10 mM) and/or have required co-treatment with the phosphatase inhibitor pervanadate to observe phosphorylation. Such observations suggest that H₂O₂ is, at best, a weak activator of PLC-1. In addition, the use of such high concentrations of H₂O₂ raises questions concerning the biologic relevance of the response, as they can be markedly cytotoxic for cells, leading to rapid necrosis. Our current studies have employed much lower concentrations of H₂O₂, and it is added to cells in the presence of serum, which contains significant anti-oxidant activity. We have demonstrated that, at concentrations as low as 200 µM, treatment with H₂O₂ results in PLC-1 phosphorylation, with concentration-dependent increases occurring over a range of 200-1000 µM in MEF (Fig. 1, A and B). Survival of MEF deficient in PLC-1 function is severely compromised over the same dose-response range of H₂O₂ (Fig. 3), which strongly supports the biologic relevance of PLC-1 phosphorylation and the role of PLC-1 in protecting cells against H₂O₂-induced cell death.

A number of questions remain concerning the mechanisms leading to PLC-1 phosphorylation in response to oxidant injury, although our studies implicate both the EGFR and Src family kinases in the process. Previously we proposed that oxidants might act to mimic the action of EGF (and perhaps other growth factors) leading to elevated EGFR kinase activity (2, 5). This in turn triggers the activation of other signaling molecules including non-receptor type tyrosine kinases, such as members of the Src family. Indeed, PLC-1 phosphorylation occurs rapidly in response to oxidant treatment, over the same time frame as that seen for phosphorylation of EGFR by EGF and H₂O₂ (2, 35). However, in response to certain stimuli, Src kinase activation has been found to lead to phosphorylation of the EGFR (36). Therefore, it is certainly possible that H₂O₂ acts in a similar fashion, first activating Src kinases, followed by activation of the EGFR.

It has been suggested that the increase in tyrosine phosphorylation is due, at least in part, to inactivation of phosphatases by H₂O₂, a mechanism that assumes a certain basal level of tyrosine kinase activity in the absence of overt stimulation. In support of this view, H₂O₂ has been shown to reversibly inactivate protein-tyrosine phosphatase 1B in cells and contribute to EGFR phosphorylation in response to EGF treatment (37). Direct activation of tyrosine kinases and inactivation of phosphatases by oxidants are not mutually exclusive events. Rather, they are both likely to influence phosphotyrosine levels and therefore the activities of upstream kinases that ultimately regulate PLC-1 phosphorylation. However, oxidant-mediated inactivation of a phosphatase involved in the regulation of tyrosine phosphorylation on PLC-1 is obviously not sufficient to produce dramatic increase in tyrosine phosphorylation of PLC-1 that results from H₂O₂ treatment.

A major conclusion of our studies is that PLC-1 has a pro-survival function in the cell's response to acute oxidative stress. Consistent with our observations, overexpression of PLC-1 in rat pheochromocytoma PC12 cells was reported to inhibit apoptosis induced by short wave length ultraviolet radiation (UVC) (38). UVC is believed to exert its cellular affects, at least in part, through generation of oxidative stress. However, a subsequent report showed no protective influence of PLC-1 overexpression against several different treatments including H₂O₂ in NIH3T3 cells, although PLC-1 overexpression did favor survival (39). Interpretation of the significance of PLC-1 expression levels in the previous studies is complicated by the fact that PLC-1 was overexpressed in cells with otherwise normal PLC-1 activity. Our approach, which utilized MEF derived from embryos in which Plcg1 expression has been eliminated through targeted gene disruption, avoided such complications and further allowed us the opportunity to evaluate how restoration of PLC-1 function in the Plcg1 null fibroblasts affected their response to H₂O₂. We observed that ectopic expression of PLC-1 in Plcg1 null MEF conferred resistance to H₂O₂ equivalent to that seen in wt MEF (Fig. 7, B and C). That PLC-1 may have a similar role in other stress circumstances is indicated by the finding that when grown in suspension, Plcg1 null are more sensitive to death than are Plcg1 null+.²

It is not clear how PLC-1 protects cells from oxidant injury. PLC-1 exhibits its influence on proliferation largely through the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol, which in turn provoke the mobilization of Ca²⁺ and activate protein kinase C, respectively. That these downstream events also contribute to PLC-1's influence on cell survival following H₂O₂ treatment was evidenced by the finding that inhibitors of PKC and Ca²⁺ mobilization rendered wt MEF more sensitive to H₂O₂ treatment, whereas pharmacologic activation of PKC and Ca²⁺ mobilization in Plcg1 null MEF enhanced their survival. Importantly, the pharmacologic agents were less effective modulators of survival in oxidant-treated cells than was manipulation of Plcg1 expression. This may be a reflection of the expected limitations of the pharmacologic approach (due to, for example, the degree of inhibition or activation achieved and the specificity of the agents actions). However, it could reflect additional, unrecognized roles of PLC-1. In this regard it is also interesting to note that wt and Plcg1 null MEF do not differ in their mitogenic response to EGF stimulation (40). The marked differences in sensitivity to oxidant injury observed in wt MEF as compared with Plcg1 null MEF may thus be attributable to a unique function of PLC-1 during stress. Attempts to identify downstream targets of PLC-1 activation that are involved in mediating stress resistance are currently under way.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

Supported by National Institutes of Health Grant CA75195.

^** To whom all correspondence should be addressed: Laboratory of Cellular and Molecular Biology, Box 12, NIA, National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8446; Fax: 410-558-8386.

Published, JBC Papers in Press, May 11, 2001, DOI 10.1074/jbc.M102693200

² A. Chattopahadhyay and G. Carpenter, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: EGFR, epidermal growth factor receptor; MEF, mouse embryonic fibroblast; PLC, phospholipase C; NAC, N-acetylcysteine; PDGFR, platelet-derived growth factor receptor; XeC, xestospongin C; TPA, 12-O-tetradecanoyl phorbol-13-acetate; IP₃, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; DAPI, 4,6-diamidino-2-phenylindole; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; PI3-K, phosphatidylinositol 3-kinase; GST, glutathione S-transferase; wt, wild type; EGF, epidermal growth factor; PP2, 4amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine; PP3, 4amino-7-phenylpyrazol[3,4-D]pyrimidine; MOPS, 4-morpholinepropanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

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