Suramin sodium

ABSTRACT

Singlet oxygen (^sup 1^O2)-induced cytotoxicity is believed to be responsible for responses to photodynamic therapy and for apoptosis of T helper cells after UV-A treatment. Other cytotoxic oxidants, such as hydrogen peroxide and peroxynitrite have been shown to stimulate cell survival signaling pathways in addition to causing cell death. Both these oxidants stimulate the Akt/protein kinase B survival signaling pathway through activation of membrane tyrosine kinase growth factor receptors. We evaluated the ability of ^sup 1^O2 to activate the Akt/ protein kinase B pathway in NIH 3T3 cells and examined potential activation pathways. Exposure of fibroblasts to ^sup 1^O2 elicited a strong and sustained phosphorylation of Akt, which occurred concurrently with phosphorylation of p38 kinase, a proapoptotic signal. Inhibition of phosphatidylinositol-3-OH kinase (PI3-K) completely blocked Akt phosphorylation. Significantly, cell death induced by ^sup 1^O2 was enhanced by inhibition of PI3-K, suggesting that activation of Akt by ^sup 1^O2 may contribute to fibroblast survival under this form of oxidative stress. ^sup 1^O2 treatment did not induce phosphorylation of platelet-derived growth factor receptor (PDGFR) or activate SH-PTP2, a substrate of growth factor receptors, suggesting that PDGFR was not activated. In addition, specific inhibition of PDGFR did not affect Akt phosphorylation elicited by ^sup 1^O2. Activation of neither focal adhesion kinase (FAK) nor Ras protein, both of which mediate responses to reactive oxygen species, appeared to be pathways for the ^sup 1^O2-induced activation of the PI3-K-Akt survival pathway. Thus, activation of Akt by ^sup 1^O2 is mediated by PI3-K and contributes to a survival response that counteracts cell death after ^sup 1^O2-induced injury. However, unlike the response to other oxidants, activation of the PI3-K-Akt by ^sup 1^O2 does not involve activation of growth factor receptors, FAK or Ras protein.

Abbreviations: cAmp, cyclic adenosine 3':5'monophosphate; CS, calf serum; DAPI, 4',6'-diamidino-2-phenylindole; DMEM, Dulbecco modified Eagle medium; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated protein kinase; FAK, focal adhesion kinase; GPCR, G-protein-coupled receptors; JNK, cJun-N-terminal kinase; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; ^sup 1^O2, singlet oxygen; ONOO^sup -^ peroxynitrite; PBS, phosphate-buffered saline; PDGFR, platelet-derived growth factor receptor; PI3-K, phosphatidylinositol-3-OH kinase; RB, rose bengal; RNS, reactive nitrogen species; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; TPA, 12-o-tetradecanoyl-phorbol-13-acetate.

INTRODUCTION

Reactions of reactive oxygen and nitrogen species (ROS-RNS) with cellular molecules can lead to oxidative stress, a condition in which the level of ROS-RNS exceeds the cell's antioxidant capacity. Cell death by apoptotic or nonapoptotic pathways is frequently caused by oxidative stress. ROS-RNS are produced by physical and chemical stresses such as ionizing and UV radiations, photosensitization or exposure to redox cycling chemicals and include hydrogen peroxide, singlet oxygen (^sup 1^O2), peroxynitrite (ONOO^sup -^) as well as superoxide anion, hydroxyl radical and nitric oxide. These species contribute to the responses of cells and tissue to environmental agents as well as mediate radiation and photodynamic therapies. Studies in the last few years have shown that oxidative stress may also stimulate cell survival responses. For example, ROS are produced in response to binding of growth factors and cytokines to their membrane receptors and activate subsequent intracellular signaling pathways leading to enhanced cell survival (1,2). The range of cellular responses to ROS-RNS from cytotoxicity to enhanced survival is attributed at least partly to differences in ROS-RNS concentrations-higher concentrations are associated with cytotoxicity, whereas lower concentrations are involved in intracellular signaling pathways that may lead to increased cell survival (2). In addition to differences in ROS-RNS concentrations, the responses of cells are influenced by differences in the chemical reactivity between types of ROS-RNS and by the subcellular location where these species are formed. Greater understanding of these differences may allow increased control of the ROS-RNS-induced responses and expand the use of these species in therapeutic strategies.

^sup 1^O2 is an ROS formed by transfer of energy from an excited state photosensitizer to ground state oxygen. The apoptotic and nonapoptotic cytotoxicity mechanisms initiated by ^sup 1^O2 are believed to be responsible for tumor cell killing in photodynamic therapy and for T helper cell death in UV-A radiation skin therapy (3,4). Many studies have probed the mechanisms whereby ^sup 1^O2 initiates apoptosis. We have demonstrated that generation of ^sup 1^O2 by photosensitization induces activation of the mitogen-activated protein kinase (MAPK) p38 and of caspase-8 and that activation of these species is required for apoptosis of HL-60 cells (5,6). Other studies have shown that the MAPK, the cJun-N-terminal kinase (JNK) as well as the p38 kinase are activated and involved in apoptosis after ^sup 1^O2 treatment in several cell types (7-10). Mitochondrial damage that is induced by ^sup 1^O2 and leads to apoptosis has recently been shown to be mediated by oxidation of Bcl-2 (11,12).

H^sub 2^O^sub 2^ and ONOO^sup -^, in addition to initiating cytotoxicity, stimulate the activation of survival signaling sequences such as the phosphatidylinositol-3-OH kinase (PIS-K)-Akt pathway (13,14) and the MAPK MEK-extracellular signal-regulated protein kinase (ERK) pathway (15-17). Activation of these pathways leads to production or activation of transcription factors such as AP-1 (via the ERK pathway) and members of the forkhead family of transcription factors and NF-[kappa]B (via the Akt pathway). Akt (also called protein kinase B) also promotes survival by inactivation of the Bcl-2 homolog Bad and of caspase-9 (18). Activation of Akt and ERK1/2 by H^sub 2^O^sub 2^ increases cell survival after injury induced by this oxidant (16,19). ONOO^sup -^ also activates these two pathways, although the functional significance remains clear (14,15). Both H^sub 2^O^sub 2^ and ONOO^sup -^ initiate these survival pathways by activating growth factor receptors such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) (14-16,19). Akt activation is subsequent to binding of PI3-K to phosphorylated growth factor receptors. PI3-K generates phosphorylated phosphoinositides in the plasma membrane, and Akt binds to these phospholipids, becomes phosphorylated and then participates in downstream signaling. Other potential pathways by which ROS-RNS might initiate these survival signaling pathways involve focal adhesion kinase (FAK) and Ras protein. FAK is a nonreceptor protein tyrosine kinase that mediates signal transduction by integrins and has also been shown to operate upstream of PI-3K-Akt signaling pathway in H^sub 2^O^sub 2^-induced apoptosis (20). Ras has been shown to associate with PI3-K and induce Akt activation in response to oxidative stress (21).

In contrast to H^sub 2^O^sub 2^, O2 generally does not activate ERK1/2. This result has been found in HL-60 cells in our studies (5) and by others using HaCaT, dermal fibroblasts and HeLa cells (7,8,22). This raises the question of whether ^sup 1^O2 is able to activate cell survival pathways. Specifically, it is not known whether ^sup 1^O2 has the ability to activate the PI3-K-Akt pathway. Support for connection between ^sup 1^O2 and Akt activation is indirect. ^sup 1^O2 has been shown to induce activation of NF-[kappa]B, a transcription factor that mediates antiapoptotic signaling following many stimuli including oxidant injury (23-25), and both PI3-K-Akt and MEK-ERK pathways mediate activation of NF-[kappa]B by multiple factors (26-28). In addition, NF-[kappa]B was identified to be a target of Akt in the antiapoptotic PDGF signaling (29).

The purpose of this study was to determine whether ^sup 1^O2 activates the PI3-K-Akt survival signaling pathway and, if so, to examine potential activation pathways. Our results in NIH 3T3 fibroblasts show that ^sup 1^O2 rapidly induces phosphorylation of Akt and that the proapoptotic p38 kinase is activated with similar kinetics. Activation of Akt by ^sup 1^O2 is dependent on PI3-K, but in marked contrast to other ROS-RNS, the activation is not dependent on growth factor receptors, FAK or Ras. Our results also demonstrate that ^sup 1^O2-induced activation of the P13-K-Akt pathway promotes cell survival but the mechanism does not involve suppression of p38 kinase activity.

MATERIALS AND METHODS

Materials. 4',6'-Diamidino-2-phenylindole (DAPI) was from Roche (Basel, Switzerland). Anti-phospho-PDGFR[beta] (Y716) and anti-phospho-FAK (Tyr397) were from Upstate Biotechnology Inc. (Lake placid, NY). Rabbit anti-phospho-p38, anti-p38, anti-phospho-Akt (serine 473), anti-Akt and PD98059 were from Cell Signaling Technology (Beverly, MA). Anti-PDGFR, anti-SH-PIP2 and anti-FAK were from Santa Cruz Biotechnology Inc (Santa Cruz, CA). PDGF-BB and EGF were purchased from R&D Systems (Minneapolis, MN). Rose Bengal (RB); wortmannin, LY294002; 12-o-tetradecanoyl-phorbol-13-acetate (TPA) and H^sub 2^O^sub 2^ were from Sigma-Aldrich (St. Louis, MO). AG 1295 and suramin were from Calbiochem-Novabiochem (San Diego, CA).

Cell culture and treatments. NIH 3T3 cells, a murine fibroblast cell line, were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in Dulbecco modified Eagle medium (DMEM) containing 7% calf serum (CS). Cells at 80% confluence were growth arrested by incubation in DMEM with 0.5% CS for 24 h before use. When required, cells were pretreated with various inhibitors before irradiation, and then the medium with and without inhibitors was collected. After irradiation, the collected medium was added back to the dishes, and cells were incubated for additional times as indicated in figure legends.

Generation of ^sup 1^O2. For generation of ^sup 1^O2, cells were incubated with 3 [mu]M RB for 15 min in phosphate-buffered saline (PBS) without Ca^sup 2+^ and Mg^sup 2+^ and then exposed to 180 mJ/cm^sup 2^ visible light (514 nm) provided by a continuous wave argon laser (Innova 100, Coherent) unless indicated otherwise. The irradiance was 30 mJ/cm^sup 2^. DMEM with 0.5% CS was added to the cells after irradiation.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cells were treated with RB plus visible light and then incubated in DMEM with 0.5% CS for the indicated times before replacement by the same medium containing 0.5 [mu]MMTT. After addition of MTT to the culture and incubation for an additional 30 min, the medium was aspirated and 2 mL of dimethyl sulfoxide was added. The optical density at 554 nm was determined with a spectrophotometer.

Determination of apoptosis. Cells were seeded on coverslips. After treatments and incubation, cells were washed once with PBS, fixed with methanol and then stained with DAPI. Cells with condensed nuclei were considered to be apoptotic. Five hundred cells remaining on the coverslips were counted for each sample under the fluorescent microscope, and the number of apoptotic cells was expressed as the percentage of the total cell population.

Western blotting. The procedures for performing Western blotting were the same as described previously (30). Briefly, equal amounts of proteins were loaded in lanes, separated on 10% polyacrylamide gels and transferred to polyvinylidene flouride membranes. After incubating with 5% skim milk over night at 4[degrees]C, the membranes were blotted with specific antibodies as needed. The immunocomplexes were detected using enhanced chemiluminescence (Amersham, Piscataway, NJ).

Immunprecipitation. Cells were washed with PBS and lysed in 200 [mu]L of ice-cold lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM phenylmethanesulfonyl flouride, 10 mM NaF, 10 [mu]g/mL apoptinin and 200 [mu]M Na^sub 3^VO^sub 4^ for 15 min. After removal of nuclei by centrifugation, supernatants were incubated with the specific antibody at 4[degrees]C for 1 h followed by addition of protein G-Sepharose for 1 h. The resulting precipitates were washed five times with lysis buffer and then boiled for 5 min in 1x sodium dodecyl sulfate (SDS) buffer. Supernatants were resolved on 10% polyacrylamide gels, and proteins on the membrane were analyzed as described previously using individual antibodies.

RESULTS

^sup 1^O2 decreases cell viability and induces apoptosis in NIH 3T3 cells

To observe the effect of ^sup 1^O2 on the survival of fibroblasts, we exposed NIH 3T3 cells to 514 nm light in the presence of 3 [mu]M RB. Cell viability was evaluated by the MTT assay. Figure 1A shows that ^sup 1^O2 caused a rapid decrease in cell viability, which occurred in a time-dependent fashion after photosensitization. After 2 h of incubation, the viability decreased to approximately 70% of the control value. Maximum decrease was observed at 4 h, the longest time examined in this study.

Cell death induced by ^sup 1^O2 showed a typical morphological characteristic of apoptosis, nuclear condensation, as observed after DAPI staining (compare Fig. 1B and D). At the end of 2 h of incubation after photosensitization with 3 [mu]M RB plus 180 mJ/cm^sup 2^, about 35% of the cells became apoptotic, whereas less than 5% cells with condensed nuclei were observed in cells treated with light only or RB only (Fig. 1C). Cleavage of DNA after ^sup 1^O2 treatment was detected by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (data not shown). These data suggest that ^sup 1^O2 can induce a rapid apoptotic process in fibroblasts.

^sup 1^O2 stimulates phosphorylation of Akt

The fate of cells after exposure to a toxic insult appears to be determined by the balance of apoptotic, necrotic and survival pathways. Some ROS such as H^sub 2^O^sub 2^ and ONOO^sup -^ activate PI3-K-Akt, a common survival pathway (14,19), and this activation by H^sub 2^O^sub 2^ has been shown to enhance survival in fibroblasts (19). To determine whether ^sup 1^O2 treatment initiates a similar response, we examined phosphorylation of Akt in NIH 3T3 cells after treatment with RB and 514 nm light. Figure 2A shows that ^sup 1^O2 induced the phosphorylation of Akt as demonstrated by immunoblotting using an antibody against Akt phosphorylated on ser473. The phosphorylation of Akt occurred in a light dose-dependent manner, with maximum effect at 180 mJ/cm^sup 2^. Higher light doses led to a gradual decrease in Akt phosphorylation. A time-course study revealed that ^sup 1^O2-induced Akt phosphorylation occurred within 5 min, reached the maximal level at 30 min and then persisted until at least 120 min (Fig. 2B). Akt phosphorylation in response to PDGP and H^sub 2^O^sub 2^ was used as positive control. A strong and rapid induction of Akt phosphorylation was observed after PDGF or ^sup 1^O2 exposure (Fig. 2C,D).

Previously, we demonstrated that p38 is activated and involved in apoptosis during oxidant injury caused by ^sup 1^O2 in HL-60 cells (5). To determine whether this proapoptotic signal is activated concurrently with Akt phosphorylation, we examined the phosphorylation status of p38 in cells exposed to ^sup 1^02 and H^sub 2^O^sub 2^. Figure 28 shows that ^sup 1^0^sub 2^ induced a strong phosphorylation of p38, which occurred with kinetics that were similar to those observed for Akt phosphorylation. H^sub 2^O^sub 2^ treatment also induced p38 phosphorylation but with slower kinetics than those observed for Akt (Fig. 2D). These results indicate that ^sup 1^0^sub 2^ can simultaneously trigger both survival and death signals in nbroblasts.

Induction of Akt phosphorylation by ^sup 1^O2 is dependent on PI3-K

Akt is generally activated through a P13-K-mediated mechanism, although it is also activated independent of P13-K under some circumstances (31). Other oxidants, H^sub 2^O^sub 2^ and ONOO^sup -^ , induce Akt activation by a PI3-K-dependent mechanism (14,19). To assess whether PI3-K is also involved in the activation of Akt by ^sup 1^02, cells were pretreated with wortmannin or LY 294002 and then exposed to ^sup 1^O2. As shown in Fig. 3A,C, both inhibitors completely blocked ^sup 1^O2-induced Akt phosphorylation. Consistent with previous observations (19), H^sub 2^O^sub 2^-induced phosphorylation of Akt was also sensitive to PI3-K inhibitors in fibroblasts (data not shown). These data suggest that the phosphorylation of Akt induced by ^sup 1^O2 is dependent on PI3-K.

Activation of Akt by ^sup 1^O2 enhances cell survival

The PI3-K-Akt pathway is well known for its role in mediating cell survival. If activation of the PI3-K-Akt pathway by ^sup 1^O2 protects cells against apoptosis, inhibition of this pathway should lead to enhancement of cell death. To test this possibility we pretreated cells with wortmannin or LY294002 before ^sup 1^O2 exposure. Figure 3B,D show that in the presence of each inhibitor, cell survival was further reduced by about 20% in comparison with cells treated identically in the absence of these inhibitors. This result indicates that the PI3-K-Akt pathway contributes to the survival response that counteracts cell death during ^sup 1^O2-induced injury.

PI3-K-Akt-mediated cell survival is not through suppression of p38 activation

Because ^sup 1^O2 stimulates both p38 and P13-K-Akt pathways simultaneously, we examined whether the antiapoptotic effect of PI3-K-Akt is through perturbation of p38 activation. As shown in Fig. 4A, cell viability was significantly increased when cells were treated with 20 [mu]M SB 203580, a specific inhibitor of p38 phosphorylation. This concentration was able to largely block phosphorylation of p38 induced by ^sup 1^O2 (Fig. 4B). However, neither wortmannin nor LY294002 treatment affected p38 phosphorylation in response to ^sup 1^O2 (Fig. 4C). Therefore, we conclude that the mechanism by which PI-3-Akt inhibits apoptosis does not involve steps upstream of p38 kinase activation.

^sup 1^O2 does not induce phosphorylation of PDGFR or SH-PTP2 tyrosine phosphatase

EGFR and PDGFR undergo phosphorylation in response to oxidants such as H^sub 2^O^sub 2^ and ONOO^sup -^, and this phosphorylation is required for activation of Akt by these oxidants (14,19). Because NIH 3T3 cells express a high level of PDGFR, we first investigated whether ^sup 1^O2 treatment alters PDGFR activity. For this purpose, cell lysates of treated and untreated control NIH 3T3 cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using anti-phospho-PDGFR[beta] at tyrosine^sup 716^. The resulting blots revealed that phospho-PDGFR[beta] was not observed in the ^sup 1^O2-treated cells but that a phosphorylated protein with a higher molecular weight than that of PDGFR was present (Fig. 5A). This band was not strong but was consistently present in lysates of ^sup 1^O2-treated cells but was not detected in untreated cells or in cells treated with PDGF or H^sub 2^O^sub 2^ (Fig. 5A,B). As expected, treatment with PDGF or H^sub 2^O^sub 2^ induced phosphorylation of PDGFR (Fig. 5B,C). Using an antibody against total PDGFR, we also detected high molecular protein that entered the gel in the ^sup 1^O2-treated but not in the PDGF- or H^sub 2^O^sub 2^-treated cells, indicating that this protein is a covalent product of PDGFR with one or more other proteins and may represent a homodimer. This presumed dimer was detected within 5 min after ^sup 1^O2, treatment and the amount increased over the 60 min incubation time (Fig. 5A).

Concurrent with the appearance of the putative dimer, the amount of PDGFR decreased in cells treated with ^sup 1^O2 (Fig. 5A). The increase in high-molecular weight protein may account for a part of this decrease. Degradation of the PDGFR may also partly contribute to the decrease, although lower-molecular weight fragments of the PDGFR were not detected with the anti-PDGFR antibody used. We have recently observed that ^sup 1^O2 treatment initiated proteolytic degradation of EGFR in human keratinocytes (32). An alternative explanation is that oxidation of the PDGFR modifies the binding site for the anti-PDGFR antibody such that the decreased staining does not truly represent a decrease in protein. The time-dependent increase in putative PDGFR dimer detected on the blot using anti-PDGFR antibody argues against this latter explanation.

Because the anti-phospho-PDGFR antibody used in this study only recognizes PDGFR[beta] when it is phosphorylated at tyrosine^sup 716^, this result did not rule out the possibility that ^sup 1^O2 activates PDGFR by phosphorylation on other tyrosine residues. To test this possibility, PDGFR was immunoprecipitated using an anti-PDGFR[beta] antibody, and the precipitated proteins were then analyzed on Western blots using an antiphosphotyrosine antibody (PY20). As shown in Fig. 5D, a band corresponding to 185 kDa was detected in PDGF- but not in ^sup 1^O2-treated cells. TPA treatment was used as a negative control. As expected, no phosphorylated PDGFR was detected. Similar results were also obtained when cell lysates were immunoprecipitated with PY20 and blotted with anti-PDGFR[beta] antibody (Fig. 5E). Taken together, these data indicate that ^sup 1^O2 treatment does not produce stable, phosphorylated monomeric PDGFR. A small amount of phosphorylated presumably dimeric PDGFR was detected (Fig. 5A) that was not observed in the immunoprecipitates.

To test whether the phosphorylated dimer of PDGFR produced by ^sup 1^O2 was functionally active, we examined the phosphorylation status of SH-PTP2. SH-PTP2, which is also called SHP-2, is a widely distributed protein tyrosine phosphatase containing Src homology 2 (SH2) domains with which it binds to activated PDGFR (33,34). Binding of SH-PTP2 leads to its phosphorylation on tyrosine residues. Thus, the phosphorylation of this protein can be used to reflect whether PDGFR is activated. Western blot analysis of cell lysates using anti-SH-PTP2 antibody was used to assess the effect of ^sup 1^O2 on phosphorylation of this protein. As shown in Fig. 5B,C, retardation of SH-PTP2 protein was seen in response to PDGF, which is consistent with the PDGFR phosphorylation in the time course indicated by PDGF treatment (Fig. 5B), suggesting that phosphorylated SH-PTP2 is being detected. A similar shift in mobility of this protein was also detected in H^sub 2^O^sub 2^-treated cells (Fig. 5C). However, no such band shift was observed in ^sup 1^O2-treated cells (Fig. 5A). This result suggests that ^sup 1^O2 did not induce activation of SH-PTP2, supporting the concept that PDGFR is not activated by treatment with ^sup 1^O2.

The role of PDGFR and other growth factor receptors in ^sup 1^O2-induced Akt phosphorylation

To assess the functional significance of the putative PDGFR dimer in ^sup 1^O2-treated cells, we tested the effect of AG1295, a specific inhibitor of PDGFR kinase activity, on Akt phosphorylation and cell survival in response to ^sup 1^O2. At 10 [mu]M, this inhibitor has been shown to be able to block ONOO^sup -^ -induced phosphorylation of Akt in human skin fibroblasts (14). As shown in Fig. 6A,B, pretreatment of cells with this inhibitor did not significantly alter ^sup 1^O2-induced Akt phosphorylation even when AG1295 completely blocked phosphorylation of PDGFR induced by PDGF. We also observed that AG1295 treatment did not affect ^sup 1^O2-induced cell death (data not shown).

Suramin, a broad inhibitor of growth factor receptors, has been reported to block UV-C-induced early growth response gene-1 activation and H^sub 2^O^sub 2^-triggered ERK phosphorylation in NIH 3T3 cells (16,34). To further evaluate the role played by PDGFR and possible participation of other growth factor receptors in activation of Akt by ^sup 1^O2, we examined the effect of 50 [mu]M suramin on Akt phosphorylation. Similar to what had been observed in the AG1295-treated cells, pretreatment of cells with suramin did not affect ^sup 1^O2-induced Akt phosphorylation. However, at 50 [mu]M, this inhibitor completely blocked phosphorylation of PDGFR by its ligand (Fig. 6D). These data suggest that PDGFR and other growth factor receptors do not mediate the activation of PI-3-Akt pathway by ^sup 1^O2.

^sup 1^O2 stimulates dephosphorylation of FAK

FAK is a tyrosine kinase that is autophosphorylated as well as activated in response to integrin-mediated signals or modulation by growth factor receptors (35,36). PDGF treatment stimulates association of PI3-K with FAK (29,37). To investigate the phosphorylation status of FAK in response to ^sup 1^O2, we analyzed the cell lysates by Western blot using anti-phospho-FAK antibody. Consistent with a previous report, in quiescent NIH 3T3 cells, FAK is highly tyrosine-phoshorylated (38). ^sup 1^O2 treatment led to a decrease in the phosphorylated FAK in a time-dependent manner. Probing of the same samples using an antibody against total FAK revealed that the intensity of the 125 kDa FAK band was not changed (Fig. 7). These results suggest that FAK activation is not responsible for activation of PI3-K-Akt pathway after ^sup 1^O2 treatment.

PI3-K is not associated with Ras after ^sup 1^O2 treatment

An additional pathway leading to activation of PI3-K involves binding to Ras. Reactive free radical species generated by nitric oxide donors were reported to lead to the recruitment of PI3-K to the plasma membrane, where it associated directly with the effector domain of Ras and became activated (21). Therefore, we assessed whether PI3-K associated with Ras in response to ^sup 1^O2 by immunoprecipitating Ras from total cell extracts with monoclonal anti-Ras antibody and by immunoblotting these immunoprecipitates with antibodies to p85[alpha], one of the adaptor subunits of PI3-K. Although a large amount of Ras was immunoprecipitated with this anti-Ras antibody in ^sup 1^O2-treated cells, p85[alpha] was not detected in the immunoprecipitates (Fig. 8A). To confirm our observation, we reversed the immunoprecipitating and the immunoblotting antibodies. Treatment of cells with ^sup 1^O2 did not lead to an increase in the amount of Ras, even though association with p85 and Ras is barely detected in untreated control cells (Fig. 8B). Similar results were obtained when cells were immunoprecipitated with p110[alpha], a catalytic subunit of PI3-K (data not shown). These results suggest that Ras does not function as an activator of PI3-K after ^sup 1^O2 treatment.

DISCUSSION

The serine-threonine kinase Akt is an important antiapoptotic protein, which mediates survival processes in response to activation of tyrosine kinase growth factor receptors. ROS-RNS such as H^sub 2^O^sub 2^ and ONOO^sup -^ stimulate activation of Akt and, at least for H^sub 2^O^sub 2^, this activation enhances cell survival (14,19). ^sup 1^O2, a moderately strong oxidizing species, has been reported to stimulate JNK and p38 kinase, signaling molecules associated with apoptosis, without affecting ERK1/2, which like Akt is associated with cell survival (8). In this study we provide evidence that ^sup 1^O2 induces activation of Akt in fibroblasts, that this activation promotes cell survival and that growth factor receptors do not mediate the Akt activation. Our experiments were carried out under conditions that produced approximately the same extent of cell death as found in studies where H^sub 2^O^sub 2^ and ONOO^sup -^ stimulated Akt activation (14,19).

Akt has been shown to act downstream of PI3-K in a wide variety of cell types. Our results using the PI3-K inhibitors, wortmannin and LY294002, lead to the same conclusion for ^sup 1^O2-induced Akt phosphorylation (Fig. 3). Stimulation of growth factor receptors is a major mechanism for activation of PI3-K, and ROS-mediated activation of Akt appears to follow this pathway because EGFR mediates activation of Akt by H^sub 2^O^sub 2^ (19) and PDGFR is required for phosphorylation of Akt by ONOO^sup -^ (14). Initially we thought that the growth factor receptor-mediated mechanism might account for activation of Akt in response to ^sup 1^O2. However, our data do not support this hypothesis. First, ^sup 1^O2 did not induce significant phosphorylation of PDGFR under the same conditions that caused substantial Akt phosphorylation, although it induced formation of putative dimers of the receptors (Fig. 5A). Second, ^sup 1^O2 treatment did not induce phosphorylation of a tyrosine phosphatase, SH-PTP2, a substrate of growth factor receptors (Fig. 5A). SH-PTP2 is activated by binding to phosphorylated growth factor receptors.

Third, treatment of cells with AG1295, a specific inhibitor for PDGFR kinase activity, did not influence O2-induced activation of Akt, although at the same concentration it blocked phosphorylation of PDGFR after stimulation by PDGF (Fig. 6A,B). The possibility that ^sup 1^O2-induced Akt phosphorylation might be mediated by other growth factor receptors was not confirmed because suramin, a general inhibitor of membrane receptors, also did not interfere with ^sup 1^O2-induced Akt phosphorylation (Fig. 6C,D). Therefore, we conclude that activation of P13-K-Akt by ^sup 1^O2 is not mediated by activation of growth factor receptors.

The putative dimer of PDGFR observed after ^sup 1^O2 treatment might be similar to the EGFR dimer formed by treatment of A431 cells with ONOO^sup -^ (39). This dimer has been proposed to involve oxidation of tyrosine by ONOO^sup -^ to form tyrosyl free radicals and subsequent covalent bonding between these free radicals on two EGFR molecules because the EGFR contains 15 tyrosines in its extracellular domain and dityrosine fluorescence was detected. This cross-linked EGFR dimer showed much lower ability to activate a downstream EGFR substrate, phospholipase C-[gamma]1 (39). ^sup 1^O2 oxidizes several amino acids in proteins, including tyrosine, and induces protein-protein cross-links but does not produce dityrosine (40). The mechanism for production of ^sup 1^O2-induced protein cross-links begins with oxidation of histidine (or other oxidizable amino acids) to form a product that subsequently reacts with an amino acid on a neighboring protein to from a covalently bonded cross-link (41). Our results indicate that monomeric phosphorylated PDGFR is not present after ^sup 1^O2 treatment and that even the phosphorylated putative PDGFR dimer does not seem to be active because a specific inhibitor of PDGFR kinase activity did not inhibit ^sup 1^O2-induced Akt activation (Fig. 5A). Thus, the covalently linked dimers of EGFR and PDGFR produced by ROS do not act in the same manner as the noncovalent dimers resulting from growth factor binding. We did not observe PDGFR dimers on treatment of NIH 3T3 cells with H^sub 2^O^sub 2^ under conditions where Akt is activated (Fig. 5C), and H^sub 2^O^sub 2^ did not produced EGFR dimers in A431 cells (39). Thus, these three ROS affect growth factor receptors, and the subsequent cellular responses, in markedly different manners. In addition, H^sub 2^O^sub 2^ activates growth factor receptors by inactivating phosphatases, which dephosphorylate the receptors (42). The mechanism for phosphatase inactivation by H^sub 2^O^sub 2^ involves oxidation of a particular amino acid, the ionized cysteine (thiolate ion) in the active site of the phosphatase (43). Apparently, ^sup 1^O2 does not have the ability to oxidize this residue in cells so that the growth factor receptors are not activated by ^sup 1^O2 treatment. ^sup 1^O2 is expected to react with thiolate anions, but its very short lifetime in cells (~100 ns) may limit its ability to diffuse to the location of the appropriate phosphatase in cells before decaying or reacting with other cellular molecules.

Because a growth factor-mediated pathway did not account for ^sup 1^O2 activation of PI3-K-Akt, other potential pathways were examined. Several previous reports indicated that integrin-mediated cell adhesion is critical in cell survival of fibroblast (38,44,45). FAK, a nonreceptor tyrosine kinase, is associated functionally with integrins and plays an important role in transducing survival signals by integrins (35). Overexpression of FAK results in resistance to apoptosis induced by many stimuli including H^sub 2^O^sub 2^ and UV (20,46). Interestingly, the ability of FAK to regulate cell survival is also related to regulation of PI3-K(20). Phosphorylated FAK can bind directly to the p85 subunit of PI3-K and contribute to its activation (47). In addition, phosphorylated FAK can also activate the Ras pathway, which could lead to the activation of PI3-K (38). If FAK were involved in activation of PI3-K-Akt pathway by ^sup 1^O2, the level of phoshorylated FAK should increase. However, the opposite result was obtained-^sup 1^O2 treatment led to a decrease in the amount of phosphorylated FAK in a time-dependent manner (Fig. 8). This result suggests that ^sup 1^O2-stimulated phosphorylation of Akt is not associated with FAK-mediated PI3-K activation and, on the contrary, the reduced tyrosine phosphorylation of FAK after ^sup 1^O2 treatment might attenuate the basal level of phosphorylated Akt. This response to ^sup 1^O2 may lead to cell detachment because FAK phosphorylation is required for cell adhesion. Consistent with this notion, we observed that the NIH 3T3 cells were much more easily detached from culture dishes after ^sup 1^O2 treatment.

An alternative mechanism for ^sup 1^O2-induced PI3-K-Akt activity involves Ras because this protein has been shown to mediate the phosphorylation of PI3-K-Akt in response to ROS (21). In that report, generation of ROS by an NO donor induced an increase in the level of Ras and p85a in the immunoprecipitate complexes using anti-p85[alpha] antibody and anti-Ras antibody, respectively, suggesting an ROS-induced interaction of Ras and PI3-K. However, we failed to detect p85[alpha] or p110[alpha] PI3-K in the Ras immunoprecipitates; we also did not find an increase in the level of Ras in PI3-K immunoprecipitates, indicating that Ras probably does not act as an upstream activator of PI3-K-Akt in response to ^sup 1^O2. Another potential mechanism for growth factor receptor-independent activation of PI3-K involves cyclic adenosine 3':5'monophosphate (cAMP) -mediated signaling because agents that raise intracellular cAMP levels can activate both PB-K and Akt (31,48). In addition, it has been reported that Akt is activated by G-protein-coupled receptors (GPCR) and that the pathway connecting G-proteins to Akt implicates signals emanating from G[alpha]^sub q^, G[alpha]^sub [iota]^, and G^sub [beta][gamma]^ dimers through a pathway that involves PI3-K activity (49,50). Interestingly, oxidants such as H^sub 2^O^sub 2^ can directly target G-proteins leading to activation of ERK1/2 and Akt, which is mediated by PI3-K (51). Whether ^sup 1^O2 targets cAMP-mediated signaling, GPCR or G-proteins to activation of Akt needs further exploration. Recently copper ions were shown to activate PI3-K-Akt by a mechanism not involving growth factor receptors, but the mechanism was not examined (52).

Regardless of the mechanism, on activation by ^sup 1^O2, the PI3-K-Akt pathway contributes to promoting cell survival after ^sup 1^O2 injury. This is clearly indicated by our findings that the specific inhibition of Akt activation by two inhibitors of PI3-K resulted in enhancement of cell death (Fig. 3B,D). Akt promotes survival by interfering with many steps in the apoptotic pathways (18). In this study, we showed that p38 mediates ^sup 1^O2-induced apoptosis in NIH 3T3 cells (Fig. 4A,B), which was consistent with our previous report in HL-60 cells (5). Cross-talk between p38 and PI3-K has also been reported in serum-deprived HeLa cells: treatment of these cells with wortmannin and overexpression of dominant negative mutants of p85 or Akt blocks the protective effect of serum and inhibits p38 activation (53). We explored the possibility that the Akt-mediated survival response was through suppression of p38 kinase activity. The results showed that PI3-K inhibitors did not inhibit p38 phosphorylation induced by ^sup 1^O2 at the concentration that potentiated cell death (Fig. 4C). Thus, Akt does not act upstream of p38 to enhance survival, but it may act downstream of p38 in inhibiting apoptotic pathways activated by ^sup 1^O2. We have shown that p38 mediates activation of several apoptotic events including Bid cleavage, caspase-3 activation, decrease in mitochondrial transmembrane potential and release of cytochrome c from mitochondria during ^sup 1^O2-induced apoptosis (5). ^sup 1^O2 also induces activation of caspase-9 and causes mitochondrial damage by process involving Bcl-2 (3,54). Because Akt activation has been shown to prevent apoptosis by phosphorylation of caspase-9 (55) and Akt activity stabilizes mitochondrial membrane potential and blockade of cytochrome c release from mitochondria (56), these mechanisms may be involved in the increased cell survival on ^sup 1^O2-induced Akt activation.

In summary, the results of this study demonstrate that exposure of NIH 3T3 cells to ^sup 1^O2 induces both phosphorylation of Akt, an antiapoptotic response, and phosphorylation of p38, a proapoptotic response. Activation of Akt by ^sup 1^O2 is mediated by PI3-K and contributes to a survival response that counteracts cell death after ^sup 1^O2-induced injury. However, unlike the response to other oxidants, activation of the PI3-K-Akt by ^sup 1^O2 does not involve growth factor receptors, FAK or Ras protein. The mechanism by which ^sup 1^O2 initiates activation of PI3-K is still unclear and is the subject of ongoing studies.

Acknowledgements-This work was supported by NIH grant GM30755.

[para] Posted on the website on 30 July 2003

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Shougang Zhuang and Irene E. Kochevar*

Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA

Received 15 May 2003; accepted July 2003

*To whom correspondence should be addressed at: Wellman Laboratories of Photomedicine, Massachusetts General Hospital WEL-224, 55 Fruit Street, Boston, MA 02114, USA. Fax: 617-726-3192; e-mail: kochevar@helix.mgh.harvard.edu

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