Neuregulin Rescues PC12-ErbB4 Cells from Cell Death Induced by H 2 O 2 REGULATION OF REACTIVE OXYGEN SPECIES LEVELS BY PHOSPHATIDYLINOSITOL 3-KINASE*

Neuregulins (NRGs), a large family of transmembrane polypeptide growth factors, mediate various cellular re-sponses depending on the cell type and receptor expression. We previously showed that NRG mediates survival of PC12-ErbB4 cells from apoptosis induced by serum deprivation or tumor necrosis factor- (cid:1) treatment. In the present study we show that NRG induces a significant protective effect from H 2 O 2 -induced death. This effect of NRG is mediated by the phosphatidylinositol 3-kinase (PI3K)-signaling pathway since NRG failed to rescue cells from H 2 O 2 insult in the presence of the PI3K inhib- itor, LY294002. Furthermore, the downstream effector of PI3K, protein kinase B/AKT, is activated by NRG in the presence of H 2 O 2 , and protein kinase B/AKT activation is inhibited by LY294002. In addition, our results demonstrate that reactive oxygen species (ROS) elevation induced by H 2 O 2 is inhibited by NRG. LY294002, which blocks NRG-mediated rescue, increases ROS levels. Moreover, both H 2 O 2 -induced ROS elevation and cell death are reduced by expression of activated PI3K. These results suggest that PI3K-dependent pathways may regulate toxic levels of ROS generated by oxidative stress.

Growth factors activate various signaling pathways that are critical for neuronal cell growth and survival (1,2). The neuregulins (NRGs) 1 are a family of growth and differentiation factors that bind to members of the epidermal growth factor family of tyrosine kinase receptors and result in many important effects on neurons and glial cell development (3)(4)(5)(6)(7)(8). NRGs along with ErbB-3 and ErbB-4 receptors are highly expressed in the developing and mature nervous system (7,9,10). After brain insult, the level of NRG (11) and ErbB-4 receptor (11,12) elevates, indicating that ErbB-4 and NRG may function in either synaptic plasticity or neuroprotection. NRG and its receptors mediate various biological effects depending on the cell type examined. Several studies exist to demonstrate that NRG can serve as a differentiation factor for astrocytes (10), oligodendrocytes (13), and neurons (14). Moreover, it was demonstrated that axon-derived neuregulin promotes oligodendrocyte survival in the developing rat optic nerve (15) and that neuregulin in the central nervous system diminishes autoimmune demyelination, promotes oligodendrocyte progenitor expansion, and enhances remyelination (16). In addition, neuregulin affects neuronal survival and neurite outgrowth of developing rat retina (17).
Reactive oxygen species (ROS) are produced by several cellular metabolic reactions. Cells also possess antioxidant systems to control the redox state, which is important for their survival. ROS that cause oxidative stress have been implicated in several diseases including cancer and neurodegenerative disorders (18,19). In addition, several studies indicate that nontoxic levels of ROS may play an essential role as signaling molecules regulating cell growth and differentiation (20 -24). Oxidative stress such as H 2 O 2 treatment induces apoptotic cell death in both PC12 cells and cultured neurons (25,26). The PC12 cell model has been extensively used to study the signaling pathways leading to neuronal differentiation induced by neurotrophins such as NGF compare with the signaling pathways leading to mitogenesis induced by growth factors such as epidermal growth factor (27)(28)(29)(30)(31). It was also demonstrated that many growth factors and neurotrophins can promote neuronal survival of several classes of neurons. Among these factors are insulin, insulin-like growth factor-1, brainderived neurotrophic factor, NGF, NT3, and NT4/5 (32)(33)(34).
The PC12 system has also been used to study the effects of neurotrophic factors on cell survival. After stimulation by tumor necrosis factor-␣ or when deprived of growth factors, these cells die apoptotically, and NGFs can maintain their long term survival (34 -36). The survival effect induced by NGFs in PC12 cells requires the activation of the PI3K-signaling pathway (37,38). It was also demonstrated that NGF protects PC12 cells and neurons from oxidative stress-induced death (39).
Recently, it has been demonstrated that ErbB-4 receptor stably expressed in PC12 cells mediate NRG-induced signals and neurite outgrowth that is indistinguishable from those mediated by NGF-activated Trk receptor (40). It was also demonstrated that NRG rescues PC12-ErbB-4 cells from apoptosis induced by serum deprivation or tumor necrosis factor treatment (41). Because in various pathological conditions oxidative stress may contribute to neuronal dysfunction and NRGs mediate similar effects as NGFs, we examined the effect of NRGs on H 2 O 2 -induced toxicity and analyzed the intracellular mechanism of the NRG-mediated protection from oxidative stress.
Cell Lines-PC12 cells and PC12 cells that express ErbB-4 (40) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 7.5% heat-inactivated fetal bovine serum (FBS), and 7.5% horse serum (HS). Cells were incubated at 37°C in 5% CO 2 in air, and the medium was changed every 3-4 days. Cells were passaged when 90% confluent using 0.5 mM EDTA in PBS. Cells were induced to differentiate by growing on collagen-coated plates at 2 ϫ 10 4 cells/ml in the presence of 50 ng/ml NGF for 7 days. Before experiments were performed, cells were washed twice with PBS.
Cell Survival Assays-Cells were resuspended and seeded on collagen-coated 96-well plates at 7.5 ϫ 10 3 cells/well in DMEM supplemented with 2.5% FBS and 2.5% HS and treated without or with H 2 O 2 for 30 min in the presence or absence of either NRG or NGF at 50 ng/ml for comparison of long term factor activity. Cell survival was determined by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium bromide (MTT) assay, which determines mitochondrial activity in living cells (42). 0.1 mg/ml MTT was incubated with the analyzed cells for 2 h at 37°C. Living cells can transform the tetrazolium ring into dark blue formazan crystals, which can be quantified by reading the optical density at 550 -650 nm after lysis of the cells with acidic isopropanol. Staining of nuclei with the fluorescent DNA dye 4Ј,6-diamidine-2Ј-phenylindole dihydrochloride (DAPI) was used to estimate the number of dying cells. Cells were scored for apoptosis by nuclear morphology. Treated cells were fixed in 4% formaldehyde, washed three times with PBS, and then stained with DAPI solution (1 g/ml) for 10 min. After washing with PBS and mounting, the cells were photographed. The instrument used was an Olympus optical inverted phasecontrast microscope model IX70 (ϫ20 magnification).
Lysate Preparation and Immunoblot-Cells were exposed to the indicated stimuli. After treatment, cells were solubilized in lysis buffer. Lysates were cleared by centrifugation. For direct electrophoretic analysis, boiling gel sample buffer was added to cell lysates. Lysates were resolved by SDS-polyacrylamide gel electrophoresis through 10% gels and electrophoretically transferred to nitrocellulose membrane. Membranes were blocked for 1 h in 0.02 M Tris HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20) containing 6% milk and blotted with 1 g/ml primary antibodies for 2 h followed by 0.5 g/ml secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected with the enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).
Measurement of Intracellular ROS Generation-Measurement of intracellular ROS generation was determined using the DCFH-DA assay (43). DCHF-DA was dissolved in Me 2 SO and diluted with DMEM lack-ing phenol red to a final concentration of 100 M. Cells were plated on collagen-coated 48-well culture plates at a density of 1 ϫ 10 5 cells/well in DMEM supplemented with 7.5% FBS and 7.5% HS. After 24 h, medium was replaced by DMEM containing 2.5% FBS and 2.5% HS for 1 h. Cells were treated for 30 min with 0.5 mM H 2 O 2 in the presence or absence of the indicated growth factors or drugs. The medium was then replaced with fresh medium containing growth factors or drugs for the indicated time periods. Cells were then loaded with 100 M DCFH-DA for 30 min at 37°C. The loading was terminated by washing the cells with DMEM lacking phenol red containing 2.5% FBS and 2.5% HS. The cells were either photographed by an Olympus optical inverted phasecontrast microscope model IX70 (ϫ100 magnification), or the DCF fluorescence was monitored using a microplate fluorescence reader (Bio-TEK Instruments, FL500) with an excitation wavelength of 485 nm and an emission wavelength 530 nm. The increase in fluorescence for each treatment was calculated by the relative fluorescence of each treatment compared with the control untreated cells normalized by the number of cells as determined by the MTT assay.
Transfection-One day before transfection, PC12-ErbB-4 cells were seeded at a density of 2 ϫ 10 5 cells/well in 24-well plates. To each well 300 l of DNA-LipofectAMINE mixture (2 g of DNA and 30 g of LipofectAMINE in 1 ml of Opti-MEM (Life Technologies, Inc.)) were added according to the manufacture's instructions. Each transfection was performed four times. After incubation of cells for 5 h with the DNA LipofectAMINE mixture, equal volume of fresh medium was added, and incubation was continued. Medium was replaced 24 h later, and cells were treated with H 2 O 2 for 30 min. The viability of the transfected cells in all experiments was monitored 72 h after transfection by measuring the activity of SEAP in the medium of the transfected cells. The ratio of the different DNA species in each transfection was 10:1 for pSG5/ 5ЈMycTp110␣CAAX or pcDNA3 and reporter plasmid (secreted alkaline phosphatase (SEAP)).
Assay for SEAP Activity-SEAP activity was assayed as described previously (45). Briefly, culture medium (200 l) from transfected cells was collected and spun for 2 min at 10,000 ϫ g. The supernatant was incubated at 65°C for 10 min, and then aliquots (25 l) from each treatment were incubated with 200 l of SEAP buffer (1 M diethanolamine, 0.5 mM MgCl 2 , and 10 mM L-homoarginine) containing 5 mg/ml p-nitrophenylphosphate at 37°C until a yellow color developed. The plates were read on a micro-enzyme-linked immunosorbent assay reader at wavelength 405 nm.  (Fig. 1). The death induced by H 2 O 2 treatment appeared to have the characteristics of apoptosis; the cells shrunk and their nuclei were condensed or fragmented (Fig. 2). Treatment of the PC12-ErbB-4 cells with NRG (100 ng/ml) completely abolished the H 2 O 2 -induced toxicity (Fig. 1). NGF (50 ng/ml) furnished a comparable protection from the H 2 O 2 -induced cell death. We next examined whether NRG can also protect differentiated PC12-ErbB-4 cells that exhibit a sympathetic neuronal cell phenotype from H 2 O 2 -induced cell death. For this aim, we performed experiments using PC12-ErbB-4 cells that were induced to differentiate by NGF treatment for 1 week. Early experiments showed that the differentiated cells were more resistant to cell death induced by H 2 O 2 (not shown). However H 2 O 2 at a concentration of 1 mM induced cell death comparable with that observed with 0.5 mM in the non-differentiated cells. As shown in Figs. 1 and 2, incubation of differentiated PC12-ErbB-4 cells with NRG or with NGF inhibited apoptotic cell death induced by 1 mM H 2 O 2 .

NRG Protects ErbB-4-expressing PC12 Cells from H 2 O 2 -induced
Identification of the Signaling Component Involved in NRGmediated Protection from H 2 O 2 -induced Apoptosis-To examine which signaling pathway is involved in the protective effect of NRG in H 2 O 2 -treated PC12-ErbB-4 cells, we used pharmacological inhibitors of PI3K (LY294002), Erk (PD98059), p38 (SB203580), and protein kinase C (GF109203X). These are prominent signaling pathways known to be activated by NRG (46). Cells were treated for 30 min with H 2 O 2 in the absence or presence of NRG (100 ng/ml) and in the presence of the indicated inhibitors. As shown in Fig. 3, none of the inhibitors except LY294002 could block the NRG-mediated protection from H 2 O 2 -induced cell death. This was apparent in both the differentiated and the non-differentiated cells (Fig. 3). We have Whole cell lysates were prepared from cells treated for the indicated time points with H 2 O 2 (0.5 mM), NRG (100 ng/ml), or both. A, lysates were analyzed by immunoblotting with a mAb to phosphorylated PKB/Akt. As a control, blots were reacted with mAb to PKB/AKT. The experiment was repeated three times with similar results. B, whole cell lysates were prepared from cells treated for 5 min with H 2 O 2 (0.5 mM), NRG (100 ng/ml), or 20 M LY294002 (LY) as indicated. Lysates were analyzed by immunoblotting with a mAb to phosphorylated PKB/ Akt. As a control, blots were reacted with mAb to PKB/AKT. The experiment was repeated three times with similar results.

FIG. 5. NRG-mediated reduction in ROS levels induced by H 2 O 2 treatment is blocked by LY294002.
A, cells were plated in 48-well collagen-coated plates at a density of 10 5 cells/well in media containing 2.5% HS and 2.5% FBS. Wells were treated with 0.5 mM H 2 O 2 with or without NRG (100 ng/ml) for 30 min. The medium was then replaced, and NRG treatment was continued for the indicated time periods. 30 min before the indicated time, medium was replaced to DMEM without phenol red, and cells were preloaded with 100 M DCFH-DA for 30 min. After two washes, DCF fluorescence intensity was measured, and the number of cells was determined by the MTT assay. The relative DCF fluorescence was determined relative to cell number. The data are presented as fold induction of DCF fluorescence at each time point compared with the DCF fluorescence at the start point (time 0) and are the mean Ϯ S.D. of 4 determinations. Each experiment was repeated at least three times with similar results. B, cells were plated as described in A. Wells were pretreated with 20 M LY294002 (LY) for 1 h and then treated with 0.5 mM H 2 O 2 with or without NRG (100 ng/ml) for 30 min. The medium was then replaced, and NRG and LY294002 treatments were continued for 48 h. After 48 h, the medium was replaced with DMEM without phenol red, and cells were preloaded with 100 M DCFH-DA for 30 min. After two washes, DCF fluorescence intensity was measured, and the number of cells was determined by the MTT assay. The relative DCF fluorescence was determined relative to cell numbers. The data are presented as the percentages of relative DCF fluorescence compare with control untreated cells and are the mean Ϯ S.D. of six determinations. Each experiment was repeated at least three times with similar results. CON, control. C, cells were treated as described in B. Panels were randomly selected and are representative fields. The photographs were taken with an Olympus optical inverted phase-contrast microscope model IX70 (ϫ20 magnification). previously shown that PI3K inhibition induces apoptosis of PC12-ErbB-4 cells, namely, cell death occurs also in the presence of NGF or NRG (41). The addition of LY294002 as well as other inhibitors (not shown) by themselves could reduce cell viability (78% cell death induced by LY294002). These results suggest that inhibition of PI3K pathway (and also other signaling pathways) has a toxic effect on the cells and that the toxic effect of PI3K inhibitor is not prevented by activation of ErbB-4. However, the toxic effect of the other inhibitors used was prevented by NRG (41). The inability of NRG to rescue cells after treatment with LY294002 indicates that PI3K activity may be required for the NRG protection from H 2 O 2 -induced cell death and encouraged us to further investigate the involvement of PI3K activation induced by NRG.
Because PI3K and its downstream effector PKB/Akt are frequently associated with cell survival, we examined whether PKB/Akt is involved in the PI3K-dependent protection of NRG from H 2 O 2 -induced cell death. As shown in Fig. 4A, NRG, but not H 2 O 2 , induced sustained PKB/Akt phosphorylation. Although in the presence of H 2 O 2 , NRG-induced activation of PKB/Akt was reduced, a significant sustained activation was observed. Furthermore, the PI3K inhibitor LY294002 inhibited the NRG-induced activation of PKB/Akt both in the presence or absence of H 2 O 2 (Fig. 4B). Taken together the results presented in Figs. 3 and 4 indicate that PI3K and PKB/Akt activation may be involved in the NRG-mediated rescue of PC12-ErbB-4 cells from H 2 O 2 -induced apoptosis.

NRG Inhibits ROS Elevation Induced by H 2 O 2 Is Mediated by PI3K-Previous studies demonstrated that H 2 O 2 could induce ROS elevation in PC12 cells and that a marked elevation
in ROS leads to cell death (47). Because the PI3K-PKB/Akt pathway is involved in NRG-mediated rescue of PC12-ErbB-4 cells from H 2 O 2 -induced death, we examined the effect of NRG on ROS generation. Cells were plated in 48-well collagencoated plates at a density of 10 5 cells/well in media containing 2.5% HS and 2.5% FBS. Wells were pretreated with 20 M LY294002 for 1 h and then treated with 0.5 mM H 2 O 2 with or without NRG (100 ng/ml) for 30 min. The medium was then replaced, and NRG and LY294002 treatments were continued for 48 h. After 48 h, medium was replaced to DMEM without phenol red, and cells were preloaded with 100 M DCFH-DA for 30 min. After two washes, DCF fluorescence intensity was measured, and the number of cells was determined by the MTT assay. The relative DCF fluorescence was determined relative to cell number. As shown in Fig. 5A, H 2 O 2 treatment induced a dramatic increase in ROS levels. NRG blocked the H 2 O 2 -induced elevation in ROS (Fig. 5). Consistent with the involvement of PI3K in the protection induced by NRG, LY294002 blocked the NRG-mediated reduction in ROS levels as measured by DCF-fluorescence (Fig. 5, B and C). These results indicate that NRG can abolish the H 2 O 2 -induced production of ROS and that PI3K mediates this effect.  n ϭ 4). The experiments were repeated three times with similar results. C, PC12-ErbB-4 cells were cotransfected with red fluorescent plasmid (pDsRed1) to visualize the transfected cells and with either control pcDNA3 vector or p110-CAAX vector. 24 h after transfection, wells were treated with 0.5 mM H 2 O 2 for 30 min, and 2 days later they were loaded with the fluorophore, DCF. Activated PI3K-expressing cells or pcDNA3-expressing cells appear in red, DCF fluorescence appears in green, and the overlap appears in yellow. Panels were randomly selected and are representative fields. The photographs were taken with an Olympus optical inverted phase-contrast microscope Model IX70 (ϫ20 magnification). D, PC12-ErbB-4 cells were cotransfected with red fluorescent plasmid (pDsRed1) and with either control pcDNA3 vector or p110-CAAX vector. 24 h after transfection, wells were treated with 0.5 mM H 2 O 2 for 30 min, and 3 h later they were loaded with the fluorophore, DCF. Activated PI3K-expressing cells or pcDNA3-expressing cells appears in red, DCF fluorescence appears in green, and the overlap appears in yellow. Panels were randomly selected and are representative fields. The photographs were taken with an Olympus optical inverted phase-contrast microscope model IX70 (ϫ20 magnification). oxidative stress induced by H 2 O 2 we transfected PC12-ErbB-4 cells with the constitutively active PI3K expression vectors (p110-CAAX). The constitutive activity of PI3K in the transfected cells was confirmed by measuring phosphorylation of Akt, as can be seen in Fig. 6A. In subsequent experiments cells were co-transfected with p110-CAAX or empty vector (pcDNA3) and SEAP reporter gene. The effect of constitutively active PI3K on viability of H 2 O 2 -treated cells was examined by SEAP activity in the co-transfected cultures. As shown in Fig.  6B, transfection with p110-CAAX increased the activity of SEAP in both H 2 O 2 -treated and in untreated cells. SEAP activity in the pcDNA3-transfected cells was reduced after H 2 O 2 treatment (Fig. 6B). These results show that constitutively active PI3K can rescue PC12-ErbB4 cells from oxidative stress. In addition, the PI3K inhibitor LY294002 blocked the survival effect of activated PI3K (Fig. 6B). In a different set of experiments, cells were transiently co-transfected with p110-CAAX expression vector or empty vector (pcDNA3) and red fluorescent plasmid (pDsRed1). This enabled visualization of the cotransfected cells. One-day post-transfections cells were treated with H 2 O 2 as described above, and 3 h or 2 days post-treatment they were loaded with the fluorophore DCFH-DA. The results of a representative experiment are shown in Fig. 6, C and D. As shown, H 2 O 2 increased ROS levels in the cells at 3 h and 2 days post-H 2 O 2 treatment, and this increase was blocked in cells that express the activated PI3K. These results suggest that PI3K activity can regulate ROS levels even in the absence of NRG. Taken together these results and the ability of PI3K inhibitor to block NRG-mediated rescue and to block NRG inhibition of ROS elevation induced by H 2 O 2 indicate that the PI3K pathway may function as a junction to regulate toxic levels of ROS in the cells. DISCUSSION Oxidative stress is thought to contribute to neuronal dysfunction under a variety of pathological conditions. Previous studies showed that NGF and brain-derived neurotrophic factor can rescue neuronal and PC12 cells from death induced by oxidative stress. In the present study, we demonstrate that NRG protects PC12-ErbB-4 cells from oxidative stress via the PI3K-PKB/Akt-dependent pathway. We show that the protective effects of NRG are mediated by modulation of ROS levels in the cells and that this modulation depends on the PI3K pathway. Specifically, we show that H 2 O 2 induces ROS elevation and death of PC12-ErbB-4 cells, where activation of PI3K by NRG inhibited ROS production and cell death. Indeed, overexpression of activated PI3K reduced the toxic levels of ROS in the cells and protected them from H 2 O 2 -induced death. These results are schematically summarized in Fig. 7.
Oxidative stress including H 2 O 2 treatment results in activation of several signaling pathways (48). These pathways include the p38, c-Jun NH 2 -terminal kinase, and Erk pathways, which can mediate a variety of cellular responses. It was previously shown that NRG can activate p38-, Erk-, and PI3Ksignaling pathways (49,50). Using the p38 inhibitor SB203580, we demonstrated that inhibition of the p38-signaling pathway does not prevent NRG-mediated rescue from H 2 O 2 -induced cell death. Several studies demonstrate that NGF, epidermal growth factor, and NRG can increase Erk activation. It was demonstrated that sustained Erk activation correlates with neuronal differentiation, and transient Erk activation correlates with cell proliferation (31,40,51,52). In the present study we found that PD98059, the MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor, did not inhibit NRG-mediated survival. Together these results indicate that in PC12-ErbB4 cells, p38 and Ras/mitogen-acti-vated protein kinase pathways do not mediate NRG-induced rescue from H 2 O 2 .
Although PI3K is a well known signaling pathway involved in cell protection under various stresses, little is known about its role in protection against oxidative stress. One of the downstream effectors of PI3K is the serine/threonine kinase PKB/ Akt. Akt is involved in promotion of cell survival through inhibition of apoptosis and possibly plays a role in PI3K-mediated neuronal cell survival (53). It was recently reported that the PI3K-PKB/Akt pathway delivers an anti-apoptotic signal in human glioblastoma against H 2 O 2 -induced apoptosis (54). It was also demonstrated that in cardiomyocytes, the PI3K-PKB/ Akt-S6K pathway promotes cell survival against oxidative stress-induced apoptosis (55). Our experiments demonstrate that NRG protects PC12-ErbB-4 cells from H 2 O 2 -induced death ( Figs. 1 and 2). The protection conveyed by NRG is mediated via the PI3K pathway because LY294002 inhibited this effect (Fig. 3). In addition, we show that NRG activates PKB/Akt in the presence of hydrogen peroxide. Also, LY294002 inhibits NRG-induced PKB/Akt activation in the presence and in the absence of H 2 O 2 (Fig. 4). These results indicate that PI3K-PKB/Akt pathways regulate NRG-mediated rescue from H 2 O 2induced cell death.
ROS such as superoxide radicals, hydroxyl radicals, and H 2 O 2 are continuously produced by the cells, and their levels are regulated by a number of enzymes and physiological antioxidants. Excessive generation of ROS has been associated with cytotoxicity in a variety of pathological conditions and in both PC12 and cultured neurons (25,39,56,57). Neurotrophins regulate neuronal survival and are neuroprotective in certain models of injury including oxidative stress (58 -60). It was previously shown that NGF is able to protect PC12 cells from oxidative stress by increasing catalase activity and GSH levels (61,62). Our experiments demonstrate that NRG reduces ROS levels in H 2 O 2 -treated PC12-ErbB-4 cells (Fig. 5). These results suggest that the effect of NRG on cell viability may be because of the reduction in ROS levels. Moreover, our results demon- strate that PI3K inhibitor, which in itself blocks NRG-mediated rescue also blocks the NRG-mediated reduction in H 2 O 2induced ROS elevation. Thus, ROS levels appear to be regulated by PI3K. Consistent with this notion, the H 2 O 2induced ROS elevation and cell death were blocked by the expression of activated PI3K (Fig. 6). Hence, we suggest that PI3K-dependent pathways may regulate toxic levels of ROS generated by oxidative stress.