NFκB Activation Is Required for the Neuroprotective Effects of Pigment Epithelium-derived Factor (PEDF) on Cerebellar Granule Neurons*

Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cells (1–3 days in vitro) against induced apoptosis and mature cells (5+ days in vitro) against glutamate toxicity, but its precise mechanism is still unknown. Because the transcription factor NFκB blocks cell death, including neuronal apoptosis, we have investigated the ability of PEDF to exert its effects via NFκB activation. PEDF induced an increased phosphorylation of IκBα, decreased levels of IκB proteins, and translocation of p65 (RelA) to the nucleus followed by a time-dependent increase of NFκB-DNA binding activity in both immature and mature neurons. The protective effects of PEDF against both induced apoptosis and glutamate toxicity were blocked by the addition of either the IκB kinase inhibitor BAY 11-7082, which inhibits the phosphorylation of IκB, orN-acetyl-Leu-Leu-norleucinal, which blocks proteosome degradation of IκB, demonstrating that NFκB is required for the neuroprotective effects of PEDF. Reverse transcription-polymerase chain reaction analysis revealed that up-regulation of the anti-apoptotic genes for Bcl-2, Bcl-x, and manganese superoxide dismutase was observed in PEDF-treated immature but not mature neurons. Up-regulation of nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor mRNA was long-lasting in mature neurons. These results suggest that PEDF promotes neuronal survival through activation of NFκB, which in turn induces expression of anti-apoptotic and/or neurotrophic factor genes.

PEDF mRNA was detected in a broad range of human fetal and adult tissues including almost all brain areas (2). Later studies demonstrate that PEDF has neurotrophic effects on cerebellar granule cell neurons (CGCs) in culture (3)(4)(5), on neurons cultured from hippocampus (6), on motor neurons from spinal cord (7)(8), and on retinal neurons (9). Moreover, PEDF has differential effects on CGCs, protecting immature (days in vitro (DIV) (2) but not mature (DIV6) cells against low K ϩ /serumfree-induced apoptosis (5) while protecting mature CGCs (DIV8) against glutamate-induced toxicity (4). PEDF most effectively blocked induced apoptosis in immature cells (DIV2) when added 24 h before the change of medium (5). These observations suggest that the responses of CGCs to PEDF may vary as neural differentiation proceeds, possibly because of changes in signal transduction pathways or gene expression as a function of cell maturity.
The signal transduction pathways that mediate the cell survival-promoting actions of neurotrophic factors are being worked out, and in many cases activation of transcription factors is involved. Recent studies show that activation of the transcription factor NFB plays a critical role in preventing neuronal death in a number of models, including glutamate toxicity (10 -11), low K ϩ -induced apoptosis (12), hypoxia/reoxygenation-induced apoptosis (13), ␤-amyloid peptide-induced toxicity (14 -15), optic nerve transection (16), IB kinase-deficient mice (17), oxidative stress (18 -20), and death of developing peripheral neurons (21)(22)(23). Furthermore, inhibition of NFB by overexpression of IB or treatment with proteosome inhibitors promotes apoptosis in neurons (13,15,16,19,22,24). These observations suggest that NFB plays an important role in neuronal survival and death. In this study, we have examined the potential role of NFB in the neuroprotective effects of PEDF.  and N-acetyl-Leu-Leu-norleucinal (ALLN) were obtained from Calbiochem. All culture reagents were obtained from Life Technologies. Polyclonal antibodies against p65 (RelA), RelB, c-Rel, p50, p52, IB␣, and IB␤ were purchased from Santa Cruz Biotechnology, Santa Cruz CA. An antibody against phospho-IB␣ was from Cell Signaling Technology, Beverly MA.
Nuclear Extract Preparation and Electrophoresis Mobility Shift Assay (EMSA)-Nuclear extracts for the EMSAs were prepared by a mini-extraction protocol (27). EMSA was performed with a commercial kit (Promega Corp., Madison, WI) according to the manufacturer's instructions. Two or 5 g of nuclear extract were incubated with a 32 P-labeled DNA sequence containing the AP-1, CRE, or NFB binding site. The DNA-protein complexes were separated from unbound oligonucleotide by electrophoresis through native 6% polyacrylamide gels using 25 mM Tris base, 25 mM borate, 0.5 mM EDTA buffer. After electrophoresis, the gels were dried and analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Competition experiments were performed by co-incubation with a 100-fold excess of unlabeled double-stranded oligonucleotide in the DNA-protein binding reaction. For supershift analysis, 2 l of antibody were preincubated with nuclear proteins for 30 min at 4°C before the binding reaction.
Immunohistochemistry for p65-After experimental treatments, CGCs were washed 3 times with phosphate-buffered saline and fixed by incubating in 4% paraformaldehyde for 30 min. Cells were then incubated in permeabilization buffer (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. To block the nonspecific antibody binding, cells were incubated in a blocking solution containing 1% bovine serum albumin and 0.3% Triton X-100 for 2 h. Next, cells were incubated with rabbit polyclonal anti-p65 antibody at a dilution of 1:200 at 4°C overnight followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody. The images were acquired using a confocal laser-scanning microscope with a 100ϫ objective (488-nm excitation and 510-nm emission).
TUNEL Assay-Apoptosis was detected by TUNEL assay using the in situ cell death detection kit according to the manufacturer's instructions (Roche Molecular Biochemicals). Briefly, CGCs were fixed in 4% paraformaldehyde for 1 h at room temperature, and then cells were incubated in permeabilization buffer (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. The cells were then incubated with TUNEL reaction medium at 37°C for 1 h. After incubation, cells were stained with propidium iodide (5 g/ml) for 5 min. DNA fragmentation was evaluated using a laser confocal microscope. TUNEL-positive cells were counted in 2-6 randomly selected fields from two different chambers. 600 -1200 cells were scored for each treatment. The percentage of TUNEL-positive cells was calculated as the number of TUNEL-positive cells divided by the total number of cells (propidium iodide-stained cells).
Lactate Dehydrogenase (LDH) Assay-LDH, a stable cytoplasmic enzyme present in all cells, is rapidly released into the cell culture medium after damage to the plasma membrane. To measure LDH activity released from CGCs, we used a commercially available cytotoxicity detection kit (Roche Molecular Biochemicals). For measurement of released LDH, culture medium was collected and centrifuged to remove contaminating cells and cellular debris. To assay total LDH activity, 100 l of 1% Triton X-100 were then added to the wells, and the cells were incubated for 30 min. The percentage of LDH release was calculated as LDH in the culture medium divided by total LDH (cellular plus medium LDH). For the actual assay, 100 l of each sample were transferred to a 96-well microtiter plate, 100 l of LDH assay reagent were added to each well and incubated for up to 30 min at room temperature, and the absorbance of samples was measured at 490 nm.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-Total RNA was extracted from CGCs according to the manufacture's instructions for TRIZOL Reagent (Life Technologies). Two g of total RNA were converted to first strand cDNA using the first-strand cDNA synthesis kit (Amersham Pharmacia Biotech). The resulting cDNA was subjected to PCR analysis. The PCR amplification mixture, in a final volume of 25 l, consisted of 1ϫ Taq DNA polymerase buffer, 0.2 mM dNTPs, 1.5 mM MgCl 2 , 0.5 M each specific primer, and 2.5 units of Taq DNA polymerase (Life Technologies) according to the manufacturer's instructions. The cycle number was chosen such that amplification of the products was in the linear range with respect to the amount of input cDNA. Each cycle consisted of 30 s at 94°C for denaturation, 30 s at 60°C for annealing, and 60 s at 72°C for extension. The PCR products were stained with ethidium bromide after agarose gel electrophoresis and photographed using Polaroid film type 667. Cyclophilin was used as the non-changing control RNA.
Statistical Analysis-For statistical analysis, data were compared by 1-way analysis of variance and Fisher's protected least square difference.

RESULTS
Effect of PEDF on NFB Binding Activity-To determine whether PEDF affects NFB transcriptional factor activity, the level of NFB binding to DNA was analyzed using EMSA in CGCs treated with or without PEDF. PEDF was added at 7 nM, a concentration previously reported to prevent death of CGCs after exposure to low K ϩ /serum-free medium or glutamate (4,5). Two NFB binding bands were detected using nuclear extracts derived from CGCs at DIV2, whereas three bands were detected in cells at DIV8 (Fig. 1, A and B). Time course studies showed that NFB binding activity increased within 1 h in immature CGCs (DIV2), with a maximal effect observed 2 h after PEDF treatment (Fig. 1A). A similar rapid induction was observed in mature CGCs (DIV8), but this induction was sustained for at least 6 h after PEDF treatment (Fig. 1B). Induction of NFB binding activity was observed over a concentration range of 0.5-10 nM PEDF (Fig. 1C). The addition of a 100-fold excess of unlabeled NFB oligonucleotide, but not AP-1, CRE, or OCT-1 oligonucleotides, displaced the binding ( Fig. 2A).

PEDF-induced NFB Contains Heterodimeric Complexes of p50 and p65
Proteins-To elucidate the nature of the NFB complex activated by PEDF, we performed antibody supershift analysis using nuclear extracts from both immature and mature neurons. As shown in Figs. 2, B and C, the addition of p50 antibody resulted in an almost complete loss of both the lower and upper bands of the NFB complex, whereas antibody to p65 (RelA) reduced the amount of the upper complex without affecting the lower one. In contrast, antibodies against RelB, cRel, or p-52 did not cross-react with the NFB-DNA complex. These analyses suggest that the PEDF-treated neurons contain nuclear NFB⅐Rel complexes composed predominantly of p50/ p65 heterodimer and p50/p50 homodimer. Interestingly, the p65/p50 heterodimer was markedly increased in mature neurons (Fig. 2C) relative to immature neurons (Fig. 2B).
Role of IB Kinase and Proteosomes in Activation of NFB by PEDF-NFB is normally sequestered in the cytoplasm by a member of the IB family of inhibitory proteins. When cells are exposed to activators of NFB, these IB proteins undergo sequential phosphorylation, ubiquitination and proteosomal degradation, thereby allowing the translocation of dimeric complexes of NFB to the nucleus. To examine whether activation of NFB by PEDF requires IB degradation by proteosomes, we analyzed the expression level of IB proteins in PEDF-treated neurons using Western blot analysis. As shown in Fig. 3A, PEDF caused a time-dependent decrease in both IB␣ and IB␤ protein levels. Furthermore, rapid phosphorylation of IB␣ protein was observed in PEDF-treated CGCs (Fig. 3B). In contrast, p65 levels in whole cell lysates of PEDF-treated cells were not changed over a 6-h period (Fig. 3C). To provide further evidence that IB phosphorylation was required and that proteosomes were involved in PEDF-induced NFB activation, CGCs were treated with 10 M BAY 11-7082, which inhibits IB kinase activity and subsequently inhibits the nuclear translocation of NFB (28), or with 10 M proteosome inhibitor ALLN, which interferes with IB proteolysis (29,30). As shown in Fig. 3D, pretreatment with either BAY-11-7082 or ALLN inhibited the ability of PEDF to activate NFB. These results suggest that PEDF-induced NFB activation requires IB phosphorylation and degradation via the proteosomes.
To further demonstrate activation of NFB protein, immunohistochemistry was used to show nuclear translocation (Fig.  3E). Untreated or PEDF-treated CGCs were fixed and stained with antibody to p65. Under basal conditions, no nuclear staining for p65 was observed. However, 2 h after PEDF exposure, neuronal nuclei were labeled with the p65 antibody. Furthermore, RT-PCR analysis revealed that PEDF induces expression in both immature and mature neurons of IB␣ mRNA (Fig. 6), which has been shown to be regulated by NFB (31).
IB Kinase and Proteosome Inhibitors Block the Neuroprotective Effects of PEDF-CGCs undergo apoptosis when they are switched from medium containing serum and 25 mM KCl to serum-free medium containing 5 mM KCl (32)(33)(34). Although glutamate toxicity can cause both apoptotic and necrotic neuronal death (35) depending on the cell type, we have found that under the conditions used in this paper to induce glutamatemediated death, the death occurs by a necrotic process. Because PEDF can protect CGCs against both K ϩ /serum deprivation (5) and glutamate toxicity (4), it was of interest to determine whether PEDF-induced NFB activity is required for both of these neuroprotective effects of PEDF. We therefore tested the effect of IB kinase and proteosome inhibitors on low K ϩ /serum-free-induced apoptosis and on glutamate toxicity.
When DIV2 cells were switched to serum-free medium containing 5 mM K ϩ , ϳ50% of the cells were TUNEL-positive 6 h later, indicative of apoptotic death (Fig. 4, A and B). Neuronal death caused by low potassium was almost completely prevented by a 20-h preincubation with 7 nM PEDF. The neuroprotective effect of PEDF was completely blocked by the addition of either ALLN (10 M) or BAY 11-7082 (10 M) 30 min before the addition of PEDF (Fig. 4, A and B). As already reported (5), PEDF did not protect CGCs against induced apoptosis at DIV8 (data not shown). Neither ALLN nor BAY 11-  Similar results were obtained when DIV8 CGCs were exposed to 100 M glutamate for 1 h (Fig. 5). Results from both the MTS assay (Fig. 5A) and the LDH release assay (Fig. 5B) revealed that the neuroprotective effect of PEDF against glutamate toxicity was also completely blocked by the IB kinase and proteosome inhibitors, whereas the inhibitors had no effect when added alone. These results suggest that PEDF-induced NFB activity is directly involved in its neuroprotective effects against both induced apoptosis and glutamate toxicity.
PEDF-induced Expression of Bcl-2, Bcl-x, and Mn-SOD in Immature CGCs (DIV2)-NFB plays important roles in regulating the expression of various genes in both the nervous system and the immune system (36). However, little is known about NFB-regulated genes that are associated with antiapoptotic effects in the nervous system. Mattson et al. (18) and Tamatani et al. (37) report that induction of Bcl family members and Mn-SOD by NFB contributes to the neuroprotective action of tumor necrosis factor-␣ (TNF-␣). Potential NFB binding sites have been identified in the promoter regions of the Bcl-x and Mn-SOD genes (38,39). We therefore examined the effect of PEDF on expression of mRNAs for Bcl-2, Bcl-x, and Mn-SOD in CGCs using RT-PCR analysis. As shown in Fig. 6, PEDF treatment led to a transient increase in Bcl-2, Bcl-x, and Mn-SOD mRNA levels at 3 h in immature CGCs (DIV2). In contrast, no induction of these mRNAs was observed in mature neurons (DIV8) at any time from 3 to 9 h after PEDF addition.

PEDF-induced Expression of NGF, BDNF, and GDNF mRNA in Both Immature and Mature
Neurons-Several researchers have reported that neurotrophic factors such as NGF, BDNF, and GDNF protect neurons against both glutamate toxicity (40 -42) and low K ϩ -induced apoptosis (43,44). A potential NFB binding site has been identified in the promoter regions of the genes for NGF and GDNF (45,46). We therefore examined the effect of PEDF on expression of mRNAs for NGF, BDNF, and GDNF in CGCs using RT-PCR analysis. As shown in Fig. 6, PEDF treatment led to a transient increase in NGF, BDNF, and GDNF mRNA levels at 3 h in immature CGCs (DIV2). In contrast, sustained mRNA expression of NGF and BDNF was observed from 3 to 9 h in mature CGCs (DIV8), whereas GDNF mRNA did not increase until 9 h after PEDF treatment. DISCUSSION NFB is composed of homo-and heterodimers of members of the Rel family of related transcription factors that control the expression of numerous genes. In general, NFB exists as a heterodimer comprising a 50 kDa (p50) and a 65-kDa (p65) subunit and is sequestered in the cytoplasm by an inhibitory protein of the IB family. Immunological activators of NFB such as TNF-␣, interleukin-1␤, or lipopolysaccharide result in the phosphorylation and degradation of the IB inhibitory protein, allowing free NFB to enter the nucleus, bind to its cognate DNA sequences, and induce target gene transcription.
The role of NFB in the regulation of neuronal survival or death is currently under intense investigation. Activation of NFB in neurons is associated with neuroprotection against death-inducing stimuli such as exposure to ␤-amyloid protein, oxidative stress, and nitric oxide (14,15,18,20,47). Cytokines (ciliary neurotrophic factor, leukemia inhibitory factor, cardiotropin-1, and interleukin-6) as well as NGF through its low affinity receptor p75 promote neuronal survival through NFB activation (21,23), but to date no one has reported that members of the classic neurotrophic factor family, acting through Trk receptors, promote neuronal survival via NFB. However, there are also reports that NFB activation can lead to neuronal apoptosis. Indirect results from Grilli et al. (48) demonstrate that aspirin inhibits both NFB activity and glutamate toxicity without showing direct evidence that inhibition of NFB activity blocks neuronal death. Post et al. (49,50) report that haloperidol-or antidepressant-induced clonal hippocampal HT22 cell death is mediated by NFB activation and that overexpression of a super-repressor form of IB inhibited death. Whether NFB inhibits or promotes neuronal death may depend on the cell type and the experimental paradigm.
In this study we demonstrate that treatment with the neurotrophic factor PEDF activates NFB in both immature (DIV2) and mature (DIV8) GCGs. PEDF treatment led to decreased levels of IB proteins and an increased level of phosphorylated IB␣. Furthermore, PEDF-mediated activation of NFB was attenuated by ALLN, which inhibits proteosome activity, and by BAY 11-7082, which inhibits the phosphorylation of IB; in turn, these inhibitors blocked the ability of PEDF to protect the cells against apoptosis or glutamate toxicity. These results demonstrate that in neurons as in other cell types phosphorylation and degradation of IB proteins are required for activation of NFB, and more importantly, that activation of NFB mediates the ability of the neurotrophic factor PEDF to protect CGCs against both apoptotic and glutamate-induced death.
In the presence of serum, the cells require the appropriate level of depolarization, achieved by inclusion of a high concentration (25 mM) of extracellular potassium (51,52). Either lowering KCl to the physiological level (32) or removing the serum (52) induces apoptosis in CGCs. In our previous results (5), PEDF most effectively blocked induced apoptosis in immature cells (DIV2) when added 24 h before the induction of apoptosis but provided some protection when added simultaneously. However, 24 h of pretreatment with PEDF had a minimal effect when apoptosis was induced in mature DIV6 cells; the addition at the same time was completely ineffective. In this paper, consistent with our previous finding, the number of TUNELpositive cells induced by low K ϩ /serum-free medium in cultures of immature neurons (DIV2) was dramatically reduced by 24 h of pretreatment with PEDF (Fig. 4). The NFB inhibitors BAY 11-7082 and ALLN completely blocked this neuroprotective effect, suggesting that PEDF protects immature cerebellar granule neurons from apoptosis through NFB activation. Several NFB-regulated genes have been identified that may play important roles in increasing cellular resistance to apoptotic cell death. Tamatani et al. (37) report that induction of Bcl-2 and Bcl-x expression through NFB activation is involved in the neuroprotective action of TNF-␣ against hypoxia-or nitric oxide-induced injury. Mattson et al. (18) report that increased expression of Mn-SOD contributes to the neuroprotective action of TNF-␣. Expression of neurotrophic factors such as NGF and GDNF is also regulated by NFB activation (46,53,54). In this study, transient induction of Bcl-2, Bcl-x, and Mn-SOD mRNA expression as well as NGF, BDNF, and GDNF mRNA was observed in PEDF-treated immature neurons (DIV2). These results suggest that PEDF may protect the CGCs against induced apoptosis through induction of the anti-apoptotic genes Bcl-2, Bcl-x, and Mn-SOD; what role the transient expression of the neurotrophic factors may play remains to be examined.
In contrast to induced apoptosis, PEDF was active against glutamate toxicity but only in mature neurons (4), at least in part because glutamate receptors are expressed only in the mature neurons (55). Glutamate neurotoxicity appears to involve an increase in intracellular calcium resulting from the opening of N-methyl-D-aspartate channels, with possible con-tributions from voltage-sensitive calcium channels activated by glutamate-induced depolarization and calcium release triggered from intracellular stores (56 -58). In this study, we analyzed protective effects of PEDF against glutamate toxicity using both the LDH release assay and the MTS assay. Results from these assays suggest that PEDF promotes cell survival against glutamate toxicity through NFB activation, since both ALLN and BAY 11-7082 block the protective effect of PEDF. Although both necrotic and apoptotic cell death have been associated with glutamate toxicity in other cell types, in our model of glutamate toxicity the primary mechanism of cell death appears to be necrosis since the number of TUNELpositive cells was not changed significantly by glutamate (data not shown). Thus, PEDF prevents necrotic cell death in mature CGCs via an NFB-mediated mechanism, as was seen with the block of apoptosis. Interestingly, PEDF does not induce the anti-apoptotic genes Bcl-2, Bcl-x, or Mn-SOD in mature CGCs but causes a long-lasting induction of the genes for NGF, BDNF, and GDNF (still elevated 9 h after PEDF treatment). No NFkB binding site has been found in the BDNF promoter, although it does have possible CRE and AP-1 binding sites (59). Because induction of BDNF gene expression was observed in both immature and mature neurons, this observation raises the possibility that induction of BDNF mRNA by PEDF was achieved through another transcription factor such as CREB (cAMP-response element-binding protein) or AP-1. We detected transient low-level induction of CRE binding and CREB (cAMP-response element-binding protein) phosphorylation as well as of AP-1 binding in PEDF-treated CGCs (results not shown).
PEDF-treated neurons contain predominantly p50/p65 heterodimers and p50/p50 homodimers of NFB. However, the mature neurons differentially expressed more p65/p50 than p50/p50 relative to the immature neurons. The NFB complex, depending on its subunit composition, may stimulate or repress target genes. For example, specific induction of p50/p50 homodimer inhibits p65/p50-induced TNF-␣ gene expression (60 -62). More recently Ravi et al. (63) report that c-Rel induces expression of the death receptors tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R1 and TRAIL-R2 and promotes TRAIL-induced cell death, whereas cytokine-mediated activation of the p65 subunit of NFB increases expression of the apoptosis inhibitor, Bcl-x, and protects cells from TRAIL. These observations raise the possibility that the difference between NFB dimers may affect gene expression and susceptibility of cells to various cytotoxic stimuli including glutamate or low K ϩ . Thus, the differential expression of NFB dimer species in mature versus immature CGCs after PEDF treatment may be responsible for the differences observed in gene expression, which in turn determine the ability of PEDF to protect against apoptotic versus glutamate-induced death.