CREB Regulates AChE-R-Induced Proliferation of Human Glioblastoma Cells

Abstract

The cyclic adenosine monophosphate (AMP) response element-binding protein, CREB, often modulates stress responses. Here, we report that CREB suppresses the glioblastoma proliferative effect of the stress-induced acetylcholinesterase variant, AChE-R. In human U87MG glioblastoma cells, AChE-R formed a triple complex with protein kinase C (PKC) ε and the scaffold protein RACK1, enhanced PKCε phosphorylation, and facilitated BrdU incorporation. Either overexpressed CREB, or antisense destruction of AChE-R mRNA, PKC, or protein kinase A (PKA) inhibitors—but not CREB combined with PKC inhibition suppressed—this proliferation, suggesting that CREB's repression of this process involves a PKC-mediated pathway, whereas impaired CREB regulation allows AChE-R-induced, PKA-mediated proliferation of glioblastoma tumors.

Introduction

Glioblastoma multiforme (GBM), the most common primary brain tumor, carries a grave prognosis, despite aggressive treatment [1]. However, the mechanisms underlying GBM pathogenesis and poor response to conventional therapy are yet unclear. GBMs commonly overexpress both the platelet-derived growth factor receptor and the epidermal growth factor receptor and their ligands [2]; the latter can activate various signaling pathways associated with glioblastoma cell survival and tumor formation [3]. Also, GBMs either overexpress or lose expression of various protein kinase C (PKC) isoforms implicated in cell proliferation and invasion [4]. In particular, various stress signals, growth factors, and kinases promote phosphorylation-mediated activation of the cyclic adenosine monophosphate (AMP) response element-binding transcription factor, CREB, involved in glial cell fate determination [5,6]. As some of these signals also induce expression of the acetylcholinesterase variant AChE-R [7] and because AChE-R is involved in glioblastoma proliferation [8], we explored the possibility that these effects are interrelated. The three 3′ splice variants of AChE have distinct noncatalytic activities (reviewed in Ref. [7]; Figure 1A). The “synaptic” (tailed) isoform, AChE-S, is the principal AChE variant in the brain and muscles and its C-terminus is encoded by the open reading frame in exon (E) 6. AChE-E, the “erythrocytic” (hydrophobic) isoform, links E4 and E5 to encode a different 43-amino acid C-terminal peptide, which is anchored through a glycophosphoinositide moiety to erythrocyte surface membranes. The “readthrough” isoform, AChE-R, expressed in embryonic and tumor cells possesses a C-terminus encoded by intron 4. AChE-R is overproduced under psychological stress in response to AChE inhibitors and in myasthenic muscles, all of which are under cholinergic imbalance [7–9]. We have recently shown that AChE mRNA accumulates in primary human astrocytomas in correlation with these tumors' grade of aggressiveness, which further associates with an mRNA splicing shift from AChE-S to the AChE-R transcript [10]. In the present study, we further investigate the function of the R-splice variant in cell proliferation and the signaling molecules that mediate AChE-R's effect. Here, we report that in human glioblastoma cells, CREB, a common downstream target for multiple signaling pathways, is an intrinsic repressor of PKCε-mediated AChE-R-induced proliferation, and demonstrate how this function may fail under drastic excess of AChE-R. Under PKC inhibition, which blocks CREB's repression, AChE-R may still promote proliferation, probably through a protein kinase A (PKA)-mediated pathway.

Figure 1.

CREB interactions with AChE splice variants affect glioblastoma proliferation. (A) Shown is a schematic presentation of the ACHE gene (as included in the reverse sequence of the cosmid inset; accession no. AF002993) and the relevant splice variants, AChE-S and AChE-R. Exons (gray boxes) and introns (white boxes) are marked. ACHE gene expression is regulated by a distal domain (DD), a proximal promoter (PP) that includes a CRE domain, and an intronic enhancer (IE). In glioblastoma cells, this gene is transcribed into AChE-R and AChE-S mRNA with distinct 3′ domains [7,8]. Expression of CREB in U87MG cells cytoplasmic (C) and nuclear (N) extracts transfected with either irrelevant DNA (Ct), AChE-R, AChE-S, or CREB expression vectors, or AChE-R and CREB together. Transfection efficacy, assessed by GFP expression, was 25 ± 8%. Lower panel shows densitometric quantification of CREB expression in the nuclear extracts. (C) AChE-R overexpression increases U87MG cell proliferation in an antisense suppressible manner: BrdU incorporation was assessed in U87MG cells, under various treatments, with or without overexpressing AChE-R (black and white columns, respectively). Treatment included transfections with AChE-S or CREB expression vectors, or treatment with the human (h) EN101 antisense oligonucleotide targeted at human AChE mRNA or with the corresponding inverse oligonucleotide INVEN101, in both cases with or without AChE-R. Notice that both CREB and EN101 were able to suppress AChE-R-induced cell proliferation. Columns show percent increase over control (= cells transfected with the irrelevant GFP plasmid) ± SEM; average of four duplicate transfections. *Statistically significant difference from control (P < .005, ANOVA). Mean absorbance value (A₄₀₅/A₄₉₂) for control was 0.3.

Materials and Methods

Cell Cultures and Transfection

Human glioblastoma U87MG and COS1 cells were grown in Dulbecco's modified Eagle's medium (Biological Industries, Beit Ha'emek, Israel) with 10% fetal calf serum (FCS) and 2 mM l-glutamine at 37°C and 5% CO₂-humidified chamber. PC12 cells were grown in Dulbecco's modified Eagle's medium with 10% FCS, 8% donor horse serum (DHS), and 2 mM l-glutamine. Transfections involved 0.4 or 12 µg of plasmid DNA per well in 48-well plates or 25-cm² flasks (using Lipofectamine Plus; Gibco BRL Life Technologies, Bethesda, MD) of either human AChE-R, AChE-S [8] or CREB expression vectors [11], or AChE-R and CREB together. The nonrelevant pEGFP-C2 plasmid (Clontech Laboratories, Palo Alto, CA) served as control.

Nuclear Protein Extractions

Nuclear and cytoplasmic protein extracts were prepared from U87MG cells 24 hours posttransfection. Cells were washed twice in phosphate-buffered saline (PBS), scraped, precipitated by centrifugation (1000 rpm, 5 minutes), then rewashed in PBS. Precipitates were incubated with complete miniprotease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and 150 µl of buffer A (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, and 1 mM EDTA) on ice (15 minutes), lysed through a 21-gauge needle, and centrifuged at 14,000 rpm (4°C, 10 minutes). Supernatants containing cytoplasmic protein extracts were removed. One hundred microliters of buffer B (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, and 1.5 mM MgCl₂), 25 µl of 5 M NaCl, and complete miniprotease inhibitor cocktail were added to the precipitate, incubated on ice (30 minutes), and centrifuged at 14,000 rpm (4°C, 10 minutes). Supernatants containing nuclear protein extracts were stored at -20°C until use.

Cell Proliferation Assay

U87MG human glioblastoma cells were grown in 48-well plates and transfected (in quadriplicates) as described above. Cell proliferation was assessed 30 hours posttransfection by measuring the incorporation of 5′-bromo-2-deoxyuridine (BrdU; Roche Diagnostics GmbH) over 6 hours, as previously described [12].

Oligonucleotides

Human (h)EN101, a 20-mer antisense oligonucleotide targeted at exon 2 of human AChE mRNA, was added to the culture medium with transfected DNA at a concentration of 2 nM, previously reported to induce preferential degradation of AChE-R mRNA [9,12,13]. Antisense penetrance into cells was previously quantified using fluoresceinlabeled EN101. At 2 nM, virtually all treated cells include EN101 molecules [14]. EN101's three 3′-terminal residues (*) were substituted with oxymethyl groups at the 2′ position (5′-CTGCGATATTTTCTT GTA*C*C*-3′). Similarly protected INVEN101, an oligonucleotide of sequence inverse to EN101, served as control [9].

Immunoprecipitation

Cell homogenates were prepared [13] and incubated (overnight, 4°C) with 2 µg of goat polyclonal antibodies targeted to the human AChE N-terminus (Santa Cruz Biotechnology, Santa Cruz, CA), then with 50 µl of suspended four Fast Flow protein G Sepharose beads (1 hour, 4°C; Amersham Pharmacia Biotech, Upsala, Sweden). Sedimented beads were washed three times in NET buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% gelatin, and complete miniprotease inhibitor cocktail; Roche Diagnostics GmbH), suspended in sample buffer, heated (95°C, 5 minutes), and centrifuged at maximal speed to remove beads.

Immunoblot Analyses

U87MG cell homogenates prepared 24 hours posttransfection, protein extracts, or immunoprecipitates prepared as described above were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; BioRad, Hercules, CA) and blotted onto nitrocellulose membranes. Membranes were incubated in a blocking solution (3% skim milk, 2% bovine serum albumin [or 5% bovine serum albumin when detecting a phosphorylated protein], 0.3% Tween-20 in Tris-buffered saline, 1 hour, room temperature). Immunodetection (4°C, overnight) was with either monoclonal mouse anti-PKCβ (P2584; Sigma Chemical Co., St. Louis, MO) diluted 1:8000, mouse anti-PKCε (Transduction Laboratories, San Diego, CA) diluted 1:1000, mouse anti-RACK1 (R20620; Transduction Laboratories, Lexington, KY) diluted 1:2500, rabbit anti-phosphorylated PKC-ε antibodies (diluted 1:300; Santa Cruz Biotechnology), rabbit anti-CREB diluted 1:800 (New England Biolabs, Beverly, MA), or rabbit anti-Ser-133 phosphorylated CREB diluted 1:600 (New England Biolabs). Development (room temperature, 2 hours) was with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies diluted 1:10,000 (Jackson Laboratories, West Grove, PA) and enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech, UK).

Specific Inhibitors

H-89, a specific PKA inhibitor, and bisindolylmaleimide (BIM), a specific PKC inhibitor (Calbiochem, San Diego, CA), were used at 10 µM. Diisopropylfluorophosphate (DFP), an organophosphate inhibitor of AChE catalytic activity, was used at 1 µM.

AChE's catalytic activity

AChE's catalytic activity, measured by accumulation of hydrolyzed acetylthiocholine (ATCh), was assessed as previously described [13].

Results

To explore the potentially interrelated role(s) of CREB and AChE-R in controlling human glioblastoma cell proliferation and to search for the mechanism(s) underlying such proliferation, we compared transfection and antisense suppression effects with those of anticholinesterases and protein kinase inhibitors by measuring cell proliferation and protein levels and properties.

CREB Can Suppress AChE-R-Induced Proliferation

In human glioblastoma U87MG cells, transfection with an AChE-R, but not an AChE-S expression vector, enhanced BrdU incorporation by 42 ± 4% (average ± SEM; Figure 1C), as compared to control cells transfected with the nonrelevant pEGFP-C2 plasmid (P = .001, ANOVA). This suggested that AChE-R's proliferative effect is independent of AChE's catalytic activity, shared by these two AChE variants.

AChE-R is a soluble protein [7]. Thus, AChE-R transfection leads to AChE-R secretion, which may spread the proliferative effect also to cells that do not contain the plasmid. Therefore, the increase in proliferation surpassed the percentage of transfected cells, which was assessed by GFP expression as 25 ± 8%.

The transcription factor, CREB, notably mediates cellular responses to various mitogens and stressors. CREB is expressed in glioblastoma cells; however, its modulation in these cells under stress was not studied extensively. CREB transfection resulted in nuclear accumulation of its protein product in U87MG cells (Figure 1B). Also, CREB transfection induced irreproducible changes in BrdU incorporation, with insignificant effects on cell proliferation. AChE activity remained unchanged in cells overexpressing CREB as compared to controls, although the ACHE promoter includes a potential CREB-binding site (data not shown). In contrast, U87MG cells cotransfected with CREB and AChE-R displayed suppressed cell proliferation to a nonsignificant difference from control (control levels ± 5%) (Figure 1C). CREB levels remained high in the cotransfected cells, suggesting an antimitogenic role for CREB over AChE-R-mediated proliferation in astrocytes. To test the alternative possibility—namely that both AChE-R and CREB (although to a lesser extent) each act independently to induce cell proliferation (but contact-mediated growth control masks this cumulative effect)—we cultured U87MG cells at lower densities (10,000 and 5000 cells/well). However, in these cultures as well, co-overexpression of AChE-R and CREB did not increase cell proliferation as compared to control cultures (data not shown), making this possibility highly unlikely.

Antisense Suppression Supports the Notion of a Selective AChE-R Proliferative Effect

EN101 selectively induces destruction of the AChE-R mRNA transcript. At 2 nM concentration, EN101 induced an inconsistent effect on cell proliferation, which did not reach statistical significance. However, in U87MG cells overexpressing AChE-R, EN101 reduced proliferation by 78% (from 42% to 11% increase in BrdU incorporation over control, P < .005). The effect was sequence-specific, as the inverse sequence, INVEN101, did not suppress AChE-R-induced proliferation (maintaining a 35 ± 4% increase in cell proliferation over control; Figure 1C). Thus, both gain of function (namely, the proliferative effect induced by transfections with an AChE-R, but not AChE-S expression vector) as well as loss of function (abolition of the AChE-R proliferative effect by a selective antisense agent) support the variant specificity of AChE-R's proliferative effect.

AChE-R Forms Triple Complexes with RACK1 and PKCε

In neuronal cells, AChE-R forms intracellular triple complexes with PKCβII and its scaffold protein, RACK1 [13]. However, immunoblot analysis failed to detect PKCβ in U87MG cell extracts (data not shown). To explore the protein-protein interactions of AChE-R in glioblastoma cells, we used antibodies targeted at the N-terminus of AChE for coimmunoprecipitation tests. In cell homogenates from AChE-R-transfected U87MG cells, these antibodies again failed to coimmunoprecipitate PKCβII, but coimmunoprecipitated both RACK1 and PKCε, a novel calcium-independent PKC isotype previously reported as involved in astrocytoma proliferation [15] (Figure 2). Antibodies to RACK1, which efficiently detect it in immunoblots, failed to pull down these multiprotein complexes. Compatible with previous reports [13], this may be due to the corresponding epitope being located in a site masked in the AChE-R/RACK1/PKC complexes. Anti-AChE antibodies failed to precipitate any of these proteins in monkey kidney COS1 cells, which do not express AChE, attesting to the specificity of these analyses. Rat pheochromocytoma (PC12) cells express both PKCβ and PKCε; however, anti-AChE antibodies pulled down RACK1 and PKCβ, but not PKCε, from homogenates of these cells (Figure 2), suggesting that PKCβ competes successfully with PKCε in the formation of such complexes and that the RACK1-mediated AChE-R interaction with PKC is cell type- and PKC isoform-specific.

Figure 2.

PKCε, but not PKCβ, forms triple complexes with AChE and RACK1 in U87MG cells. Shown are results of immunodetection of RACK1, PKCε, and PKCβ in either U87MG, PC12, or COS1 cells immunoprecipitated with antibodies targeted to the human AChE N-terminus or PC12 whole cell homogenates. Schematic presentations of the protein complexes specific for each cell type, as suggested by the immunoprecipitations, are drawn.

AChE-R Overexpression in U87MG Cells Facilitates PKCε Phosphorylation

To test the functional significance of the in vitro-observed AChE-R-PKCε interaction, we studied the effect of AChE-R overexpression on PKCε phosphorylation by using selective antibodies (Figure 3A).

Figure 3.

Enhanced PKCε phosphorylation under an excess of AChE-R, but not of AChE-S. (A) Western blot analysis showing immunodetection with anti-phosphorylated PKCε antibodies in U87MG extracts from three different transfections with either AChE-R or AChE-S vectors. Densitometric quantification of the p-PKCε signal is presented in the lower panel. (B) AChE's catalytic activity (measured by accumulation of hydrolyzed acetylthiocholine, ATCh) in corresponding cell extracts.

Facilitated PKCε interaction with antibodies specific for phosphorylated PKCε was reproducibly observed in extracts of U87MG cells overexpressing AChE-R, as compared to control cells or cells overexpressing AChE-S. This effect, as well, appeared to be independent of AChE's catalytic activity. Although AChE expression is well documented in primary human glioblastoma tumors [16,17], U87MG cells showed only minimal endogenous AChE catalytic activity. Also, AChE protein levels were negligible as immunolabeling of AChE in U87MG extracts was very faint (data not shown), likely reflecting residual levels of the protein from the serum component of the medium the cells were grown in. Overexpressing the AChE variants in these cells makes this model somewhat more biologically relevant. AChE activity was higher in cell homogenates overexpressing AChE-S; however, PKCε phosphorylation was much more limited, as compared with AChE-R-overexpressing cells (Figure 3B). Thus, these data support our hypothesis that the RACK1-AChE-R interaction, but not acetylcholine hydrolysis, facilitates the AChE-R-induced proliferation.

AChE-R-Induced Proliferation Involves PKC and PKA Phosphorylation, But Not Cholinergic Signaling

We treated AChE-R-transfected U87MG cells with 1 µM DFP, an inhibitor of AChE's catalytic activity; H-89, a selective PKA inhibitor [18]; or BIM, which inhibits PKC activities [19]. DFP did not suppress the AChE-R-induced proliferation of AChE-R transfected U87MG cells (data not shown), supporting our conclusion that this effect is noncatalytic. In contrast, at 10 µM, either H-89 or BIM completely suppressed the AChE-R proliferative effect (Figure 4A), suggesting that both PKC and PKA signaling pathways are involved in the AChE-R-induced proliferation of U87MG cells.

Figure 4.

CREB release of AChE-R-induced proliferation under PKC inhibition. (A) Shown is the outcome of U87MG cell transfections with CREB, AChE-R, or AChE-S vectors, alone (top) or together (bottom), following treatment with either 10 µM H-89, a specific PKA inhibitor (hatched bars), or 10 µM BIM, a specific PKC inhibitor (filled bars). Columns represent percent increase of BrdU incorporation over control ± SEM; average of four duplicate transfections. *Statistically significant difference from control (P < .05, ANOVA). (B) CREB phosphorylation increases in U87MG cells under cotransfection with CREB and AChE-R. Immunoblot analysis was with antibodies specific for Ser-133-phosphorylated CREB. A densitometric quantification of the p-CREB signal is shown. (C) Proposed mechanism for AChE-R-induced glioblastoma cell proliferation.

AChE-R Proliferative Effect Under PKC Inhibition of CREB Signaling

Neither BIM nor H-89 affected U87MG proliferation under AChE-S/CREB co-overexpression. Also, the PKA inhibitor, H-89, had no apparent effect on U87MG cell proliferation under AChE-R/CREB co-overexpression. In contrast, cell proliferation increased significantly (46 ± 1.5%, P < .005) over control in BIM-treated cells co-overexpressing AChE-R and CREB (Figure 4A), demonstrating that BIM revoked CREB's suppression of AChE-R-induced proliferation, retrieving the full scope of the AChE-R proliferative effect. This was compatible with the assumption that the CREB-suppressive effect over AChE-R-induced U87MG cell proliferation depends on PKC activation. Indeed, CREB phosphorylation increased in cells cotransfected with AChE-R and CREB, suggesting an interaction between these two signaling pathways (Figure 4B). Nevertheless, under PKC inhibition, which prevents the suppressive effect of CREB, AChE-R proliferative effects could be transduced through the PKA-dependent pathway (Figure 4, left upper panel).

Discussion

Using U87MG cells, we found that the transcription factor, CREB, and the stress-induced variant of acetylcholinesterase, AChE-R, contribute together to the balance between signals promoting and suppressing the proliferation of glioblastoma cells. AChE-R enhances proliferation in a manner independent of its catalytic activity, probably transduced by either PKC- or PKA-mediated signaling pathways, and suppressible by CREB as well as by an AChE-R-targeted antisense agent. In glioblastoma cells, AChE-R interacts with RACK1 and PKCε in a triple complex that differs from the PKCβII-including complex of PC12 cells. Our findings are compatible with the assumption that in glioblastoma cells under acute situations, associated with extreme excess of AChE-R, CREB's regulation may fail to prevent uncontrolled proliferation.

Transcriptional Regulation Of AChE-R-Induced Proliferation

Our findings suggest an antimitogenic role for CREB in astrocytes and point to an intrinsic transcriptional regulation mechanism over AChE-R-mediated proliferation. CREB is a plasticity-associated transcription factor, mediating responses to various neurotransmitters, mitogenic factors, and differentiating factors [6]. CREB promotes proliferation and survival of neurons and glia in the injured brain [20] and mediates cell viability during early embryonic development [21]. However, in smooth muscle cells, CREB activation (by Ser-133 phosphorylation) associates with suppressed expression of multiple cell cycle regulatory genes and reduced proliferation [6,22]. Thus, CREB may operate either as an inducer or as a suppressor of gene expression, depending on the signal pathway promoting its activation.

Antisense Suppression of the AChE-R Proliferative Effect

EN101 is a 2′-oxymethylated antisense oligonucleotide, which targets a common site on the exon 2-encoded part of AChE mRNA. EN101 selectively induces destruction of the unstable AChE-R mRNA transcript, possibly because it can interact only with newly transcribed AChE mRNA chains. Whereas the relatively stable AChE-S mRNA is protected from degradation in translatable complexes, the rapidly emerging AChE-R mRNA transcripts are destroyed before having the chance to get protected. Selective AChE-R mRNA destruction by EN101 was demonstrated in the mouse [13], rat [9], and human clinical studies [23]. Nanomolar doses of such antisense agents attenuated cell proliferation in cultured osteosarcoma cells (SaOs-2) [12] and human hematopoietic progenitor cells [24]. Here, we report that EN101 was able to significantly suppress the AChE-R proliferative effect in cultured glioblastoma cells, suggesting a role for AChE in the pathogenesis of various tumors. Although EN101 is currently being tested in the UK and Israel for its capacity to improve neuromuscular functioning in myasthenic patients [23], over a dozen anti-tumor antisense drugs are currently being tested for treating different tumors, at different phases of clinical trials [25]. Further research will be required to test the anti-neoplastic utility of EN101 in the treatment of glioblastoma and/or other tumors.

Attributing AChE-R-Induced Proliferation to PKCε

AChE-R interaction with PKCβII and its scaffold protein, RACK1, was recently reported to mediate extended conflict behavior [13]. In glioblastoma U87MG cells, PKCε, but not PKCβ, forms triple complexes with RACK1 and AChE. The RACK1-mediated interaction between AChE and a specific PKC isoform thus appears to be cell type-restricted. Although both PKCβ and PKCε were detected in PC12 cells, only PKCβ formed AChE-RACK1 complexes in these cells, suggesting that PKCε would interact with AChE-RACK1 complexes only in the absence of PKCβ. Another possibility is that because the complexes were formed with endogenous AChE, rather than with an overexpressed specific variant, difference in the composition of AChE variants may contribute to the formation of these triple complexes.

In human glioblastoma cells, induction of a dominant-negative PKCε mutant blocked cell proliferation [15]. PKCε, furthermore, contributes to tumor development and cell invasion in prostate cancer [26] and induces glial cell metastasis by activating Erk to mediate integrin-dependent adhesion and cell migration [27]. RACK1 links PKCε to integrin β chains [28], suggesting its involvement with these events.

PKCε's activation and ability to respond to secondary messengers require specific phosphorylation [29]. In this study, we show an association between PKCε phosphorylation and AChE-R overexpression in glioblastoma cells. The AChE-R-PKCε interaction in glioblastoma cells, as well as PKCε's facilitated phosphorylation, thus highlight AChE-R's involvement in a signaling pathway associated with tumor cell proliferation and aggressiveness, supporting our notion of AChE-R's role in glioblastoma tumorigenesis.

AChE-R-Induced Proliferation Involves Phosphorylation, But Not Cholinergic Signaling

Acetylcholine stimulates proliferation of rat cortical astrocytes and human astrocytoma cells by activating muscarinic acetylcholine receptors [30,31]. MAPK, PKCε, and ζ were also implicated in this process [32,33]. However, the more aggressive glioblastoma cells lack functioning acetylcholine receptors [34]. Together with our finding that AChE-R-induced proliferation is refractory to DFP inhibition of AChE's catalytic activity, this suggests that glioblastoma proliferation dissociates from cholinergic pathways. Within transfected cells, the C-terminus of AChE-R confines it to the cytoplasm [8]. There is also recent immunocytochemical evidence demonstrating the cytosolic localization of neuronal AChE-R [35]. To promote cell proliferation, signal transduction pathways are hence required for inducing nuclear activation of cell cycle effectors. We found that AChE-R-induced proliferation involves both PKA and PKC signals. The fact that both PKC and PKA inhibition completely abolished AChE-R-induced proliferation raises a possibility that calls for further exploration—that these pathways are being activated in succession following AChE-R's signal. Moreover, as signaling pathways can be activated and inactivated by a number of intermediates, other pathways and mediators should be further investigated as well.

Suppressed AChE-R Proliferative Effect Under PKC-Induced CREB Signaling

Various growth factors, stress signals, and kinases, including PKA, PKC, calcium/calmodulin kinase 2, and MAPK-activated protein (MAPKAP), promote CREB activation by Ser-133 phosphorylation, resulting in complex, at times diverse, cellular outcomes, including cell proliferation or quiescence, which are context- and activator-dependent [6,11,36]. PKC-mediated CREB activation induced proliferation of early oligodendrocytes [37]. Although PKA-dependent CREB activation promotes astroglia differentiation [5], it is required for Schwann cells proliferation [38]. CREB's suppression of AChE-R-induced proliferation was associated with CREB/Ser-133 phosphorylation and was revoked by PKC—but not by PKA—inhibitors, suggesting that CREB's antimitogenic effects are PKC-mediated, but also that AChE-R may induce proliferation through a PKA-dependent pathway. This is compatible with the assumption that AChE-R-induced PKA-mediated proliferation, which would be redundant in the presence of effective PKC-mediated CREB activation and phosphorylation under low endogenous CREB levels, can become fully expressed under PKC inhibition and CREB overexpression: an increase in AChE-R levels results in a PKC-mediated (according to our data, PKCε-mediated) cell proliferation. This PKC activation is probably PKA-dependent, as both PKC and PKA inhibitors abolished AChE-R's effects, suggesting that these pathways are activated in succession. However, because PKC may be activated through other pathways as well, this calls for further investigation in order to show direct PKA-dependent PKC activation upon AChE-R signal.

PKA may also mediate cell proliferation through a CREB-dependent pathway, as reported by others [39,40]. Our data suggest that when activated by PKC, CREB suppresses AChE-R-induced cell proliferation, unless AChE-R is in extreme excess as compared to CREB's endogenous levels. Under PKC inhibition, both AChE-R-PKC-mediated proliferation as well as PKC-mediated CREB's inhibitory effect are attenuated. Yet, under AChE-R excess, PKC inhibition results in a net proliferative effect probably because AChE-R induces proliferation through a non-PKC-dependent—perhaps a PKA-mediated—pathway. Nevertheless, other signaling pathways may be involved in this effect as well (Figure 4, scheme).

In conclusion, our findings are compatible with the hypothesis that CREB's basal levels are insufficient to block the AChE-R proliferative effect in cells with extreme excess of AChE-R compared to CREB (e.g., under AChE-R transfection). This may increase the risk for glial tumor growth in individuals exposed to anticholinesterases or head trauma, both shown to induce massive AChE-R overexpression [7].