cAMP-responsive Element-binding Protein (CREB) and cAMP Co-regulate Activator Protein 1 (AP1)-dependent Regeneration-associated Gene Expression and Neurite Growth*

Background: The inability to up-regulate “regeneration-associated genes” contributes to the failure of axonal regeneration after injury. Results: Expression of activated CREB and cAMP up-regulation co-regulate AP1-dependent regeneration-associated gene expression and enhance axon growth. Conclusion: Targeting transcription factor activity and the associated second messengers facilitates regenerative growth. Significance: This transcription factor plus small molecule approach may be an efficient means to stimulate axon regeneration. To regenerate damaged axons, neurons must express a cassette of regeneration-associated genes (RAGs) that increases intrinsic growth capacity and confers resistance to extrinsic inhibitory cues. Here we show that dibutyrl-cAMP or forskolin combined with constitutive-active CREB are superior to either agent alone in driving neurite growth on permissive and inhibitory substrates. Of the RAGs examined, only arginase 1 (Arg1) expression correlated with the increased neurite growth induced by the cAMP/CREB combination, both of which were AP1-dependent. This suggests that cAMP-induced AP1 activity is necessary and interacts with CREB to drive expression of RAGs relevant for regeneration and demonstrates that combining a small molecule (cAMP) with an activated transcription factor (CREB) stimulates the gene expression necessary to enhance axonal regeneration.

The capacity of peripheral axons of sensory neurons to regenerate after injury is highly dependent on the transcriptional response initiated by axon damage (1). This response includes the induction of regeneration-associated genes (RAGs) 2 (2)(3)(4) such as the activating transcription factor 3 (ATF3), argi-nase 1 (Arg1), galanin (Gal), and interleukin 6 (IL6). Moreover, although CNS-projecting axons do not regenerate after injury, the induction of RAGs by peripheral axon injury in sensory neurons facilitates the growth of subsequently lesioned CNSprojecting axons of the same neurons (5). The increase of cAMP levels after peripheral axon injury is thought to initiate regeneration-associated transcription (6); however, some studies indicate that cAMP up-regulation alone is less effective than peripheral nerve injury in driving axon regeneration (3,7,8). This suggests that regeneration-associated transcription requires additional biochemical effectors of peripheral nerve injury for optimal efficacy. Among the different transcription factors involved in axon regeneration (9,10), the cAMP-responsive element binding protein (CREB) has received significant attention given the ability of cAMP analogues to facilitate regeneration of some CNS axons in vivo and to promote neurite growth in the presence of myelin-derived inhibitors in vitro (6,11,12). Furthermore, expression of a constitutive-active CREB fusion protein allows dorsal root ganglion (DRG) neurons to overcome myelin-derived inhibitors of axonal growth in vitro and central axon regeneration of sensory neurons following a dorsal column lesion in vivo (13); however, the specific contribution of cAMP-signaling and CREB to regeneration-associated transcription remains unclear.
To address the roles of cAMP and CREB in promoting axon growth and regeneration-associated transcription, we targeted CREB activity by expressing constitutive-active (CREB-CA) and dominant-negative (CREB-DN) CREB variants in DRG neurons. We show that CREB-CA and dibutyryl-cAMP (Bt 2 cAMP) have both common and distinct targets among the RAGs that we investigated. Of note, CREB-CA and Bt 2 cAMP synergized to enhance neurite growth in an AP1-dependent fashion on inhibitory substrates, which mirrored the expression pattern of Arg1. Our findings indicate that maximum axon growth requires both CREB and cAMP-regulated AP1 activity. These results further reveal the complexity of regenerationassociated transcription and suggest that a combinatorial strategy affecting both CREB and AP1 activation may be optimal to trigger the regenerative phenotype.

EXPERIMENTAL PROCEDURES
Use of animals conformed to the Burke-Cornell IACUC guidelines under approved protocols.
Primary Neuron and Cell Culture-Mouse cortical neurons were cultured from E14.5 embryos as previously described and used for experiments 1 day later (14). Mouse DRG neurons were cultured from E12.5 embryos as previously described and plated in Neurobasal/B27 media supplemented with 50 ng/ml NGF (EMD Millipore) ϩ 10 M 5-fluorodeoxyuridine. Media were changed at 4 days in vitro, and cultures were used at 7 days in vitro when nearly all Schwann cells were killed. CHO cells stably expressing myelin-associated glycoprotein (MAG) or the empty pSJL vector were cultured as previously described (15). All cell culture media components were obtained from Invitrogen unless otherwise noted.
Real Time RT-PCR-RNA was harvested with TriReagent (Sigma), column-purified, and treated with DNase (Directzol kit, Zymo Research). cDNA libraries were generated (iScript, Bio-Rad) from which gene expression was assayed by real time PCR using TaqMan FAM-labeled probes (Invitrogen) with VIC-labeled ␤-actin or GAPDH included in duplex as endogenous controls. For Arg1, the endogenous controls were run in parallel. Relative expression levels of each gene are summarized in Table 1 EMSA-Nuclear proteins were prepared from DRG neuron cultures after treatment with Bt 2 cAMP (NE-PER kit; Pierce). 50 fmol of 5Ј IRDye700-labeled double-stranded DNA probes were combined with 5 g of nuclear protein, incubated at room temperature for 20 min, and resolved on a gradient acrylamide mini gel (Bio-Rad) at 100 V for 2 h. For supershift experiments, 5 g of each antibody was added to the nuclear protein/probe mixture before incubation. The gels were imaged using the LiCor gel imaging system (LiCor). The antibodies used were: rabbit IgG (12-370, Millipore), c-fos (SC-253X, Santa Cruz Biotechnology), c-Jun (SC-45X, Santa Cruz), JunD (SC-74X, Santa Cruz), and FosB (SC-28213X, Santa Cruz). Probe sequences are listed in Table 2.
ChIP-After treatments, cultures were fixed in 1% formalin (Sigma) in PBS for 9 min at room temperature and then quenched with 125 mM glycine in PBS for 5 min. Cells were collected in ice-cold harvesting buffer (100 mM Tris-HCl, 10 mM DTT, pH 9.4), pelleted, and lysed. The resulting chromatin was sheared by bath sonication (Bioruptor, Diagenode) and immunoprecipitated with 3 g of antibody using the low cell number ChIP kit (Diagenode). Enrichment of promoter regions was probed using SYBR-Green real time PCR (Invitrogen). The antibodies used were as with the EMSA supershift assays, CREB (17-600; Millipore) and acetyl-H3 (06-599; Millipore). The primer sequences are listed in Table 2.
Lentiviral Vector Generation-The coding sequences of modified CREB and Fos proteins were cloned into the LentiLox 3.7 backbone under the control of the synapsin I promoter, restricting expression to neurons. The viruses were produced by the University of Iowa Gene Transfer Vector Core (Iowa City, IA). Viruses were added at a multiplicity of infection of 5 directly to culture media at 4 days in vitro, and the neurons were treated or trypsinized at 7 days in vitro.
MAG Inhibition Assays-Untreated or lentivirus-transduced neurons were trypsinized and replated upon MAG-and R2-CHO cell monolayers in clear-bottom 96-well plates at ϳ150 neurons/well. Bt 2 cAMP (final 2 mM) or forskolin (final 10 M) was added 1 h later. 24 h later the co-cultures were fixed with 4% paraformaldehyde and then processed for fluorescence immunocytochemistry with an anti-␤III-tubulin antibody (1:500, rabbit monoclonal; Epitomics). Neurons were imaged using a flash cytometer (Trophos) that captures images of entire wells of the 96-well plate. The total neurite length/neuron was automatically measured using the neurite outgrowth application in Metamorph (Molecular Devices). Neurite length data are expressed as total neurite length per neuron. Representative higher magnification images were taken with an inverted Motic AE31 epifluorescence microscope with a 20ϫ objective.
Statistical Analysis-Data were plotted as the mean Ϯ S.E. unless otherwise indicated from at least three independent experiments from separate primary cultures. The statistical tests used are indicated in the figure legends. For mRNA expression, the data are plotted as box and whiskers to show the range, median, and mean obtained between independent experiments.

RESULTS
Increasing neuronal cAMP levels can promote neurite growth and reverse growth cone repulsion to inhibitory cues in vitro (3, 11, 18 -20). To assess neurite growth, we replated primary DRG neurons upon CHO cell monolayers stably expressing either MAG or a control vector (R2) in media lacking NGF to limit basal neurite growth. Neurons plated on MAG-CHO cells exhibited significant inhibition of neurite growth compared with those on R2-CHO cells, consistent with the inhibi-tory properties of MAG as a component of CNS myelin (Fig. 1, A-C) (15). The addition of Bt 2 cAMP, a cell-permeable cAMP analog, increased neurite length on both cell types, indicating that Bt 2 cAMP stimulates neurite growth on both inhibitory and permissive substrates (Fig. 1, A-C).
The transcription-dependent axon growth effects of cAMP have been attributed to the activation of CREB (13). To assess the role of CREB in neurite growth, we expressed a constitutiveactive CREB fusion protein (VP16CREB; referred to as CREB-CA), a dominant-negative CREB protein (ACREB; referred to as CREB-DN), or GFP control using lentiviruses to manipulate CREB transcriptional activity (21,22). At a multiplicity of infection of 5, this yielded 100% transduction efficiency. In DRG neurons, the expression of CREB-CA further increased Bt 2 cAMP-induced CRE reporter activity, whereas CREB-DN blocked CRE activity induced by Bt 2 cAMP (Fig. 1D). DRG neurons transduced with these viruses were replated upon the CHO cell monolayers to assess neurite growth. Consistent with previous studies, CREB-CA enhanced basal neurite growth on both MAG-and R2-CHO cells (Fig. 1, E-G) (13). Interestingly, CREB-CA showed marked synergy with Bt 2 cAMP, causing a greater than additive increase in neurite growth on MAG-CHO cells. Surprisingly, we found that the Bt 2 cAMP-mediated neurite growth persisted in neurons expressing CREB-DN ( Fig. 1, E-G). These results indicate that although activation of CREBdependent gene expression can by itself promote neurite growth, it is not necessary for cAMP-triggered growth, which signifies the existence of an alternative growth-promoting pathway downstream of cAMP.
To examine the specific contribution of CREB and its upstream second messenger cAMP to RAG expression, we measured the transcript levels of several candidate RAGs (2,3,9,10,(23)(24)(25)(26)(27) in neurons expressing either GFP, CREB-CA, or CREB-DN in the presence or absence of Bt 2 cAMP (Fig. 1H). Consistent with previous reports, Bt 2 cAMP increased the expression levels of a subset of these RAGs in GFP-transduced neurons (Fig. 1H); however, the behavior of these Bt 2 cAMPresponsive genes in neurons expressing either CREB-CA or CREB-DN were markedly different. We observed several distinct gene expression patterns. Of note are the following. 1) Vasoactive-intestinal peptide (VIP) was highly and maximally induced (ϳ47-fold) by Bt 2 cAMP. 2) ATF3 behaved as a canon- ical CREB target; CREB-DN prevented ATF3 induction by Bt 2 cAMP, whereas CREB-CA strongly induced its expression regardless of Bt 2 cAMP treatment. 3) IL6 transcription is completely CREB-independent; neither CREB-DN nor CREB-CA altered the induction of IL6 by Bt 2 cAMP. 4) Arg1 and Gal were up-regulated by Bt 2 cAMP in a CREB-independent manner (i.e. CREB-DN did not block their induction), although CREB-DN significantly decreased basal and, thus, Bt 2 cAMP-induced lev-els of Gal. CREB-CA alone also up-regulated their basal expression, but this was greatly increased in neurons that were also treated with Bt 2 cAMP (ϳ17-50-fold). The expression levels of each gene are summarized as a heat map (Fig. 1H) and in Table 1.
We next correlated RAG expression levels to neurite length on MAG-CHO cells under each CREB manipulation to determine the genes associated with neurite growth. By this analysis, Arg1 and Gal were the Bt 2 cAMP-induced genes with the highest correlation between expression levels and neurite length (R 2 ϭ 0.954 and 0.958, respectively; Fig. 1H). Because Gal expression was attenuated by CREB-DN, we focused on the transcriptional regulation of Arg1, which was unaltered by CREB-DN, to further explore the synergy between Bt 2 cAMP and CREB-CA in driving specific RAG expression.
To identify the promoter regions critical for Bt 2 cAMP responsiveness, we used a series of Arg1 promoter-luciferase reporter constructs with progressively shorter promoter regions (Ϫ4.8 kb, Ϫ3.3 kb, Ϫ2.8 kb, Ϫ1.4 kb, Ϫ0.8 kb, and Ϫ112 bp). Luciferase activity was equally up-regulated by Bt 2 cAMP (2 mM for 24 h) with all constructs, indicating that the minimum-essential promoter region resided in the initial 112 bp ( Fig. 2A). FIGURE 2. Arg1 transcription is mediated by AP1. A, promoter-reporter constructs containing different lengths of the Arg1 promoter were equally activated by Bt 2 cAMP in DRG neurons, indicating that the initial 112 bp contains the minimum essential promoter. p Ͻ 0.05, Bt 2 cAMP versus control for each construct, Mann-Whitney test, n ϭ 4. B, top, the initial 112 bp of the Arg1 promoter contains putative CREB and AP1 binding sites. Bottom, site-directed mutagenesis of the putative Arg1-AP1 binding site. C, chromatin from cortical neurons treated with Bt 2 cAMP was subjected to ChIP with a CREB antibody. CREB occupancy in the initial 112 bp of the Arg1 promoter was unchanged by Bt 2 cAMP, whereas occupancy was increased at the c-fos promoter, a CREB target gene. *, p Ͻ 0.05; n.s. ϭ p Ͼ 0.05, Mann-Whitney test, n ϭ 4. D, mutation of the putative Arg1-AP1 site abrogates promoter activation by Bt 2 cAMP (db-cAMP). *, p Ͻ 0.01; two-way ANOVA with Bonferroni post test, n ϭ 3. E, AP1 nuclear protein complexes bind to the Arg1 promoter. Nuclear proteins from Bt 2 cAMP-treated DRG (left and middle panels) and cortical (cort) neurons (right panel) were assayed by EMSA with an AP1 consensus (asterisk) or putative Arg1-AP1 (arrowheads) binding site probes. Bt 2 cAMP induced prominent mobility shifts of both probes. Cross competition with a 180-fold excess of unlabeled AP1 probe eliminated binding of the Arg1 probe, indicating that the same nuclear protein complexes bind to both probes. Representative gels from 3 independent experiments are shown. To determine whether CREB binds to this region, we assessed the occupancy of CREB by ChIP from neurons treated with Bt 2 cAMP (2 mM) for 2 h, a time point we have previously shown as maximal CREB activation (14). The initial 112-bp region of the Arg1 promoter was not enriched by CREB ChIP, indicating that CREB does not directly bind this minimal Arg1 promoter (Fig. 2C). By contrast, the CRE region of the Fos promoter, an established CREB target gene, was significantly enriched (28).
To determine which other transcription factor might bind to this region, we searched for binding sites using the Transfac 7.0 public database and identified a putative AP1 binding site (Fig.  2B, top panel). To assess its necessity, we mutated two nucleotides within this site of the Arg1-112-bp construct (Fig. 2B,  lower panel), which attenuated Bt 2 cAMP-induced reporter activity, indicating that the site was essential for promoter activation (Fig. 2D). We then used an EMSA with a consensus AP1 site probe to establish that Bt 2 cAMP (2 mM for 24 h) increased AP1 DNA binding activity of nuclear proteins from DRG neurons (Fig. 2E, left panel). Similarly, Bt 2 cAMP increased binding to a probe containing the putative Arg1-AP1 site (Fig. 2E, middle panel). Bt 2 cAMP also induced a mobility shift of the Arg1-AP1 probe with nuclear proteins from cortical neurons, indicating that binding to this site is regulated by Bt 2 cAMP in multiple neuron types (Fig. 2E, right panel). As expected, an excess of an unlabeled AP1 probe eliminated the binding of both labeled probes, indicating that the Bt 2 cAMP-induced mobility shifts were specific to the AP1 probe sequence and that the same nuclear protein complexes may bind both the Arg1 and consensus AP1 probes (Fig. 2E). Conversely, an excess of an unlabeled probe with a mutated AP1 sequence did not eliminate binding to the labeled probes (data not shown). To identify the AP1 subunits that are active in DRG neurons, we used antibodies against c-fos, FosB, JunD, and c-jun to supershift the consensus AP1 probe. The JunD antibody exhibited a prominent supershift, whereas the c-fos and c-jun antibodies attenuated binding of nuclear protein complexes to the probe, indicating that these subunits may be active after Bt 2 cAMP treatment (Fig. 2F).
We then assessed the occupancy of c-fos, c-jun, and JunD at the Arg1 promoter using ChIP analysis. Under basal conditions, the occupancy of only JunD was evident; treatment with Bt 2 cAMP released JunD while simultaneously inducing c-jun occupancy (Fig. 2G). These antibodies did not enrich chromatin from a gene desert used as a negative control. To determine whether the changes in AP1 activity were due to the transcriptional regulation of AP1 subunits, we measured the expression of Jun and JunD at 2, 8, and 24 h and found no change associated with Bt 2 cAMP exposure (Fig. 2H).
We next addressed the sufficiency and necessity of AP1 activity in driving Arg1 transcription by directly modulating AP1 activity using lentiviruses to express dominant-negative (AFos; referred to as Fos-DN) or constitutive-active (FosVP16; referred to as Fos-CA) variants of the Fos AP1 subunit (29,30). The expression of Fos-DN blocked Bt 2 cAMP-induced expression of an AP1 response element-driven luciferase reporter, whereas Fos-CA increased basal and Bt 2 cAMP-induced reporter activity (Fig. 2I). Importantly, Arg1 mRNA levels mirrored this pattern, as Fos-DN attenuated Arg1 induction by Bt 2 cAMP, whereas Fos-CA increased basal Arg1 levels and potentiated Bt 2 cAMP-mediated Arg1 expression (Fig. 2J). These data indicate that AP1 activity is a necessary mediator of Bt 2 cAMP-induced Arg1 transcription.
We next asked whether CREB-CA and Bt 2 cAMP induced Arg1 expression by activating AP1. The expression of CREB-CA increased AP1 reporter activity, indicating that it may directly drive AP1-mediated transcription (Fig. 3A). To determine whether AP1 activity was necessary, we co-expressed CREB-CA and Fos-DN to drive CREB activity while blocking AP1. This reversed the synergy between CREB-CA and Bt 2 cAMP, indicating that AP1 activity was necessary for Arg1 induction and that CREB gene products may interact with AP1 to further drive RAG transcription (Fig. 3B). As AP1 subunits can be CREB target genes, we measured the expression levels of two prominent subunits, Fos (Fig. 3C) and Jun (Fig. 3D), in neurons transduced with CREB-CA. The expression of Fos was markedly induced by CREB-CA (15-fold), whereas Jun levels were modestly, but significantly, up-regulated (1.5-fold). These data suggest that CREB-CA may increase AP1 activity by increasing the expression of AP1 subunits. To further probe the role of AP1 in RAG transcription, we measured the expression levels of all 12 RAGs under each manipulation of CREB and AP1 to see which other genes were also AP1-dependent (Fig.  3E, left panels, and Table 1). Interestingly, only Arg1 showed AP1 dependence for both Bt 2 cAMP and CREB-CA-induced expression (Table 1).
Because Bt 2 cAMP is a cell membrane permeant analog of cAMP that requires hydroylzation of the butyrate side chains for activity (31), we tested whether off-target actions of this compound contributed to our gene expression changes. We used forskolin (10 M), an activator of cellular adenylate cyclase, to generate intracellular cAMP in neurons transduced with GFP, CREB-CA, and CREB-CA ϩ Fos-DN (Fig. 3E, right panel, and Table 1). Interestingly, only IL6 expression deviated from that observed with Bt 2 cAMP, as IL6 levels were not induced by forskolin (Fig. 3). This congruence indicates that the gene expression effects and interaction of CREB-CA with Bt 2 cAMP are indeed cAMP-dependent.
We next assessed whether the manipulation of CREB and AP1 altered neurite growth of DRG neurons. Blocking AP1 activity with Fos-DN inhibited Bt 2 cAMP-mediated neurite growth on both MAG-and R2-CHO cells while driving AP1 activity with Fos-CA increased neurite growth in combination with Bt 2 cAMP only on MAG (Fig. 4, A-C); however, both basal and Bt 2 cAMP-induced neurite growth was greater in CREB-CA-expressing neurons. This effect of CREB-CA was reversed in cells co-transduced with CREB-CA and Fos-DN, indicating that AP1 mediated these effects of CREB activation (Fig. 4,  A-C). This necessity of AP1 was also evident on the permissive R2-CHO cells, although the effect of CREB-CA was additive. These data indicate that these manipulations drive both intrinsic growth and counteract the extrinsic inhibitory signals from MAG (Fig. 4, A-C). These experiments excluded NGF from the replating media to limit basal neurite growth from the trophic effects of NGF; thus, expression of Fos-CA and CREB-CA may simply reconstitute some of the transcriptional effects of NGF. We repeated these experiments with a low concentration of NGF (1 NOVEMBER 21, 2014 • VOLUME 289 • NUMBER 47 ng/ml) to determine whether neurite growth was still affected by manipulation of CREB and AP1 activity. The inclusion of NGF increased both basal and Bt 2 cAMP-mediated neurite growth on MAG-and R2-CHO cells; however, blocking AP1 with Fos-DN only partially abrogated the effect of Bt 2 cAMP on MAG-CHO cells, indicating that NGF can activate AP1-independent pathways that act with Bt 2 cAMP to promote neurite growth (Fig. 4, D and E). Despite the presence of NGF, the AP1-dependent interaction between CREB-CA and Bt 2 cAMP persisted, indicating that the benefit derived from further activating CREB required AP1 activity (Fig. 4, D and E). Finally, we used forskolin (10 M) to verify that the neurite growth effects of Bt 2 cAMP were due to the elevation of intracellular cAMP levels. Similar to gene expression, the neurite growth-promoting effect and interaction with CREB-CA mirrored that of Bt 2 cAMP (Fig. 4, F and G); however, the magnitude of growth was greater with forskolin, indicating that it may generate higher local levels of free cAMP than the Bt 2 cAMP analog.

CREB and cAMP Regulate Neurite Growth and Gene Transcription
To evaluate the relationship between the expression levels of the candidate RAGs and neurite growth on MAG-CHO cells with each manipulation of AP1 and CREB activity, we normalized the Bt 2 cAMP-induced expression levels of each gene and the corresponding neurite length data, assigning the levels obtained with GFP as the baseline (0) and the manipulation with highest gene expression/neurite growth as the maximum (100) and asked whether gene expression correlated with neurite length (Fig. 5, A and B). We found significant correlation between these factors only for Arg1 (R 2 ϭ 0.9699, p ϭ 0.0003, two-tailed Pearson test; Fig. 5A), indicating that the transcriptional program regulating Arg1 and possibly other RAGs not queried in our study may be the most relevant to neurite growth in DRG neurons. These findings also suggest that Arg1 expression may be a good biomarker for the activation of this program. Together, these data indicate that these RAGs may not be maximally up-regulated by Bt 2 cAMP alone and that further activation of transcription pathways downstream of cAMP (CREB and AP1) concurrent with cAMP up-regulation may provide superior RAG expression and neurite growth (Fig. 5C).

DISCUSSION
Analysis of the peripheral nerve injury-induced transcriptome has identified individual genes and pathways that contribute to axon regeneration. These transcriptional responses func- tion to increase intrinsic growth capacity and confer resilience to inhibitory cues, allowing neurons to regenerate damaged peripheral nervous system and CNS axons. The convergent nature of these responses stresses the importance of engaging these pathways sufficiently upstream of the terminal effectors (2,3,9,10,(32)(33)(34). Although cAMP-activated pathways are important components of this machinery, a number of studies show that cAMP elevation alone may insufficiently drive RAG transcription and is only effective at improving regeneration in some settings (3,6,8,12,20,35). This complexity is further exemplified by our disparate results regarding the efficacy of constitutive-active and dominant-negative versions of CREB and Fos to influence Bt 2 cAMP induction of RAGs and promote or interfere with Bt 2 cAMP-dependent neurite growth. In our studies we found that exposure to Bt 2 cAMP promoted neurite growth in DRG neurons, and this ability was further potentiated by the expression of a CREB-CA variant in the same cells. Moreover, our experiments identified the transcription factor AP1 as a likely candidate to synergize with CREB to enhance neurite growth on inhibitory substrates through the regulation of specific RAGs, such as Arg1 (Fig. 5C).
Although the expression of CREB-CA was sufficient to drive Arg1 transcription and neurite growth, we could not block the effects of Bt 2 cAMP by inhibiting CREB activity with CREB-DN ( Fig. 1H and Table 1). Instead, we found that both the Bt 2 cAMP-and CREB-CA-mediated transcription of Arg1 was AP1-dependent in DRG neurons, indicating that both manipulations act by inducing AP1 activity (Figs. 2J and 3B). Together, this suggests that Bt 2 cAMP activates both AP1 and CREB in parallel and that CREB-activated gene products likely interact with AP1 to further increase the expression of certain RAGs, including Arg1. Although we focused on an AP1 site in the initial 112 bp of the Arg1 promoter (Fig. A-G), other putative AP1 binding sites have been described further upstream and are functional in non-neuronal cell types; whether these sites act cooperatively in DRG neurons remains to be determined (36,37). Both Bt 2 cAMP and CREB-CA increased AP1mediated transcriptional activity in our study (Figs. 2I and 3A). Multiple mechanisms can increase AP1 activity, including the transcriptional up-regulation of AP1 subunits, changes in subunit composition, and subunit phosphorylation (38). In our study both Bt 2 cAMP and, to a greater extent, CREB-CA increased Fos mRNA levels ( Fig. 3C), whereas only CREB-CA increased Jun mRNA (Fig. 3D). Although the incorporation of Fos subunits can increase AP1 DNA binding and transcriptional activity, we did not find changes in Fos occupancy at the Arg1 promoter after Bt 2 cAMP treatment in control cells within the detection limits of our assay (Fig. 2G); however, along with the induction of Fos by Bt 2 cAMP, the inhibition Arg1 expression by Fos-DN and the effect of the c-Fos antibody on AP1 EMSA probe binding suggest a role for Fos in regulating Arg1 transcription (Fig. 2, F and J). With CREB-CA, the high levels of Fos expression and Jun induction may indeed drive the AP1dependent transcription, including that of Arg1 (Fig. 3, C and  D). AP1 transcriptional activity is enhanced by the phosphorylation of the N-terminal transactivation domain of Jun by c-Jun-N-terminal kinases (JNK) (39). This may be a point of AP1 regulation by cAMP, as cross-talk between cAMP-activated pathways (both protein kinase A and EPAC-mediated) and MAPK/JNK pathways has been described in other cell types and suggests the importance of the non-transcriptional actions of cAMP (40). We found a concurrent increase in Jun and decrease in JunD occupancy at the Arg1 promoter after Bt 2 cAMP treatment (Fig. 2G), that was not associated with changes in mRNA levels of either subunit (Fig. 2H). JunD is an atypical member of the AP1 family that can act as both a tran- , indicating that the CREB-sufficient but AP1-dependent transcriptome is relevant for promoting axon growth. C, scheme for the transcriptional regulation of cAMP-induced RAG expression. Bt 2 cAMP activates the transcription of Arg1 in a CREB-sufficient but AP1-dependent manner. Activated CREB synergizes with cAMP-induced AP1 activity to drive higher levels of Arg1 transcription as well as neurite growth (red arrows). scriptional activator and repressor (41). Together our data suggest that JunD binding to the Arg1 promoter in the basal state may interfere with CREB-CA-driven AP1-dependent transcription; upon Bt 2 cAMP treatment, the activation of AP1 may release JunD to enable strong transcription of AP1 targets downstream of CREB-CA.
Importantly, AP1 was necessary for Bt 2 cAMP-mediated neurite growth on both permissive and inhibitory substrates (Fig. 4, B and C), which is consistent with the prominent role for AP1 in mediating peripheral axon regeneration in vivo. AP1 activity is up-regulated in DRG neurons after peripheral nerve injury and remains elevated until target reinnervation (42), whereas targeted deletion of c-Jun in neurons impedes regeneration after axotomy (43), indicating that AP1 is necessary to maintain RAG expression to facilitate regeneration. On a network level, AP1 subunits have been identified as central hubs of injury-induced gene transcription in DRG mRNA profiling studies (3,9,32). Although other regeneration-associated AP1 targets have been identified, our data suggest that up-regulating any single gene product, including Arg1, is unlikely to harness the full effect of activating upstream transcription factors to drive the regenerative "program." This is evidenced by the inability of S-(2-boronoethyl)-L-cysteine (BEC), a pharmacological inhibitor of Arg1 enzymatic activity, to block the neurite growth effect of CREB-CA and Bt 2 cAMP (data not shown). As Arg1 has been previously shown to play important roles in nervous system repair by promoting polyamine synthesis, inhibiting nitric oxide production, and regulating protein translation, the lack of necessity for Arg1 does not preclude an important role for Arg1 in axonal regeneration but suggests that other genes and pathways likely compensate for the loss of Arg1 activity.
An important finding in this study is that the expression of Arg1, Gal, and NPY were further increased by directly activating CREB and/or AP1 transcriptional activity in conjunction with Bt 2 cAMP ( Fig. 3H and Table 1). This suggests that the activation of either transcription factor by Bt 2 cAMP is insufficient to drive maximal RAG expression and neurite growth or, alternatively, that Bt 2 cAMP triggers an inhibitory feedback loop such as the inducible cAMP early repressor (ICER) that can be overcome by the constitutive-active transcription factor variants (30). Notably, endogenous CREB mRNA levels were not altered by Bt 2 cAMP, and total levels of CREB transcripts (both from endogenous CREB and CREB-CA) were increased to 5-fold over GFP-transduced neurons by CREB-CA expression. This could indicate that the endogenous levels of CREB may be limiting in mediating RAG transcription in response to Bt 2 cAMP. By contrast, the combined Fos levels were 275-fold increased in transduced Fos-CA neurons ( Fig. 3E and Table 1), indicating that high Fos expression alone is insufficient to recapitulate the CREB-CA effect. Together this suggests that other direct CREB-CA target genes are required in addition to the activation of AP1.
In the context of peripheral nerve injury, many pathways are simultaneously activated within neurons and in surrounding non-neuronal cells. These include the retrograde transport of protein complexes from inured axons, including transcription factors (i.e. STAT3) and activators of injury signaling (i.e. JNK and ERK) that ultimately lead to transcriptional changes at the soma (9, 44 -46). Additionally, perturbation of the normal injury response of macrophages and Schwann cells can occlude axon regeneration and the peripheral lesion conditioning effect (47)(48)(49)(50). Some of these factors, such macrophage-released oncomodulin, work in synergy with cAMP to promote both peripheral nervous system and CNS axon regeneration (50,51). It is likely that these effectors converge with cAMP-mediated signaling and gene transcription to drive axonal regeneration.
After injury, CNS axons are exposed to factors that inhibit regeneration, including myelin proteins such as MAG. Although this may alter the transcriptional landscape of the associated neurons, previous CNS axon injury of DRG neurons does not preclude the induction of RAGs after peripheral axon injury, suggesting that the injury-induced transcriptional programs are not repressed by inhibitory substrates (27). In the context of our studies, we expect that similar transcriptional changes occur in the neurons replated onto MAG-CHO cells as those plated upon laminin.
Our findings suggest that efforts to promote axonal regeneration by increasing neuronal cAMP levels alone may yield limited success. Rather, we identify a CREB-mediated, AP1-dependent transcriptional "module" that functions in concert with cAMP to promote robust neurite growth under inhibitory and permissive conditions. Although our data reaffirm the importance of CREB for axonal regeneration, they indicate that cAMP in conjunction with CREB activation is necessary to maximally activate the transcriptional program that recapitulates the regenerative phenotype. This interdependence between CREB and cAMP suggests that a strategy targeting more than a single transcription pathway (i.e. both CREB and AP1) may be more efficient than the genetic activation of a single transcriptional factor. This idea is consistent with the observed superior efficacy of multimodal combinatorial approaches utilizing cAMP elevation in combination with neurotrophic factors to promote regeneration (52)(53)(54). Additionally, as Arg1 mRNA expression is regulated in a CREB-sufficient and AP1-dependent fashion, Arg1 levels may serve as a relevant biomarker for pharmacological or genetic manipulations aimed at promoting regenerative gene transcription.