{'fraction_non_alphanumeric': 0.03667527884554792, 'fraction_numerical': 0.024261138067931187, 'mean_word_length': 4.7458363504706735, 'pattern_counts': {'":': 0, '<': 5, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
AMP N1-oxide, a unique compound of royal jelly, induces neurite outgrowth from PC12 cells via signaling by protein kinase A independent of that by mitogen-activated protein kinase

Earlier we identified adenosine monophosphate (AMP) N1-oxide as a unique compound of royal jelly (RJ) that induces neurite outgrowth (neuritegenesis) from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK) but also that of cAMP/calcium-response element-binding protein (CREB) in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA) by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.


Introduction
Royal jelly (RJ), which is fed to the queen honeybee, has been reported to have a variety of biological activities towards various types of cells (1)(2)(3)(4). Although there are few reports so far showing the effects of RJ on the nervous system, we recently found that an extract of RJ induces neurites from cultured PC12 cells, a cell line of rat pheochromocytoma, and identified adenosine monophosphate (AMP) N 1 -oxide as one of the active components (5). AMP N 1 -oxide is a unique compound not found in natural products other than RJ; and it suppresses the proliferation of PC12 cells and stimulates the expression of neurofilament M, a protein of mature neurons, thus demonstrating that AMP N 1 -oxide induces neuronal differentiation of PC12 cells (5), as does nerve growth factor (NGF), a well-known neurotrophic factor that affects PC12 cells. In response to the binding of NGF to receptors p75 and TrkA, PC12 cells stop dividing and extend neurites, differentiating into neurons similar to those found in sympathetic neurons (6). NGF triggers mainly two cascades of cellular signaling that mediate neurite outgrowth, neurofilament M expression and cell survival, i.e. mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1 or 2 (ERK1/2) and phosphatidylinositol 3-kinase/Akt pathways (6,7).
The neurite outgrowth-promoting activity of AMP N 1 -oxide was found to be mediated by adenylate cyclase-coupled adenosine A 2A receptors (5). Adenosine A 2A receptor activation leads to an increase in the cAMP level followed by activation of protein kinase A (PKA) and extracellular signal-regulated kinase 1 or 2 (ERK1/2) in PC12 cells (8,9). Activation of ERK1/2 is a checkpoint to assess the activation of the Ras/MAPK cascade. Moreover cAMP elevation induces the development of neurites similarly as treatment with NGF (9). Forskolin, an activator of adenylate cyclase, increases the cAMP level, whose increase is in turn followed by neuronal differentiation (10). Also, the rescuing effect of A 2A receptor-mediated cAMP/PKA signals was reported in the case of NGF-induced neurite outgrowth from PC12 cells impaired by suppression of the MAPK cascade (11).
Therefore, in this study, we focused on elucidating the intracellular signaling by AMP N 1 -oxide responsible for stimulation of neurite outgrowth and/or suppression of cell growth. Also, we speculated about the action of AMP N 1 -oxide in RJ toward the nervous system.

Materials and Methods
Materials AMP N 1 -oxide was chemically synthesized (5). The adenosine A 2A receptor antagonist ZM241385 and epidermal growth factor (EGF) were purchased from Sigma (St. Louis, MO). The PKA inhibitor KT5720 was purchased from Biomol GmbH (Hamburg, Germany). The MEK inhibitor PD98059 came from Wako, and the Trk inhibitor K252a, from Santa Cruz Biotechnology (Santa Cruz, CA). NGF was purified from mouse submaxillary glands as described previously (12).

Assessments of Neurite Outgrowth and Cell Growth
PC12 cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% horse serum and 5% fetal bovine serum (6). For assessment of neurite outgrowth, cells were plated at 10 4 -10 5 cells/well in six well plates coated with collagen. The cells were exposed to test agents 1 day after plating, and morphological changes were observed under a phase-contrast microscope. Neurite-bearing cells were defined as those with processes longer than the length of the cell body. Four areas, each containing 100-200 cells, were randomly selected in each well, and the neurite-bearing cells were counted. Cell proliferation was evaluated by counting the total cell number, or by incorporation of 5-bromodeoxyuridine (BrdU) (Sigma) as described (13). Cells were cultured on cover glasses coated with polyl-lysine and collagen in medium containing test samples. The cultures were incubated for another 4 h after the addition of BrdU (10 mM), fixed with 4% paraformaldehyde, and reacted with Alexa Fluor TM -conjugated anti-BrdU antibody (Sigma). For estimating total cell number, cell nuclei were stained with propidium iodide (Invitrogen, Carlsbad, CA).

Immunoprecipitation
AMP N 1 -oxide or NGF was added to PC12 cell cultures, which were then incubated for 10 min, 1 h or 2 h. The cells were washed with ice-cold PBS, and lysed with the lysis buffer. The cell lysates were incubated overnight at 4 C with anti-Trk antibody (C-14; Santa Cruz Biotechnology), followed by incubation with protein A-Sepharose beads (Amersham Pharmacia Biotech, IL) for 1 h at 4 C. The pellet was then collected, washed with the lysis buffer, and subjected to SDS-PAGE. Phosphorylated TrkA was detected by Western blotting with anti-phosphotyrosine antibody (Santa Cruz Biotechnology).

Correlation Between Antimitotic Activity and Neurite
Outgrowth-promoting Activity AMP N 1 -oxide at concentrations between 1 and 33 mM induced neurite outgrowth from PC12 cells in a dosedependent manner (Fig. 1A) similarly as shown earlier (5). On the other hand, when the antimitotic effect of AMP N 1 -oxide on PC12 cells was examined by using the BrdU-labeling method, the ratio of BrdU-positive cells to total cells decreased with an increase in the concentration of AMP N 1 -oxide (Fig. 1B). At the highest concentration tested (33 mM), AMP N 1 -oxide reduced the ratio of BrdU-positive cells to nearly 2%, but induced the maximal neurite outgrowth. Namely, the rate of cell proliferation was inversely related to the degree of neurite outgrowth. In other words, the antimitotic activity of AMP N 1 -oxide may be critical for the neurite outgrowthpromoting activity by this molecule.

Phosphorylation of CREB and MAPK by AMP N 1 -oxide
We first examined the phosphorylation of CREB in response to AMP N 1 -oxide, since we had earlier shown that the actions of AMP N 1 -oxide were mediated by the adenylcyclase-coupled adenosine receptor A 2A (5). As Fig. 2A shows, phosphorylation of CREB was increased dose dependently by AMP N 1 -oxide up to 500 mM, and diminished by the selective A 2A receptor antagonist ZM241385 (Fig. 2B), suggesting that the phosphorylation of CREB was evoked by AMP N 1 -oxide through A 2A receptor-mediated PKA activation.
MAPK was also phosphorylated in a dose-dependent manner by AMP N 1 -oxide from 4 to 500 mM (Fig. 3A).
The MEK inhibitor PD98059 or A 2A receptor antagonist ZM241385 clearly inhibited the phosphorylation of MAPK, whereas the PKA inhibitor KT5720 did not affect it even when administered at concentrations higher than 2 mM (Fig. 3B), demonstrating that MAPK phosphorylation was evoked through A 2A receptor-mediated MEK activation independent of PKA activity.
Involvement of MAPK or PKA Signaling in the Action Mechanism of AMP N 1 -oxide Involvement of MAPK activation in neurite outgrowth was next examined. The MEK inhibitor PD98059 did not inhibit AMP N 1 -oxide-induced neurite outgrowth (Fig. 4A), suggesting the induced outgrowth to be independent of the MAPK signaling pathway. The suppression  of cell growth was not dependent on MAPK, either, since the growth of PC12 cells was also slow when cells were treated with AMP N 1 -oxide and PD98059 at the same time. However, the PKA inhibitor KT5720 significantly reduced a percentage of the cells with the AMP N 1 -oxideinduced neurites (Fig. 4A), and simultaneously attenuated the AMP N 1 -oxide-induced suppression of cell proliferation (Fig. 4B). These observations suggest the participation of PKA signaling not only in the neuritegenic activity of AMP N 1 -oxide but also in its cell growthsuppressing activity.
Interaction of the Action Mechanism of AMP N 1 -oxide with TrkA Signal Pathway Transactivation of mitogenic kinase receptors through G protein-coupled receptors was described earlier (14,15). Namely, adenosine and adenosine receptor agonists were reported to activate Trk receptors through a mechanism that requires the adenosine A 2A receptor (16). Therefore, we examined whether TrkA was activated during neurite outgrowth by AMP N 1 -oxide. However, we could not obtain any positive results indicating activation (data not shown). Furthermore, treatment of cells with a selective TrkA inhibitor, K252a, greatly reduced the phosphorylation level of MAPK of cells incubated with NGF, but the level obtained with AMP N 1 -oxide was unaffected (data not shown). The neurite outgrowth by AMP N 1 -oxide was also unaffected by K252a. Thus, TrkA activation seems not to be involved in the response of cells to AMP N 1 -oxide.  N 1 -oxide (B). The cells were precultured for 30 min in medium containing KT5720 (2 mM) or PD98059 (100 mM). Then, AMP N 1 -oxide (20 or 100 mM) was added, and the cells were cultured for another 1 day. A: the number of process-bearing cells was counted, and the ratio of them to total cells was calculated. The values are expressed as the mean AE standard error (n = 6). Significant difference from the nontreated cells was determined by one-way ANOVA with Tukey's test, *P < 0.05. 'ns' means non-significant relationship. B: the cells were cultured for 4 days in medium containing the indicated concentrations of AMP N 1 -oxide and KT5720. The number of cells was then counted, and the values expressed as the mean AE standard error (n = 3). Significant differences from the values of the corresponding cells untreated with the inhibitor were determined by one-way ANOVA with Tukey's test, ***P < 0.001, **P < 0.01, *P < 0.05. Significance of differences in values between non-treated control cells and the cells treated with 20 mM AMP N 1 -oxide was determined by Student's t-test, ***P < 0.001.

Discussion
Adenosine plays an essential role in modulating neuronal function via adenosine receptors (17). In the central nervous system (CNS), the A 2A receptor gene is strongly expressed and is suggested to be involved in the regulation of synaptic plasticity and to play a critical role in early neuronal development (18). In such a context, adenosine derivatives may have various effects on the CNS, and be expected to modulate brain functions.
Here, we demonstrated that MAPK phosphorylation was evoked through A 2A receptor-mediated MEK activation in a manner independent of PKA signaling (Fig. 3). Recently, Kim et al. (19) reported that secretin-induced neurite outgrowth of PC12 cells and that the activity was dependent on a cAMP-MAPK pathway. According to them, a PKA inhibitor suppressed the phosphorylation of MAPK, which is inconsistent with our results. MAPK can be phosphorylated in response to cAMP in a manner independent of PKA, whose phosphorylation is mediated by the Rap1-B-Raf pathway (20) or an Src family kinase (21). This pathway might be involved in the mechanism of AMPN 1 -oxide-evoked MAPK activation.
Integrin signals activate the MAPK signaling pathway, which is required for Mn 2+ -induced neurite outgrowth of PC12 cells (22). TrkA signals evoked by NGF also cause MEK activation followed by MAPK phosphorylation necessary for neurite outgrowth (23). Thus, it is obvious that both Mn 2+ -and NGF-induced neurite outgrowth require activation of MAPK. However, AMP N 1 -oxideinduced neurite outgrowth was independent of the activation of the MAPK signaling pathway (Fig. 4). This mechanism may be supported by A 2A signalmediated activation of CREB, which is known to induce neurite outgrowth without MAPK signaling (24). In this study, we found that the AMP N 1 -oxide-induced CREB phosphorylation dependently on A 2A receptormediated PKA signaling (Fig. 2). However, we have not yet had direct evidence whether the CREB phosphorylation is correlated with neurite outgrowth or cell growth suppression elicited by AMP N 1 -oxide.
Neuronal differentiation is accompanied by suppressed cell proliferation as reported earlier (25). Although the mechanism underlying cell growth suppression by AMP N 1 -oxide is not known, we showed earlier that activities of both cell growth suppression and neurite outgrowth are mediated by the adenosine A 2A receptor (5). Other investigators recently identified translin-associated protein X (TRAX) as a novel protein that interacts with the C-terminal part of the adenosine A 2A receptor, and considered TRAX to be an essential molecule for mediating A 2A receptor-induced inhibition of cell proliferation (26). It is likely that AMP N 1 -oxide interacts with TRAX at higher affinity than does AMP. This hypothesis may explain the preferential neurite outgrowth by AMP N 1 -oxide. Thus, AMP N 1 -oxide may modulate the activities of various neurotrophic factors through TRAX and A 2A receptors to a greater extent than AMP. Therefore, AMP N 1 -oxide would be expected to have beneficial effects on the CNS when used in combination with other agents such as favorable neurotrophic factors.
AMP N 1 -oxide is a unique compound not found in natural resources so far other than RJ. Therefore, it may be the molecule responsible for RJ-specific physiological actions on the CNS. Our study provides molecular-based evidence that RJ regulates neuronal functions through A 2A receptor signaling enhanced by AMP N 1 -oxide. We expect neuroprotective effects of AMP N 1 -oxide on the brain as a potent agonist of A 2A receptor, because particular ligands of A 2A receptors including selective agonists are reported to protect neurons against kainateinduced excitotoxicity in vivo (27) or to attenuate the A 1 receptor-mediated synaptic depression in the CA1 area of the hippocampus in vitro (28). In addition, A 2A receptor antagonists can also reduce damage produced by combinations of sub-threshold doses of the endogenous excitotoxin quinolinic acid and free radicals (29). These observations suggest that up or down regulation of A 2A receptor-linked signaling pathway appear to be promising for the prevention of neuronal damage (30). As A 2A receptors are predominantly expressed in dendrites of medium spiny neurons of the striatum in rats, Cabeza et al. (31) demonstrated that A 2A receptor agonist, CGS-21680, activated both ERK1/2 and CREB in the caudateputamen, suggesting that AMP N 1 -oxide modulates neuronal signaling in various brain regions including caudate-putamen.
Elucidation of the physiological roles of AMP N 1 -oxide in brain function is important to develop RJ as an evidence-based complementary and alternative medicine. Taking RJ as a source of AMP N 1 -oxide may be promising to maintain healthy brain functions.