Limonene Enhances the cAMP Response Element (CRE)-Dependent Transcriptional Activity Activated via Adenosine A2 A Receptor in a Neural-Crest Derived Cell Line, PC-12

Abstract

Prevention and treatment of Alzheimerʼs disease are urgent problems for elderly people in developed countries. To seek useful compounds that treat, delay the onset, or prevent the development of Alzheimerʼs disease, we screened natural resources using the cyclic AMP response element-dependent transcription in the PC12 cell line. We previously found that honey bee royal jelly stimulates the cyclic AMP response element-dependent transcription activity in PC12 cells. Here, we report that R-limonene, a monoterpene, enhanced the royal jelly-induced cyclic AMP response element-dependent transcription activity, whereas R-limonene showed no effect in absence of royal jelly. The enhancement was inhibited by ZM241385, an antagonist for adenosine A2 A receptor. R-limonene also enhanced the cyclic AMP response element-dependent transcription activity activated by adenosine and AMP. Thus, R-limonene represents a novel type of modulator that enhances the CRE-dependent transcription via adenosine A2 A receptor pathway.

Key words

adenosine receptor - CRE-dependent transcription - limonene - memory - royal jelly

Abbreviations

AD: Alzheimerʼs disease

cAMP: cyclic AMP

CRE: cAMP response element

LTP: long-term potentiation

RJ: royal jelly

Neurodegenerative disorders, including AD, have a devastating impact on both patients and society [1]. Due to this expanding threat to our society, disease management and therapy are urgently needed. The biological basis of learning and the memory depends on the establishment of LTP in the hippocampus [2]. Biochemically, LTP is formed by activation of cAMP-dependent signaling and subsequent transcription of the target genes of the CRE-binding protein. Our screening of natural resources lead to the finding that nobiletin, a citrus flavonoid, activates the CRE-dependent transcription in PC12D cells, a rat pheochromocytoma cell line [3]. Our next studies proved that nobiletin improves the memory deterioration in beta-amyloid infused AD model rats [4], bulbectomy-induced cholinergic neurodegenerative mice [5], a transgenic mouse model of AD [6], and Parkinson disease model mice [7]. Using the same strategy, we found that honeybee RJ stimulates the CRE-dependent transcription in PC12D cells [8]. RJ contains AMP and AMP-N1-oxide that induces the neurite outgrowth via adenosine A2 A receptor [9].

Adenosine A2 A receptor is a G-protein coupled receptor that distributes in the central nervous system [10]. The role of adenosine A2 A receptor in the formation of LTP is puzzling [11]. Although an agonist of the receptor increased LTP in the rat hippocampus [12], mice deficient in the receptor exhibited the improved spatial recognition [13]. Thus, modulators of the receptor are potential pharmacological tools to treat AD, although there is a controversy over the function of adenosine A2 A receptor in the brain activity.

To find a substance that potentiates the antidementia activity of RJ, we screened extracts derived from herbal plants. Preliminary screening identified limonene as a candidate for the activator of the RJ-induced stimulation of CRE-dependent transcription in PC12 cells. To confirm this finding, we tested commercially available limonenes for the CRE-dependent transcription induced by RJ in PC12 cells. As shown in [Fig. 1 A], R-limonene dose-dependently enhanced the CRE-dependent transcription activity induced by 10 µg/mL RJ. Interestingly, R-limonene had no effect on the CRE-dependent transcription activity in the absence of RJ. R-limonene was effective on both low and high concentrations of RJ ([Fig. 1 B]).

Fig. 1 Limonene enhances the CRE-dependent transcription induced by RJ in PC12 cells. A Limonene dose-dependently enhanced the CRE-dependent transcription induced by RJ. Note that limonene alone had no effect on the CRE-dependent transcription. B Limonene was effective on various concentrations of RJ in enhancing the CRE-dependent transcription. PC12 cells were transfected with a CRE reporter plasmid and incubated with RJ in the presence or absence of 100 µM limonene for 5 h. The luciferase activity was relative to the control value. The assay was performed in quadruplicate. **P < 0.05.

We next examined the effect of R-limonene on RJ along the time-course of its stimulation of CRE-dependent transcription ([Fig. 2]). RJ at 10 µg/mL significantly increased the CRE-dependent transcription activity 1–5 h after administration. The activating effect of RJ diminished at 24 and 48 h after administration. The addition of 100 µM R-limonene to RJ significantly enhanced the CRE-dependent transcription activity induced by RJ from 1 to 24 h. Again, R-limonene alone had no effect on the CRE-dependent transcription activity at all time points examined.

Fig. 2 Time-course of the effect of limonene on the CRE-dependent transcription induced by RJ. PC12 cells were transfected with a CRE reporter plasmid and stimulated with RJ and/or limonene for the indicated times. The luciferase activity was relative to the control value (1 h without any additives). •, none; ○, 100 µM limonene; ■, 10 µg/mL RJ; □, 10 µg/mL RJ + 100 µM limonene. The assay was performed in quadruplicate. **Significantly different from the value with RJ at the same time point, p < 0.05.

We next tested the effect of several analogues of R-limonene on the CRE-dependent transcription induced by RJ ([Fig. 3]). (S)-(−)-limonene and (4R)-limonene oxide enhanced the RJ-induced CRE-dependent transcription comparable to the level of R-limonene. (4 S)-limonene oxide significantly increased the CRE-dependent transcription induced by RJ, but the potency of the limonene oxide was weaker than that of R-limonene. Interestingly, all these limonene analogues had no effect on the CRE-dependent transcription in absence of RJ. These results suggest the propenyl moiety that attaches to the cyclohexene or cyclohexane might be important for the activiating effect of limonenes.

Fig. 3 Effect of limonene analogues on the CRE-dependent transcription induced by RJ in PC12 cells. PC12 cells were transfected with a CRE reporter plasmid and incubated with RJ and/or various types of limonene analogues for 5 h. The luciferase activity was relative to the control value. 1, control; 2, R-limonene; 3, (S)-(−)-limonene; 4, (4R)-limonene oxide; 5, (4 S)-limonene oxide; 6, 10 µg/mL RJ; 7, 10 µg/mL RJ + R-limonene; 8, 10 µg/mL RJ + (S)-(−)-limonene; 9, 10 µg/mL RJ + (4R)-limonene oxide; 10, 10 µg/mL RJ + (4 S)-limonene oxide. Limonene analogues were tested at 100 µM. The assay was performed in quadruplicate. **Significantly different from the value with RJ alone, p < 0.05.

Next, we examined the effect of the adenosine receptor A2 A antagonist ZM241385 on the RJ-induced stimulation of CRE-dependent transcription. The antagonist dose-dependently suppressed the CRE-dependent transcription induced by RJ ([Fig. 4]). The limonene-induced enhancement of the CRE-dependent transcription was also inhibited by ZM241385 in a similar manner. These results suggest that the action of RJ on the CRE-dependent transcription is probably mediated via the adenosine receptor A2 A.

Fig. 4 Effect of ZM241385, an antagonist of adenosine A2 A receptor, on the CRE-dependent transcription induced by RJ and limonene. PC12 cells were transfected with a CRE reporter plasmid and incubated with 10 µg/mL RJ and/or 100 µM limonene in the presence of various concentrations of ZM241385 for 5 h. The luciferase activity was relative to the control value. The assay was performed in quadruplicate. **Significantly different from the value without ZM241385, p < 0.05.

We examined the effect of limonene on the CRE-dependent transcription induced by adenosines. Both adenosine and AMP activated the CRE-dependent transcription in PC12 cells in a dose-dependent manner (Fig. 1S, Supporting Information). Adenosine was more potent than AMP in activating the CRE-dependent transcription. The addition of limonene dose-dependently enhanced the CRE-dependent transcription induced by adenosine (Fig. 2A S, Supporting Information). (S)-(−)-limonene and (4R)-limonene oxide significantly enhanced the adenosine-induced CRE-dependent transcription like R-limonene (Fig. 2B S, Supporting Information). (4 S)-Limonene oxide slightly increased the CRE-dependent transcription induced by adenosine, but the increase was insignificant. These results confirmed the effect of limonenes on RJ and that limonene acts on the signaling pathway that includes adenosine receptor A2 A.

We have shown here that limonene enhanced the CRE-dependent transcription induced by RJ and adenosine via adenosine receptor A2 A in PC12 cells. RJ has been known to induce the neurite outgrowth mediated by the adenosine receptor in PC12 cells [9]. Both the neurite outgrowth and the CRE-dependent transcription induced by RJ appear to share the similar signal transduction pathways. In this regard, it is interesting that limonene competitively decreased the binding of [³H]CGS 21 680, an A2 A receptor agonist, to a rat brain cerebral cortex membrane, suggesting that limonene is a direct ligand for A2 A receptor [14]. But limonene alone failed to stimulate the CRE-dependent transcription in PC12 cells ([Figs. 1 A], [2] and [3]), whereas AMP and adenosine activated the CRE-dependent transcription (Fig. 1S, Supporting Information). Thus, the interaction of limonene with the adenosine receptor may be different from that of the authentic ligands. Limonene may act on the adenosine A2 A receptor in an allosteric manner.

That limonene itself has no activity to induce the CRE-dependent transcription, while it augments the activity of ligands of adenosine A2 A receptor, makes limonene a unique compound. A comparison of the activities of limonene analogues revealed that the propenyl moiety that attaches to the cyclohexene or cyclohexane might be important in modulating the activity of ligands of the adenosine A2 A receptor ([Fig. 3]). The structure will become a lead compound to develop a novel type of agonists with higher activity than limonene.

Materials and Methods

Materials

Native RJ was obtained by the Yamaguchiʼs organic bee culture method [15]. The RJ used in this study was prepared from frozen powder of native RJ as described [8]. R-limonene was purchased from Nacalai Tesque. (S)-(−)-Limonene, (4R)-Limonene oxide (mixture of cis and trans), (4 S)-Limonene oxide (mixture of cis and trans), adenosine 5′-monophosphate, adenosine, and ZM 241 385 (purity > 98 %) were obtained from Sigma-Aldrich. PC12 cells were obtained from the Riken BioResource Center.

Measurements of CRE-dependent transcriptional activity in PC12 cells

To measure the CRE-dependent transcriptional activity in PC12 cells, we adopted the reporter gene assay as described previously [8]. Details are provided in the Supporting Information. The concentration of limonene and its analogues used in the present study (100 µM) had little effect on the morphology of PC 12 cells.

Statistical analysis

Data are expressed as the means ± SD. Comparison between the groups were performed using paired Studentʼs t-test using Microsoft Excel.

Supporting information

Details on the measurement of the CRE-dependent transcriptional activity in PC12 cells and on the effects of adenosine and AMP on the CRE-dependent transcription in PC12 cells, and of limonene on the CRE-dependent transcription induced by adenosine are available as Supporting Information.

Acknowledgements

We thank Ms. Michi Kawada for her excellent technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

^* These authors contributed equally to this work.

• References

• 1 Tanzi RE, Bertram L. Twenty years of the Alzheimerʼs disease amyloid hypothesis: a genetic perspective. Cell 2005; 120: 545-555
• 2 Kandel ER. The molecular biology of memory storage: a dialogue between genes and synapses. Science 2001; 294: 1030-1038
• 3 Nagase H, Omae N, Omori A, Nakagawasai O, Tadano T, Yokosuka A, Sashida Y, Mimaki Y, Yamakuni T, Ohizumi Y. Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities. Biochem Biophys Res Commun 2005; 337: 1330-1336
• 4 Matsuzaki K, Yamakuni T, Hashimoto M, Haque AM, Shido O, Mimaki Y, Sashida Y, Ohizumi Y. Nobiletin restoring beta-amyloid-impaired CREB phosphorylation rescues memory deterioration in Alzheimerʼs disease model rats. Neurosci Lett 2006; 400: 230-234
• 5 Nakajima A, Yamakuni T, Haraguchi M, Omae N, Song SY, Kato C, Nakagawasai O, Tadano T, Yokosuka A, Mimaki Y, Sashida Y, Ohizumi Y. Nobiletin, a citrus flavonoid that improves memory impairment, rescues bulbectomy-induced cholinergic neurodegeneration in mice. J Pharmacol Sci 2007; 105: 122-126
• 6 Onozuka H, Nakajima A, Matsuzaki K, Shin RW, Ogino K, Saigusa D, Tetsu N, Yokosuka A, Sashida Y, Mimaki Y, Yamakuni T, Ohizumi Y. Nobiletin, a citrus flavonoid, improves memory impairment and Abeta pathology in a transgenic mouse model of Alzheimerʼs disease. J Pharmacol Exp Ther 2008; 326: 739-744
• 7 Yabuki Y, Ohizumi Y, Yokosuka A, Mimaki Y, Fukunaga K. Nobiletin treatment improves motor and cognitive deficits seen in MPTP-induced Parkinson model mice. Neuroscience 2014; 259: 126-141
• 8 Fujiwara H, Kogure A, Sakamoto M, Yamakuni T, Mimaki Y, Murata K, Hitomi N, Yamaguchi K, Ohizumi Y. Honeybee royal jelly and nobiletin stimulate CRE-mediated transcription in ERK-independent and -dependent fashions, respectively, in PC12D cells. J Pharmacol Sci 2011; 116: 384-387
• 9 Hattori N, Nomoto H, Mishima S, Inagaki S, Goto M, Sako M, Furukawa S. Identification of AMP N1-oxide in royal jelly as a component neurotrophic toward cultured rat pheochromocytoma PC12 cells. Biosci Biotechnol Biochem 2006; 70: 897-906
• 10 Stone TW, Ceruti S, Abbracchio MP. Adenosine receptors and neurological disease: neuroprotection and neurodegeneration. Handb Exp Pharmacol 2009; 193: 535-587
• 11 Düster R, Prickaerts J, Blokland A. Purinergic signaling and hippocampal long-term potentiation. Curr Neuropharmacol 2014; 12: 37-43
• 12 de Mendonça A, Ribeiro JA. Endogenous adenosine modulates long-term potentiation in the hippocampus. Neuroscience 1994; 62: 385-390
• 13 Wang JH, Ma YY, van den Buuse M. Improved spatial recognition memory in mice lacking adenosine A2 A receptors. Exp Neurol 2006; 199: 438-445
• 14 Park HM, Lee JH, Yaoyao J, Jun HJ, Lee SJ. Limonene, a natural cyclic terpene, is an agonistic ligand for adenosine A(2 A) receptors. Biochem Biophys Res Commun 2011; 404: 345-348
• 15 Yamaguchi K. Innovation of natural beekeeping and quality control of bee products for promising apiculture. 1. Current problems of beekeeping and recurrence of natural bee-culture for resolution of the problems. J Yunnan Agric Univ 2004; 19: 322-329