5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavo?ne(5-OH-HxMF), a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43). 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB) phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501), a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA) inhibitor, but not MEK1/2, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) or calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action.
References
[1]
Bui NT, Konig HG, Culmsee C, Bauerbach E, Poppe M, et al. (2002) p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones. J Neurochem 81: 594–605.
[2]
Schulte-Herbruggen O, Jockers-Scherubl MC, Hellweg R (2008) Neurotrophins: from pathophysiology to treatment in Alzheimer's disease. Curr Alzheimer Res 5: 38–44.
[3]
Levy YS, Gilgun-Sherki Y, Melamed E, Offen D (2005) Therapeutic potential of neurotrophic factors in neurodegenerative diseases. BioDrugs 19: 97–127.
[4]
Poduslo JF, Curran GL (1996) Increased permeability of superoxide dismutase at the blood-nerve and blood-brain barriers with retained enzymatic activity after covalent modification with the naturally occurring polyamine, putrescine. J Neurochem 67: 734–741.
[5]
Spedding M, Gressens P (2008) Neurotrophins and cytokines in neuronal plasticity. Novartis Found Symp 289: 222–233;discussion on 233-240.
[6]
Price RD, Milne SA, Sharkey J, Matsuoka N (2007) Advances in small molecules promoting neurotrophic function. Pharmacol Ther 115: 292–306.
[7]
Greene LA, Aletta JM, Rukenstein A, Green SH (1987) PC12 pheochromocytoma cells: culture, nerve growth factor treatment, and experimental exploitation. Methods Enzymol 147: 207–216.
[8]
Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 73: 2424–2428.
[9]
Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, et al. (1991) ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65: 663–675.
[10]
Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80: 179–185.
[11]
Boss V, Roback JD, Young AN, Roback LJ, Weisenhorn DM, et al. (2001) Nerve growth factor, but not epidermal growth factor, increases Fra-2 expression and alters Fra-2/JunD binding to AP-1 and CREB binding elements in pheochromocytoma (PC12) cells. J Neurosci 21: 18–26.
[12]
Spencer JP, Vauzour D, Rendeiro C (2009) Flavonoids and cognition: the molecular mechanisms underlying their behavioural effects. Arch Biochem Biophys 492: 1–9.
[13]
Zhao L, Brinton RD (2003) Vasopressin-induced cytoplasmic and nuclear calcium signaling in embryonic cortical astrocytes: dynamics of calcium and calcium-dependent kinase translocation. J Neurosci 23: 4228–4239.
[14]
Vitolo OV, Sant'Angelo A, Costanzo V, Battaglia F, Arancio O, et al. (2002) Amyloid beta -peptide inhibition of the PKA/CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling. Proc Natl Acad Sci U S A 99: 13217–13221.
[15]
Impey S, Obrietan K, Wong ST, Poser S, Yano S, et al. (1998) Cross talk between ERK and PKA is required for Ca2+ stimulation of CREB-dependent transcription and ERK nuclear translocation. Neuron 21: 869–883.
[16]
Honda K, Shimohama S, Sawada H, Kihara T, Nakamizo T, et al. (2001) Nongenomic antiapoptotic signal transduction by estrogen in cultured cortical neurons. J Neurosci Res 64: 466–475.
[17]
Leinninger GM, Backus C, Uhler MD, Lentz SI, Feldman EL (2004) Phosphatidylinositol 3-kinase and Akt effectors mediate insulin-like growth factor-I neuroprotection in dorsal root ganglia neurons. FASEB J 18: 1544–1546.
[18]
Fox K (2002) Anatomical pathways and molecular mechanisms for plasticity in the barrel cortex. Neuroscience 111: 799–814.
[19]
Miyamoto E (2006) Molecular mechanism of neuronal plasticity: induction and maintenance of long-term potentiation in the hippocampus. J Pharmacol Sci 100: 433–442.
[20]
Spencer JP (2010) The impact of fruit flavonoids on memory and cognition. Br J Nutr 104: Suppl 3S40–47.
[21]
Lotito SB, Frei B (2006) Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free Radic Biol Med 41: 1727–1746.
[22]
Yen JH, Weng CY, Li S, Lo YH, Pan MH, et al. (2011) Citrus flavonoid 5-demethylnobiletin suppresses scavenger receptor expression in THP-1 cells and alters lipid homeostasis in HepG2 liver cells. Mol Nutr Food Res 55: 733–748.
[23]
Li S, Lo CY, Ho CT (2006) Hydroxylated polymethoxyflavones and methylated flavonoids in sweet orange (Citrus sinensis) peel. J Agric Food Chem 54: 4176–4185.
[24]
Manthey JA, Grohmann K, Guthrie N (2001) Biological properties of citrus flavonoids pertaining to cancer and inflammation. Curr Med Chem 8: 135–153.
[25]
Lai CS, Tsai ML, Cheng AC, Li S, Lo CY, et al. (2011) Chemoprevention of colonic tumorigenesis by dietary hydroxylated polymethoxyflavones in azoxymethane-treated mice. Mol Nutr Food Res 55: 278–290.
[26]
Benavente-Garcia O, Castillo J (2008) Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. J Agric Food Chem 56: 6185–6205.
[27]
Nagase H, Yamakuni T, Matsuzaki K, Maruyama Y, Kasahara J, et al. (2005) Mechanism of neurotrophic action of nobiletin in PC12D cells. Biochemistry 44: 13683–13691.
[28]
Nagase H, Omae N, Omori A, Nakagawasai O, Tadano T, et al. (2005) Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities. Biochem and Biophys Res Commun 337: 1330–1336.
[29]
Matsuzaki K, Miyazaki K, Sakai S, Yawo H, Nakata N, et al. (2008) Nobiletin, a citrus flavonoid with neurotrophic action, augments protein kinase A-mediated phosphorylation of the AMPA receptor subunit, GluR1, and the postsynaptic receptor response to glutamate in murine hippocampus. Eur J Pharmacol 578: 194–200.
[30]
Nakajima A, Yamakuni T, Haraguchi M, Omae N, Song SY, et al. (2007) Nobiletin, a citrus flavonoid that improves memory impairment, rescues bulbectomy-induced cholinergic neurodegeneration in mice. J Pharmacol Sci 105: 122–126.
[31]
Yamamoto Y, Shioda N, Han F, Moriguchi S, Nakajima A, et al. (2009) Nobiletin improves brain ischemia-induced learning and memory deficits through stimulation of CaMKII and CREB phosphorylation. Brain Res 1295: 218–229.
[32]
Onozuka H, Nakajima A, Matsuzaki K, Shin RW, Ogino K, et al. (2008) Nobiletin, a citrus flavonoid, improves memory impairment and Abeta pathology in a transgenic mouse model of Alzheimer's disease. J Pharmacol Exp Ther 326: 739–744.
[33]
Al Rahim M, Nakajima A, Saigusa D, Tetsu N, Maruyama Y, et al. (2009) 4′-Demethylnobiletin, a bioactive metabolite of nobiletin enhancing PKA/ERK/CREB signaling, rescues learning impairment associated with NMDA receptor antagonism via stimulation of the ERK cascade. Biochemistry 48: 7713–7721.
[34]
Das KP, Freudenrich TM, Mundy WR (2004) Assessment of PC12 cell differentiation and neurite growth: a comparison of morphological and neurochemical measures. Neurotoxicol Teratol 26: 397–406.
[35]
Costello B, Meymandi A, Freeman JA (1990) Factors influencing GAP-43 gene expression in PC12 pheochromocytoma cells. J Neurosci 10: 1398–1406.
[36]
Dworkin S, Mantamadiotis T (2010) Targeting CREB signalling in neurogenesis. Expert Opin Ther Targets 14: 869–879.
[37]
Mayr B, Montminy M (2001) Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2: 599–609.
[38]
Scholzke MN, Schwaninger M (2007) Transcriptional regulation of neurogenesis: potential mechanisms in cerebral ischemia. J Mol Med 85: 577–588.
[39]
Best JL, Amezcua CA, Mayr B, Flechner L, Murawsky CM, et al. (2004) Identification of small-molecule antagonists that inhibit an activator: coactivator interaction. Proc Natl Acad Sci U S A 101: 17622–17627.
[40]
Meinkoth JL, Alberts AS, Went W, Fantozzi D, Taylor SS, et al. (1993) Signal transduction through the cAMP-dependent protein kinase. Mol Cell Biochem 127-128: 179–186.
[41]
Finkbeiner S (2000) CREB couples neurotrophin signals to survival messages. Neuron 25: 11–14.
[42]
Persengiev SP, Green MR (2003) The role of ATF/CREB family members in cell growth, survival and apoptosis. Apoptosis 8: 225–228.
[43]
Riccio A, Pierchala BA, Ciarallo CL, Ginty DD (1997) An NGF-TrkA-mediated retrograde signal to transcription factor CREB in sympathetic neurons. Science 277: 1097–1110.
[44]
Spencer JP (2009) Flavonoids and brain health: multiple effects underpinned by common mechanisms. Genes Nutr 4: 243–250.
[45]
Rice-Evans CA, Miller NJ (1996) Antioxidant activities of flavonoids as bioactive components of food. Biochem Soc Trans 24: 790–795.
[46]
Pollard SE, Kuhnle GG, Vauzour D, Vafeiadou K, Tzounis X, et al. (2006) The reaction of flavonoid metabolites with peroxynitrite. Biochem and Biophys Res Commun 350: 960–968.
[47]
Spencer JP (2007) The interactions of flavonoids within neuronal signalling pathways. Genes Nutr 2: 257–273.
[48]
Spencer JP (2009) The impact of flavonoids on memory: physiological and molecular considerations. Chem Soc Rev 38: 1152–1161.
[49]
Spencer JP, Vauzour D, Rendeiro C (2009) Flavonoids and cognition: the molecular mechanisms underlying their behavioural effects. Arch Biochem Biophys 492: 1–9.
[50]
Qiu P, Dong P, Guan H, Li S, Ho CT, et al. (2010) Inhibitory effects of 5-hydroxy polymethoxyflavones on colon cancer cells. Mol Nutr Food Res 54: S244–S252.
[51]
Li S, Pan MH, Lai CS, Lo CY, Dushenkov S, et al. (2007) Isolation and syntheses of polymethoxyflavones and hydroxylated polymethoxyflavones as inhibitors of HL-60 cell lines. Bioorg Med Chem 15: 3381–3389.
[52]
Pan MH, Lai YS, Lai CS, Wang YJ, Li S, et al. (2007) 5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavo?neinduces apoptosis through reactive oxygen species production, growth arrest and DNA damage-inducible gene 153 expression, and caspase activation in human leukemia cells. J Agric Food Chem 55: 5081–5091.
[53]
Lai CS, Li S, Chai CY, Lo CY, Ho CT, et al. (2007) Inhibitory effect of citrus 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavo?neon 12-O-tetradecanoylphorbol 13-acetate-induced skin inflammation and tumor promotion in mice. Carcinogenesis 28: 2581–2588.
[54]
Van Hooff CO, Holthuis JC, Oestreicher AB, Boonstra J, De Graan PN, et al. (1989) Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells. J Cell Biol 108: 1115–1125.
[55]
Jap Tjoen San ER, Schmidt-Michels MH, Spruijt BM, Oestreicher AB, Schotman P, et al. (1991) Quantitation of the growth-associated protein B-50/GAP-43 and neurite outgrowth in PC12 cells. J Neurosci Res 29: 149–154.
[56]
Mosevitsky MI (2005) Nerve ending "signal" proteins GAP-43, MARCKS, and BASP1. Int Rev Cytol 245: 245–325.
[57]
Vossler MR, Yao H, York RD, Pan MG, Rim CS, et al. (1997) cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 89: 73–82.
[58]
Sands WA, Palmer TM (2008) Regulating gene transcription in response to cyclic AMP elevation. Cell Signal 20: 460–466.
[59]
Katoh S, Mitsui Y, Kitani K, Suzuki T (1997) Hyperoxia induces the differentiated neuronal phenotype of PC12 cells by producing reactive oxygen species. Biochem Biophys Res Commun 241: 347–351.
[60]
Lin CW, Wu MJ, Liu IY, Su JD, Yen JH (2010) Neurotrophic and cytoprotective action of luteolin in PC12 cells through ERK-dependent induction of Nrf2-driven HO-1 expression. J Agric Food Chem 58: 4477–4486.
[61]
Dijkmans TF, van Hooijdonk LWA, Schouten TG, Kamphorst JT, Fitzsimons CP, et al. (2009) Identification of new nerve growth factor-responsive immediate-early genes. Brain Res 1249: 19–33.