Nobiletin, a citrus flavonoid, activates Vasodilator-stimulated phosphoprotein in human platelets through non-cyclic nucleotide-related mechanisms

Thanasekaran Jayakumar, Kao Chang Lin, Wan-Jung Lu, Chia Ying Lin, Geraldine Pitchairaj, Jiun Yi Li, Joen-Rong Sheu

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Nobiletin, a bioactive polymethoxylated flavone, has been described to possess a diversity of biological effects through its antioxidant and anti-inflammatory properties. Vasodilator-stimulated phosphoprotein (VASP) is a common substrate for cyclic AMP and cyclic GMP-regulated protein kinases [i.e., cyclic AMP-dependent protein kinase (PKA; also known as protein kinase A) and cyclic GMP-dependent protein kinase (PKG; also known as protein kinase G)] and it has been shown to be directly phosphorylated by protein kinase C (PKC). In the present study, we demonstrate that VASP is phosphorylated by nobiletin in human platelets via a non-cyclic nucleotide-related mechanism. This was confirmed by the use of inhibitors of adenylate cyclase (SQ22536) and guanylate cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)], since they prevented VASP phosphorylation induced by nobiletin. Furthormore, this event was also not affected by specific inhibitors of PKA (H-89), PKG (KT5823) and PKC (Ro318220), representing cyclic nucleotide-dependent pathways upon nobiletin-induced VASP phosphorylation. Similarly, inhibitors of p38 mitogen-activated protein kinase (MAPK; SB203580), extracellular signal-regulated kinase 2 (ERK2; PD98059), c-Jun N-terminal kinase 1 (JNK1; SP600125), Akt (LY294002) and nuclear factor-κB (NF-κB; Bay11-7082) did not affect nobiletin.induced VASP phosphorylation. Moreover, electron spin resonance, dichlorofluorescein fluorescence and western blotting techniques revealed that nobiletin did not affect hydroxyl radicals (OH.), intracellular reactive oxygen species (ROS) and on protein carbonylation, respectively. Furthermore, the nobiletin.induced VASP phosphorylation was surprisingly reversed by the intracellular antioxidant, N-acetylcysteine (NAC), but not by the inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI). It was surprising to observe the differential effects of nobiletin and NAC on VASP phosphorylation in human platelets, since they both have been reported to have antioxidant properties. The likely explanation for this discrepancy is that NAC may bind to allosteric sites on the receptor different from those that nobiletin binds to in human platelets. Taken together, our findings suggest that nobiletin induces VASP phosphorylation in human platelets through non-cyclic nucleotide-related mechanisms. Nevertheless, the exact mechanisms responsible for these effects need to be further confirmed in future studies.

Original languageEnglish
Pages (from-to)174-182
Number of pages9
JournalInternational Journal of Molecular Medicine
Volume39
Issue number1
DOIs
Publication statusPublished - Jan 1 2017

Fingerprint

Citrus
Flavonoids
Blood Platelets
Nucleotides
Phosphorylation
Acetylcysteine
Cyclic GMP-Dependent Protein Kinases
flavone
Antioxidants
Cyclic AMP-Dependent Protein Kinases
Protein Kinase C
MAP Kinase Kinase 2
Mitogen-Activated Protein Kinase 8
Protein Carbonylation
vasodilator-stimulated phosphoprotein
nobiletin
Allosteric Site
Quinoxalines
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
NADPH Oxidase

Keywords

  • Cyclic amp-dependent protein kinase/cyclic gmp-dependent protein kinase
  • Mitogen-activated protein kinases
  • N-acetylcysteine
  • Nobiletin
  • Nuclear factor-κB
  • Protein carbonylation
  • Reactive oxygen species
  • Vasodilator-stimulated phosphoprotein

ASJC Scopus subject areas

  • Genetics

Cite this

Nobiletin, a citrus flavonoid, activates Vasodilator-stimulated phosphoprotein in human platelets through non-cyclic nucleotide-related mechanisms. / Jayakumar, Thanasekaran; Lin, Kao Chang; Lu, Wan-Jung; Lin, Chia Ying; Pitchairaj, Geraldine; Li, Jiun Yi; Sheu, Joen-Rong.

In: International Journal of Molecular Medicine, Vol. 39, No. 1, 01.01.2017, p. 174-182.

Research output: Contribution to journalArticle

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AU - Lin, Kao Chang

AU - Lu, Wan-Jung

AU - Lin, Chia Ying

AU - Pitchairaj, Geraldine

AU - Li, Jiun Yi

AU - Sheu, Joen-Rong

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AB - Nobiletin, a bioactive polymethoxylated flavone, has been described to possess a diversity of biological effects through its antioxidant and anti-inflammatory properties. Vasodilator-stimulated phosphoprotein (VASP) is a common substrate for cyclic AMP and cyclic GMP-regulated protein kinases [i.e., cyclic AMP-dependent protein kinase (PKA; also known as protein kinase A) and cyclic GMP-dependent protein kinase (PKG; also known as protein kinase G)] and it has been shown to be directly phosphorylated by protein kinase C (PKC). In the present study, we demonstrate that VASP is phosphorylated by nobiletin in human platelets via a non-cyclic nucleotide-related mechanism. This was confirmed by the use of inhibitors of adenylate cyclase (SQ22536) and guanylate cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)], since they prevented VASP phosphorylation induced by nobiletin. Furthormore, this event was also not affected by specific inhibitors of PKA (H-89), PKG (KT5823) and PKC (Ro318220), representing cyclic nucleotide-dependent pathways upon nobiletin-induced VASP phosphorylation. Similarly, inhibitors of p38 mitogen-activated protein kinase (MAPK; SB203580), extracellular signal-regulated kinase 2 (ERK2; PD98059), c-Jun N-terminal kinase 1 (JNK1; SP600125), Akt (LY294002) and nuclear factor-κB (NF-κB; Bay11-7082) did not affect nobiletin.induced VASP phosphorylation. Moreover, electron spin resonance, dichlorofluorescein fluorescence and western blotting techniques revealed that nobiletin did not affect hydroxyl radicals (OH.), intracellular reactive oxygen species (ROS) and on protein carbonylation, respectively. Furthermore, the nobiletin.induced VASP phosphorylation was surprisingly reversed by the intracellular antioxidant, N-acetylcysteine (NAC), but not by the inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI). It was surprising to observe the differential effects of nobiletin and NAC on VASP phosphorylation in human platelets, since they both have been reported to have antioxidant properties. The likely explanation for this discrepancy is that NAC may bind to allosteric sites on the receptor different from those that nobiletin binds to in human platelets. Taken together, our findings suggest that nobiletin induces VASP phosphorylation in human platelets through non-cyclic nucleotide-related mechanisms. Nevertheless, the exact mechanisms responsible for these effects need to be further confirmed in future studies.

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KW - Vasodilator-stimulated phosphoprotein

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