A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation
© Chang et al; licensee BioMed Central Ltd. 2011
Received: 12 August 2011
Accepted: 14 December 2011
Published: 14 December 2011
Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function.
Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study.
NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM)-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization.
Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when these types of drugs are considered for the treatment of cancer and various inflammatory diseases.
Sesamol (3,4-methylenedioxyphenol) is a constituent of roasted sesame seeds, Sesamum indicum L., an important oilseed crop . Sesamol is a potent phenolic antioxidant contained only in processed sesame oil. Several beneficial effects of sesamol were reported including antioxidation, chemoprevention, antimutagenic, and antihepatotoxic properties [2–5]. Traditionally, sesame seed oil was used to remove wrinkles and prevent aging, when applied in a facial massage to the skin . Recently, sesamol was found to induce growth arrest and apoptosis in cancer and cardiovascular cells . Sesamol was also found to enhance vascular fibrinolytic capacity through regulating gene expression of a plasminogen activator and nitric oxide (NO) release in endothelial cells [7, 8].
Arterial thrombosis is quite distinct from venous thrombosis in that arterial thrombosis is mostly composed of platelets that adhere to ruptured endothelial surfaces . Venous thrombosis, which is enriched in fibrin and erythrocytes, can occur in the absence of vessel wall damage. Therefore, platelet aggregation may play a crucial role in the atherothrombotic process .
Despite the very important roles of platelets in the development of acute thrombosis, coronary heart diseases (CHDs), and atherosclerosis, no data are available concerning the effect of sesamol on platelet activation. Recently, we reported that sesamol exhibited potent activity of inhibiting platelet aggregation . Its mechanism may involve an increase in the cAMP-endothelial NO synthase (eNOS)/NO-cGMP pathway, followed by inhibition of the phospholipase Cγ2 (PLCγ2)-protein kinase C (PKC)-p38 mitogen-activated protein kinase (MAPK)-thromboxane A2 cascade, thereby leading to inhibition of [Ca2+]i mobilization, and finally inhibition of platelet aggregation .
In the present study, we further investigated the mechanisms of sesamol in inhibiting platelet activation in greater detail, and found that sesamol obviously suppressed nuclear factor-kappa B (NF-κB)-mediated signaling events in washed human platelets. NF-κB, a transcription factor, regulates diverse cell functions ranging from inflammation to cell death. As the term, "nuclear factor" implies, the actions of NF-κB require its translocation from the cytosol to the nucleus to bind cognate nuclear DNA sequences. Platelets are anucleated, do not differentiate or proliferate, and thus are a good model for studying non-genomic functions of NF-κB in sesamol-mediated inhibition of NF-κB activation. We therefore for the first time examined the cellular NF-κB signaling events associated with sesamol-mediated inhibition of platelet activation.
Sesamol, collagen (type I), prostaglandin E1 (PGE1), heparin, (E)-3-(4-methylphenylsulfonyl)-2-propenenitrile (BAY11-7082), 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), and 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were purchased from Sigma Chemical (St Louis, MO, USA); Fura 2-AM was from Molecular Probe (Eugene, OR, USA); the anti-phospho-IKKα (Ser180)/IKKβ (Ser181) polyclonal antibody (pAb), anti-IκBα (44D4) pAb, anti-PLCγ2, anti-phospho (Tyr759) PLCγ2 monoclonal antibodies (mAbs), anti-phospho (Ser) PKC substrate (p47) pAb, and the anti-phospho-NF-κB p65 (Ser536) pAb were from Cell Signaling (Beverly, MA, USA); the anti-α-tubulin mAb was from NeoMarkers (Fremont, CA, USA); and the Hybond-P polyvinylidene difluoride (PVDF) membrane, enhanced chemiluminescence (ECL) Western blotting detection reagent and analysis system, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG), and sheep anti-mouse IgG were from Amersham (Buckinghamshire, UK). Sesamol was dissolved in 0.5% dimethyl sulfoxide (DMSO) and stored at 4°C until used.
Human platelet suspensions were prepared as previously described . This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the Institutional Review Board of Taipei Medical University, and all human volunteers provided informed consent. In brief, blood was collected from healthy human volunteers who had taken no medicine during the preceding 2 weeks, and was mixed with acid/citrate/glucose (9:1; v/v). After centrifugation at 120 g for 10 min, the supernatant (platelet-rich plasma; PRP) was supplemented with PGE1 (0.5 μM) and heparin (6.4 IU/ml), and then incubated for 10 min at 37°C and centrifuged at 500 g for 10 min. The platelet pellets were suspended in 5 ml of Tyrode's solution, pH 7.3 [containing (mM) NaCl 11.9, KCl 2.7, MgCl2 2.1, NaH2PO4 0.4, NaHCO3 11.9, and glucose 11.1], then apyrase (1.0 U/ml), PGE1 (0.5 μM), and heparin (6.4 IU/ml) were added, and the mixture was incubated for 10 min at 37°C. After centrifugation of the suspensions at 500 g for 10 min, the washing procedure was repeated. The washed platelets were finally suspended in Tyrode's solution containing bovine serum albumin (BSA) (3.5 mg/ml) and adjusted to about 4.5 × 108 platelets/ml. The final concentration of Ca2+ in the Tyrode's solution was 1 mM.
A turbidimetric method was applied to measure platelet aggregation , using a Lumi-Aggregometer (Payton, Scarborough, Ontario, Canada). Platelet suspensions (3.6 × 108 platelets/ml) were preincubated with various concentrations of sesamol or inhibitors for 3 min before the addition of collagen (1 μg/ml). The reaction was allowed to proceed for 6 min, and the extent of aggregation was expressed in light-transmission units.
Measurement of platelet [Ca2+]i mobilization by Fura 2-AM fluorescence
Citrated whole blood was centrifuged at 120 g for 10 min. The PRP was incubated with Fura 2-AM (5 μM) for 1 h. Washed platelets (8 × 108 platelets/ml) were then prepared as described above. Finally, the external Ca2+ concentration of the platelet suspensions was adjusted to 1 mM. The rise in the [Ca2+]i was measured using a fluorescence spectrophotometer (CAF 110, Jasco, Tokyo, Japan) with excitation wavelengths of 340 and 380 nm, and an emission wavelength of 500 nm .
Washed platelets (1.2 × 109/ml) were preincubated with sesamol (2.5~25 μM) or various inhibitors for 3 min, followed by the addition of collagen (1 μg/ml) to trigger platelet activation. The reaction was stopped, and platelets were immediately re-suspended in 200 μl of lysis buffer (50 mM Hepes, 5 mM EDTA, 50 mM NaCl, 1% triton X-100, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 10 mM NaF, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, and 2 mM dithiothreitol) for 1 h. Lysates were centrifuged at 5000 g for 5 min. Samples containing 80 μg of protein were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%); proteins were electrotransferred by a semidry transfer method (Bio-Rad, Hercules, CA). Blots were blocked with TBST (10 mM Tris-base, 100 mM NaCl, and 0.01% Tween 20) containing 5% BSA for 1 h and then probed with various primary antibodies (diluted 1:1000 in TBST). Membranes were incubated with HRP-linked anti-mouse IgG or anti-rabbit IgG (diluted 1:3000 in TBST) for 1 h. Immunoreactive bands were detected by an ECL system. The bar graph depicts the ratios of semiquantitative results obtained by scanning reactive bands and quantifying the optical density using videodensitometry (Bio-profil; Biolight Windows Application V2000.01; Vilber Lourmat, France).
Determination of lactate dehydrogenase (LDH)
In brief, washed platelets (3.6 × 108/ml) were preincubated with Tyrode's solution, solvent control (0.5% DMSO), and various concentrations of sesamol (5~100 μM) for 20 min at 37°C, a 10-μl aliquot of supernatant was deposited on a Fuji Dri-Chem slide LDH-PIII (Fuji, Tokyo, Japan), and the absorbance wavelength was read at 540 nm using an ultraviolet-visible recording spectrophotometer (UV-160; Shimazu, Japan). A maximal value (MAX) of LDH was observed from sonicated platelets.
The experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations (n). Values of n refer to the number of experiments, each made with different blood donors. All experiments were assessed by an analysis of variance (ANOVA). If this analysis indicated significant differences among group means, then each group was compared using the Newman-Keuls method. p < 0.05 was considered statistically significant.
Concentration- and time-dependent effects of sesamol on collagen-induced NF-κB activation in washed human platelets
The roles of cyclic nucleotides in sesamol-mediated inhibition of NF-κB signaling
The effects of cyclic nucleotides are mediated via their respective protein kinase (i.e., PKA, a specific cAMP-dependent protein kinase), which phosphorylates substrate proteins involved in platelet inhibitory pathways . To investigate whether sesamol's inhibition of NF-κB was regulated by PKA, a PKA inhibitor (H89) that inhibits ATP binding to PKA catalytic subunits (PKAc) was used. As shown in the Figure 2D, H89 (5 μM) exhibited a similar effect as SQ22536 (100 μM) in reversing the sesamol-mediated inhibition of IκBα degradation.
The roles of NF-κB in regulating the PLCγ2-PKC cascade in platelets
The functional relevance of NF-κB in [Ca+2]i mobilization and platelet aggregation
The function of NF-κB has been extensively studied in nucleated cells. Diverse stimuli, including cytokines, viral infection, UV radiation, and free radicals, can induce NF-κB activation. Genes regulated by NF-κB include those involved in inflammation, cell survival, differentiation, and proliferation responses . Therefore, NF-κB is an attractive target for therapeutic interventions against cancer and inflammatory diseases. Platelets are anucleated cells; however, several studies found that platelets express transcription factors such as steroid/nuclear receptors , a glucocorticoid receptor , and peroxisome proliferator-activated receptors (PPARs) . Those findings suggest that transcription factors can exert non-genomic functions on platelets.
In human platelets, cAMP or cGMP plays a crucial role in platelet inhibition. The effect of cAMP is mediated via cAMP-dependent protein kinase (PKA). PKA is a tetrameric holoenzyme consisting of a regulatory (PKAr) subunit dimer and two catalytic (PKAc) subunits. Elevation of cAMP levels and binding of cAMP to PKAr causes dissociation of the kinase complex and release of free active catalytic subunits . Although PKA is mainly activated by cAMP, a fraction of total cellular PKA forms a complex with NF-κB-IκB proteins and may be released upon NF-κB activation by different stimuli [26, 27]. Recently, Gambaryan et al.  have reported that PKA is also activated through cAMP-independent mechanisms, which involves to be dissociated the PKAc from NF-κB-IκB-PKAc complex by triggering IKKβ phosphorylation in thrombin- and collagen-activated platelets. This effect is taken as a novel feedback inhibitory mechanism for prevention of undesired platelet activation. In a previous study , we showed that sesamol increases cAMP formation and phosphorylates vasodilator-stimulated phosphoprotein (VASP), which was obviously reversed in the presence of SQ22536. In the present study, sesamol markedly inhibited NF-κB activation (i.e., IKKβ phosphorylation) (Figure 1) in collagen-stimulated platelets. These results suggested that sesamol activates PKA through a classical cAMP-dependent mechanism, which phosphorylates substrate proteins involved in platelet inhibitory pathways. Herein, we propose a novel platelet inhibitory pathway of inhibiting NF-κB activation by cAMP/PKA (Figure 5). However, our experiments do not completely rule out the possibility that other, yet-unidentified kinases besides cAMP/PKA are involved in sesamol-mediated inhibition of NF-κB activation.
In conclusion, the most important findings of this study demonstrate for the first time that the antiplatelet activity of sesamol may involve an increase in cAMP/PKA, followed by inhibition of NF-κB-PLC-PKC signaling events, which leads to inhibition of [Ca2+]i mobilization, and finally inhibition of platelet aggregation. Therefore, sesamol may represent an increased therapeutic potential to treat such thromboembolic disorders. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our present data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when these types of drugs are considered for treating cancer and various inflammatory diseases.
This work was supported by grants from the National Science Council of Taiwan (NSC97-2320-B-038-016-MY3) and Cathay General Hospital (96-CGH-TMU-01, 98-CGH-TMU-01-4, and CGH-MR-9702 and MR-9616).
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