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Epicatechin, catechin, and dimeric procyanidins inhibit PMA-induced NF-B activation at multiple steps in Jurkat T cells¹

GERARDO G. MACKENZIE^*, FERNANDO CARRASQUEDO, JOSÉ M. DELFINO^*, CARL L. KEEN^,||, CÉSAR G. FRAGA and PATRICIA I. OTEIZA^*,,,2

^* Departamento de Química BiolÓgica, Instituto de Química y Fisicoquímica Biológicas,
Fisicoquímica, Programa de Radicales Libres, (Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, C1113AAD, Buenos Aires, Argentina; and Departments of
Nutrition,
Environmental Toxicology and
^|| Internal Medicine, University of California Davis, Davis, California 95616, USA

²Correspondence: Department of Nutrition, University of California, Davis, CA 95616. E-mail: poteiza{at}ucdavis.edu

SPECIFIC AIMS

The objective of this work was to investigate the possible modulation of the transcription factor NF-B activation by flavan-3-ol monomers and dimers, and to characterize the step(s) in the activation cascade at which these compounds could act. We pretreated Jurkat cells with (–)-epicatechin (EC), (+)-catechin (CT), or B-type dimeric procyanidins oligomers of the above (DP-B), and evaluated their effects on PMA-induced NF-B activation.

PRINCIPAL FINDINGS

1. EC, CT, and DP-B are incorporated into the cells and EC, and DP-B accumulated in the nuclei
The incubation for 24 h with varying concentrations of EC, CT, or DP-B (1.7–17.2 µM) lead to a dose-dependent incorporation of these compounds in the cells with a minor cleavage of DP-B. A dose-dependent accumulation of EC and DP-B in the nuclear fraction was observed, while CT was undetectable. The nuclear/total cell content was 10-times higher in the cells treated for 24 h in the presence of DP-B compared with EC.

2. EC, CT, and DP-B treatment of Jurkat T-cells inhibited PMA-induced activation of transcription factor NF-B
A dose-response inhibition of PMA-induced NF-B-DNA binding activity, measured by gel shift of nuclear fractions, was observed in cells pre-treated for 24 h with EC, CT, or DP-B (1.7–17.2 µM). The lowest DNA binding activity was reached at concentrations of 8.6 µM EC, 17.2 µM CT, and 17.2 µM DP-B (65, 64, and 75% reduction, respectively), compared with that observed in cells treated only with PMA). The inhibition of the PMA-induced increase in NF-B DNA binding activity resulted in a reduced transactivation of the NF-B driven gene IL-2, as determined by a decreased production of IL-2 (53, 48, and 59% reduction of IL-2 protein levels in the cells pretreated with 8.6 µM EC, 17.2 µM CT, and 17.2 µM DP-B, respectively).

3. EC, CT, and DP-B inhibit the cytosolic events of NF-B activation and regulate oxidant concentrations
One of the required steps in the activation of NF-B is the phosphorylation and further degradation of the IB peptides that prevent the translocation of the active NF-B into the nucleus. IB is phosphorylated at two serines (S32 and S36) by specific IB kinases (IKK) which are also activated by phosphorylation. After incubation (2.5 min) with PMA, IKKß phosphorylation was lower in the cells pretreated with 8.6 µM EC, 17.2 µM CT, or 17.2 µM DP-B (51, 42, and 47% of nonpretreated cells, respectively). The phosphorylation of IB (p-IB) revealed a maximum value 10 min after the addition of PMA. At that time, the ratio p-IB/IB was 32, 28, and 31% lower in the cells preincubated with 8.6 µM EC, 17.2 µM CT, and 17.2 µM DP-B, respectively, compared with the cells incubated with PMA alone.

One of the signals that lead to the activation of the NF-B pathway is an increase in intracellular oxidants. After 2.5 min of incubating cells in the presence of PMA, a significant increase in cell oxidant levels (evaluated with the probe 5(or 6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate which, after oxidation, is converted into a fluorescent compound) was observed. This increase was inhibited in cells pretreated with 8.6 µM EC, 17.2 µM CT, or 17.2 µM DP-B for 24 h at all the times measured (2.5–30 min).

4. EC, CT, and DP-B also act at the nuclei by blocking the binding of the active NF-B to its DNA consensus sequence
The treatment with PMA determined a significant increase in RelA and p50 (components of the active NF-B) nuclear content, after 4 h incubation. This increase was of similar extent in the cells preincubated with EC, CT or DP-B and the cells treated only with PMA. However, a lower NF-B nuclear binding activity was observed in cells pretreated with EC, CT, or DP-B. One mechanism to explain these results would be the direct inhibition, by EC, CT or DP-B, of the binding of NF-B to the B-sites. Nuclear fractions isolated from cells incubated in the absence of flavanols and dimer, and treated with PMA for 4 h, were added in vitro, during the binding reaction, with variable concentrations of DP-B (0.1–100 nM) (Fig. 1 A), EC (0.2–200 nM) or CT (0.2–200 nM) for 30 min. The addition of 20 nM EC, 20 nM CT, or 10 nM DP-B reduced (78, 81 and 78% respectively) the binding of NF-B to DNA (Fig. 1B ). DP-B did not affect the DNA binding of transcription factors OCT-1 and CREB (Fig. 1C ).

View larger version (42K): [in this window] [in a new window]   Figure 1. EC, CT, and DP-B prevent in vitro NF-B, but neither CREB nor OCT-1 binding to DNA. NF-B binding activity was measured by EMSA in nuclear fractions isolated from cells incubated for 4 h with100 ng/ml PMA. Nuclear fractions were incubated with 0.2-200 nM EC, 0.2-200 nM CT or 0.1-100 nM DP-B for 30 min prior to the EMSA assay. NF-B-DNA binding activity of nuclear fractions incubated (A) with 0.1-100 nM DP-B, (B) with 20 nM EC, 20 nM CT or 10 nM DP-B. C) Nuclear fraction samples from PMA-treated cells were incubated with 0.1–100 nM DP-B for 30 min prior to the binding assay for CREB (full bars) or OCT-1 (empty bars). After quantitation results are shown as means ± SE of 5 independent experiments. *Significantly different compared with PMA group (P<0.001, one way ANOVA test).

5. DP-B could interact with NF-B proteins and prevent its binding to B sites?
To define a possible interaction between NF-B proteins and DP-B a molecular model of DP-B was initially constructed allowing free rotation around dihedral angles that constitute the major determinants of its structure. Values for these angles in the conformer corresponding to the global minimum led to a folded structure where ring B' stacks onto ring A orienting the hydroxyl groups toward the same edge of the molecule. Figure 2 depicts the superimposition of the base specific guanines of the B-DNA consensus sequence and DP-B, and their interaction through hydrogen bonds with the pairs of Arg residues present in the DNA binding region of both p50 (Arg 54 and Arg 56) and RelA (Arg 33 and Arg 35). Stacked rings Bacute; and A of DP-B lie very close to the positions occupied by the two guanine rings (0.41 and 0.47 Å RMS deviation for the guanine pairs binding p50 and RelA, respectively). Moreover, the polar atoms of DP-B are favorably placed for giving rise to a similar hydrogen bonding pattern to those observed in the complex (Fig. 2) . Overall, in both cases, DP-B appears to behave as a very reasonable mimic of the guanine pairs. To a lesser extent, this situation could be extended to EC and CT, both compounds sharing the same hydrogen bonding group distribution critical for binding, but lacking the covalent bridge present in DP-B.

View larger version (29K): [in this window] [in a new window]   Figure 2. Superimposition between DP-B and guanine pairs. Superimposition of the minimum energy conformer of DP-B (in yellow) upon the guanine pairs (in green) taken from the double dodecamer in the complex 1VKX. Putative hydrogen bonding interactions (in red) mediated by Arg residues of p50 subunit (top) or RelA subunit (bottom) are shown.

CONCLUSIONS

The present work investigated the regulation of transcription factor NF-B by the flavan-3-ols, EC and CT, and a dimeric fraction (DP-B). EC, CT, and DP-B are present in plasma after the consumption of flavonoid-rich foods and their capacity to modulate NF-B may contribute to their reported antiinflammatory and immunomodulatory actions. Our results show that EC, CT and DP-B are incorporated in Jurkat T cells and inhibit PMA-induced NF-B activation at different levels in the NF-B activation cascade. We present novel evidence of the possible regulatory effect of EC, CT, and a DP-B acting through a direct and specific interaction with the active NF-B.

Inhibition of the early steps in the NF-B activation cascade could be partially mediated by the well described antioxidant action of EC, CT, and DP-B, since we observed that these flavonoids inhibited PMA-induced increase in the steady state level of cell oxidants. However, other mechanisms of action, such as a direct effect of EC, CT and DP-B on IKK, could also be operating.

When characterizing the direct action of EC, CT and DP-B on NF-B-DNA binding, the building of the molecular model of DP-B revealed its possible mode of interaction with NF-B proteins. The described previously crystal structure of NF-B bound to DNA shows the presence of two consecutive guanines in the B-site that are essential for the base-specific binding in both p50 and RelA of the active NF-B to its cognate sequence. Arg 54 and Arg 56 of p50, and Arg 33 and Arg 35 of RelA are the residues interacting with the guanines –4 and –3, and +3 and +4, respectively. The global minimum energy conformer of DP-B is proposed here as a mimic for the guanine-guanine pair. Consistently, none of the two other transcription factors tested (OCT-1 and CREB) present two consecutive guanines in their DNA consensus motifs, and their binding to DNA is not altered by EC, CT, and DP-B. As both pairs of Arg present in p50 and RelA are essential for the base-specific contact, binding of DP-B to these residues would prevent its interaction with DNA.

The present work demonstrates that the previously observed inhibition of IL-2 and IL-1ß production by cocoa procyanidins can be attributed in part to the inhibition of the NF-B activation cascade. Regulation of IL-2 at the level of transcription is critically involved in the control of T cell expansion and in the normal immune response. In this context, the regulation of NF-B by the flavan-3-ols, EC and CT, and DP-B may contribute to the reported antiinflammatory and immunomodulatory actions proposed for these compounds.

In summary, the flavanols, EC and CT, and the B type dimeric oligomers, can regulate the immune response in part by modulating the oxidants-responsive transcription factor NF-B. This modulation can occur at the early steps of the NF-B activation cascade, i.e., regulation of oxidant levels, IKK activation and subsequent IB phosphorylation, and at later stages, through a direct interaction of EC, CT and DP-B with NF-B proteins inhibiting the binding of active NF-B to B sites.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram. The scheme summarizes the inhibition of PMA-induced NF-B activation by DP-B. DP-B enters the cell and in part accumulates in the nuclei. In the cytosol it prevents the increase in oxidant species generated by PMA, one signal for NF-B activation. Other inhibitory actions of DP-B in PMA-induced triggering of the initial cytosolic events of the NF-B cascade are also possible. DP-B could selectively interact with the active NF-B, preventing its binding to B sites at the promoter/enhancer regions of NF-B-dependent genes such us IL-2. Similar effects were observed for the flavan-3-ols EC and CT.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0402fje

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