Okadaic acid induces sustained activation of NFκB and degradation of the nuclear IκBα in human neutrophils

doi:10.1016/S0003-9861(03)00336-9

Archives of Biochemistry and Biophysics

Volume 417, Issue 1, 1 September 2003, Pages 44-52

https://doi.org/10.1016/S0003-9861(03)00336-9 Get rights and content

Abstract

Human neutrophils differ from other cells by containing high amount of IκBα in the nucleus, and this increased nuclear IκBα accumulation is associated with the inhibition of NFκB activity and increased apoptosis. However, the mechanisms regulating NFκB activation and IκBα degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFκB activity and IκBα degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFκB and degradation of the nuclear IκBα, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-δ (PKCδ) and IκB kinase (IKK) inhibit the OA-induced activation of NFκB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFκB is regulated by a continuous phosphorylation and degradation of the nuclear IκBα.

Section snippets

Materials

Ficoll-Paque PLUS, Dextran T-500, T4 polynucleotide kinase, poly(dI–dC), and Sephadex G25 spin columns were purchased from Pharmacia (Piscataway, NJ). Hanks’ Balanced Salt Solution, RPMI 1640 medium, and endotoxin tested, heat-inactivated fetal calf serum were obtained from Life Technologies (Grand Island, NY). Escherichia coli expressed purified recombinant human TNFα and the ELISA kit for IL-8 measurement were purchased from R&D Systems (Minneapolis, MN). [γ-³²P]ATP was purchased from

Okadaic acid, inhibitor of phosphatases PP1 and PP2A, induces sustained activation of NFκB in human neutrophils

Initially, we investigated the effect of OA on NFκB activity in TNF-stimulated neutrophils. As we have described previously [15], [33], and as shown in Fig. 1, neutrophils stimulated with TNF contain NFκB p50/50 homodimer as well as the inducible p50/65 heterodimer. At early time points (30 min and 1 h), OA (0.5 μM) did not significantly increase the extent of NFκB DNA binding in TNF-stimulated neutrophils. However, after prolonged neutrophil stimulation with TNF (2, 3, and 4 h), OA dramatically

Discussion

Human neutrophils differ from other cells by containing high amount of IκBα in the nucleus of resting cells [15], [16]. The increased amount of IκBα in the nucleus inhibits NFκB activity in the neutrophils and increases apoptosis of these terminally differentiated cells [16]. However, the mechanisms regulating IκBα degradation and activation of NFκB in human neutrophils are little understood. In this study, we used the inhibitor of serine/threonine phosphatases PP1 and PP2A, okadaic acid, to

Note added in proof

Since the completion of this work. Kettritz and co-workers reported involvement of IκB kinase in the NFκB activation in human neutrophils [57]

Acknowledgements

This work was supported by National Institutes of Health Grant HD39643 and North Shore—Long Island Jewish Research Institute Faculty Research Award (to I. Vancurova).

References (57)

A.B. Lentsch et al.
Respir. Physiol.
(2001)
C.A. Pettersen et al.
Chest
(2002)
P.A. Baeuerle et al.
Cell
(1996)
N. Mukaida et al.
J. Biol. Chem.
(1990)
P.P. McDonald et al.
Blood
(1997)
C. Ward et al.
J. Biol. Chem.
(1999)
I. Vancurova et al.
J. Biol. Chem.
(2001)
J. Ding et al.
J. Biol. Chem.
(1992)
S. Revan et al.
J. Biol. Chem.
(1996)
J.C. Gay et al.
Biochim. Biophys. Acta
(1997)

B. Djerdjouri et al.

Biochem. Biophys. Res. Commun.

(1994)

N.J. Avdi et al.

J. Biol. Chem.

(2002)

P. Kreienbuhl et al.

Blood

(1992)

M. Carballo et al.

J. Biol. Chem.

(1999)

Y. Yamamoto et al.

J. Biol. Chem.

(1999)

D.D. Browning et al.

J. Biol. Chem.

(1997)

Y. Sonoda et al.

J. Biol. Chem.

(1997)

K.N. Schmidt et al.

J. Biol. Chem.

(1995)

D.X. Fu et al.

J. Biol. Cem.

(2003)

Y.M.W. Janssen-Heininger et al.

Free Radic. Biol. Med.

(2000)

S. Ghosh et al.

Cell

(2002)

J.W. Christman et al.

Chest

(2000)

E. Abraham

Crit. Care Med.

(2003)

R.M. Strieter et al.

Am. J. Pathol.

(1992)

F. Bazzoni et al.

J. Exp. Med.

(1993)

Z. Zentay et al.

Pediatr. Res.

(1999)

A.S. Baldwin

J. Clin. Invest.

(2001)

R.D. Ye

J. Leukoc. Biol.

(2001)

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Okadaic acid induces sustained activation of NFκB and degradation of the nuclear IκBα in human neutrophils

Abstract

Section snippets

Materials

Okadaic acid, inhibitor of phosphatases PP1 and PP2A, induces sustained activation of NFκB in human neutrophils

Discussion

Note added in proof

Acknowledgements

Respir. Physiol.

Chest

Cell

J. Biol. Chem.

Blood

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Blood

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Cem.

Free Radic. Biol. Med.

Cell

Chest

Crit. Care Med.

Am. J. Pathol.

J. Exp. Med.

Pediatr. Res.

J. Clin. Invest.

J. Leukoc. Biol.