Dipyridamole activation of mitogen-activated protein kinase phosphatase-1 mediates inhibition of lipopolysaccharide-induced cyclooxygenase-2 expression in RAW 264.7 cells
Introduction
Dipyridamole is a drug that is frequently used in nephrology clinics because it improves proteinuria in a variety of glomerulonephritis (Camara et al., 1991). Dipyridamole co-formulated with aspirin significantly reduces proteinuria in patients with membranous proliferative glomerulonephritis (Harmankaya et al., 2001), and reduces the risk of a second stroke in patients with prior stroke/transient ischemic attack (Lenz and Hilleman, 2000). Furthermore, dipyridamole is a useful agent when combined with warfarrin in certain patient groups, such as those with prosthetic heart valves (Gibbs and Lip, 1998).
Dipyridamole is a non-selective phosphodiesterase (PDE) inhibitor, which inhibits degradation of cAMP and cGMP (Beavo, 1995), and an inhibitor of nucleoside transport (Hammond et al., 1985). Dipyridamole also inhibits platelet function by blocking adenosine reuptake and degradation (Stafford et al., 2003). Increasing evidence has indicated that many phosphodiesterases possess anti-inflammatory effects. At least 11 phosphodiesterase isozymes have been cloned. The rank order of the inhibition of phosphodiesterases by dipyridamole is PDE5 > PDE4 > PDE2 > PDE1 and 3 (Beavo, 1995). Recently it was shown that dipyridamole inhibits the nuclear factor kappa B (NF-κB)-mediated inflammatory effect (Weyrich et al., 2005).
NF-κB comprises a family of transcription factors including subunit members of p50, p65 (Rel A), c-Rel, p52, and Rel B, that are involved in the inducible expression of a variety of genes that regulate inflammatory responses (Baldwin, 1996). NF-κB exists in a latent form in the cytoplasm of unstimulated cells comprising a transcriptionally active dimer bound to the inhibitory protein, the inhibitor of NF-κB (IκB). The multiple forms of IκB are IκB-α, -β, -γ (p105), -δ (p100), -ε, and Bcl-3 (Baldwin, 1996). A kinase complex known as IκB kinase (IKK) rapidly phosphorylates IκB-α. The IKKs are part of a large multiprotein complex called the IKK signalsome, which is important for NF-κB activation. The IKK isoforms, IKK-α and IKK-β, phosphorylate IκB, which leads to IκB degradation and the translocation of NF-κB from the cytosol to the nucleus (Brown et al., 1995, Chen et al., 1995). IKK kinase activity is stimulated when cells are treated with cytokines or by overexpression of the kinases, MAPK/Erk kinase kinase-1 and NF-κB-inducing kinase (Malinin et al., 1997, Yin et al., 1998). Recently, dipyridamole has been shown to attenuate NF-κB translocation and block MMP-9 and monocyte chemoattractant protein-1 (MCP-1) generated by lipopolysaccharide (LPS)-treated monocytes (Weyrich et al., 2005).
Activation of the NF-κB signaling pathway is closely linked to activation of mitogen-activated protein kinases (MAPKs). The p38 MAPK signaling pathway is involved in LPS-induced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions in macrophages (Chen and Wang, 1999, Dean et al., 1999, Paul et al., 1999). Activation of MAPKs requires phosphorylation of both the threonine and tyrosine residues, and phosphorylation is strictly controlled in part by a family of about 12 dual-specificity phosphatases or MAPK phosphatases, which inactivate MAPKs by dephosphorylation of both the threonine and tyrosine residues within the activation motif (Camps et al., 2000). Glucocorticoids inhibit the expression of proinflammatory genes and are widely used in the treatment of inflammatory diseases. Several lines of evidence indicate that glucocorticoids mediate anti-inflammatory effects via induction and activation of mitogen-activated protein kinase phosphatase 1 (MKP-1) (Clark, 2003, Kassel et al., 2001). MKP-1 dephosphorylates and inactivates p38 MAPK (Lasa et al., 2002), and subsequently suppresses proinflammatory cytokine biosynthesis in LPS-stimulated macrophages (Chen et al., 2002).
In this study, we investigated the role of MKP-1 in dipyridamole inhibition of iNOS and COX-2 expressions in LPS-stimulated RAW 264.7 cells. We demonstrate that dipyridamole exerts potent inhibitory effects on LPS-induced interleukin-6 (IL-6), MCP-1, iNOS, and COX-2 expressions in RAW 264.7 macrophages via activation of MKP-1. We further linked MKP-1 activation to dephosphorylation and inactivation of p38 MAPK which regulates IKK activation and the NF-κB signaling pathway. Taken together, our data reveal that dipyridamole stimulates MKP-1 activation which negatively regulates p38 MAPK and NF-κB signaling pathways, and subsequently suppresses LPS-induced COX-2 expression in RAW 264.7 macrophages.
Section snippets
Materials
Affinity-purified mouse polyclonal antibodies to COX-2, iNOS, IκB-α, IKK-β, and p65 NF-κB, protein A beads, and horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were obtained from Transduction Laboratory (Lexington, KY). All materials for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Bio-Rad (Hercules, CA). LPS, 2′-amino-3′-methoxyflavone (PD 98059), pyrrolidine dithiocarbamate (PDTC), and
Dipyridamole inhibits LPS-induced iNOS, COX-2, IL-6 and MCP-1 expression in RAW 264.7 cells
To evaluate the effects of dipyridamole on the inflammatory responses in macrophages, we monitored LPS-induced iNOS, COX-2, IL-6 and MCP-1 expressions, and nitrite production in RAW 264.7 cells. When cells were treated with 1 μg/ml of LPS, the expression levels of iNOS and COX-2 significantly increased (data not shown). Treatment of cells with dipyridamole prior to the addition of LPS inhibited LPS-induced iNOS and COX-2 expressions in RAW 264.7 cells (Fig. 1A). Dipyridamole also inhibited
Discussion
Dipyridamole is a non-selective PDE inhibitor, which has been shown to improve proteinuria in membranous glomerulonephritis, mesangial IgA glomerulonephritis, and focal and segmental glomerulonephritis (Harmankaya et al., 2001). However, the molecular basis of its beneficial effect on renal disease is still not clear. LPS is a bacterial endotoxin which induces the expression of a number of proteins associated with inflammation. LPS-induced COX-2 expression is a key mediator of inflammatory
Acknowledgments
HML was supported by a grant (NSC93-2314-B-038-032) from the National Science Council, Taipei, Taiwan. The authors wish to thank Shu-Ting Tsai for her skilled technical assistance.
References (31)
- et al.
Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries
Biochem. Pharmacol.
(2000) - et al.
p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes
J. Biol. Chem.
(1999) - et al.
Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages
Biochem. Biophys. Res. Commun.
(1991) - et al.
Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways
J. Biol. Chem.
(1999) - et al.
Pimobendan inhibits the activation of transcription factor NF-kappaB: a mechanism which explains its inhibition of cytokine production and inducible nitric oxide synthase
Life Sci.
(2000) - et al.
Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages
Cell. Signal.
(1999) - et al.
Role of protein kinase C in BSA-AGE-mediated inducible nitric oxide synthase expression in RAW 264.7 macrophages
Biochem. Pharmacol.
(2003) - et al.
HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation
Cell
(1998) - et al.
Cross-talk between pro-inflammatory transcription factors and glucocorticoids
Immunol. Cell Biol.
(2001) The NF-kappa B and I kappa B proteins: new discoveries and insights
Annu. Rev. Immunol.
(1996)