Role of CREB in modulation of TNFα and IL-10 expression in LPS-stimulated RAW264.7 macrophages
Introduction
The pro-inflammatory cytokine tumor necrosis factor (TNF)α and the anti-inflammatory cytokine interleukin (IL)-10 are key players in inflammation, initiated, among other stimuli, upon toll-like receptor 4 (TLR4)-mediated detection of lipopolysaccharide (LPS), a molecular component of Gram-negative bacteria. Tight regulation of cytokine production is essential for the balance between proper immune reaction against the pathogen and prevention of excessive damage to the host (Beutler, 2004).
Activation of the cAMP pathway in macrophages by a multitude of G protein-coupled receptor (GPCR) ligands, such as agonists of the β-adrenergic receptor (β-AR), serves to down-regulate production of TNFα, as well as to up-regulate production of IL-10 (Kast, 2000, Szelenyi et al., 2000, Zidek, 1999). The canonical cAMP pathway propagates via protein kinase A (PKA)-mediated phosphorylation of CREB (Gonzalez and Montminy, 1989), a transcription factor which binds to the CRE sites, present at both TNFα (Kuprash et al., 1999) and IL-10 (Platzer et al., 1999) promoters.
Phosphorylation and activation of CREB following treatment of LPS-stimulated RAW264.7 macrophages with a cAMP inducer results in transcriptional activation at the IL-10 promoter (Avni et al., 2010, Goldsmith et al., 2009a). Consistently, mutations at CRE sites in the human IL-10 promoter result in loss of transcriptional activity (Platzer et al., 1999). These observations thus indicate that cAMP-activated CREB can positively regulate IL-10 transcription in LPS-stimulated macrophages.
The role of CREB in TNFα transcription is far less understood. On the one hand, cAMP inducers negatively regulate TNFα expression (Goldsmith et al., 2009b, Kast, 2000). On the other hand, mutations at the CRE site in the mouse TNFα promoter result in loss of transcriptional activity in LPS-stimulated (O’Donnell and Taffet, 2002) or mycobacteria-stimulated (Roach et al., 2005) mouse RAW264.7 macrophages. Thus, while cAMP is considered a negative regulator of TNFα transcription, the latter studies suggested that CREB may be a positive regulator of TNFα transcription.
In addition to the controversy regarding the role of CREB in TNFα transcription, it is unclear whether LPS itself activates CREB. First, some groups (Chen et al., 1999, Osawa et al., 2006), but not others (Avni et al., 2010, Greten et al., 1996), have been able to observe a cAMP increase in LPS-stimulated RAW264.7 macrophages. The apparent contradictory may be explained by the different incubation times; long and short, respectively. Second, LPS has been shown to stimulate phosphorylation of CREB on Ser-133 via MSK1, a kinase which is downstream to p38 (Caivano and Cohen, 2000, Eliopoulos et al., 2002). Yet, in these studies it was not directly demonstrated whether CREB was activated by this phosphorylation event.
Therefore, the goal of our research was to evaluate the role of CREB in transcriptional regulation of TNFα and IL-10, in LPS-stimulated macrophages. We show here that isoproterenol induced PKA-mediated phosphorylation of CREB as well as transcriptional activation of a cAMP-dependent luciferase reporter, whereas LPS induced MSK1-mediated phosphorylation of CREB, but failed to activate it. Furthermore, PKA and CREB activation was found to be required for transcriptional regulation of TNFα and IL-10 by isoproterenol, while it was found to be irrelevant for LPS stimulation of cytokine expression.
Section snippets
Reagents, plasmids, and cell culture
Lipopolysaccharide (LPS; Escherichia coli serotype 055:B5), H89, rolipram, isoproterenol, propranolol, PMSF and DMSO, were purchased from Sigma–Aldrich (St. Louis, MO). Ro318220 was purchased from Calbiochem (Darmstadt, Germany). SB203580 was purchased from A.G. Scientific (San Diego, CA). l-Glutamine, penicillin and streptomycin were purchased from Biological Industries (Beit Haemek, Israel). DMEM and FBS were purchased from Gibco (Carlsbad, CA). BSA was purchased from Amresco (Solon, OH).
LPS and isoproterenol differentially elevate intra-cellular cAMP level
In order to probe CREB activation by LPS, we initially measured the ability of LPS to elevate the intra-cellular level of cAMP in RAW264.7 macrophages. Fig. 1 shows that there was no apparent increase in cAMP level in response to LPS treatment at early time points of less than 30 min, in agreement with our previous results (Avni et al., 2010). Then however, a 1.8-fold increase in basal cAMP level was measured at 30–60 min of LPS treatment. In contrast to the modest and delayed effect of LPS, the
Discussion
The role of CREB in LPS signaling has been poorly defined. The results presented here indicate that LPS stimulates MSK1-mediated phosphorylation, but not activation, of CREB. Accordingly, CREB appears not to be involved in TNFα and IL-10 production in LPS-stimulated macrophages. Yet, cAMP inducers such as β-AR agonists suppress TNFα production and elevate IL-10 production via PKA and CREB.
We have demonstrated here that isoproterenol sequentially activates the signaling components of the cAMP
Concluding note
We have demonstrated here that the outcome of CREB phosphorylation on Ser-133 is context-dependent, as isoproterenol both phosphorylated and activated CREB in a cAMP-dependent manner, whereas LPS phosphorylated CREB in a cAMP-independent manner, which bears no transcriptional activity. The p38–MSK1 pathway mediates high expression of TNFα and only minimal expression of IL-10 in LPS-stimulated RAW264.7 macrophages, in a CREB-independent manner. Agonists of the β-AR activate the cAMP–PKA–CREB
Acknowledgments
The research was supported by grants from the Israel Science Foundation (#907/07) and from the Public Committee for Allocation of Estate Funds at Israel's Ministry of Justice (#3223). We are grateful to Mrs. Nava Silberstein for superb technical assistance, to Dr. Marc Montminy for the gifts of pCREB antibody, dominant negative CREB and CRE-luciferase construct, and to Dr. Chundong Yu for the gift of TNFα promoter-luciferase vector. Finally, thanks to Meir Goldsmith and Yifat Glucksam for
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These authors contributed equally.