In this report, we investigated the mechanisms underlying crosstalk between the TLR4 and cAMP pathways in macrophages. The suppressive effect of cAMP on the activation of immune cells has been known for many years (12
). In monocyte-derived cells, cAMP attenuates LPS-induced production of proinflammatory cytokines such as TNF-α (13
) and MIP-1α (17
), whereas it enhances the production of mediators with more anti-inflammatory effects on macrophages, such as IL-10 (51
) and G-CSF (52
). Some studies have used analogs of cAMP to address the relative contributions of the most likely cAMP effectors: PKA and EPAC, to inhibiting the production of TNF-α (20
); however, most previous studies have used the inhibitor H-89 to implicate PKA in cAMP-mediated effects. Although potent in its inhibition of PKA, H-89 also shows strong inhibitory activities against other kinases, including MSK1, Rho-associated kinase 2 (ROCK2), S6 kinase 1, and Akt (55
). Our data from experiments with selective cAMP analogs and RNAi specific for catalytic subunits of PKA showed that the effects of cAMP on LPS-induced expression of TNF
, and G-CSF
in RAW 264.7 cells were all PKA dependent; however, we found that H-89 inconsistently reversed the effects of cAMP in repeated experiments in RAW cells. This may have been caused by inhibition of some kinases (mentioned above) that can influence macrophage activation, and emphasizes that caution should be taken in interpreting results from experiments with inhibitors that can act on multiple kinases.
The ability to express miRNA-based shRNAs specific for both catalytic isoforms of PKA from a single retrovirus (27
) was vital to determining the contribution of PKA to the effects of cAMP on LPS-induced expression of cytokine genes (). Although the reversal in the enhanced expression of IL-10
was incomplete in these experiments, this might have been due to incomplete knockdown of PKA and it is consistent with the observation that PKA-Cα knockout mice are viable despite the abundance of PKA-C in certain tissues of these mice being <10% of that of WT mice (56
). It is also noteworthy that the fold induction in the abundance of TNF-α mRNA induced by LPS alone was higher in the PKA-C-deficient RAW 264.7 cell line than in control cells (). Because the cells were stimulated for 2 hours, LPS would be expected to induce the expression of COX-2
and the release of endogenous prostaglandins, including PGE2
. Autocrine stimulation of EP2 receptors, EP4 receptors, or both by PGE2
could then lead to PKA-dependent attenuation of the expression of TNF
). Accordingly, knockdown of PKA-C would limit this feedback and lead to the observed higher induction of TNF
-α expression in the PKA-C-depleted cells compared to that in control cells. This is also consistent with the reduced abundance of LPS-induced TNF-α observed in PDE4B knockout mice (58
), presumably caused by prolonged activation of PKA in the absence of LPS-induced expression of PDE4B
. The physiological relevance of this negative feedback by prostaglandins is supported by the observation that resolution of pulmonary inflammation is compromised in COX-2 knockout mice (59
Previous studies have suggested roles for AKAPs in various immune cell functions (34
), and a report identified AKAP13 as a regulator of TLR2-induced signaling in the human monocyte cell line THP-1 (63
). The use of selective PKA-anchoring inhibitor peptides enabled us to assess whether specific cellular localization of either type I or type II PKA was required for cAMP-mediated modulation of the expression of LPS-induced cytokine genes. Suppression of the expression of genes encoding proinflammatory cytokines by cAMP signaling was specifically reversed by delocalization of type II PKA, whereas the cAMP-mediated enhancement of the expression of G-CSF
was specifically reversed by the delocalization of type I PKA. This suggests that divergent patterns of gene transcription could result from differential localization of type I and type II PKA holoenzymes, through their association with specific AKAPs (fig. S2
), to substrates that activate different regulatory circuits and influence different transcriptional outcomes.
On the other hand, we showed that the PKA-dependent enhancement of LPS-induced expression of IL-10 did not depend on localization of the kinase, but, in contrast to the other cytokine genes tested, did require the presence of CREB. CREB is activated by its phosphorylation by PKA at Ser133; however, the PKA-mediated enhancement of LPS-induced expression of IL-10 did not appear to be simply a summation of its expression induced by LPS and that induced by cAMP alone. Although expression of IL-10 was CREB dependent, we detected negligible expression of IL-10 in response to cAMP induced in response to PGE2 (). Thus, it appears that the effect of cAMP on IL-10 expression was to specifically enhance expression induced by a stronger activating stimulus, such as LPS.
To address potential points of crosstalk between cAMP and the TLR4 pathway, we assessed the activation state of established intermediates in the TLR4 signaling cascade. Although previous reports from studies in THP-1 cells suggested that p38 MAPK is a potential site of crosstalk (16
), we found no effect of increased cAMP concentration on the LPS-induced phosphorylation of p38, JNK, or ERK proteins in RAW 264.7 cells (fig. S4
). Similarly, another point of interaction between these pathways proposed from studies in THP-1 cells, cAMP-dependent attenuation of IKK activity and the subsequent degradation of IκBα (16
), was not observed in our studies (). This suggests that mechanisms of cAMP-mediated regulation of the TLR4 pathway may vary between different monocyte-derived cell types.
In RAW 264.7 cells, our data suggest that cAMP had specific effects on the activation dynamics of NF-κB proteins. A slowed nuclear accumulation of the p50–p65 heterodimer in the first hour after LPS stimulation was shown by both time-lapse microscopy () and EMSA experiments (), whereas there was a later cAMP-dependent enhancement of p50–p50 homodimer formation after 4 hours of stimulation with LPS and an agonist of the cAMP pathway (). Because the p50–p50 homodimer is proposed to be a transcriptional repressor at NF-κB-dependent promoters, both of these early and late effects of cAMP on NF-κB activity could contribute to cAMP-mediated suppression of LPS-induced expression of proinflammatory cytokine genes.
Our data on AKAP95 and p105 provide insight into the mechanistic basis of the early effect of cAMP on gene expression, which is of particular relevance to the amount of TNF-α released from activated macrophages in the early stages of infection. This is especially important in sepsis, in which high concentrations of TNF-α have serious pathological consequences (64
). Our RNAi screen of the AKAPs found in RAW cells suggested that the targeting of PKA by AKAP95 was necessary for PGE2
to mediate suppression of LPS-induced expression of TNF
-α, and the data from experiments with BMDMs derived from AKAP95GT
mice, which expresses a truncated AKAP95 protein that cannot bind to PKA (45
), supported the hypothesis that it was the PKA-targeting function of AKAP95 that was important in this regard. It is noteworthy that only the early suppression (30 and 60 min) by PGE2
of the expression of TNF
-α was lost in the AKAP95GT
mouse, whereas the suppressive effect of cAMP in BMDMs from these mice was comparable to that of BMDMs from WT mice at 2 and 4 hours (). It is possible that these later effects of cAMP depended on the enhanced formation of the NF-κB p50–p50 complexes observed by EMSA () and that this occurs through an additional mechanism that is independent of AKAP95.
The initial identification of p105 as a putative binding partner for AKAP95 in the Alliance for Cellular Signaling (AfCS) yeast two-hybrid screen emphasizes the value of this data resource (http://www.signaling-gateway.org/data/Y2H/cgi-bin/y2h.cgi
). Moreover, the coordinates of the p105 and AKAP95 protein fragments isolated from the yeast two-hybrid screen provided additional insight to the nature of the interaction between them (). Whereas AKAP95 is primarily a nuclear protein, p105 is almost exclusively cytoplasmic; however, the region of AKAP95 involved in its interaction with p105 spans the nuclear localization sequences. The presence of a cytoplasmic pool of AKAP95 in RAW 264.7 cells suggested that this may have been dependent on its interaction with p105. The identification of a similar pool of AKAP95 in Jurkat cells (65
) suggests that AKAP95 may have cytoplasmic functions specific to hematopoietic lineages.
The identification of a PKA phosphorylation site in the C-terminal region of p105 suggests that p105 is a candidate substrate for AKAP95-targeted PKA. This PKA site is adjacent to several well-known IKK target sites (48
), phosphorylation of which promote degradation of p105. Although p105 is a member of the IκB family (66
), it has not been conclusively shown that it can function as a genuine IκB; that is, it has not been shown to undergo stimulus-dependent degradation to release transcription-promoting NF-κB dimers (67
). Although we did not directly show an IκB-type function for p105 in our study, we provided evidence that the stimulus-dependent phosphorylation of p105 by IKK was inhibited by PGE2
, and that the loss of this inhibitory effect in the AKAP95GT
mouse () was coincidental with the loss of cAMP-dependent suppression of LPS-induced, early TNF
-α expression (). This is suggestive of a mechanism whereby the earliest NF-κB complexes entering the nucleus after stimulation of macrophages by LPS have been released from inhibition by p105.
The possibility that these early complexes are not inhibited by the canonical IκB proteins is supported by the observation that PGE2
had no effect on LPS-induced degradation of IκB in primary macrophages (), despite substantially suppressing the LPS-induced expression of TNF
-α (). This mechanism is supported by a report from Libby and colleagues that identified an interaction between p105 and the EP4-associated protein, EPRAP (50
). This report also showed that PGE2
inhibits LPS-induced phosphorylation of IKK and degradation of p105 in macrophages, and that EPRAP recruits p105 into a complex with EP4. In mice lacking EP4, the early attenuation (up to 60 min) by PGE2
of LPS-induced expression of TNF
-α was lost, but the later effect of PGE2
was maintained, which is analogous to the phenotype that we observed in BMDMs from the AKAP95GT
mouse (). Together, these data suggest a mechanism whereby p105 is constitutively part of a complex with AKAP95 in the cytoplasm of macrophages and is then recruited to EP4 by EPRAP on stimulation with PGE2
. This would bring the p105-AKAP95 complex closer to the site of cAMP production, which could facilitate the activation of AKAP95-anchored PKA to phosphorylate p105 at Ser940
In summary, our results provide substantial insight into the mechanisms by which cAMP promotes its anti-inflammatory effects in macrophages. They suggest that production of this second messenger leads to a pattern of signaling that can modulate the activation of macrophages through the TLR4 pathway at multiple levels (). The existence of different classes of PKA-scaffolding proteins that bind selectively to different sub-types of PKA provides another layer of control to enable different modulatory effects of PKA on the cytokine profile of the activated macrophage. Our data suggest that type II PKA is targeted by AKAP95 to p105 to specifically suppress the early expression of TNF
-α and to another component of the canonical MyD88-dependent TLR4 pathway to attenuate LPS-induced expression of MIP-1
α. A pool of type I PKA is presumably localized to another substrate to specifically mediate enhancement of the expression of G-CSF
. It is tempting to suggest that this enhancement might be through the MyD88-independent, TRIF-dependent component of the TLR4 pathway, because G-CSF
has been implicated as one of the genes whose expression is induced through this pathway (69
). The targeting of different pools of PKA to either arm of the TLR4 pathway would allow an increase in PKA activity to have opposite modulatory effects on different LPS-induced transcripts. We suspect that this requirement for scaffold proteins might be a recurrent theme in the analysis of signaling complexity, as scaffold proteins provide a mechanism through which the cell, armed with a limited toolbox of pleiotropic signaling effector enzymes, can organize different combinations of components to allow for context-dependent signaling (70
). The anti-inflammatory mechanisms of cAMP described here may thus provide a therapeutic target for certain inflammatory disorders.
Fig. 9 A proposed schematic for the mechanisms through which PKA may modulate LPS-induced expression of cytokine genes in macrophages. LPS induces the expression of genes encoding both proinflammatory (TNF-α, MIP-1α) and anti-inflammatory (IL-10, (more ...)