IL-10 plays a pivotal regulatory role in inflammation. Many chronic diseases, such as psoriasis, rheumatoid arthritis, inflammatory bowel diseases, multiple sclerosis, and asthma are linked to improper regulation of inflammatory responses. Furthermore, a frequent correlation between chronic inflammation and tumor development has been observed (10
). Thus, the regulation of IL-10 is of clinical interest due to its anti-inflammatory and immunosuppressive properties. We showed previously that depletion of AUF1 in THP-1 monocyte cells suppresses the LPS-mediated induction of IL10
without significantly affecting the degradation of IL10
mRNA and that ectopic expression of the p40 AUF1 isoform selectively rescues LPS-mediated IL10
). This led to the (correct) hypothesis that AUF1 regulates IL10
expression by modulating signaling pathways that regulate the transcription of IL10
during inflammatory responses.
Activation of both the NF-κB and p38 MAPK pathways is essential for the induction of inflammatory genes in response to LPS exposure. Indeed, blocking either pathway with small-molecule inhibitors blocks the induction of IL10
expression (Fig. ). Also, knockdown of AUF1 impairs both these pathways (Fig. ), consistent with the aforementioned hypothesis. As the importance of the NF-κB pathway for IL10
expression is well documented (7
), we focused in this study on the mechanisms by which AUF1 controls NF-κB signaling.
LPS interaction with CD14 promotes dimerization of Toll-like receptor 4 (TLR4) and the subsequent recruitment of adaptor molecule MyD88. MyD88 then recruits the serine/threonine kinases interleukin-1 receptor-associated kinase 4 (IRAK4) and IRAK1 to initiate the activation of a cascade of kinases. IRAK4 phosphorylates IRAK1, which mediates the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6) to the receptor complex. The IRAK1-TRAF6 complex then dissociates from the receptor to interact with and activate TAK1. TRAF6 is a ubiquitin ligase that may catalyze autopolyubiquitination by Lys63-linked ubiquitin chains. TAK1-interacting proteins TAB1 and TAB2 then bind these chains, initiating the formation and activation of the TAK1 complex by phosphorylation of TAK1. TAK1 in turn phosphorylates the IKKβ subunit of IKK, which phosphorylates IκB, leading to its destruction within the NF-κB/IκB complex by proteasomes. NF-κB subsequently translocates to the nucleus, where it activates its target genes, including IL10
. Indeed, cells from cells from Tak1−/−
mice present impaired activation of the IKK complex when exposed to proinflammatory cytokines compared to the activation of the IKK complex in cells from wild-type mice. Furthermore, ectopic expression of TAK1 restores the activation of the IKK complex in Tak1−/−
). TAK1 functions as a branching signal transducer downstream of MyD88, IRAK1, and TRAF6 in the NF-κB and MAPK pathways.
Our key initial finding was that knockdown of AUF1 reduces the levels of phosphorylated IκBα in response to LPS exposure compared to the levels in cells expressing a control shRNA (Fig. and ). This would serve to maintain NF-κB in the cytoplasm and dampen the activation of its target genes (e.g., IL10). We thus worked our way backward through the signaling pathway to determine the upstream protein(s) whose expression is affected by AUF1 knockdown and could therefore account for the reduced IκBα phosphorylation. Indeed, AUF1 knockdown significantly reduced the levels of the kinase TAK1 (Fig. ) without significantly affecting its associated proteins, TAB1 and TAB2, or its upstream activator, TRAF6. As noted above, the TAK1 kinase normally phosphorylates the IKKα/β subunit of IKK, but AUF1 knockdown reduced phospho-IKKα/β levels and, thus, reduced both IκBα phosphorylation and NF-κB binding. Complementation with a TAK1 expression vector in cells with AUF1 knockdown restored NF-κB activation in these cells. This indicates that the regulation of TAK1 gene expression is the focal point of AUF1 control of NF-κB signaling.
We note that, in extracts of cells expressing either shAUF1 alone or shAUF1 plus TAK1 cDNA, DNA binding by NF-κB is less transient than that in control cells (Fig. and ). In shAUF1-expressing cells, low-level but sustained binding may be due to the failure of IκB to remove NF-κB from the nucleus. Upon LPS binding to TLR4, IκB, encoded by a canonical NF-κB-responsive gene, is phosphorylated and degraded, allowing NF-κB to translocate to the nucleus. IκB is resynthesized later in a NF-κB-dependent manner and transports NF-κB back to the cytoplasm. It is notable that the level of IκB is reduced in untreated shAUF1-expressing cells (Fig. , IκBα). Due to impaired NF-κB signaling in shAUF1-expressing cells, IκB is not resynthesized following degradation. As a result, NF-κB remains DNA bound longer upon AUF1 knockdown. Complementation of TAK1 expression upon AUF1 knockdown restores NF-κB DNA binding that is also less transient than that of shCTRL-expressing cells. The transfected TAK1 transgene is likely not regulated by AUF1, probably due to our intentional exclusion of the 3′-UTR. Together, these results suggest the importance of an AUF1-TAK1 mRNA interaction for the regulation of LPS-mediated NF-κB signaling. Thus, this regulation cannot take place, presumably due to reduced abundance of AUF1 in shAUF1-expressing cells and the lack of a TAK1 3′-UTR in the TAK1 expression vector in cells expressing the shAUF1 plus TAK1 open reading frame.
How does AUF1 control the levels of TAK1? While knockdown of AUF1 reduces TAK1 abundance, it does not affect the levels of TAK1
mRNA (Fig. and data not shown) or TAK1 protein stability (Fig. ). However, mRNP immunoprecipitation with AUF1 antibody revealed that TAK1
mRNA is an AUF1-associated target (Fig. ). The simplest interpretation of these results is that AUF1 promotes the translation of TAK1
mRNA. Indeed, AUF1 affects the translation of TAK1
mRNA by promoting the initiation step(s) (Fig. ). This is not without precedence, since AUF1 promotes the translation of MYC
mRNA by binding the AU-rich element (ARE) within its 3′-UTR (18
). However, future work will be required to fully elucidate the mechanisms by which AUF1 controls the translation of TAK1
Nonetheless, transfection of an shRNA-refractory p40AUF1
cDNA but not of p37AUF1
selectively restores the LPS-mediated induction of IL10
). Significantly, p40AUF1
elevates TAK1 protein levels and, consequently, the levels of phosphorylated TAK1 (Fig. ). As expected, p40AUF1
also restores the LPS-induced activation of IKKα/β (Fig. ) and IL10
expression (Fig. ). Also, p40AUF1
elevates p38 MAPK signaling (i.e., phospho-MAPKAPK-2) (Fig. ). However, identification of AUF1 targets within the p38 MAPK pathway and how AUF1 controls these signaling events will require future work. Finally, p40AUF1
does not alter the cytoplasmic levels of TAK1
mRNA; also, AUF1 knockdown does not alter the stability of TAK1 protein. These observations are consistent with the aforementioned translational-activation mechanism.
Overproduction of proinflammatory cytokines due to mRNA stabilization occurs in AUF1 knockout mice (22
). Thus, AUF1 plays a critical role in limiting proinflammatory cytokine production upon LPS challenge. Our research has led to a novel mechanism of AUF1-mediated regulation of inflammatory responses by modulation of the NF-κB and p38 MAPK pathways through regulation of the upstream mediator TAK1. Proinflammatory stimuli like cytokines and microbial products activate TAK1 upstream of IKK and MAPKs (JNK and p38 MAPK). The inducible transcription factors NF-κB, which is activated by IKK, and AP-1, which is activated by JNK and p38 MAPK, are crucial mediators of multiple aspects of inflammatory responses. Thus, p40AUF1
, via its interaction with TAK1
mRNA, regulates inflammatory responses. Further studies will address issues such as how LPS affects this interaction. In conclusion, our data indicate that functional interactions between RNA-binding protein AUF1 and TAK1, a critical component of inflammatory signaling pathways, has profound effects upon cytokine production during inflammatory responses.