Mounting evidence indicate that GCs modulate gene expression far beyond transcriptional regulation, but the molecular signature of GC effect on posttranscriptional regulation has been only partially revealed. In particular, little is known on the effect of GCs on the complex biology of the ARE-binding proteins, which act downstream the kinase signaling cascades and ultimately convey the stimulus-driven changes in mRNA turnover, by recruiting or inhibiting the enzymatic complexes responsible for mRNA deadenylation and decay.
In the present study we report that the ARE-binding protein TTP is a critical mediator of GC action, as global gene regulation exerted by GCs was severely blunted in MEFs cells isolated from TTP−/− mice while conserved in the WT littermates. Similar effects on the expression of select genes were observed by targeted disruption of TTP by siRNA in WT MEFs.
We found TTP to be inducible by GCs in primary and immortalized airway epithelium, providing evidence for a more direct control of posttranscriptional gene regulation by GC in a relevant therapeutic cell target. Induction of TTP was reproducible in MEFs, in line with data showing upregulation of TTP in mice tissues after GC administration in vivo
). This allowed us to use MEFs from TTP−/−
mice to characterize the relevance of TTP in the global
changes in gene expression mediated by GCs. It is interesting to note that TTP is inducible by both proinflammatory stimuli like TNF-α as well as by anti-inflammatory signals like GCs, likely due to its function as a central regulator in the expression of genes involved in inflammation.
Comparison of the gene expression profile elicited by GCs in WT and TTP−/−
MEFs revealed for the first time a striking dependence of the GCs response on TTP, indicating that the impact of posttranscriptional regulation in the mechanism of action of GCs is larger than ever appreciated, and supporting the relevance of the GC-driven induction of anti-inflammatory genes (6
Inhibition of chemokine expression is considered a major factor in the suppressive action of GCs on adaptive immune responses (1
). The strong antagonistic effect of GCs on chemokine expression, which was detected in GC-treated WT MEFs both in TNFα-stimulated and at baseline, was one of the most affected by the lack of TTP. Loss of significant GC inhibition of adhesion molecules, like VCAM-1, further points at TTP as an essential molecular component of one of the main mechanism of anti-inflammatory action of GCs, the inhibition of inflammatory cell recruitment (1
Based on the hypothesis that TTP would mediate mRNA destabilization, it was somewhat expected that lack of TTP would impact, to some degree, the ability of GCs to inhibit gene expression. With regard of genes induced
by GCs, according to this proposed mode of action, loss of TTP could be expected to increase
their steady-state levels due to lack of mRNA destabilization. Instead, in TTP−/−
MEFs we found a loss, or else a significant decrease, in the expression of genes induced by GCs in WT cells. Some of the genes for which GC-driven induction was lost in TTP−/−
MEFs are involved in the response of the acute phase of inflammation, such as the serine protease inhibitors (serpins) and orosomucoid, or display potent anti-inflammatory activity like metallothionein-2, which acts as a potent cytoprotective and antioxidant agent and inhibits the expression of inflammatory chemokines and cytokines (52
). This target profile underlines how TTP participates also to the modulation of the innate immune response by GCs (5
The mechanism by which TTP mediates the GC response is bound to be complex, involving both direct effects, likely mediated by binding of TTP to discrete AREs, as well as indirect effects. In probing such complex system, we found the association of TTP with just six out of eleven of the transcripts whose differential response to GCs in TTP−/−
cells had been validated. These transcripts - CXCL1, CCL7, CCL2, IL-6, CXCL5, and MMP-9 – all contain AUUUA pentamers or UUAUUUAUU nonamers in the 3’ UTRs that are compatible with TTP binding (54
), although in different number and sequence contexts (Table S1 in the online Supplementary Material
). The transcripts showing no association with TTP by mRNP-IP, despite profound changes in GC sensitivity in TTP−/−
cells, displayed instead only one or none of these sequences in the 3’UTRs, with the noticeable exception of EGR-1 mRNA, which bears two UAUUUAU heptamers in a highly A/U-rich milieu (Table S1
). As RBPs mediate changes in mRNA turnover in a dynamic fashion, it is likely that association of TTP with its targets would change over time and therefore could be missed in a single-timepoint experiment. It is well established that the context of the 3’UTR sequence where AREs are embedded, the secondary structure of the entire transcript and the ionic milieu of the cell environment are key determinants of the specific binding and the on-off rate of regulatory proteins. Consideration of all these factors will be needed to proceed to more in-depth investigations to define the molecular interface between TTP and its targets and its role in mediating GC response.
Along the same lines, the results from our experiments with Act D support only in part, at least in the condition tested, the assumption that association with TTP would accelerate the decay of the targeted transcripts upon GC treatment, but rather implicate that TTP would participate to a GC-induced remodeling of the RNP complex leading to different functional outcomes. For CCL7 and CCL2 mRNA, the acceleration of mRNA decay induced by GC in WT MEFs was indeed lost in TTP−/−
cells, in line with the original hypothesis; however, CXCL5 mRNA displayed in DMSO-treated WT MEFs a much longer half-life as compared to that in TTP−/−
cells, indicating that in this condition, TTP participates to a turnover mechanism with a final outcome of relative stabilization. The inhibitory effect of GC on CXCL5 seems to be lost in TTP−/−
cells as a consequence to the loss of this function. Yet another different outcome was observed for the turnover of IL-6 mRNA, which was not modified by either GC treatment or by lack of TTP despite significant enrichment in the RNP-IP assay and the presence of a 3’UTR extremely rich in AREs (Table S1
). For the latter reason, as well as for the clear TTP dependence of the GC responsiveness of this transcript we deem unlikely that IL-6 mRNA would become associated with TTP only during the preparation of cell extracts for the RNP-IP analysis.
Such differences might be in part due to the limitation of the experimental model used. Actinomycin D arrests the global transcriptional activity of the cell, and inducible or labile regulatory factors, possibly transcript-specific, may be absent or degraded, altering the physiological mRNA turnover rates. Moreover, nuclear export of mRNA is coupled to ongoing gene transcription in mammalian cells (55
), as it is the nuclear reaccumulation following nucleocytoplasmic shuttling of several important mRNA-binding proteins, including HuR (56
). These factors may have critically affected the decay rate of CXCL7, CCL5 and serpina3n mRNAs following Actinomycin D treatment.
Besides the assay's limitations, however, our data clearly underscore complex interactions between GC treatment and the role of TTP in regulating gene expression. The results obtained for CXCL5 and IL-6 mRNA turnover, as well as the concomitant loss
gene expression in TTP−/−
cells raises clearly the possibility that TTP could have a more pleiotropic function than previously appreciated. Dynamic interplay of TTP with other RNA-binding factors, rather than direct binding to AREs, may ultimately be responsible for a role of TTP in increased transcript stabilization. In a recent study, TTP has been found unable to bind to the ARE-bearing INOS mRNA, and to interact instead with the KH-type splicing regulatory protein (KSRP), another ARE-binding protein that bound to INOS mRNA, mediating its decay. Under proinflammatory stimulation, such protein-protein interaction would therefore ‘dislodge’ KSRP and the associated exosome and allow binding for HuR, which mediates INOS mRNA stabilization (31
). An RNA-independent, mRNA stabilizing function of TTP had been first observed in TTP mutants lacking RNA-binding function, which were found to increase the half-life of known TTP targets such as TNFα (60
). Many of the transcripts found in this study to be upregulated by GCs in a TTP-dependent manner do display AREs and could be therefore suitable candidates for testing the hypothesis that TTP mediates GC action through a complex remodeling of ribonucleoprotein complexes. Along the same lines, the lack of AREs in other genes whose sensitivity to GCs was significantly affected in TTP−/−
cells indicate the presence of yet undiscovered mechanisms of GC-driven gene regulation by TTP, either dependent from binding to cis
-elements distinct from AREs or due to protein-protein regulatory interactions. Along these lines, it is important to underscore the participation of TTP to the formation of processing bodies, which are distinct cytoplasmic sites of ribonucleoprotein complexes where mRNAs targeted for decay are transiently sequestered from translation (61
). In this case, TTP could promote mRNA sequestration and decay independently from direct contact with the mRNA.
Regardless the mechanism, the TTP-mediated effect can also be vastly amplified indirectly
, for example in case the affected gene is a regulatory protein - a transcription factor, a signaling molecule, etc.-, by the loss of its target's downstream function. Transcription factors are often expressed as early-response genes and display fast transcript decay rates (62
). We found that GC inhibition of pro-inflammatory transcription factors, like the NFκB molecular species NFκB1 and Early growth response (Egr)-1 (63
) was abolished in TTP−/−
cells (). The early response, ARE-bearing gene Egr-1 encodes for a transcription factor that mediates tissue inflammation and remodeling by promoting the expression of multiple genes involved in inflammation, apoptosis and matrix production (64
). Consistent with a potential regulatory role of TTP, inhibition of Egr-1 by dexamethasone was reported to occur posttranscriptionally in a myelomonocytic cell line (66
). The role of TTP in mediating the inhibition of Egr-1 by GCs would indirectly determine the GC-mediated inhibition of multiple Egr-1-dependent pro-inflammatory genes, indicating how regulation of mRNA turnover can also indirectly affect transcriptional control of multiple genes. More studies will be needed to elucidate the pathways mediating such indirect TTP actions.
Despite the different effect of the lack of TTP on targets' mRNA turnover, the loss of GC-induced repression of those genes - CCL2, CCL7, CXCL5, and IL-6 - that demonstrated TTP binding was also reproducible, although by different degree, following silencing of TTP by siRNA in WT MEFs. Despite differences between this model and the TTP KO-derived cells, due different level of TTP repression and to the diverse phenotype of cells carrying chronic vs. acute factor depletion, these data indicate the relevance of TTP in GC action beyond the TTP−/−
mouse model. In line with these results and in additional support of the potential relevance of this mechanism beyond the model we tested and in human biology, Smoak and Cidlowski report the partial loss of dexamethasone-induced downregulation of TNFα in human airway epithelial cell line A549 where TTP was suppressed by stable transfection of a TTP shRNA (35
). Although induction of TTP after in vivo
administration of GC has been documented in several mouse tissues (35
), in vitro
treatment of mouse macrophages with GC is reported to inhibit LPS-induced TTP expression(67
), suggesting tissue-specificity of TTP regulation possibly linked to the protective effect of GC treatment on innate immune responses.
Along with studies on the mechanism of TTP-mediated GC response, it is pressing to identify TTP regulation in clinical settings where GCs are administered, and to investigate disregulation of GC-driven TTP expression or function as a possible mechanism of steroid resistance. It can be envisioned that mutations affecting the levels of TTP or its ARE-binding ability could greatly impair GC action. To this end, multiple single-nucleotide polymorphisms have been identified in the human TTP gene (68
Our genome-wide approach provides convincing evidence that the role of posttranscriptional gene regulation in GC response is much larger than previously appreciated, and point at TTP as a key mediator in this process, hence having far-reaching implications in our understanding of the pathogenesis and treatment of inflammatory diseases.