Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway.

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor alpha secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3'-untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cell-free assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor alpha.


Introduction
Lipolysaccharide (LPS 1 )-induced production of tumor necrosis factor α (TNFα) by monocyte/macrophages is regulated at both transcriptional and post-transcriptional levels. Posttranscriptional regulation of TNFα synthesis occurs in part by modulation of its mRNA stability. This in turn is dependent upon a so-called class II AU-rich element (ARE) found in the 3'-untranslated region (3'-UTR) of TNFα transcripts (1). This ARE has been implicated in the regulation of both TNFα mRNA stability and its translation (2,3). Targeted deletion of the TNFα mRNA ARE in mice (∆ARE mice) results in the overproduction of TNFα and the development of a systemic inflammatory syndrome (4). A role for the protein serine/threonine For western blotting, confluent cultures of BMMφ were stimulated with LPS (1 µg/ml) for the indicated times, and processed as described (22). l00 µg of protein was separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose, and sequentially probed with antibodies against phosphorylated p38 (Cell Signaling, Beverly, MA) or against total p38 (A-12) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Detection was performed with the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).
For kinase assays, GST-p38 was purchased from Calbiochem (Calbiochem-Novabiochem Corporation, San Diego, CA) and used in a cell-free kinase assay using 5 µg of the MBP-hTTP (maltose-binding protein-human TTP) or MBP-mTTP (maltose-binding protein-mouse TTP) fusion proteins as substrates. These recombinant proteins were produced in E. coli as amino terminal fusions of TTP with maltose-binding protein, and affinity purified on amylose resin (New England Biolabs, Beverly, MA) as previously described (23). We also expressed and purified the MBP protein as a control. The details of fusion protein expression and purification will be described elsewhere. The conditions for p38 phosphorylation of the purified fusion proteins were as recommended by the manufacturer. Where indicated, SB203580 or SB220025 was used to inhibit p38 kinase activity at 5 µM. Phosphorylated proteins were resolved by 10% SDS-PAGE, followed by autoradiography. For kinase assays in which endogenous p38 was used, BMMφ were stimulated with LPS (1 µg/ml) for 15 min, and the cells were harvested as described (22), with the following modifications: sodium deoxycholate and SDS were omitted from the wash buffer, and the kinase reaction was performed at 37°C for 30 min. MBP-mTTP (2.5 µg) was used as the substrate. Phosphorylated proteins were resolved by 11% SDS-PAGE, followed by autoradiography.
To evaluate TTP phosphorylation in intact cells, confluent 60-mm dishes of BMMφ were washed with phosphate-free Dulbecco's modified Eagle's medium and incubated for 4 h in the presence of 200 µCi/ml of 32 P-orthophosphoric acid (NEN Life Science Products, Boston, MA). The p38 inhibitors SB203580 and SB220025 (5 µM) were then added for 30 min, followed by 1 µg/ml LPS for another 30 min. Cells were harvested and lysed as described above, and equal amounts of TCA-precipitable counts were immunoprecipitated using a specific antiserum against TTP (19). Phosphorylated proteins were resolved by 12% SDS-PAGE, followed by autoradiography and quantification using a PhosphorImager Typhoon 8600 and in 2-6 µl of the reaction mixture described above containing CIAP and Na 2 HPO 4 were added to 25 µl of the lysis buffer described above, without protease inhibitors, containing 2 x 10 5 cpm of RNA probe, and used in mobility shift assays as described (24,26).

Effect of p38 inhibitors on TNF production by WT and TTPKO bone marrow-derived macrophages
We first evaluated the effect of two p38 inhibitors, SB203580 and SB220025, on the secretion of TNFα by BMMφ derived from either WT or TTPKO mice. Both p38 inhibitors decreased LPS-stimulated TNFα synthesis in WT BMMφ, albeit at higher concentrations than required to inhibit TNFα synthesis by LPS-stimulated human monocyte/macrophages. When SB220025 was used, the compound inhibited the production of TNFα by the WT BMMφ with an IC 50 of approximately 2 µM (Fig. 1B), similar to the concentrations required previously 3 . However, in the TTPKO BMMφ, the effective inhibitory concentration was shifted markedly to the right, with an IC 50 of approximately 30 µM (Fig. 1B). Similar results were observed when the cells were stimulated with a lower concentration of LPS, 0.1 µg/ml, and then exposed to the p38 inhibitors (data not shown).
The specificity of this resistance of TTPKO-derived BMMφ to p38 inhibitors was tested by using a completely unrelated inhibitor of TNFα secretion, the TACE inhibitor Marimastat

Activation of p38 in bone marrow-derived macrophages
To determine whether LPS could activate p38 to the same extent in the WT and TTPKO BMMφ, the cells were stimulated with LPS (1 µg/ml), followed by immunoblotting for phosphorylated (activated) and total p38. This resulted in the rapid (within 5 min) phosphorylation of p38, which peaked at 15-30 min, and was still apparent after 60 min (Fig. 2).
The pattern of phosphorylation was very similar in WT and TTPKO cells (Fig. 2), suggesting that activation of p38 occurred normally, even in the absence of TTP.

Phosphorylation of a recombinant MBP-TTP fusion protein by recombinant p38
To determine whether recombinant TTP could serve as a substrate for p38, we performed cell-free kinase assays using commercially available p38, in the form of a GST-p38 fusion protein, and recombinant mouse and human TTP (as fusion proteins with maltose binding protein or MBP) as substrates. As shown in Fig This phosphorylation was also abolished by the p38 inhibitors.

Phosphorylation of TTP in intact cells
To determine whether activation of the p38 kinase pathway could lead to TTP phosphorylation in intact cells, we labeled BMMφ derived from both WT and TTPKO mice ruling out the possibility that significant concentrations of nonspecific kinases or substrates were present in the reactions. These results suggested that p38 kinase could be activated by LPS in both the TTPKO and the WT macrophages, and that in both cases the kinase was sensitive to the action of p38 inhibitors in the cell-free kinase assay.

Effect of dephosphorylation of cell-expressed TTP on ARE binding.
We next evaluated the effect of "global" TTP dephosphorylation on its binding to a GM-CSF ARE probe. We first developed experimental conditions that would result in extensive dephosphorylation of TTP expressed in transfected 293 cells labeled with 32 P. That these conditions would be adequate for dephosphorylating the phosphorylated protein are demonstrated in Fig. 6A. In this experiment, 32 P-labeled TTP was the most prominent phosphoprotein in a crude cell extract; almost all of the 32 P-labeling could be removed by the incubation at 30 o with CIAP, and the decrease in 32 P label was also accompanied by a shift to a lower apparent molecular weight in the SDS gel (Fig. 6A).
We next performed similar dephosphorylations on non-radioactive 293 cell extracts containing similar amounts of expressed TTP from six independent but similar transfections.
Because the CIAP protein migrated close to the M r of highly phosphorylated TTP in these SDS gels and for other reasons, we took great pains to ensure that the extracts containing phosphorylated and dephosphorylated TTP contained otherwise identical concentrations of reactants after the incubation with CIAP. To do this, we added the phosphatase inhibitor  Fig. 6C, but was apparent at all three protein concentrations used. When evaluated by probe disappearance, the same trend was seen, i.e., more probe was shifted by the dephosphorylated protein in each pair. This is especially apparent when comparing the probe radioactivity in lanes 5 and 6 and in lanes 7 and 8. Similar results were seen in the other 11 gels consisting of the duplicate gel for the one pictured in Fig. 6C as well as the duplicate gels for the remaining five pairs of extracts.
The data from all of these experiments were quantitated by Phosphorimager analysis, and subjected to three types of statistical comparisons. First, we treated all 12 sets of Phosphorimager raw data for the TTP-probe complex as independent data points, and compared the phosphorylated to the dephosphorylated samples by a paired t test. Using this approach, when the data equivalent to lane 3 in Fig. 6C were compared to the data equivalent to lane 4, the dephosphorylated protein bound 2.3-fold more probe than the phosphorylated protein (p = 0.0018). A similar comparison between the samples corresponding to lanes 5 and 6 in Fig. 6C resulted in a 62% average increase in probe binding by the dephosphorylated protein (p = 0.001).
At the highest protein concentrations, corresponding to lanes 7 and 8, the difference was not statistically significant (67% increase, p = 0.08).
In a second approach, we expressed each Phosphorimager value of the protein -probe of probe remained unshifted by the dephosphorylated protein compared to 53% by the phosphorylated protein ( p = 0.0034).

Discussion
One of the major findings in these studies was that macrophages derived from TTPKO mice were less sensitive than normal cells to the effects of p38 inhibitors on LPS-stimulated TNFα release. These data in turn suggest that TTP is part of the p38 signal transduction cascade, initiated by LPS in macrophages, that leads ultimately to stimulated TNFα secretion.
In the TTPKO mouse model, macrophages produce excess amounts of TNFα secondary to stabilization of TNFα mRNA (15,17). This occurs because of the absence of TTP, which ordinarily binds directly to the ARE in the TNFα mRNA, destabilizing it by a still unknown mechanism (15,24) P38 has been implicated previously in the regulated synthesis of several inflammatory cytokines; in the case of TNFα, p38 is thought to exert its effects primarily on the regulation of TNFα mRNA stability These results suggest that TTP may be a component of the cascade by which p38 regulates TNFα production. To exclude the possibility that p38 activation could be impaired in the absence of TTP, we showed that LPS stimulated the phosphorylation of p38 with a very similar time-course in both WT and TTPKO cells, and that levels of immunoreactive p38 were very similar in the two cell types. Cell-free kinase assays with commercial p38 and recombinant human and mouse MBP-TTP fusion proteins showed that p38 could catalyze the phosphorylation of TTP in a manner inhibited by both SB203580 and SB220025. In addition, TTP phosphorylation in intact macrophages was stimulated when BMMφ were exposed to LPS, and this stimulated phosphorylation was prevented by the use of the p38 inhibitors. These results strongly suggest that TTP can be phosphorylated in macrophages after activation of p38, even though they do not prove that TTP is directly phosphorylated by p38 in intact cells.
Finally, we showed that the activated p38 present in the TTPKO cells after exposure to LPS was able to phosphorylate the MBP-mTTP fusion protein normally in a cell-free kinase assay, a reaction that was again sensitive to the p38 inhibitor SB220025. This is additional evidence against the possibility of abnormal activation or expression of p38 in the absence of TTP.

One important difference between the WT and TTPKO cells is that the TTPKO cells
produced about five times more TNFα after LPS stimulation than their WT counterparts, as we have shown previously (17). However, both cell types were similarly responsive to Marimastat, which inhibits TNFα production by inhibiting TACE (21) through a mechanism that is not thought to involve p38 kinases (32). This difference should be kept in mind when evaluating the comparative effects of the inhibitors in these cells.
Another concern is the differential expression of p38 isotypes in macrophages. Human macrophages have been shown to express the p38α and p38δ isotypes (33). These two isotypes differ in their substrate specificity, reactivity with p38 antibodies, mechanisms of activation, and sensitivity to p38 inhibitors (33,34). The inhibitors used in the present study are thought to be specific for p38α and p38β, but to have no effect on p38δ (20,34). In addition, the antibodies used for both immunoblotting and immunoprecipitation in the present study are thought to recognize only p38α and p38β. We demonstrated that expression of p38, presumably α, was essentially identical in the WT and TTPKO cells used in the present study; however, it was at least theoretically possible that p38δ could be overexpressed in the TTPKO cells, thus providing a mechanism for their resistance to p38 inhibition. However, the expression of p38δ mRNA was at very low levels and was equivalent in the WT and TTPKO cells 5 , suggesting that the relevant isotype in our system is p38α.
P38 plays an important role in the regulation of the inflammatory response. It has been shown that p38 activation can increase the stability of mRNAs encoding several proinflammatory cytokines (29,35). P38 activation can also induce the transcription of genes encoding pro-inflammatory factors, and the translation of their mRNAs (36,37). The use of p38 inhibitors in the treatment of inflammatory diseases has been proposed, and several inhibitors of this type have been used successfully in experimental models of inflammatory diseases (7,20,28).
However, little is known to date about the downstream components of the signaling pathways that actually regulate this response. The results reported here suggest the possible involvement of TTP downstream from p38. TTP is a phosphoprotein that contains several consensus phosphorylation sites for proline-directed, stress-activated kinases (19), and it can be phosphorylated in vivo and in vitro by some of these kinases. Moreover, we found that cell-expressed TTP "globally" dephosphorylated with alkaline phosphatase bound an ARE probe with greater apparent avidity than did the native, phosphorylated protein. TTP appears to be mostly cytosolic in macrophages (15), but can shuttle between nucleus and cytosol in fibroblasts (38); this is similar to the behavior of P38 itself, which is thought to be transported from the nucleus to the cytoplasm by its phosphorylated substrate MAPKAPK2 (39).
A possible model for a P38-TTP linkage is shown in Fig. 8. According to this proposed model, once TTP was phosphorylated by p38 or another kinase downstream from p38, TTP could change its subcellular localization and/or exhibit decreased binding to the TNFα mRNA ARE, thus increasing the stability of TNFα mRNA and increasing the synthesis and secretion of this cytokine (Fig. 8A). In contrast, decreased TTP phosphorylation resulting from the use of the p38 inhibitors could promote TTP binding to the TNFα ARE, resulting in the rapid destabilization and degradation of the TNFα mRNA, and consequently, reduction in the levels of secreted protein (Fig. 8B). The situation in the complete absence of TTP would more closely resemble that seen in Fig. 8A, i.e., the lack of TTP would promote TNFα mRNA stability and increased TNFα secretion in both the presence and absence of p38 inhibitors (Fig. 8C,D). This Although their experimental design differed from ours in several respects, including our use of longer exposure of the cells to the inhibitors, a different source for the inhibitors, and a wider range of inhibitor concentrations, we cannot provide a satisfactory explanation for this difference in results. Considerably more work will be needed to account for some of these disparate data.