Effect of p38 Inhibitors on TNF
α 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. Differences in sensitivity to various p38 inhibitors among animal species have been reported previously (27
As shown in , SB203580 effectively inhibited the LPS-stimulated secretion of TNFα by BMMϕ derived from WT mice (solid circles
). The IC50
for this compound was ~5 μM, similar to the value obtained in previous studies with mouse splenocytes3
and thioglycollate-elicited peritoneal macrophages (4
). However, when the same compound was used in parallel experiments in BMMϕ derived from animals deficient in TTP (open circles
), there was no inhibition of TNFα production at any concentration of SB203580 used ().
Fig. 1 Resistance of TTP-deficient BMMϕ to p38 inhibitors. A,SB203580; B, SB220025; C, Marimastat. BMMϕ derived from WT (solid circles) or TTPKO (open circles) mice were incubated in the presence of increasing concentrations of p38 inhibitors (more ...)
When SB220025 was used, the compound inhibited the production of TNFα by the WT BMMϕ with an IC50
of ~2 μM (), similar to the concentrations required previously.3
However, in the TTPKO BMMϕ, the effective inhibitory concentration was shifted markedly to the right, with an IC50
of ~30 μM (). 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 (21
). As seen in , both WT and TTPKO BMMϕ responded similarly to Marimastat, with the IC50
for inhibition of TNFα secretion being 0.6 μM in the WT and 2 μM in the TTPKO cells, respectively.
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 (). The pattern of phosphorylation was very similar in WT and TTPKO cells (), suggesting that activation of p38 occurred normally, even in the absence of TTP.
Fig. 2 p38 phosphorylation in TTP-deficient and control BMMϕ. BMMϕ were stimulated with LPS (1 μg/ml) for the indicated times. The cells were then washed, harvested, homogenized, and centrifuged as described under “Experimental (more ...)
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 , p38 was able to phosphorylate both MBP-mTTP (lane 2) and MBP-hTTP (lane 6) (Mr ~75,000 for both fusion proteins) in this cell-free system. Both SB203580 (5 μM, lanes 3 and 7) and SB220025 (5 μM, lanes 4 and 8) inhibited this phosphorylation. No phosphorylation was observed when the substrate was the same amount (5 μg) of recombinant MBP alone (lane 10) (predicted Mr 41,000). No phosphorylation of either fusion protein or MBP was observed when the p38 kinase was omitted from the reaction (lanes 5, 9, and 11), ruling out the possibility of contaminating kinases in the MBP-TTP preparations. The GST-p38 kinase fusion protein itself appeared to be phosphorylated in these reactions and could be seen as a radioactive band of Mr 68,000 (, lanes 1, 2, 6, and 10). This phosphorylation was also abolished by the p38 inhibitors.
Fig. 3 GST-p38 phosphorylation of mouse and human TTP in a cell-free assay. 1 μg of commercial recombinant GST-p38 (p38) was used in cell-free phosphorylation assays using recombinant MBP-mTTP or MBP-hTTP (5 μg) as substrates. GST-p38 could phosphorylate (more ...) 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 with [32
P]orthophosphate, and we stimulated them with LPS (1 μg/ml for 30 min) in the presence or absence of the p38 inhibitors (5 μm
). Under these conditions (30 min stimulation with LPS), TTP protein levels were not altered.4
As shown in , a specific antiserum against TTP immunoprecipitated a protein of approximately Mr
43,000, whose phosphorylation was increased approximately 3-fold after LPS stimulation (compare lane 2
with lane 1
). Preincubation with either SB203580 (lane 3
) or SB220025 (lane 4
) decreased the level of TTP phosphorylation by ~50%. The immunoprecipitated, 32
P-labeled protein was completely absent in the TTPKO cells (lanes 5
), confirming its identity as TTP.
Fig. 4 Effect of p38 inhibitors on LPS-stimulated phosphorylation of TTP in BMMϕ. BMMϕ labeled with [32P]orthophosphoric acid were treated as described under “Experimental Procedures,” and cell extracts were immunoprecipitated (more ...)
p38 from TTPKO Bone Marrow-derived Macrophages Is Active and Sensitive to p38 Inhibitors—BMMϕ derived from both WT and TTPKO mice were stimulated with LPS (1 μg/ml) for 15 min, and p38 was immunoprecipitated with a specific antibody for total p38. This immunoprecipitate was then used in a cell-free protein kinase assay, using recombinant MBP-mTTP as the substrate, in the presence or absence of 5 μm SB220025. As shown in , p38 immunoprecipitated from either LPS-stimulated WT or TTPKO cells could phosphorylate MBPmTTP to approximately the same extent (lanes 2 and 5), and this phosphorylation could be inhibited by SB220025 (lanes 3 and 6). No phosphorylated proteins were observed in the absence of immunoprecipitated p38 (lane 9) or in the absence of MBP-mTTP (lanes 7 and 8), 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.
Fig. 5 Phosphorylation of recombinant TTP by endogenous BMMϕ p38. BMMϕ were stimulated with LPS (1 μg/ml) for 15 min, and then p38 was immunoprecipitated as described under “Experimental Procedures.” Immune complexes were (more ...)
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 32P. That these conditions would be adequate for dephosphorylating the phosphorylated protein are demon-strated in . In this experiment, 32P-labeled TTP was the most prominent phosphoprotein in a crude cell extract; almost all of the 32P-labeling could be removed by the incubation at 30 °C with CIAP, and the decrease in 32P label was also accompanied by a shift to a lower apparent molecular weight in the SDS gel ().
Fig. 6 Effect of TTP dephosphorylation on its binding to an ARE probe. A, the indicated amounts of protein from 32P-labeled cellular extracts from 293 cells transfected with CMV.(his)6N.hTTP were incubated with (+) or without (−) active CIAP as indicated (more ...)
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 Mr 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 Na2HPO4 (final concentration in the extract, 0.1 mm) to both halves of otherwise identical reaction mixtures containing the cellular extract and CIAP; in one case, the inhibitor was added before the 2-h incubation with CIAP and in the other case after the incubation. In this way, the two halves of the extract contained identical concentrations of all reactants, the only difference being the timing of the addition of the phosphatase inhibitor.
The results of a Western blot of these samples are shown in , in which TTP was recognized by an antiserum directed against a bacterially expressed human TTP fusion protein with maltose-binding protein.2
In each case, treatment with active CIAP led to extensive dephosphorylation of immunoreactive TTP, resulting in the collapse of the multiple bands or smear representing the normally phosphorylated protein to a single immunoreactive band of approximate Mr
34,000. For convenience, we will refer to these pairs of TTP-containing extracts as phosphorylated and dephosphorylated, respectively.
These same pairs of samples from the six independent cell extracts were then used in RNA mobility shift experiments, using as a probe the ARE from mouse GM-CSF. This was used in preference to a TNFα probe because the single major complex formed in the absence of expressed TTP is usually minor in intensity and does not migrate to the same location on the non-denaturing gels as the TTP-probe complex, in contrast to TNFα ARE probes.4
These gel shift assays were performed with gradually increasing concentrations of cellular protein, so that subtle differences in RNA binding activity could be detected. Each pair of samples was analyzed in parallel on the same gel, and a second identical gel was performed in each case. An example of an autoradiograph from one such electro-phoretic mobility shift experiment is shown in . The GM-CSF ARE RNA probe migrates as two bands near the bottom of the gel. When an extract (1 μg of protein) from 293 cells transfected with vector alone was used in the gel shift assay, there was almost no detectable shift of the probe, along with the appearance of a minor radioactive band, labeled complex I; this unidentified ARE-binding protein is routinely observed in extracts from untransfected 293 cells4
when this ARE probe is used. When extracts containing progressively increasing concentrations of protein containing the paired phosphorylated and dephosphorylated TTP were evaluated, there was a clear increase in the ability of the dephosphorylated protein to shift the radioactive ARE probe into the gel. This is most obvious when comparing lanes 5
in 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 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 we compared the phosphorylated to the dephosphorylated samples by a paired t test. By using this approach, when the data equivalent to lane 3 in were compared with 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 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 complex as a percentage of the maximum value, in all cases represented by the samples corresponding to lane 8 in . Results from each pair of gels representing a single sample were averaged, and then the six sets were again averaged. These mean values ± S.E. are illustrated in . Although these means could not be compared statistically because of the normalization procedure, it is once again apparent that the dephosphorylated protein shifted more probe at each concentration of protein extract than the phosphorylated protein: 2.5-fold more at the 0.4 μg concentration, 2-fold more at the 0.8 μg concentration, and 45% more at the 1.2 μg concentration.
Fig. 7 Quantitation of the effect of TTP dephosphorylation on ARE binding. Six pairs of control and dephosphorylated TTP-containing protein extracts were used at the indicated protein concentrations to quantitate the effect of dephosphorylation on the amount (more ...)
A final analysis was performed on the ability of the protein extracts to retard probe migration to its normal location at the bottom of the gel. In this case, the results of the PhosphorImager values were normalized by expressing them as a percentage of the values obtained from the probe alone lane, corresponding to lane 1 in . In each case, the values normalized in this way were averaged for each pair of duplicate gels, and then these individual averages were compared by a paired t test. These results are shown in . At the lowest protein amount of 0.4 μg, the dephosphorylated protein shifted slightly more probe than the phosphorylated protein (89% of probe remaining versus 97%, p = 0.026). At 0.8 μg of protein, the difference was more obvious: 56% of probe remained unshifted by the dephosphorylated protein versus 76% by the phosphorylated protein (p = 0.0027). Finally, at 1.2 μg of protein, 32% of probe remained unshifted by the dephosphorylated protein compared with 53% by the phosphorylated protein (p = 0.0034).