Acetylation of mitogen-activated protein kinase phosphatase-1 inhibits Toll-like receptor signaling

Abstract

The mitogen-activated protein kinase (MAPK) pathway plays a critical role in Toll-like receptor (TLR) signaling. MAPK phosphatase-1 (MKP-1) inhibits the MAPK pathway and decreases TLR signaling, but the regulation of MKP-1 is not completely understood. We now show that MKP-1 is acetylated, and that acetylation regulates its ability to interact with its substrates and deactivate inflammatory signaling. We found that LPS activates acetylation of MKP-1. MKP-1 is acetylated by p300 on lysine residue K57 within its substrate-binding domain. Acetylation of MKP-1 enhances its interaction with p38, thereby increasing its phosphatase activity and interrupting MAPK signaling. Inhibition of deacetylases increases MKP-1 acetylation and blocks MAPK signaling in wild-type (WT) cells; however, deacetylase inhibitors have no effect in cells lacking MKP-1. Furthermore, histone deacetylase inhibitors reduce inflammation and mortality in WT mice treated with LPS, but fail to protect MKP-1 knockout mice. Our data suggest that acetylation of MKP-1 inhibits innate immune signaling. This pathway may be an important therapeutic target in the treatment of inflammatory diseases.

Figure 1.

Deacetylase inhibitors decrease LPS activation of NO synthesis and NOS2 expression. (A) TSA inhibits LPS-induced NO production in a dose-dependent manner. RAW cells were pretreated with increasing amounts of TSA for 1 h, and then treated with or without LPS 100 ng/ml for 16 h, and the amount of NO₂⁻ was measured in the supernatant by the Griess reaction. (n = 3 ± the SD). (B) Sodium butyrate inhibits LPS-induced NO production in RAW cells. RAW cells were pretreated with increasing amounts of sodium butyrate for 1 h and treated with or without LPS 100 ng/ml for 16 h, and the amount of NO₂⁻ was measured in the supernatant by the Griess reaction (n = 3 ± the SD). (C) TSA inhibits the LPS-induced increase in NOS2 RNA levels (dose–response). RAW cells were pretreated with increasing amounts of TSA for 1 h and treated with LPS 100 ng/ml for 6 h. Total RNA was analyzed by Northern blotting with a cDNA probe for NOS2 (top) or a ribosomal phosphoprotein RNA 36B4 as a control (bottom). (D) TSA inhibits LPS-induced increase in NOS2 RNA levels (time course). RAW cells were pretreated with TSA 30 ng/ml for 1 h and treated with LPS 100 ng/ml for 0–7 h. Total RNA was analyzed by Northern blotting with a cDNA probe for NOS2. (E) TSA inhibits LPS-induced increase in NOS2 protein levels (dose–response). RAW cells were pretreated with increasing amounts of TSA for 1 h, LPS was added for 6 h, and cell lysates were immunoblotted with antibody to NOS2 (top) or ERα (bottom). (F) TSA decreases LPS-induced inflammatory cytokine RNA levels. RAW cells were treated with LPS and TSA for 4 h. Total RNA was analyzed by Northern blotting with probes for TNF-α, IL-1β, IL-6, or GAPDH as a control. (G) TSA decreases LPS-, PGN-, and poly(I:C)-induced NOS2 protein levels. RAW cells were treated with the TLR agonists PGN, poly(I:C), LPS, or E. coli for 16 h. Cell lysates were immunoblotted with antibody to NOS2. (H) TSA slightly increases expression of COX-2 and CXCL2. RAW cells were treated with LPS, TSA, or both as in F, and analyzed for COX-2 and CXCL2 expression by RT-PCR.

Figure 2.

Deacetylase inhibition blocks MAPK signaling. (A) The MAPK pathway regulates NOS2 expression. RAW cells were treated for 1 h with 10 μM of the p38 MAPK inhibitor SB203580, 10 μM of the MEK1/2 inhibitor UO126, or 5 μM of the JNK inhibitor SP600125, and then 100 ng/ml LPS was added for 6 h and cell lysates were immunoblotted with antibody to NOS2 (top) or MEK1/2 (bottom left) of HuR (bottom right). (B) TSA inhibits phosphorylation of the MAPKs p38 and ERK1, but not JNK. RAW cells were pretreated with 30 ng/ml TSA for 1 h, and then treated with 100 ng/ml LPS for 30 min, and cell lysates were immunoblotted with antibodies to MAPK pathway members. (C) TSA inhibits the MKK3/6–p38–Elk-1 signaling cascade between MKK3/6 and p38. RAW cells were treated with TSA and LPS as in B, and cell lysates immunoblotted with antibodies to MAPK members. (D) TSA does not inhibit MKK3 kinase activity. RAW cells were treated with LPS and TSA as in B and C, and lysates were immunoprecipitated with antibody to MKK3. The kinase activity of MKK3 was assayed by adding recombinant GST-p38 and ATP to immunoprecipitated MKK3 for 30 min at 30°C, and then immunoblotting for phospho-p38 (top) and total p38 (bottom).

Figure 3.

MKP-1 is acetylated. (A) MKP-1 interacts with the histone acetyl transferase p300 in RAW cells. RAW cells were treated with TSA and LPS. Cell lysates were immunoprecipitated with antibody to p300, fractionated by SDS-PAGE, and immunoblotted with antibodies to MKP-1. Immunoblots of total MKP-1 and p300 are shown in the middle and bottom gels. (B) TSA increases MKP-1 acetylation. RAW cells were pretreated with [³H]sodium acetate for 1 h and TSA for 0.5 h and with 100 ng/ml LPS for 0–3 h, and then cell lysates were immunoprecipitated with antibody to MKP-1 and autoradiographed (top) or immunoblotted with antibody to MKP-1 (bottom). (C) p300 and PCAF acetylate MKP-1 on residue K57 in vitro. A peptide encoding the MAPK docking domain of WT (WT) MKP-1 (amino acids 47 to 76) was incubated with recombinant p300 HAT domain or recombinant PCAF in the presence of [¹⁴C]-acetyl CoA. A mutant MKP-1 peptide with lysine replaced by arginine (K57R) was used as a control. The reactions were fractionated by 16.5% Tris-Tricine gel and autoradiographed. (D) MKP-1 is acetylated on residue K57 in cells. HEK293 cells were transfected with vectors expressing a fragment of WT HA-MKP-1(WT) or mutant HA-MKP-1(K57R), and then treated with TSA and LPS as above. Cell lysates were immunoprecipitated with antibody to acetyl-lysine, fractionated by SDS-PAGE, and immunoblotted with antibody to HA (top). Total lysates were also immunoblotted for HA-MKP-1 (bottom).

Figure 4.

MKP-1 acetylation increases the interaction between MKP-1 and p38. (A) TSA increases MKP-1 interaction with p38 in cells. RAW cells were treated with LPS and TSA for 1 h, cell lysates were immunoprecipitated with antibodies to p38 or MKP-1, and immunoprecipitates were immunoblotted with antibodies as shown. (B) Acetylation increases MKP-1 peptide interaction with p38 in vitro. Biotinylated peptides from the MKP-1 docking domain (containing aa residues 46–74) were synthesized with lysine (K57) or with acetyl-lysine (K57Ac). MKP-1 peptides were incubated with recombinant p38, precipitated with streptavidin–agarose beads, and precipitants were immunoblotted with antibody to p38. (top) p38 precipitant with MKP-1 peptide. (bottom) Total p38 input. (C) Acetylation increases MKP-1 peptide interaction with p38 in vitro. Biotin-MKP-1(47–76) (blue) and biotin-MKP-1(47-K57Ac-76) (red) were immobilized onto a streptavidin sensor chip. Recombinant p38 was injected into the flow cell, and changes in SPR were measured over 15 min. This experiment was repeated twice with similar results. (D) Acetylation increases WT MKP-1(WT), but not mutant MKP-1(K57R), interaction with p38 in cells. HeLa cells were transfected with plasmids expressing MKP-1(WT)-ERα or MKP-1(_K57R)-ERα. The cells were treated with 4-HT to induce MKP-1-ERα expression, and then treated with LPS and TSA for 1 h. Cell lysates were immunoprecipitated and immunoblotted with antibodies to ERα and to p38 as indicated. (E) Acetylation increases WT MKP-1(WT) interaction with ERK, but not mutant MKP-1(K57R) interaction with ERK. HeLa cells were transfected with plasmids expressing MKP-1(WT)-ERα or MKP-1(K57R)-ERα and His₆-ERK1. The cells were treated with 4-HT to induce MKP-1-ERα expression, and then treated with LPS and TSA for 1 h. Cell lysates were immunoprecipitated, and then immunoblotted with antibody to the ERα tag of MKP-1 and antibody to the (His)₆ tag of (His)₆-ERK1.

Figure 5.

MKP-1 acetylation decreases phosphorylation of p38. (A) Recombinant MKP-1. Recombinant MKP-1 was treated with p300 or control, incubated with recombinant phospho-p38, and levels of phospho-p38 were measured by immunoblotting. (B) MKP-1 derived from cells. RAW cells were treated with LPS and TSA or control for 4 h, and cell lysates were immunoprecipitated with antibody to MKP-1. Immunoprecipitates were incubated with recombinant phospho-p38 or phospho-ERK1, and then immunoblotted with antibodies to phospho-p38 or phospho-ERK. The total amount of phospho-p38 or phospho-ERK1 added to the reaction is shown as input above. (C) MKP-1 catalytic assay. Recombinant MKP-1 was pretreated or not with p300 and acetyl-CoA for 30 min at 30°C, and then recombinant p38 or control was added for 1 h at 30°C. The artificial substrate OMFP was added and the A 477 nm was measured at 30°C for 0–40 min (n = 2 ± the SD; error bars too small to see).

Figure 6.

MKP-1 mediates the effects of acetylation upon phosphorylation of p38 and NOS2 expression in cells. (A) MKP-1 siRNA decreases MKP-1 expression in RAW cell lines. RAW cells were stably transfected with a vector encoding an siRNA hairpin sequences directed against MKP-1 nucleotides 67–85 (clone #1) or MKP-1 nucleotides 743–761 (clones #2-3) or a control siRNA sequence. Three separate stably transfected clones were isolated, and cell lysates were immunoblotted with antibody to MKP-1. (B) Knockdown of MKP-1 restores phospho-p38 levels and NOS2 expression in RAW cells treated with TSA. RAW cells stably transfected with siRNA directed against MKP-1 were treated with LPS and TSA, and cell lysates were immunoblotted with antibodies to NOS2 or MAPK family members. (C) Cells from MKP-1^−/− mice maintain levels of phospho-p38 after treatment with TSA. MEFs from MKP-1^−/+ or MKP-1^−/− mice were treated with LPS and TSA for 30 min, and cell lysates were immunoblotted with antibodies to p38 as above. (D) Effects of TSA on p38 are restored by rescue of MKP-1^−/− cells with MKP-1(WT), but not with MKP-1(K57R). Cells from MKP-1^−/− were immortalized with SV40 T-antigen, and then transfected with plasmids expressing MKP-1(WT)-ERα or MKP-1(K57R)-ERα. The cells were treated with 4-HT to induce MKP-1-ERα expression, and then treated with LPS and TSA for 1 h. Cell lysates were immunoblotted with antibody to phospho-p38 (top), total p38 (middle), and the ER tag of MKP-1 (bottom). (E) Antiinflammatory effects of TSA are restored by rescue of MKP-1^−/− cells with MKP-1(WT), but not with MKP-1(K57R). Cells from MKP-1^−/− were transfected with plasmids expressing MKP-1(WT)-ERα or MKP-1(K57R)-ERα, treated with 4-HT, and then treated with LPS and TSA for 1 h. Total cell RNA was analyzed by RT-PCR for IL-6, TNF-α, and GAPDH. (F) MKP-1 mediates TSA inhibition of p38 in primary macrophages. Peritoneal macrophages were isolated from WT and MKP-1^−/− mice, stimulated with LPS, TSA, or both, as above, and cell lysates were immunoblotted with antibody to total or phosphorylated p38.

Figure 7.

HDAC inhibition decreases LPS induced mortality and inflammation in mice. (A) Mortality. Mice were injected with 1 mg/kg TSA daily for 5 d starting on day −2, injected with 50 mg/kg LPS on day 0, and their mortality was recorded each day after LPS treatment (n = 5; P < 0.00001 for LPS vs. LPS with TSA). (B) TSA decreases LPS-induced liver inflammation. Mice were injected with saline (control) or TSA each day starting on day −2, injected with LPS on day 0, and livers were harvested 2 h (top) or 2 d (bottom) after LPS, sectioned, and stained with hematoxylin and eosin. (C) TSA decreases cytokine levels in serum. WT and MKP-1^−/− mice were injected with TSA each day starting on day −2 and injected with LPS on day 0, and serum was collected 2 h after LPS treatment and analyzed by ELISA for TNF-α and IL-1β (n = 2 ± the SD; *, P < 0.05 ± the SD). (D) Cytokine from primary macrophages. Macrophages were prepared from WT and MKP-1^−/− mice, stimulated with TSA, LPS, or both, and after 4 h TNF-α and IL-1β levels measured in the media by ELISA (n = 2 ± SD). (E) TSA suppresses cytokine RNA in vivo. Mice were injected with TSA each day starting on day −2, injected with LPS on day 0, and liver RNA was harvested 4 h after LPS treatment and analyzed by RT-PCR for TNF-α and IL-1β. Data are shown for two representative mice. (F) TSA slightly increases COX-2 and CXCL2 in vivo. Mice were injected with TSA and LPS as above, and liver RNA was harvested 4 h after LPS treatment and analyzed by RT-PCR for COX-2 and CXCL2 (n = 2). (G) Mouse NOS2 protein expression in liver. Mice were pretreated with increasing amounts of TSA for 4 h, and then treated with LPS for 16 h. Liver was harvested and immunoblotted with antibodies to NOS2 and MEK1. (H) MKP-1 is acetylated in mice. Mice were pretreated with TSA for 4 h, and then treated with LPS for 16 h as above. Liver lysates were immunoprecipitated with antibody to MKP-1 and immunoblotted with antibody to Ac-Lys or MKP-1. (I) MKP-1 mediates the protective effects of TSA after LPS. MKP-1 KO mice and their WT littermate controls were pretreated with TSA as above, and then injected with LPS 20 mg/kg, and their mortality was recorded (n = 5; P = 0.17 for LPS vs. LPS with TSA). (J) Proposed schematic of MKP-1 acetylation and regulation of innate immune signaling.