Mitogen-activated protein kinase p38 controls the expression and posttranslational modification of tristetraprolin, a regulator of tumor necrosis factor alpha mRNA stability

Abstract

Signal transduction pathways regulate gene expression in part by modulating the stability of specific mRNAs. For example, the mitogen-activated protein kinase (MAPK) p38 pathway mediates stabilization of tumor necrosis factor alpha (TNF-alpha) mRNA in myeloid cells stimulated with bacterial lipopolysaccharide (LPS). The zinc finger protein tristetraprolin (TTP) is expressed in response to LPS and regulates the stability of TNF-alpha mRNA. We show that stimulation of RAW264.7 mouse macrophages with LPS induces the binding of TTP to the TNF-alpha 3' untranslated region. The p38 pathway is required for the induction of TNF-alpha RNA-binding activity and for the expression of TTP protein and mRNA. Following stimulation with LPS, TTP is expressed in multiple, differentially phosphorylated forms. We present evidence that phosphorylation of TTP is mediated by the p38-regulated kinase MAPKAPK2 (MAPK-activated protein kinase 2). Our findings demonstrate a direct link between a specific signal transduction pathway and a specific RNA-binding protein, both of which are known to regulate TNF-alpha gene expression at a posttranscriptional level.

FIG. 1

LPS-inducible binding of a factor to human and mouse TNF-α 3′ UTRs. M-CSF-treated human peripheral blood monocytes or RAW264.7 cells were stimulated with 10 ng of LPS per ml for 2 h, and then cytoplasmic extracts were prepared. EMSAs were performed using 10 μg of cytoplasmic extract and 20 fmol of ³²P-labeled human or mouse full-length TNF-α 3′ UTR probes. Lanes 1 and 2, cytoplasmic extract of unstimulated or LPS-stimulated, M-CSF-treated human peripheral blood monocytes probed with ³²P-labeled human full-length TNF-α 3′ UTR probes; lanes 3 and 4, cytoplasmic extract of unstimulated or LPS-stimulated RAW264.7 cells probed with mouse full-length TNF-α 3′ UTR probes; lanes 5 and 6, cytoplasmic extract of unstimulated or LPS-stimulated RAW264.7 cells probed with ³²P-labeled human full-length TNF-α 3′ UTR probes. The well-resolved complexes evident in lanes 5 and 6 are labeled C1 to C6.

FIG. 2

Mapping of protein interactions with the TNF-α 3′ UTR. (A) Schematic of truncated TNF-α 3′UTR probes. (B) Results for EMSAs in which 20 fmol of each probe was used with 10 μg of cytoplasmic extract prepared from untreated RAW264.7 cells (−) or from cells stimulated for 2 h with 10 ng of LPS per ml (+).

FIG. 3

Specificity of protein interactions with the TNF-α 3′ UTR. EMSAs were performed as described for Fig. 1 and 2, using a full-length human TNF-α 3′ UTR probe and 10 μg of cytoplasmic extract from LPS-stimulated RAW264.7 cells. Competitor RNA was added to the binding reaction mixtures 20 min prior to the addition of labeled probe. (A) Protein interactions with specific RNA competitors present, as indicated, in 1- to 100-fold molar excess over the probe. (B) Protein interactions with poly(U) or poly(A) RNA present, as indicated, in 1- to 1,000-fold excess (by mass) over the probe.

FIG. 4

LPS-induction of TNF-α 3′ UTR binding activity requires de novo gene expression. (A) Cytoplasmic extracts prepared from RAW264.7 cells at the indicated times after stimulation with 10 ng of LPS per ml. Ten micrograms of each extract was used in an EMSA with 20 fmol of full-length human TNF-α 3′ UTR probe. The first lane (marked with a minus sign) contains RNA probe but no protein. (B) RAW264.7 cells preincubated with vehicle, with cycloheximide (CHX [10 μg/ml]) or with actinomycin D (AmD [1 μg/ml]) for 15 min and then stimulated with LPS for 1 h prior to the preparation of cytoplasmic extracts. Ten micrograms of each extract was used in an EMSA with 20 fmol of full-length human TNF-α 3′ UTR probe.

FIG. 5

LPS-induced complex contains TTP. Cytoplasmic extracts of LPS-stimulated RAW264.7 cells were prepared, and EMSAs were performed as described for Fig. 4, except that the probe was murine, and 1 μl of preimmune serum or anti-TTP serum was present in each 20-μl binding reaction mixture. The supershifted C4 is indicated with an asterisk.

FIG. 6

MAPK p38 activity is required for the expression of TTP in response to LPS. (A) RAW264.7 cells preincubated for 15 min with vehicle or 1 μM SB203580 and then stimulated with LPS for the times indicated. Cytoplasmic extracts were prepared and separated by SDS-polyacrylamide gel electrophoresis on a 10% gel and then analyzed by Western blotting using an anti-N-terminal TTP antiserum. The letters at the right (a, b, and c) indicate TTP bands of differing mobility. The positions of molecular size markers are indicated at the left of the gel. (B) RAW264.7 cells preincubated for 15 min with vehicle or 0.1 to 10 μM SB203580, as indicated, and then stimulated with 10 ng of LPS per ml for 2 h. Cytoplasmic extracts were prepared and used in EMSAs with 20 fmol of a full-length human TNF-α 3′ UTR probe. (C) RAW264.7 cells treated as described for panel B, with RNA then extracted and subjected to Northern blotting using a 1-kb TTP cDNA probe. As a loading control, 28S rRNA was visualized by staining of the gel with Sybr green prior to transfer and Northern blotting.

FIG. 7

TTP is differentially phosphorylated following an stimulation with LPS. RAW264.7 cells were stimulated with 10 ng of LPS per ml for 0 to 2 h, as indicated, and then cytoplasmic extracts were prepared. A 200-μg quantity of extract was treated with recombinant PP2A (72 mU) or calf intestinal phosphatase (CIP [50 U]) in the presence or absence of the phosphatase inhibitor microcystin (PI [10 μM]). The extracts were then subjected to Western blotting as described for Fig. 6A. The letters to the right (a, b, and c) indicate TTP bands of differing mobility.

FIG. 8

TTP is a substrate of MAPKAPK2. (A) Purified recombinant kinases MKK6, p38, and MAPKAPK2 (MK2) were mixed with the substrate GST-TTP (TTP) or hsp27 in the combinations indicated. Phosphorylation reactions were allowed to proceed for 30 min, and then the phosphorylated products were separated by SDS-PAGE and visualized by phosphorimaging. Lane 1, TTP alone; lane 2, MK2 and TTP; lane 3, p38 and TTP; lane 4, MKK6 and TTP; lane 5, MKK, p38, and TTP; lane 6, p38, MK2, and TTP; lane 7, MKK6, MK2, and TTP; lane 8, MKK6, p38, MK2, and TTP; lane 9, MKK6, p38, MK2, and hsp27. (B) RAW264.7 cells were left untreated (−) or stimulated with 10 ng of LPS per ml for 2 h (+), and then lysates were prepared, p38 or MAPKAPK2 was immunoprecipitated, and immune-complex kinase assays were performed using hsp27, His₆-tagged MAPKAPK2 or GST-TTP as substrate (Subst). Phosphorylated products were separated by SDS-PAGE and visualized by phosphorimaging. (C) RAW264.7 cells were stimulated with 10 ng of LPS per ml for the times indicated, and then immune-complex assays of MAPKAPK2 activity were performed as described for panel B, using recombinant hsp27 as the substrate. Phosphorylated products were separated by SDS-PAGE and visualized by phosphorimaging.