MSKs are required for the transcription of the nuclear orphan receptors Nur77, Nurr1 and Nor1 downstream of MAPK signalling

Abstract

MSK (mitogen- and stress-activated protein kinase) 1 and MSK2 are kinases activated downstream of either the ERK (extracellular-signal-regulated kinase) 1/2 or p38 MAPK (mitogen-activated protein kinase) pathways in vivo and are required for the phosphorylation of CREB (cAMP response element-binding protein) and histone H3. Here we show that the MSKs are involved in regulating the transcription of the immediate early gene Nur77. Stimulation of mouse embryonic fibroblasts with PMA, EGF (epidermal growth factor), TNF (tumour necrosis factor) or anisomycin resulted in induction of the Nur77 mRNA. The induction of Nur77 by TNF and anisomycin was abolished in MSK1/2 double-knockout cells, whereas induction was significantly reduced in response to PMA or EGF. The MSK responsive elements were mapped to two AP (activator protein)-1-like elements in the Nur77 promoter. The induction of Nur77 was also blocked by A-CREB, suggesting that MSKs control Nur77 transcription by phosphorylating CREB bound to the two AP-1-like elements. Consistent with the decrease in Nur77 mRNA levels in the MSK1/2-knockout cells, it was also found that MSKs were required for the induction of Nur77 protein by PMA and TNF. MSKs were also found to be required for the transcription of two genes related to Nur77, Nurr1 and Nor1, which were also transcribed in a CREB- or ATF1 (activating transcription factor-1)-dependent manner. Downstream of anisomycin signalling, a second ERK-dependent pathway, independent of MSK and CREB, was also required for the transcription of Nurr1 and Nor1.

Figure 1. NR4A gene transcription requires CREB

(A) The promoter region bp −2000 to bp +1000 of the transcriptional start of the Nur77, Nor1 and Nurr1 transcription factors were analysed by GALA prediction software (http://gala.cse.psu.edu/). Potential CRE (closed circle) or AP-1-like (grey rectangle in human, mouse, rat and canine promoters, black rectangle indicating a non-conserved region in the canine promoter) as well as potential sites for MEF cells (black diamond), Elk (star), USF (upstream stimulatory factor; black triangle) and nuclear factor κB (grey triangle) were identified. Sequences predicted as CRE sites were TGACGTAG and TGACGTCT in Nor1, and TGACG.CA and TGACGTCA in Nurr1, against a concensus CRE site of TGACGT(A/C)A. (B) HeLa cells were transfected with either an empty vector (light grey bars) or A-CREB expression vector (dark grey bars). After 24 h transfected cells were serum-starved for 16 h and left unstimulated (control) or stimulated for 60 min with 400 ng/ml PMA, 100 ng/ml EGF, 10 ng/ml TNF or 10 μg/ml anisomycin. Cells were then lysed and RNA isolated and analysed by real-time PCR. Levels of Nur77 mRNA were determined relative to β₂-microglobulin controls as described in the Materials and methods section. Error bars represent the S.E.M. of three individual stimulations. (C) As in (B) except a dominant-negative junD vector (black bars) was used in place of A-CREB.

Figure 2. Reporter analysis of the murine Nur77 promoter

(A) Fragments from bp −533, bp −274 and bp −100 to bp +66 of the murine Nur77 promoter were cloned into pGL3 to give Nur77 promoter luciferase reporters. Two proximal AP-1 sites were also mutated. Grey circles indicate AP-1-like elements while white circles indicate the mutated sites. (B) The 533 (cross-hatched bars), 274 (black bars) and 100 (grey bars) Nur77 reporter vectors were transfected into wild-type immortalized MEF cells. After 24 h transfected cells were serum-starved for a further 16 h and then stimulated with PMA (400 ng/ml, 60 min), EGF (100 ng/ml, 60 min) or TNF (10 ng/ml, 60 min). Cells were then lysed and luciferase activity measured as described in the Materials and methods section. (C) The 274 (black bars), AP1–1 (cross hatched bars), AP1–2 (horizontal hatched bars) and AP1–1/2 (grey bars) vectors were transfected into wild-type fibroblasts. After 24 h transfected cells were serum-starved for a further 16 h and then stimulated with PMA (400 ng/ml, 60 min) EGF (100 ng/ml, 60 min) or TNF (10 ng/ml, 60 min). Cells were then lysed and luciferase activity measured as described in the Materials and methods section. (D) The bp −274 Nur77 reporter vectors were co-transfected with either empty vector (black bars), A-CREB (white bars) or dominant-negative junD (cross-hatched bars) expression vectors into wild-type MEF cells. After 24 h transfected cells were serum-starved for a further 16 h and then stimulated with PMA (400 ng/ml, 60 min) EGF (100 ng/ml, 60 min) or TNF (10 ng/ml, 60 min). Cells were then lysed and luciferase activity measured as described in the Materials and methods section.

Figure 3. NR4A transcription requires MAPK signalling

Wild-type primary MEF cells were serum-starved for 16 h and then treated for 1 h with 2 μM PD 184352 (PD), 5 μM SB 203580 (SB), or no inhibitor as indicated. Cells were then left unstimulated (control) or stimulated (st) for 60 min with 400 ng/ml PMA (A), 100 ng/ml EGF (B), 10 ng/ml TNF (C) or 10 μg/ml anisomycin (D). Cells were lysed and the RNA isolated. Nur77 (grey bars), Nurr1 (white bars) and Nor1 (black bars) mRNA levels were determined by real-time PCR as described in the Materials and methods section. Error bars represent the S.E.M. of three individual stimulations.

Figure 4. ERK1/2 and p38α are required for NR4A induction by anisomycin

Immortalized p38α knockout (white bars) and wild-type (black bars) MEF cells (A–C) were serum-starved for 16 h, incubated with 5 μM SB 203580 (SB) for 1 h where indicated and then left unstimulated (control) or stimulated with 10 μg/ml anisomycin (Anis) for 1 h. After stimulation, total RNA was isolated and the expression of Nur77, Nurr1 and Nor1 determined by real-time PCR (A). Levels of MSK1 activity were determined by immunoprecipitation kinase assay as under the Materials and methods section (B). To determine if the ERK1/2 and p38 pathways were activated, lysates were run on SDS/polyacrylamide gels. The levels of phospho-CREB, phospho-p38, (p-p38), phospho-ERK1/2, total p38α and total ERK1/2 were determined by immunoblotting with specific antibodies (C). (D) Primary wild-type MEF cells were serum-starved for 16 h, incubated for 1 h with 5 μM SB 203580 (SB) and/or 2 μM PD 184352 (PD) where indicated and then stimulated with 10 μg/ml anisomycin (Anis) for 1 h. Cells were then lysed and the levels of phospho-CREB, phospho-p38, phospho-ERK1/2, total p38α and total ERK1/2 were determined by immunoblotting with specific antibodies. (E) Primary wild-type MEF cells were serum-starved for 16 h, incubated for 1 h with 10 μM U0126 (grey bars) or DMSO control (black bars) and then stimulated with 10 μg/ml anisomycin for 1 h. The induction of Nur77, Nurr1 and Nor1 mRNA was then determined by real-time PCR as described in the Materials and methods section. Error bars represent the S.E.M. of three individual stimulations.

Figure 5. MSKs are required for the transcription of Nur77

(A–D) Wild-type (solid bars), MSK1/MSK2^−/− (hatched bars) and primary MEF cells were serum-starved for 16 h. Cells were then stimulated with (A) PMA (400 ng/ml), (B) EGF (100 ng/ml), (C) TNF (10 ng/ml) or (D) anisomycin (10 μg/ml) for 0, 30, 60 or 120 min. Cells were lysed and the RNA isolated. Nur77, Nurr1 and Nor1 levels were determined by real-time PCR as described in the Materials and methods section. Error bars represent the S.E.M. of three individual stimulations. (E) Wild-type (WT) or MSK1/MSK2^−/− cells were serum-starved (KO) for 16 h, and then stimulated with 400 ng/ml PMA or 10 ng/ml TNF for the times indicated. Cells were then lysed and Nur77 immunoprecipitated from 3 mg of cell lysate. Immunoprecipitates were then blotted for Nur77. Cell lysates were also blotted for total ERK.

Figure 6. Expression of MSK1 in MSK1/MSK2^−/− cells rescues NR4A expression

(A) Wild-type immortalized MEF cells were serum-starved for 16 h and then incubated with 5 μM Ro 318220 (RO) or 10 μM H89 were indicated. Cells were then left unstimualed, or stimulated (St) with EGF (100 ng/ml) for 1 h. The induction of Nur77, Nurr1 and Nor1 mRNA was measured by real-time PCR as described in the Materials and methods section. Error bars represent the S.E.M. of three individual stimulations. (B) As in (A) except that cells were stimulated with anisomycin (10 μg/ml) for 1 h. (C) Immortalized MSK1/MSK2^−/− MEF cells were transfected with either empty vector (white bars) or wild-type (light grey bars) or kinase-dead (black bars) MSK1 expression vectors. Cells were then serum-starved for 16 h and left unstimulated (control), or stimulated with either EGF (100 ng/ml) or anisomycin (10 μg/ml), for 1 h. The induction of Nur77, Nurr1 and Nor1 mRNA was measured by real-time PCR as described in the Materials and methods section. Error bars represent the S.E.M. three individual stimulations. (D) Immortalized wild-type (wt) or MSK1/MSK2^−/− (ko) MEF cells were transfected with a bp −274 to bp +66 Nur77 promoter luciferase reporter vector, together with either an empty vector, wild-type or kinase-dead (KD) MSK1 expression vector, as indicated. Cells were then serum-starved for 16 h and left unstimulated (white bars), or stimulated with PMA (400 ng/ml) for 1 h. Luciferase activity was measured as described in the Materials and methods section. Error bars represent the S.E.M. of three individual stimulations.