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. 2013 Apr;33(7):1456-67.
doi: 10.1128/MCB.01690-12. Epub 2013 Feb 4.

MSK1 and MSK2 inhibit lipopolysaccharide-induced prostaglandin production via an interleukin-10 feedback loop

Kirsty F MacKenzie  1 , Mirjam W M Van Den Bosch, Shaista Naqvi, Suzanne E Elcombe, Victoria A McGuire, Alastair D Reith, Perry J Blackshear, Jonathan L E Dean, J Simon C Arthur
Affiliations

MSK1 and MSK2 inhibit lipopolysaccharide-induced prostaglandin production via an interleukin-10 feedback loop

Kirsty F MacKenzie et al. Mol Cell Biol. 2013 Apr.

Abstract

Prostaglandin production is catalyzed by cyclooxygenase 2 (cox-2). We demonstrate here that MSK1 and MSK2 (MSK1/2) can exert control on the induction of cox-2 mRNA by Toll-like receptor (TLR) agonists. In the initial phase of cox-2 induction, MSK1/2 knockout macrophages confirmed a role for MSK in the positive regulation of transcription. However, at later time points both lipopolysaccharide (LPS)-induced prostaglandin and cox-2 protein levels were increased in MSK1/2 knockout. Further analysis found that while MSKs promoted cox-2 mRNA transcription, following longer LPS stimulation MSKs also promoted degradation of cox-2 mRNA. This was found to be the result of an interleukin 10 (IL-10) feedback mechanism, with endogenously produced IL-10 promoting cox-2 degradation. The ability of IL-10 to do this was dependent on the mRNA binding protein TTP through a p38/MK2-mediated mechanism. As MSKs regulate IL-10 production in response to LPS, MSK1/2 knockout results in reduced IL-10 secretion and therefore reduced feedback from IL-10 on cox-2 mRNA stability. Following LPS stimulation, this increased mRNA stability correlated to an elevated induction of both of cox-2 protein and prostaglandin secretion in MSK1/2 knockout macrophages relative to that in wild-type cells. This was not restricted to isolated macrophages, as a similar effect of MSK1/2 knockout was seen on plasma prostaglandin E2 (PGE2) levels following intraperitoneal injection of LPS.

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Figures

Fig 1
Fig 1
Prostaglandin secretion from LPS stimulated BMDMs. (A to D) Wild-type and MSK1/2 knockout (ko) BMDMs were prepared and stimulated with 100 ng/ml LPS for the indicated times. The levels of PGE (A), PGJ (B), PGF1a (C), and PGF2a (D) were determined by ELISA. Error bars represent the standard deviations of independent stimulations of BMDMs from 4 mice per genotype, and the results are representative of three independent experiments. (E) Wild-type or MSK1/2 knockout mice were given an intraperitoneal injection of 1.8 mg/kg LPS in 100 μl of PBS or a PBS control. After 3 h, blood samples were taken from the mice and the serum levels of PGE determined by ELISA. Open symbols represent the measurements on individual wild-type mice, and closed symbols represent the measurements on individual MSK1/2 knockout mice. Black bars represent the average and standard deviation for each group. Student's t tests were carried out on all results: *, P < 0.05; **, P < 0.01.
Fig 2
Fig 2
MSK knockout results in elevated levels of cox-2 protein following LPS stimulation. Wild-type and MSK1/2 knockout (ko) BMDMs were prepared and stimulated with 100 ng/ml LPS (A) or 2 μM CpG (B) for the indicated times. Cells were lysed and the levels of cox-2 and GAPDH determined by immunoblotting. Example blots are shown in the upper panel. Graphs represent the relative intensities of the cox-2 bands using GAPDH levels to correct for loading. Quantification was carried out as described in Materials and Methods. Error bars represent the standard deviation on blots from 3 independent preparations of BMDMs per genotype. Student's t tests were carried out on all results: ns, nonsignificant; *, P < 0.05; **, P < 0.01.
Fig 3
Fig 3
LPS-stimulated cox-2 mRNA transcription requires MAPK signaling pathways. (A) Wild-type BMDMs were preincubated for 1 h with 2 μM PD184352 or 5 μM SB203580 where indicated. Cells were then stimulated with 100 ng/ml LPS for 1 h, total RNA was isolated, and the levels of cox-2 mRNA were determined by quantitative PCR (Q-PCR). (B) BMDMs were preincubated with 10 μM SB-747651A for 30 min where indicated and then stimulated for 30 min with 100 ng/ml LPS or left unstimulated (control). Levels of phospho-CREB/ATF1, -MSK1, -ERK1/2, and -p38 as well as total p38 were determined by immunoblotting. (C) As for panel B except that cells were stimulated with for 1 h or 8 h with LPS, total RNA was isolated, and cox-2 mRNA levels determined by Q-PCR. Error bars represent the standard deviation on blots from 4 independent preparations of BMDMs per genotype. Student's t tests were carried out: **, P < 0.01.
Fig 4
Fig 4
Effect of MSK knockout (ko) on cox-2 mRNA levels. Wild-type or MSK1/2 knockout BMDMs were prepared and stimulated with 100 ng/ml LPS (A), 2 μM CpG (B), or 10 μg/ml poly(I·C) (C) for the indicated times. Cells were then lysed, total RNA was isolated, and the levels of cox-2 mRNA were determined by quantitative PCR (Q-PCR). Fold induction of cox-2 mRNA levels was determined relative to the 0-h wild-type BMDMs using 18S rRNA as a reference. Error bars represent the standard deviation of independent preparations of BMDMs from 4 mice per genotype. Student's t tests were carried out on all results: ns, nonsignificant; *, P < 0.05; **, P < 0.01.
Fig 5
Fig 5
Effect of CREB Ser133Ala knock-in on cox-2 mRNA levels. Wild-type and CREB Ser133Ala knock-in BMDMs were prepared and stimulated with 100 ng/ml LPS (A), 2 μM CpG (B), or 10 μg/ml poly(I·C) (C) for the indicated times. Cells were then lysed, total RNA was isolated, and the levels of cox-2 mRNA were determined by quantitative PCR (Q-PCR). Fold induction of cox-2 mRNA levels was determined relative to the 0-h wild-type BMDMs using 18S rRNA as a reference. Error bars represent the standard deviation of independent preparations of BMDMs from 4 mice per genotype. Student's t tests were carried out on all results: ns, nonsignificant; *, P < 0.05; **, P < 0.01.
Fig 6
Fig 6
Regulation of TLR-induced IL-10 production by MSKs. (A) Wild-type BMDMs were isolated and stimulated with 100 ng/ml LPS, 2 μM CpG, or 10 μg/ml poly(I·C) for 8 h. IL-10 levels were then determined by a Luminex-based assay. (B) Wild-type and IL-10 knockout (ko) BMDMs were isolated and also stimulated with 100 ng/ml LPS, 2 μM CpG, or 10 μg/ml poly(I·C) for 8 h. cox-2 mRNA levels were determined by quantitative PCR (Q-PCR). Error bars represent the standard deviation of stimulations from 4 independent preparations of BMDMs per genotype. Student's t tests were carried out: ns, nonsignificant; **, P < 0.01.
Fig 7
Fig 7
Role of IL-10 in cox-2 mRNA induction and PGE release. (A) BMDMs were stimulated with 100 ng/ml LPS or 100 ng/ml IL-10 as indicated for 8 h. Cells were then lysed, total RNA was isolated, and the levels of cox-2 mRNA were determined by quantitative PCR (Q-PCR). (B) Wild-type or IL-10 knockout (ko) BMDMs were stimulated with 100 ng/ml LPS for the indicated times, and cox-2 mRNA levels were determined by Q-PCR. (C) Wild-type or MSK1/2 knockout BMDMs were prepared and stimulated with 100 ng/ml LPS either on its own or in the presence of either an IL-10 neutralizing antibody or isotype control (both 1 μg/ml) for 16 h, and cox-2 mRNA levels were determined by Q-PCR. (D) IL-10 knockout (black bars) or IL-10/MSK1/2 triple-knockout (gray bars) BMDMs were stimulated with 100 ng/ml LPS for 8 h, and cox-2 mRNA levels were determined by Q-PCR. For each panel, fold stimulation for cox-2 mRNA was calculated relative to wild-type unstimulated (control) cells, using 18S rRNA as a reference. (E) Wild-type or MSK1/2 knockout BMDMs were prepared and stimulated with 100 ng/ml LPS either on its own or in the presence of endogenous IL-10 (100 ng/ml) for 16 h, and PGE secretion levels in the medium were determined by ELISA. Error bars represent the standard deviation of independent preparations of BMDMs from 4 mice per genotype. Student's t tests were carried out on all results: ns, nonsignificant; *, P < 0.05; **, P < 0.01.
Fig 8
Fig 8
MSKs regulate cox-2 mRNA stability. (A) Wild-type or MSK1/2 knockout (Ko) BMDMs were prepared and stimulated with 100 ng/ml LPS for the indicated times. Cells were then lysed, total RNA was isolated, and the levels of the cox-2 primary transcript were determined by quantitative PCR (Q-PCR). (B) Wild-type BMDMs were stimulated with 100 ng/ml LPS for 6 h before 1 μg/ml actinomycin D was added and the cells were incubated for the indicated times after actinomycin addition. Cells were then lysed, total RNA was isolated, and the levels of the cox-2 primary transcript and mRNA were determined by Q-PCR. (C) Wild-type or MSK1/2 knockout BMDMs were stimulated for 6 h with LPS. Then, 1 μg/ml actinomycin D was added and the cells were incubated for the indicated times after actinomycin addition. Where indicated, 100 ng/ml of IL-10 was added at the same time as actinomycin D. Cells were then lysed, total RNA was isolated, and the levels of the cox-2 mRNA were determined by Q-PCR. (D) As for panel C except that BMDMs were stimulated for only 1 h with LPS before actinomycin D was added. (E) Wild-type or IL-10 knockout BMDMs were stimulated for 6 h with LPS before 1 μg/ml actinomycin D was added and the cells were incubated for the indicated times after actinomycin addition. Cells were then lysed, total RNA was isolated, and the levels of the cox-2 mRNA were determined by Q-PCR. (F) IL-10 knockout BMDMs were stimulated for 6 h with LPS before 1 μg/ml actinomycin D was added and the cells were incubated for the indicated times after actinomycin addition. Where indicated, 0.5 μM INCB-018424 (Rux) was added at the same time as actinomycin D. Cells were then lysed, total RNA was isolated, and the levels of the cox-2 mRNA were determined by Q-PCR. (G) Wild-type or IL-10 knockout BMDMs were stimulated with 100 ng/ml LPS for the indicated times. Total RNA was then isolated, and the levels of the primary transcript for cox-2 were determined by Q-PCR. Error bars in each panel represent the standard deviation of independent stimulations on BMDMs from 4 mice per genotype. Student's t tests were carried out on all results: ns, nonsignificant; *, P < 0.05; **, P < 0.01.
Fig 9
Fig 9
IL-10 regulates cox-2 mRNA stability via p38 and TTP. (A) Wild-type or TTP knockout (Ko) BMDMs were stimulated for 6 h with LPS. Then, 1 μg/ml actinomycin D was added and the cells were incubated for the indicated times after actinomycin addition. Where indicated, 100 ng/ml of IL-10 was added at the same time as actinomycin D. Cells were then lysed, total RNA was isolated, and the levels of the cox-2 mRNA were determined by quantitative PCR (Q-PCR). (B) IL-10 knockout BMDMs were stimulated for 6 h with LPS before 1 μg/ml actinomycin D was added and the cells were incubated for the indicated times after actinomycin addition. Where indicated, 5 μM SB203580 was added 30 min prior to actinomycin D. Cells were then lysed, total RNA was isolated, and the levels of the cox-2 mRNA were determined by Q-PCR. (C) IL-10 knockout BMDMs were stimulated for 6 h with LPS before 1 μg/ml actinomycin D was added and the cells were incubated for the indicated times after actinomycin addition. Where indicated, 2 μM PF3644022 was added 30 min prior to actinomycin D. Cells were then lysed, total RNA was isolated, and the levels of the cox-2 mRNA were determined by Q-PCR. Error bars represent the standard deviation on blots from 4 independent preparations of BMDMs per genotype. (D) Wild-type BMDMs were pretreated with 0.5 μM INCB-018424 (Rux) for 30 min and then stimulated with 100 ng/ml LPS or 100 ng/ml IL-10 as indicated for 6 h. Cells were lysed, and total and phospho-p38 MAPK levels were determined by immunoblotting.
Fig 10
Fig 10
MSKs regulate cox-2 mRNA transcription via CREB and cox-2 mRNA stability via an IL-10 feedback mechanism. Upon early TLR activation, downstream of TLR4, MSK1/2 phosphorylate the transcription factor CREB, leading to cox-2 and IL-10 mRNA transcription. Concurrently activation of p38 MAPK and its subsequent activation of MK2 lead to inhibition of the zinc finger protein TTP, and thus less mRNA degradation. However, at later time points of TLR activation, IL-10 which has been secreted from the cell feeds back in an autocrine and paracrine manner stimulating the IL-10 receptor. This leads to downstream activation of the Jak pathway which inhibits p38 and MK2, releasing TTP from its inhibitory state. This, in turn, leads to increased TTP mediated turnover of cox-2 mRNA. In our MSK1/2 knockout macrophage system we see decreased IL-10 secretion leading to attenuation of this feedback mechanism resulting in reduced cox-2 mRNA turnover and higher levels of cox-2 protein and prostaglandin release than in wild-type macrophages.
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References

    1. Marnett LJ. 2009. The COXIB experience: a look in the rearview mirror. Annu. Rev. Pharmacol. Toxicol. 49: 265–290 - PubMed
    1. Matsuoka T, Narumiya S. 2007. Prostaglandin receptor signaling in disease. ScientificWorldJournal 7: 1329–1347 - PMC - PubMed
    1. Smith WL, DeWitt DL, Garavito RM. 2000. Cyclooxygenases: structural, cellular, and molecular biology. Annu. Rev. Biochem. 69: 145–182 - PubMed
    1. Rajakariar R, Yaqoob MM, Gilroy DW. 2006. COX-2 in inflammation and resolution. Mol. Interv. 6: 199–207 - PubMed
    1. Kang YJ, Mbonye UR, DeLong CJ, Wada M, Smith WL. 2007. Regulation of intracellular cyclooxygenase levels by gene transcription and protein degradation. Prog. Lipid Res. 46: 108–125 - PMC - PubMed

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