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Functional regulation of Zfp36l1 and Zfp36l2 in response to lipopolysaccharide in mouse RAW264.7 macrophages.

Wang KT, Wang HH, Wu YY, Su YL, Chiang PY, Lin NY, Wang SC, Chang GD, Chang CJ - J Inflamm (Lond) (2015)

Bottom Line: Biochemical and functional analyses exhibited that the decrease of Zfp36l2 mRNA was cross-regulated by Ttp.Knockdown of Zfp36l1 and Zfp36l2 increased the basal level of Mkp-1 mRNAs by prolonging its half-life.Zfp36l1 and Zfp36l2 are important regulators of the innate immune response.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No.1 Sec. 4 Roosevelt Road, Taipei, 10617 Taiwan.

ABSTRACT

Background: The tristetraprolin (TTP) family of mRNA-binding proteins contains three major members, Ttp, Zfp36l1, and Zfp36l2. Ttp down-regulates the stability of AU-rich element-containing mRNAs and functions as an anti-inflammation regulator.

Methods: To examine whether other TTP family proteins also play roles in the inflammatory response, their expression profiles and the possible mRNA targets were determined in the knockdown cells.

Results: Ttp mRNA and protein were highly induced by lipopolysaccharide (LPS), whereas Zfp36l1 and Zfp36l2 mRNAs were down-regulated and their proteins were phosphorylated during early lipopolysaccharide stimulation. Biochemical and functional analyses exhibited that the decrease of Zfp36l2 mRNA was cross-regulated by Ttp. Knockdown of Zfp36l1 and Zfp36l2 increased the basal level of Mkp-1 mRNAs by prolonging its half-life. Increasing the expression of Mkp-1 inhibited the activation of p38 MAPK under lipopolysaccharide stimulation and down-regulated Tnfα, and Ttp mRNA. In addition, hyper-phosphorylation of Zfp36l1 might stabilize Mkp-1 expression by forming a complex with the adapter protein 14-3-3 and decreasing the interaction with deadenylase Caf1a.

Conclusions: Our findings imply that the expression and phosphorylation of Zfp36l1 and Zfp36l2 may modulate the basal level of Mkp-1 mRNA to control p38 MAPK activity during lipopolysaccharide stimulation, which would affect the inflammatory mediators production. Zfp36l1 and Zfp36l2 are important regulators of the innate immune response.

No MeSH data available.


Related in: MedlinePlus

Induction of Mkp-1 mRNA early during LPS stimulation is post-transcriptionally modulated by Zfp36l1 and Zfp36l2. a mRNA expression profile of Mkp-1 in RAW264.7 cells stimulated with LPS for 0, 15, 30, 45, 60, or 120 min. b Levels of Mkp-1 mRNA in different knockdown cells after LPS stimulation for 15 min. RNA was isolated and performed the real-time PCR analysis. c Analysis of Mkp-1 mRNA half-life in different knockdown cells after LPS stimulation for 15 min. Actinomycin D (10 μg · mL-1) was added to stop transcription after 0, 10, or 20 min. The remaining mRNA was detected by quantitative PCR. Mkp-1 mRNA half-life was calculated by exponential regression, 32 min, 55 min, 68 min, and 42 min in control, Zfp36l1, Zfp36l2, and dual-knockdown cells, respectively. All of experiments were carried out at least three times
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Fig5: Induction of Mkp-1 mRNA early during LPS stimulation is post-transcriptionally modulated by Zfp36l1 and Zfp36l2. a mRNA expression profile of Mkp-1 in RAW264.7 cells stimulated with LPS for 0, 15, 30, 45, 60, or 120 min. b Levels of Mkp-1 mRNA in different knockdown cells after LPS stimulation for 15 min. RNA was isolated and performed the real-time PCR analysis. c Analysis of Mkp-1 mRNA half-life in different knockdown cells after LPS stimulation for 15 min. Actinomycin D (10 μg · mL-1) was added to stop transcription after 0, 10, or 20 min. The remaining mRNA was detected by quantitative PCR. Mkp-1 mRNA half-life was calculated by exponential regression, 32 min, 55 min, 68 min, and 42 min in control, Zfp36l1, Zfp36l2, and dual-knockdown cells, respectively. All of experiments were carried out at least three times

Mentions: To further investigate the regulation of Mkp-1 mRNA during early LPS stimulation in RAW264.7 cells, we examined the expression of its mRNA (Fig. 5a). Mkp-1 mRNA increased significantly after LPS stimulation from 15 to 30 min but decreased rapidly after 45 min. To verify the functions of Zfp36l1 and Zfp36l2 immediately following LPS treatment, we examined the level of Mkp-1 mRNA in Zfp36l1- and Zfp36l2-knockdown cells after LPS stimulation for 15 min, in this time point Ttp protein was not induced significantly and the lowest level of brought-down Caf1a by Mkp-1 3’UTR was observed in Fig. 4a. The observed marked rise in the mRNA level in all knockdown cells compared with control knockdown cells implied that the decrease in Zfp36l1 and Zfp36l2 protein expression facilitated the expression of Mkp-1 mRNA in response to LPS (Fig. 5b); furthermore, the relative Mkp-1 mRNA half-life at LPS-stimulation 15 min in control, Zfp36l1, Zfp36l2, and dual-knockdown cells was 32 min, 55 min, 68 min, and 42 min, respectively (Fig. 5c). Our results indicate that knockdown of Zfp36l1, Zfp36l2, or both proteins also cause the increase of Mkp-1 mRNA half-life at early LPS stimulation for 15 min like at the resting status. Moreover, Mkp-1 mRNA at early LPS stimulation in control shLuc cells appeared more stable (half-life is 32 min) than which at the resting condition (half-life is 19 min) showed in Fig. 3c. It might be due to phosphorylation of Zfp36l1 and Zfp36l2 upon LPS stimulation leading to protein inactivation.Fig. 5


Functional regulation of Zfp36l1 and Zfp36l2 in response to lipopolysaccharide in mouse RAW264.7 macrophages.

Wang KT, Wang HH, Wu YY, Su YL, Chiang PY, Lin NY, Wang SC, Chang GD, Chang CJ - J Inflamm (Lond) (2015)

Induction of Mkp-1 mRNA early during LPS stimulation is post-transcriptionally modulated by Zfp36l1 and Zfp36l2. a mRNA expression profile of Mkp-1 in RAW264.7 cells stimulated with LPS for 0, 15, 30, 45, 60, or 120 min. b Levels of Mkp-1 mRNA in different knockdown cells after LPS stimulation for 15 min. RNA was isolated and performed the real-time PCR analysis. c Analysis of Mkp-1 mRNA half-life in different knockdown cells after LPS stimulation for 15 min. Actinomycin D (10 μg · mL-1) was added to stop transcription after 0, 10, or 20 min. The remaining mRNA was detected by quantitative PCR. Mkp-1 mRNA half-life was calculated by exponential regression, 32 min, 55 min, 68 min, and 42 min in control, Zfp36l1, Zfp36l2, and dual-knockdown cells, respectively. All of experiments were carried out at least three times
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4502546&req=5

Fig5: Induction of Mkp-1 mRNA early during LPS stimulation is post-transcriptionally modulated by Zfp36l1 and Zfp36l2. a mRNA expression profile of Mkp-1 in RAW264.7 cells stimulated with LPS for 0, 15, 30, 45, 60, or 120 min. b Levels of Mkp-1 mRNA in different knockdown cells after LPS stimulation for 15 min. RNA was isolated and performed the real-time PCR analysis. c Analysis of Mkp-1 mRNA half-life in different knockdown cells after LPS stimulation for 15 min. Actinomycin D (10 μg · mL-1) was added to stop transcription after 0, 10, or 20 min. The remaining mRNA was detected by quantitative PCR. Mkp-1 mRNA half-life was calculated by exponential regression, 32 min, 55 min, 68 min, and 42 min in control, Zfp36l1, Zfp36l2, and dual-knockdown cells, respectively. All of experiments were carried out at least three times
Mentions: To further investigate the regulation of Mkp-1 mRNA during early LPS stimulation in RAW264.7 cells, we examined the expression of its mRNA (Fig. 5a). Mkp-1 mRNA increased significantly after LPS stimulation from 15 to 30 min but decreased rapidly after 45 min. To verify the functions of Zfp36l1 and Zfp36l2 immediately following LPS treatment, we examined the level of Mkp-1 mRNA in Zfp36l1- and Zfp36l2-knockdown cells after LPS stimulation for 15 min, in this time point Ttp protein was not induced significantly and the lowest level of brought-down Caf1a by Mkp-1 3’UTR was observed in Fig. 4a. The observed marked rise in the mRNA level in all knockdown cells compared with control knockdown cells implied that the decrease in Zfp36l1 and Zfp36l2 protein expression facilitated the expression of Mkp-1 mRNA in response to LPS (Fig. 5b); furthermore, the relative Mkp-1 mRNA half-life at LPS-stimulation 15 min in control, Zfp36l1, Zfp36l2, and dual-knockdown cells was 32 min, 55 min, 68 min, and 42 min, respectively (Fig. 5c). Our results indicate that knockdown of Zfp36l1, Zfp36l2, or both proteins also cause the increase of Mkp-1 mRNA half-life at early LPS stimulation for 15 min like at the resting status. Moreover, Mkp-1 mRNA at early LPS stimulation in control shLuc cells appeared more stable (half-life is 32 min) than which at the resting condition (half-life is 19 min) showed in Fig. 3c. It might be due to phosphorylation of Zfp36l1 and Zfp36l2 upon LPS stimulation leading to protein inactivation.Fig. 5

Bottom Line: Biochemical and functional analyses exhibited that the decrease of Zfp36l2 mRNA was cross-regulated by Ttp.Knockdown of Zfp36l1 and Zfp36l2 increased the basal level of Mkp-1 mRNAs by prolonging its half-life.Zfp36l1 and Zfp36l2 are important regulators of the innate immune response.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No.1 Sec. 4 Roosevelt Road, Taipei, 10617 Taiwan.

ABSTRACT

Background: The tristetraprolin (TTP) family of mRNA-binding proteins contains three major members, Ttp, Zfp36l1, and Zfp36l2. Ttp down-regulates the stability of AU-rich element-containing mRNAs and functions as an anti-inflammation regulator.

Methods: To examine whether other TTP family proteins also play roles in the inflammatory response, their expression profiles and the possible mRNA targets were determined in the knockdown cells.

Results: Ttp mRNA and protein were highly induced by lipopolysaccharide (LPS), whereas Zfp36l1 and Zfp36l2 mRNAs were down-regulated and their proteins were phosphorylated during early lipopolysaccharide stimulation. Biochemical and functional analyses exhibited that the decrease of Zfp36l2 mRNA was cross-regulated by Ttp. Knockdown of Zfp36l1 and Zfp36l2 increased the basal level of Mkp-1 mRNAs by prolonging its half-life. Increasing the expression of Mkp-1 inhibited the activation of p38 MAPK under lipopolysaccharide stimulation and down-regulated Tnfα, and Ttp mRNA. In addition, hyper-phosphorylation of Zfp36l1 might stabilize Mkp-1 expression by forming a complex with the adapter protein 14-3-3 and decreasing the interaction with deadenylase Caf1a.

Conclusions: Our findings imply that the expression and phosphorylation of Zfp36l1 and Zfp36l2 may modulate the basal level of Mkp-1 mRNA to control p38 MAPK activity during lipopolysaccharide stimulation, which would affect the inflammatory mediators production. Zfp36l1 and Zfp36l2 are important regulators of the innate immune response.

No MeSH data available.


Related in: MedlinePlus