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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

mRNA stability regulation of Zfp36l2 by Ttp during LPS-stimulation. a mRNA half-life determination. RAW264.7 cells stimulated with LPS for 0, 20 and 50 min, and actinomycin D (10 μg · mL-1) was added for 0, 10, 30, and 50 min to stop transcription for Zfp36l2 mRNA detection, and added for 0, 1, 2 and 4 h for Zfp36l1 mRNA detection. RNAs was isolated for quantitative PCR by using primers of β-actin, Zfp36l1 and Zfp36l2. The remaining Zfp36l1 and Zfp36l2 mRNA levels were shown after normalized with the level of β-actin. The mRNA half-lives were calculated by exponential regression: 5.2 h, 4.2 h, and 3.5 h for Zfp36l1 at 0, 20 min, and 50 min of LPS treatment, respectively; 39 min, 13 min and 33 min for Zfp36l2 at 0, 20 min, and 50 min of LPS treatment, respectively. b TTP family mRNA analysis under BAY treatment. RAW264.7 cells were pretreated with or without 20 μM of BAY for 0.5 h followed by adding 100 ng · mL-1 LPS for 0.5 and 2 h. Total RNAs were isolated for quantitative PCR. c BAY treatment stabilizes Zfp36l2 mRNA. RAW264.7 cells pretreated with or without 20 μM of BAY for 0.5 h followed by adding 100 ng · mL-1 LPS for 20 min, and actinomycin D was added for 0, 0.5, 1, 2, and 4 h to stop transcription. RNAs was isolated for quantitative PCR and Zfp36l1 and Zfp36l2 mRNA half-lives were determined. d RNA pull-down analysis. Biotin labeled Zfp36l1 3’UTR, Zfp36l2 3’UTR and control 18S RNA were incubated with cytosolic extracts from RAW264.7 cells treated with LPS for 0, 0.5, 1 and 2 h, respectively. After extensive washes, the RNA-protein complexes were analyzed by western blotting with anti-Ttp. e Luciferase reporter analysis. 293 T cells were cotransfected with of 0.25 μg Zfp36l1 3’UTR- or Zfp36l2 3’UTR-containing luciferase reporter and different amounts of Flag-tagged Ttp expression plasmid. After normalized with internal control of Renilla luciferase activity, the relative firefly luciferase activity was shown. All experiments were independently repeated at least two times
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Fig2: mRNA stability regulation of Zfp36l2 by Ttp during LPS-stimulation. a mRNA half-life determination. RAW264.7 cells stimulated with LPS for 0, 20 and 50 min, and actinomycin D (10 μg · mL-1) was added for 0, 10, 30, and 50 min to stop transcription for Zfp36l2 mRNA detection, and added for 0, 1, 2 and 4 h for Zfp36l1 mRNA detection. RNAs was isolated for quantitative PCR by using primers of β-actin, Zfp36l1 and Zfp36l2. The remaining Zfp36l1 and Zfp36l2 mRNA levels were shown after normalized with the level of β-actin. The mRNA half-lives were calculated by exponential regression: 5.2 h, 4.2 h, and 3.5 h for Zfp36l1 at 0, 20 min, and 50 min of LPS treatment, respectively; 39 min, 13 min and 33 min for Zfp36l2 at 0, 20 min, and 50 min of LPS treatment, respectively. b TTP family mRNA analysis under BAY treatment. RAW264.7 cells were pretreated with or without 20 μM of BAY for 0.5 h followed by adding 100 ng · mL-1 LPS for 0.5 and 2 h. Total RNAs were isolated for quantitative PCR. c BAY treatment stabilizes Zfp36l2 mRNA. RAW264.7 cells pretreated with or without 20 μM of BAY for 0.5 h followed by adding 100 ng · mL-1 LPS for 20 min, and actinomycin D was added for 0, 0.5, 1, 2, and 4 h to stop transcription. RNAs was isolated for quantitative PCR and Zfp36l1 and Zfp36l2 mRNA half-lives were determined. d RNA pull-down analysis. Biotin labeled Zfp36l1 3’UTR, Zfp36l2 3’UTR and control 18S RNA were incubated with cytosolic extracts from RAW264.7 cells treated with LPS for 0, 0.5, 1 and 2 h, respectively. After extensive washes, the RNA-protein complexes were analyzed by western blotting with anti-Ttp. e Luciferase reporter analysis. 293 T cells were cotransfected with of 0.25 μg Zfp36l1 3’UTR- or Zfp36l2 3’UTR-containing luciferase reporter and different amounts of Flag-tagged Ttp expression plasmid. After normalized with internal control of Renilla luciferase activity, the relative firefly luciferase activity was shown. All experiments were independently repeated at least two times

Mentions: We are interested in the molecular mechanism of down-regulation of Zfp36l1 and Zfp36l2 mRNA in response to LPS treatment. Their mRNA 3’UTR contains potential AREs (Additional file 1: Figure S1), we examined whether their mRNA half-life was regulated by LPS. As shown in Fig. 2a, the half-life of Zfp36l1 mRNA was longer than that of Zfp36l2 mRNA, which was 5.2 h for Zfp36l1 and 39 min for Zfp36l2. The half-life of Zfp36l2 mRNA was significantly shortened to 13 min at 20 min of LPS treatment, and then restored to 33 min at 50 min treatment. Although the half-lives of Zfp36l1 mRNA were also decreased upon LPS treatment, they still maintained to near 4 h (Fig. 2a). It was known that Ttp induced by LPS can destabilize ARE-containing mRNA [13, 25]. We have demonstrated that NF-κB signaling pathway was required for Ttp induction [28]. To examine whether Ttp expression plays roles in the down-regulation of Zfp36l1 and Zfp36l2 mRNA, we compared the mRNA expression of Ttp, Zfp36l1and Zfp36l2 in the presence of the inhibitor (BAY) of NF-κB pathway. The pretreatment with BAY inhibited the mRNA level of Ttp in LPS-stimulation for 0.5 h and 2 h, which was consistent with the increases of Zfp36l2 mRNA level (Fig. 2b), but no correlation with the levels of Zfp36l1 mRNA. The increase of Zfp36l2 mRNA under BAY treatment was due to the increase of mRNA half-life, whereas BAY treatment would shorten Zfp36l1 mRNA half-life (Fig. 2c). Moreover, the biotin-labeled partial 3’UTR from Zfp36l1 or Zfp36l2 associates with Ttp proteins in RNA pull-down analysis (Fig. 2d), and the 3’UTR-mediated luciferase activity was down-regulated when cotransfection with Ttp expression vector (Fig. 2e). Although both Zfp36l1 and Zfp36l2 3’UTR showed physical and functional interaction with ectopic expressive Ttp, only endogenous Zfp36l2 mRNA was decreased by LPS-induced NF-κB signals. The results suggest that in addition to Ttp there are other factors involved in the regulation of Zfp36l1 mRNA stability during LPS stimulation.Fig. 2


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)

mRNA stability regulation of Zfp36l2 by Ttp during LPS-stimulation. a mRNA half-life determination. RAW264.7 cells stimulated with LPS for 0, 20 and 50 min, and actinomycin D (10 μg · mL-1) was added for 0, 10, 30, and 50 min to stop transcription for Zfp36l2 mRNA detection, and added for 0, 1, 2 and 4 h for Zfp36l1 mRNA detection. RNAs was isolated for quantitative PCR by using primers of β-actin, Zfp36l1 and Zfp36l2. The remaining Zfp36l1 and Zfp36l2 mRNA levels were shown after normalized with the level of β-actin. The mRNA half-lives were calculated by exponential regression: 5.2 h, 4.2 h, and 3.5 h for Zfp36l1 at 0, 20 min, and 50 min of LPS treatment, respectively; 39 min, 13 min and 33 min for Zfp36l2 at 0, 20 min, and 50 min of LPS treatment, respectively. b TTP family mRNA analysis under BAY treatment. RAW264.7 cells were pretreated with or without 20 μM of BAY for 0.5 h followed by adding 100 ng · mL-1 LPS for 0.5 and 2 h. Total RNAs were isolated for quantitative PCR. c BAY treatment stabilizes Zfp36l2 mRNA. RAW264.7 cells pretreated with or without 20 μM of BAY for 0.5 h followed by adding 100 ng · mL-1 LPS for 20 min, and actinomycin D was added for 0, 0.5, 1, 2, and 4 h to stop transcription. RNAs was isolated for quantitative PCR and Zfp36l1 and Zfp36l2 mRNA half-lives were determined. d RNA pull-down analysis. Biotin labeled Zfp36l1 3’UTR, Zfp36l2 3’UTR and control 18S RNA were incubated with cytosolic extracts from RAW264.7 cells treated with LPS for 0, 0.5, 1 and 2 h, respectively. After extensive washes, the RNA-protein complexes were analyzed by western blotting with anti-Ttp. e Luciferase reporter analysis. 293 T cells were cotransfected with of 0.25 μg Zfp36l1 3’UTR- or Zfp36l2 3’UTR-containing luciferase reporter and different amounts of Flag-tagged Ttp expression plasmid. After normalized with internal control of Renilla luciferase activity, the relative firefly luciferase activity was shown. All experiments were independently repeated at least two times
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: mRNA stability regulation of Zfp36l2 by Ttp during LPS-stimulation. a mRNA half-life determination. RAW264.7 cells stimulated with LPS for 0, 20 and 50 min, and actinomycin D (10 μg · mL-1) was added for 0, 10, 30, and 50 min to stop transcription for Zfp36l2 mRNA detection, and added for 0, 1, 2 and 4 h for Zfp36l1 mRNA detection. RNAs was isolated for quantitative PCR by using primers of β-actin, Zfp36l1 and Zfp36l2. The remaining Zfp36l1 and Zfp36l2 mRNA levels were shown after normalized with the level of β-actin. The mRNA half-lives were calculated by exponential regression: 5.2 h, 4.2 h, and 3.5 h for Zfp36l1 at 0, 20 min, and 50 min of LPS treatment, respectively; 39 min, 13 min and 33 min for Zfp36l2 at 0, 20 min, and 50 min of LPS treatment, respectively. b TTP family mRNA analysis under BAY treatment. RAW264.7 cells were pretreated with or without 20 μM of BAY for 0.5 h followed by adding 100 ng · mL-1 LPS for 0.5 and 2 h. Total RNAs were isolated for quantitative PCR. c BAY treatment stabilizes Zfp36l2 mRNA. RAW264.7 cells pretreated with or without 20 μM of BAY for 0.5 h followed by adding 100 ng · mL-1 LPS for 20 min, and actinomycin D was added for 0, 0.5, 1, 2, and 4 h to stop transcription. RNAs was isolated for quantitative PCR and Zfp36l1 and Zfp36l2 mRNA half-lives were determined. d RNA pull-down analysis. Biotin labeled Zfp36l1 3’UTR, Zfp36l2 3’UTR and control 18S RNA were incubated with cytosolic extracts from RAW264.7 cells treated with LPS for 0, 0.5, 1 and 2 h, respectively. After extensive washes, the RNA-protein complexes were analyzed by western blotting with anti-Ttp. e Luciferase reporter analysis. 293 T cells were cotransfected with of 0.25 μg Zfp36l1 3’UTR- or Zfp36l2 3’UTR-containing luciferase reporter and different amounts of Flag-tagged Ttp expression plasmid. After normalized with internal control of Renilla luciferase activity, the relative firefly luciferase activity was shown. All experiments were independently repeated at least two times
Mentions: We are interested in the molecular mechanism of down-regulation of Zfp36l1 and Zfp36l2 mRNA in response to LPS treatment. Their mRNA 3’UTR contains potential AREs (Additional file 1: Figure S1), we examined whether their mRNA half-life was regulated by LPS. As shown in Fig. 2a, the half-life of Zfp36l1 mRNA was longer than that of Zfp36l2 mRNA, which was 5.2 h for Zfp36l1 and 39 min for Zfp36l2. The half-life of Zfp36l2 mRNA was significantly shortened to 13 min at 20 min of LPS treatment, and then restored to 33 min at 50 min treatment. Although the half-lives of Zfp36l1 mRNA were also decreased upon LPS treatment, they still maintained to near 4 h (Fig. 2a). It was known that Ttp induced by LPS can destabilize ARE-containing mRNA [13, 25]. We have demonstrated that NF-κB signaling pathway was required for Ttp induction [28]. To examine whether Ttp expression plays roles in the down-regulation of Zfp36l1 and Zfp36l2 mRNA, we compared the mRNA expression of Ttp, Zfp36l1and Zfp36l2 in the presence of the inhibitor (BAY) of NF-κB pathway. The pretreatment with BAY inhibited the mRNA level of Ttp in LPS-stimulation for 0.5 h and 2 h, which was consistent with the increases of Zfp36l2 mRNA level (Fig. 2b), but no correlation with the levels of Zfp36l1 mRNA. The increase of Zfp36l2 mRNA under BAY treatment was due to the increase of mRNA half-life, whereas BAY treatment would shorten Zfp36l1 mRNA half-life (Fig. 2c). Moreover, the biotin-labeled partial 3’UTR from Zfp36l1 or Zfp36l2 associates with Ttp proteins in RNA pull-down analysis (Fig. 2d), and the 3’UTR-mediated luciferase activity was down-regulated when cotransfection with Ttp expression vector (Fig. 2e). Although both Zfp36l1 and Zfp36l2 3’UTR showed physical and functional interaction with ectopic expressive Ttp, only endogenous Zfp36l2 mRNA was decreased by LPS-induced NF-κB signals. The results suggest that in addition to Ttp there are other factors involved in the regulation of Zfp36l1 mRNA stability during LPS stimulation.Fig. 2

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