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

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 Tnf?, 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.

SUBMITTER: Wang KT

PROVIDER: S-EPMC4502546 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Wang Kuan-Ting KT Wang Hsin-Hui HH Wu Yan-Yun YY Su Yu-Lun YL Chiang Pei-Yu PY Lin Nien-Yi NY Wang Shun-Chang SC Chang Geen-Dong GD Chang Ching-Jin CJ

Journal of inflammation (London, England) 20150716

<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>Ttp mRNA and protein were highl ...[more]

PMID: 26180518

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Project description:Context Torin2 has various pharmacological properties. However, its anti-inflammatory activity has not been reported. Objective This study focused on the potential anti-inflammatory properties of Torin2 in lipopolysaccharide (LPS)-evoked RAW264.7 murine macrophages. The study aimed to shed light on the molecular mechanisms that ameliorate these effects. Methods Torin2 was applied to 100 ng/mL lipopolysaccharide-induced RAW 264.7 macrophages in vitro. Nitric oxide (NO) levels were detected using the Griess reagent kit. Prostaglandin E2 (PGE2), pro-inflammatory cytokines interleukin (IL)-1β, interleukin (IL)-6, and tumor necrosis factor in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Gene expression of pro-inflammatory cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were tested using real-time quantitative polymerase chain reaction (qPCR). Cyclooxygenase-2 and inducible nitric oxide synthase proteins, phosphorylation of mitogen-activated protein kinase (MAPK) subgroups, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, I-kappa-B-alpha (IκBα), and nuclear factor-kappa-B (NF-κB), and activation in extracts were detected via western blotting. Nuclear factor-kappa-B/p65 nuclear translocation was tested using an immunofluorescence assay. Results The results demonstrated that pre-treatment with Torin2 profoundly attenuated the lipopolysaccharide-stimulated levels of nitric oxide and prostaglandin E2, pro-inflammatory cytokines, messenger ribonucleic acid (mRNA), and protein expression of cyclooxygenase-2 and inducible nitric oxide synthase. Collectively, Torin2 pre-treatment notably weakened lipopolysaccharide-induced damage by reducing the phosphorylation of nuclear factor-kappa-B, p38, c-Jun N-terminal kinase, extracellular signal-regulated kinase proteins, and nuclear factor-kappa-B/p65 nuclear translocation. Conclusion Numerous pieces of evidence indicated that Torin2 reversed inflammatory activation by regulating nuclear factor-kappa-B and mitogen-activated protein kinase signaling pathways and provided a tentative potential candidate for preventing and treating inflammatory diseases. Torin2; RAW264.7 murine macrophages; Lipopolysaccharide; Anti-inflammation; Mechanisms.

Project description:Mammalian macrophages differ in their basal gene expression profiles and response to the toll-like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS). In human macrophages, LPS elicits a temporal cascade of transient gene expression including feed forward activators and feedback regulators that limit the response. Here we present a transcriptional network analysis of the response of sheep bone marrow-derived macrophages (BMDM) to LPS based upon RNA-seq at 0, 2, 4, 7, and 24 h post-stimulation. The analysis reveals a conserved transcription factor network with humans, and rapid induction of feedback regulators that constrain the response at every level. The gene expression profiles of sheep BMDM at 0 and 7 h post LPS addition were compared to similar data obtained from goat, cow, water buffalo, horse, pig, mouse and rat BMDM. This comparison was based upon identification of 8,200 genes annotated in all species and detected at >10TPM in at least one sample. Analysis of expression of transcription factors revealed a conserved transcriptional millieu associated with macrophage differentiation and LPS response. The largest co-expression clusters, including genes encoding cell surface receptors, endosome-lysosome components and secretory activity, were also expressed in all species and the combined dataset defines a macrophage functional transcriptome. All of the large animals differed from rodents in lacking inducible expression of genes involved in arginine metabolism and nitric oxide production. Instead, they expressed inducible transporters and enzymes of tryptophan and kynurenine metabolism. BMDM from all species expressed high levels of transcripts encoding transporters and enzymes involved in glutamine metabolism suggesting that glutamine is a major metabolic fuel. We identify and discuss transcripts that were uniquely expressed or regulated in rodents compared to large animals including ACOD1, CXC and CC chemokines, CD163, CLEC4E, CPM, CSF1, CSF2, CTSK, MARCO, MMP9, SLC2A3, SLC7A7, and SUCNR1. Conversely, the data confirm the conserved regulation of multiple transcripts for which there is limited functional data from mouse models and knockouts. The data provide a resource for functional annotation and interpretation of loci involved in susceptibility to infectious and inflammatory disease in humans and large animal species.

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

Publications

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

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