Abstract
After antigen and/or different cytokine stimulation, CD4+ T cells activated and differentiated into distinct T helper (Th) cells via differential T cell signaling pathways. Transcriptional regulation of the activation and differentiation of naïve CD4+ T cells into distinct lineage Th cells such as Th17 cells has been fully studied. However, the role of RNA-binding protein HuR in the signaling pathways of their activation and differentiation has not been well characterized. Here, we used HuR conditional knockout (HuR KO) CD4+ T cells to study mechanisms underlying HuR regulation of T cell activation and differentiation through distinct signaling pathways. Our work showed that, mechanistically, HuR positively promoted CD3g expression by binding its mRNA and enhanced the expression of downstream adaptor Zap70 and Malt1 in activated CD4+ T cells. Compared to WT Th0 cells, HuR KO Th0 cells with reduced Bcl-2 expression are much more susceptible to apoptosis than WT Th0 cells. We also found that HuR stabilized IL-6Rα mRNA and promoted IL-6Rα protein expression, thereby upregulating its downstream phosphorylation of Jak1 and Stat3 and increased level of phosphorylation of IκBα to facilitate Th17 cell differentiation. However, knockout of HuR increased IL-22 production in Th17 cells, which was due to HuR deficiency in reducing IL-22 transcription repressor c-Maf expression. These results highlight the importance of HuR in TCR signaling and IL-6/IL-6R axis driving naïve CD4+ T cell activation and differentiation into Th17 cells.
Highlights
After antigen stimulation, CD3 complex associated with T cell receptor (TCR) and the z chain activates and enhances the signaling cascades that determine T cell fate [1]
We compared HuR-deficient CD4+ T cells from conditional knockout (KO) mice with WT CD4+ T cells under anti-CD3 and anti-CD28 stimulation for Th0 cell differentiation, and we identified that the expression of several molecules in the signaling pathway of TCR such as CD3g, Zap70, and Malt1 was impaired in HuR-deficient Th0 cells by RNA Isolation and Quantitative RT-PCR (RT-qPCR) assay (Figure 1(a))
Western blot assays showed that the protein levels of Zap70 and Malt1 had decreased (Figure 1(d)), which is consistent with a previous study that HuR conditional knockout (HuR KO) CD4+ T cells are less activated and proliferate less than WT control cells [24]
Summary
CD3 complex associated with T cell receptor (TCR) and the z chain (zeta-chain) activates and enhances the signaling cascades that determine T cell fate [1]. Zeta-chain-associated protein kinase (Zap70) is one of the two members of the cytoplasmic Syk tyrosine kinase family and recruited to the TCR/CD3 complex and activated. Zap plays an essential role in T cell development and activation [1, 2], which is dependent on Lck to initiate the downstream signaling pathway [3]. Malt plays an important role in T cell activation and proliferation [4, 5]. TCR engagement leads to the formation of an oligomeric CBM complex (Carma, Bcl, and Malt proteins), which is required for activation of downstream canonical NF-κB signaling [6]. Previous studies provided evidence that Malt is essential for development of pathogenic T helper 17 (Th17) cells during experimental autoimmune encephalomyelitis (EAE) induction [9]
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