The Regulation of GATA-3 Expression and Function During Th2 Differentiation

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2010.The key to maintaining an effective immune system is the balance between protecting against foreign agents and prevention of attacks upon one’s self. A major cell class that controls this balance is the CD4+ T helper cell. Naïve CD4+ T cells differentiate into functionally distinct subsets and are central to both the protection against pathogens and the prevention of autoimmunity. The differentiation process is governed by master regulator transcription factors, which establish characteristic cytokine producing phenotypes by regulating the accessibility of the cytokine gene loci to the transcriptional machinery. In Th2 differentiation, this chromatin remodeling is controlled by GATA-3, a protein that is both necessary and sufficient for Th2 differentiation. Because of the critical role of GATA-3 in Th2 differentiation, it is important to understand the signaling factors that control its expression. Stimulation of T cells in the presence of IL-4 has been shown to promote Th2 differentiation and GATA-3 expression. Signaling molecules downstream of both the TCR and the IL-4R have been implicated in transcriptional upregulation of GATA-3. It has been difficult to identify individual roles of each signaling pathway though, as TCR signaling is also required for survival and expansion of naïve T cells in vitro. To bypass the dependence of T cells on TCR signaling for survival and expansion we have created a system using previously activated cells, which allows us to determine the specific contributions of IL-4R and TCR signaling towards GATA-3 and Th2 differentiation. We have found that stimulation of activated, undifferentiated T cells with IL-4 results in the upregulation of GATA-3 mRNA, yet fails to induce Th2 differentiation. The addition of TCR signaling has no effect on the level of GATA-3 mRNA, but increases the production of GATA-3 protein. These data show that TCR signaling specifically enhances the translation rate of GATA-3 protein, without affecting the rate of degradation or the global translational efficiency. Thus, the expression of GATA-3 is a key point of signal integration during Th2 differentiation, with IL-4R signaling inducing GATA-3 mRNA and TCR signaling enhancing GATA-3 translation. The TCR-dependent increase in GATA-3 translation is mediated by signaling through the phosphatidylinositol 3-kinase (PI3K) / mammalian target of rapamycin (mTOR) pathway. Specific inhibition of either of these proteins reduces the GATA-3 translation rate and the subsequent level of GATA-3 protein. The reduction in GATA-3 protein in the absence of PI3K or mTOR activation correlates with a failure to differentiation into Th2 effector cells. Thus, TCR signaling through PI3K may play a critical role in Th2 differentiation by the specific enhancement of GATA-3 translation. Gene-specific translational control is often mediated by the 5’ untranslated region (UTR) of an mRNA. The folding of mRNA into complex secondary structures and the binding of sequence-specific proteins can inhibit the translational machinery from being recruited to the mRNA, or from scanning through the 5’UTR to identify the initiation codon. To identify the regions of GATA-3 mRNA that influence its translational control, we made luciferase reporter constructs containing the GATA-3 5’UTR. The transcription of GATA-3 mRNA can be driven from two different promoters, resulting in mRNA molecules that differ only in the exon 1 portion of their 5’UTR. We show that there is a decrease in translation of mRNA reporters containing the 5’UTR that is driven from the downstream exon 1B promoter. This promoter accounts for greater than 95 percent of the total GATA-3 mRNA in T cells. Structural prediction of the GATA-3 5’UTR reveals a hairpin structure that is present in the 5’UTR B, but not 5’UTR A, that could inhibit the movement of the translational machinery. Ribosome scanning through stable secondary structures requires the activity of the RNA helicase, eIF4A. Interestingly, regulation of eIF4A helicase activity is affected by signaling through PI3K and mTOR, the same signaling pathway important for GATA-3 translational control. The expression of PDCD4, a negative regulator of eIF4A, is decreased following TCR signaling and this decrease is dependent on PI3K and mTOR signaling. Additionally, the phosphorylation of eIF4B, which promotes eIF4A activity, is also dependent on TCR signaling through PI3K and mTOR. Thus, the TCR-dependent increase in GATA-3 translation may be mediated by modulation of eIF4A helicase activity. In the process of identifying the signaling molecules required for the translation of GATA-3, we observed that inhibition of ERK signaling strongly reduced Th2 differentiation. However, this effect on Th2 differentiation is not mediated by an effect on the expression of GATA-3, as inhibition of ERK has no effect on GATA-3 mRNA induction, protein synthesis, or protein stability. ERK signaling is instead required for altering the functional capacity of GATA-3. Both the specific ability of GATA-3 to trans-activate the IL-13 promoter and the transformation into Th2 effector cytokine producing cells are lost in the absence of ERK signaling. Control of the expression and function of GATA-3 is a tightly regulated process involving many signaling events. In addition to transcriptional control, GATA-3 is regulated via translational and post-translational methods. We have identified the functions of PI3K/mTOR and ERK signaling that are required for the regulation of GATA-3 and subsequent Th2 differentiation