New Programming of IL-4 Receptor Signal Transduction in Activated T Cells: Stat6 Induction and Th2 Differentiation Mediated by IL-4Rα Lacking Cytoplasmic Tyrosines

Abstract

Signaling by the IL-4 receptor α-chain (IL-4Rα) is a key determinant of the development of the Th2 lineage of effector T cells. Studies performed in tissue culture cell lines have indicated that tyrosines of the IL-4Rα cytoplasmic tail are necessary for the induction of Stat6, a transcription factor required for Th2 differentiation. Surprisingly, we have found that in activated T cells, IL-4Rα chains lacking all cytoplasmic tyrosines promote induction of this IL-4-specific transcription factor and efficient commitment to the Th2 lineage. Mutagenesis of a tyrosine-free cytoplasmic tail identifies a requirement for the serine-rich ID-1 region in this new program of IL-4R signal transduction observed in activated T cells. Additional findings suggest that an extracellular signal-regulated kinase pathway can be necessary and sufficient for the ability of such tyrosine-free IL-4Rα chains to mediate Stat6 induction. These results provide novel evidence that the molecular mechanisms by which a cytokine specifically induces a Stat transcription factor can depend on the activation state of T lymphoid cells. Furthermore, the data suggest that one pathway by which such new programming may be achieved is mediated by extracellular signal-regulated mitogen-activated protein kinases.

T-bet antagonizes mSin3a recruitment and transactivates a fully methylated IFN-γ promoter via a conserved T-box half-site

“…For GAPDH amplification, the primers used were 5Ј-ACCACAGTCCATGCCATCAC-3Ј (forward) and 5Ј-TCCACCACCCTGTTGCTGTA-3Ј (reverse), and the PCR consisted of 23 cycles of 1 min at 94°C; 1 min at 48°C; and 1 min at 72°C. For Western blotting, whole cell, cytoplasmic, and nuclear extracts were prepared as described (39,40). Proteins (25 g per lane) were separated by SDS͞PAGE, transferred to poly(vinylidene difluoride) membranes, and probed with antibodies against T-bet, C͞EBP␤, mSin3a, or MeCP2 according to the manufacturer's recommendations.…”

“…Jurkat T cells were cultured exactly as described (37,39). A T cell variant stably expressing T-bet was created by infection of Jurkat with retrovirus-containing supernatant collected 48 h after transfection of NX amphitropic packaging cells with T-bet-MiT retrovector and centrifugation in the presence of polybrene (8 g͞ml) as described (39). Thy1.1ϩ cells were purified by positive selection using anti-CD90.1 (Thy1.1)-phycoerythrin (PE) antibody and anti-PE magnetic beads (Miltenyi Biotec).…”

Sequence Motifs in IL-4Rα Mediating Cell-Cycle Progression of Primary Lymphocytes

“…Phoenix cells were cultured in complete DMEM supplemented as for IMDM/10F and with 0.1ϫ MEM amino acids. Inhibitors of ERK activation, U0126 and PD98059, were obtained and used, as described previously (17), at concentrations of 10 and 50 M, respectively.…”

“…Our laboratory has provided evidence that TCR signaling leads to new programming of the IL-4R complex (17). This reprogramming permits tyrosine-deficient receptors to induce STAT6 signaling and Th2 development, events that were dependent on an intact ID-1 region (17). Together, these findings suggest that the structural elements of the IL-4R␣ that are necessary for proliferative signaling in activated T cells may differ from what has been found in model cell lines.…”

“…In developing and committed Th2 cells, for example, IL-4 stimulation induces STAT5 signaling (10,11). Our laboratory has provided evidence that TCR signaling leads to new programming of the IL-4R complex (17). This reprogramming permits tyrosine-deficient receptors to induce STAT6 signaling and Th2 development, events that were dependent on an intact ID-1 region (17).…”