p38 Alternative activation is required for T-cell antigen receptor (TCR)-induced expression of NFAT2 via c-Fos.
(A, left panel) Purified mouse T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and phorbol myristate acetate (PMA) plus ionomycin for 16 hours and then immunoblotted. (A, right panel) Purified mouse T cells were stimulated as in panel A, and interleukin (IL)-2 was measured in the culture supernatant (S1 Data). (B) Purified T cells from wild-type (WT) mice were stimulated with anti-CD3/CD28 or PMA/ionomycin for the indicated times, and then the lysates were immunoblotted. (C) Purified T cells from WT or double knock-in (DKI) mice were stimulated with anti-CD3/CD28 for the indicated times, and then the lysates were immunoblotted. (D) Purified WT T cells were stimulated with anti-CD3/CD28 or PMA/ionomycin for the indicated times, and binding of c-Fos to the NFAT2 promoter was analyzed by chromatin immunoprecipitation (ChiP) (S1 Data). (E) pGL3-basic luciferase reporter construct containing the nfat2 promoter consensus AP-1 binding site or its mutant (left panel) was transfected into Jurkat cells, which were stimulated overnight with anti-CD3/CD28, and then a luciferase assay was performed (right panel) (S1 Data). (F) Purified T cells from 2 WT or 2 DKI mice were infected with retrovirus carrying empty vector (EV) or c-fos. The T cells were pooled per group and stimulated with anti-CD3/CD28 for 24 hours, and the expression of nfat2 mRNA was determined by quantitative real-time PCR. nfat2 levels in WT EV-transduced and unstimulated samples were defined as 1 (S1 Data). (G) Purified murine T cells were infected and stimulated with anti-CD3/CD28 or PMA/ionomycin, and nfat2 mRNA expression was quantitated by real-time PCR as in panel F (S1 Data). The results are representative of 3 independent experiments, and the bar graphs in the right panel show the mean ± SEM of all 3. *p < 0.05, **p <0.01, ***p < 0.001. NS, not significant.