Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT)

Abstract

The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-alpha-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2-/- T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2-/- T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.

Figure 1

Resistance of junAA-deficient thymocytes to death caused by apoptotic stimuli in vitro and in vivo. (A and B) Thymocytes from wild-type (black symbols) and junAA (gray symbols) mice were cultured on 24-well plates with various concentrations of immobilized anti-CD3ɛ antibody for 24 h (A) or for the indicated periods (B) in the presence of 100 μg/ml anti-CD3ɛ antibody, and their viability was determined. (C and D) Cell death was induced by addition of various concentrations of TNFα to the medium, and cell viability was determined after 24 h (C) or after 24 and 48 h postaddition of 50 ng/ml TNFα (D). (E and F) Thymocyte viability was determined after treatment with various concentrations of anti-Fas antibody for 24 h (E) or with 1 μg/ml anti-Fas antibody after 24 and 48 h (F). One representative result of four independent experiments, each using three pairs of mice, is shown. (G) Death of CD4⁺CD8⁺ double positive thymocytes was induced by i.p. injection of the indicated doses of anti-CD3ɛ antibody in wild-type (black symbols) and junAA (white symbols) mice, and the number of CD4⁺CD8⁺ double positive thymocytes was determined after 45 h. Statistical significance was analyzed by Student's t test. *, P < 0.05; **, P < 0.01.

Figure 2

Analysis of cytokine production in effector Th1 and Th2 cells from wild-type and junAA mice. (A) IFNγ production by Th0 and Th1 cells differentiated from wild-type (filled bar) and junAA (empty bar) T cells was determined by ELISA. (B) IL-4 production by Th0 and Th2 cells differentiated from wild-type (filled bar) and junAA (empty bar) T cells was determined by ELISA. One representative result of three independent experiments, each using three pairs of mice, is shown.

Figure 3

Normal proliferation of junAA B and T cells. (A) Purified B cells from spleens of 8-to 10-wk-old mice were cultured in media or in the presence of plate-bound anti-CD40 antibody, IL-4, or lipopolysaccharide. (B) Purified T cells from spleens of 8-to 10-wk-old mice were cultured in media or in the presence of various concentrations of plate-bound anti-CD3ɛ antibody and soluble anti-CD28 antibody, or in the presence of Con A. (C) IL-2 production was measured by ELISA after 24-h stimulation of purified T cells as described above. One representative result of four independent experiments, each using three pairs of mice, is shown.

Figure 4

Reduced NF-AT DNA-binding activity jnk2-deficient T cells. (A) JNP and c-Jun protein levels were measured by using nuclear extracts from untreated or anti-CD3/anti-CD28 stimulated splenocytes. (B) Cytoplasmic and nuclear extracts from untreated (−) and PMA/ionomycin-stimulated (for 3 h, indicated by +) wild-type and jnk2−/− lymph node T cells were prepared, and NF-ATc1 protein was detected by Western analysis. (C) Nuclear extracts from PMA/ionomycin-stimulated (for 3 h) wild-type and jnk2−/− lymph node T cells were prepared 3 h after stimulation, and NF-AT and AP-1 DNA binding was detected by electrophoretic mobility shift assay.

Figure 5

Overexpression of JNK2 results in elevated NF-AT transcriptional activity. (A) NF-AT activity assay of Jurkat T cells transfected with control vector and a JNK expression construct together with NF-AT luciferase reporter construct. Protein extracts were prepared 24 h after transfection, and luciferase activity was measured in duplicate. (B) JNK2 protein levels in Jurkat T cells transfected with control vector and a JNK expression construct were determined by Western analysis.