Author(s): Elena M. Gallo [1, 2]; Monte M. Winslow [1, 2, 5]; Kirsten Canté-Barrett [1, 2, 5]; Amy N. Radermacher [1, 2]; Lena Ho [1, 2]; Lisa McGinnis [1, 2]; Brian Iritani ; Joel R. Neilson [1, 4, 5]; Gerald R. Crabtree (corresponding author) [1, 2]
At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the [alpha][beta] T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection) [1, 2]. Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.
The calcineurin/NFAT [3, 4] and the Raf-MEK-ERK [5, 6, 7] pathways have been shown to be required for positive selection of thymocytes but not for their negative selection. Calcineurin B1 (Cnb1 )-deficient CD4+ CD8+ double-positive thymocytes lack calcineurin activity, fail to dephosphorylate NFATc transcription factors and are not positively selected (Fig. 1a and ref. 3). Cnb1 -deficient thymocytes have normal phosphorylation of JNK (c-Jun N-terminal kinase), p38, protein kinase C-[theta], protein kinase D and glycogen synthase kinase 3[alpha] after crosslinking of the T-cell receptor (TCR) (Fig. 1b and ref. 3). Actin polymerization and Ca2+ influx were also normal in these cells (Supplementary Fig. 2a, b). Collectively, these results indicated that Cnb1 -deficient thymocytes did not have widespread signalling defects downstream of the TCR. However, Cnb1 -deficient thymocytes showed a specific and severe defect in ERK1/2 phosphorylation (Fig. 1c), resulting in almost undetectable induction of the ERK/Elk4 target gene Egr1 (ref. 6) after engagement of the TCR (Fig. 1d). In addition, both MAP-kinase kinase (MEK)1/2 and Raf activation were defective in Cnb1 -deficient double-positive thymocytes (Fig. 1c, e, f) after stimulation by crosslinking of the TCR.
To test whether calcineurin activity was directly required for Raf activation, we stimulated thymocytes in the presence of the calcineurin inhibitor cyclosporin A (CsA) or the MEK1/2 inhibitor UO126. Acute in vitro inhibition of MEK1/2 with UO126 but not inhibition of calcineurin activity with CsA impaired ERK1/2 phosphorylation and Egr1 induction (Fig. 2a, b). In contrast, 10-day...