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The threshold pattern of calcineurin-dependent gene expression is altered by loss of the endogenous inhibitor calcipressin

Nature Immunology volume 4, pages 874–881 (2003)Cite this article

An Editorial Expression of Concern to this article was published on 11 March 2024

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Abstract

Calcineurin links calcium signaling to transcriptional responses in the immune, nervous and cardiovascular systems. To determine the function of the calcipressins, a family of putative calcineurin inhibitors, we assessed the calcineurin-dependent process of T cell activation in mice engineered to lack the gene encoding calcipressin 1 (Csp1). Csp1 regulated calcineurin in vivo, and genes triggered in an immune response had unique transactivation thresholds for T cell receptor stimulation. In the absence of Csp1, the apparent transactivation thresholds for all these genes were shifted because of enhanced calcineurin activity. This unbridled calcineurin activity drove Fas ligand expression, which normally requires high T cell receptor stimulation and results in the premature death of T helper type 1 cells. Thus, calcipressins modulate the pattern of calcineurin-dependent transcription, and may influence calcineurin activity beyond calcium to integrate a broad array of signals into the cellular response.

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Figure 1: Csp1 is a calcineurin inhibitor.
Figure 2: Csp1-deficient T cell responses during activation.
Figure 3: Csp1-deficient TH1 cells express Fas ligand during primary activation.
Figure 4: Rescue of Csp1-deficient TH1 cell death.
Figure 5: Pattern of calcineurin-dependent gene transactivation.

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Acknowledgements

We thank members of the McKeon and Sharpe laboratories for help and advice during this work. This work was supported by grants from the National Institutes of Health (A.H.S. and F.M.) and the Phillip Morris Foundation (F.M.), a fellowship from the Juvenile Diabetes Foundation (R.J.G.) and a National Research Service Award fellowship (S.R.).

Author information

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Sandra Ryeom & Frank McKeon

  2. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 221 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Rebecca J Greenwald & Arlene H Sharpe

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Corresponding author

Correspondence to Frank McKeon.

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Supplementary information

Supplementary Fig. 1.

Calcineurin activity in Csp1-deficient T cells. (a) Anti-NFATc2 immunoblot of lysates from equivalent numbers of CD4+ T cells from WT and KO unstimulated or stimulated with 200 nM PMA and the indicated concentrations of ionomycin for 1 h. The phosphorylated isoform of NFATc2 is indicated by the closed arrows while the dephosphorylated species is indicated by the open arrows. (b) Anti-NFATc2 immunoblot of lysates from equivalent numbers of CD4+ T cells from WT and KO animals stimulated with 2.0 μM ionomycin and 200 nM PMA with 250 nM cyclosporin A. The phosphorylated isoform of NFATc2 is indicated by the closed arrows. (PDF 23 kb)

Supplementary Fig. 2.

Csp1-deficient T cell responses during activation. (a) Comparison of IL-4 production by purified CD4+ cells from WT and KO animals after activation. (b) Anti-TNP IgM responses in Csp1 WT and KO mice following immunization with TNP-KLH. Error bars represent one s.d. of the mean. (PDF 7 kb)

Supplementary Fig. 3.

Annexin V staining of WT and KO cells. (a) FACS analysis of WT and KO cells stained with Annexin V-FITC and propidium iodide (PI) 54 h after activation with anti-CD3 and anti-CD28 antibodies. Percentage of Annexin V positive cells are indicated in the lower right quadrant. (b) Time course of Annexin V-positive WT and KO cells during primary activation. (c) Comparison of activation-induced cell death between WT and KO CD4+ T cells during secondary stimulation at the indicated times. (PDF 21 kb)

Supplementary Fig. 4.

TH1 and TH2-skewed WT and KO cells. (a) FACS profile of TH1-skewed cells stained with anti-CD4 and anti-IFN-γ antibodies. (b) Immunoblot analysis of T-bet and GATA-3 expression in TH1 and TH2-skewed Csp1 WT and KO cells. (PDF 34 kb)

Supplementary Fig. 5.

T cell responses with cyclosporin A and neutralizing fas ligand antibody. (a) IFN-γ production by Csp1-deficient CD4+ T cells was measured in the presence of increasing concentrations of cyclosporin A. (b) Proliferation of CD4+ T cells after activation with anti-CD3/CD28 for 48 h and measured by [3H]-thymidine incorporation in WT cells or WT cells with low dose cyclosporin A (2.5 nM) or neutralizing Fas ligand antibody (αFasL Ig). (c) Comparison of IFN-γ production by CD4+ T cells after activation. (d) KO T cell viability was assessed by cellular ATP content during primary activation. (e) FACS analysis of Fas ligand expression on KO (pink and blue) and WT (green) T cells during primary stimulation in the presence of 2.5 nM cyclosporin A. Histograms are overlaid on data from KO T cells stained with an IgG control antibody (yellow). (PDF 28 kb)

Supplementary Fig. 6.

Analysis of Csp1 mutant mice. Southern blot analysis of BgIII-digested genomic DNA from DSCR1 (Csp1) heterozygote crosses. (PDF 12 kb)

Supplementary Fig. 7.

Characterization of sp1 monoclonal antibody. (a) Immunoblot of GST-Csp1, 2 and 3 fusion proteins with anti-Csp1 mouse monoclonal antibody. (b) Anti-GST immunoblot of GST-Csp1, 2, and 3 fusion proteins. (PDF 19 kb)

Supplementary Table 1 (PDF 25 kb)

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Ryeom, S., Greenwald, R., Sharpe, A. et al. The threshold pattern of calcineurin-dependent gene expression is altered by loss of the endogenous inhibitor calcipressin. Nat Immunol 4, 874–881 (2003). https://doi.org/10.1038/ni966

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