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. 2011 Apr 29;286(17):14795-803.
doi: 10.1074/jbc.M111.220582. Epub 2011 Feb 16.

Selective activation of the transcription factor NFAT1 by calcium microdomains near Ca2+ release-activated Ca2+ (CRAC) channels

Pulak Kar  1 , Charmaine Nelson, Anant B Parekh
Affiliations

Selective activation of the transcription factor NFAT1 by calcium microdomains near Ca2+ release-activated Ca2+ (CRAC) channels

Pulak Kar et al. J Biol Chem. .

Abstract

NFATs are a family of Ca(2+)-dependent transcription factors that play a central role in the morphogenesis, development, and physiological activities of numerous distinct cell types and organ systems. Here, we visualize NFAT1 movement in and out of the nucleus in response to transient activation of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in nonexcitable cells. We show that NFAT migration is exquisitely sensitive to Ca(2+) microdomains near open CRAC channels. Another Ca(2+)-permeable ion channel (TRPC3) was ineffective in driving NFAT1 to the nucleus. NFAT1 movement is temporally dissociated from the time course of the Ca(2+) signal and remains within the nucleus for 10 times longer than the duration of the trigger Ca(2+) signal. Kinetic analyses of each step linking CRAC channel activation to NFAT1 nuclear residency reveals that the rate-limiting step is transcription factor exit from the nucleus. The slow deactivation of NFAT provides a mechanism whereby Ca(2+)-dependent responses can be sustained despite the termination of the initial Ca(2+) signal and helps explain how gene expression in nonexcitable cells can continue after the primary stimulus has been removed.

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Figures

FIGURE 1.
FIGURE 1.
NFAT1 migrates to the nucleus following CRAC channel opening. A, cytoplasmic Ca2+ signals to thapsigargin in the presence (n = 18 cells) and absence (n = 21 cells) of external Ca2+. B, NFAT1 movement following store depletion (2 μm thapsigargin) in Ca2+-free solution. Rest denotes nonstimulated condition. Times indicate exposure to thapsigargin (Thap). C, NFAT1 movement to thapsigargin stimulation in the presence of 2 mm external Ca2+. D, NFAT1 movement co-localizes with DAPI staining of the nucleus. E, nuclear movement of NFAT1 in response to thapsigargin is suppressed by a CRAC channel blocker (Synta compound, upper panel), cyclosporin A (Cyclo. A, middle panel), and antimycin A/oligomycin (A/O, lower panel). F, aggregate data from several cells for each condition is compared (2Ca2+ = 4 cells, 0Ca2+ = 5 cells, Synta, cyclosporin A, and A/O, antimycin A and oligomycin = 4, 3, and 5 cells, respectively). The y axis represents the nuclear/cytosolic ratio of NFAT1-GFP, measured initially in nonstimulated cells and then after 40 min treatment with thapsigargin in external Ca2+ solution.
FIGURE 2.
FIGURE 2.
Time course of NFAT1 movement following CRAC channel opening. A, time course of the Ca2+ signal following readmission of external Ca2+ to cells with depleted stores is shown (average of 17 cells). B, NFAT1 movement into the nucleus is shown following Ca2+ readmission for different times. C, time course of NFAT1-GFP movement (average of five cells) is plotted against Ca2+ readmission time. The y axis shows the change in nuclear/cytoplasmic (N/C) ratio from the resting state. D, protocol evoking a pulse of Ca2+ entry is shown (average of 18 cells). E, NFAT1 movement is shown before and after Ca2+ removal. F, histogram summarizes aggregate data for the different conditions shown (four cells for each bar). Thap., thapsigargin.
FIGURE 3.
FIGURE 3.
Local Ca2+ signals drive NFAT1 migration. A, Ca2+ signals following thapsigargin (Thap.) stimulation in different external Ca2+ concentrations (n = 18 for 2 Ca2+, n = 23 for 0.5 Ca2+, and n = 16 for 0.25 Ca2+). B, NFAT1 movement in response to stimulation with thapsigargin in 0.25 mm (upper panel), 0.5 mm (middle), and 2 mm Ca2+ (lower panel). C, aggregate data for each condition is summarized (n is between 4 and 7 cells for each point). NFAT ratio is shown for 40 min stimulation. D, Ca2+ signals are compared between control cells (n = 17) and those preloaded with EGTA-AM (n = 19). E, NFAT1 movement in the presence of cytoplasmic EGTA. F, time course of NFAT1 movement is compared for control cells and those loaded with EGTA (n = 7 for each condition). N/C, nuclear/cytoplasmic ratio.
FIGURE 4.
FIGURE 4.
NFAT1 movement is tightly coupled to CRAC channels. A, Ca2+ signals evoked by CRAC channels and TRPC3 channels (activated by OAG) are compared. B, images show the lack of effect of OAG on NFAT1 movement. C, aggregate data are shown comparing the basal nuclear/cytoplasmic (N/C) NFAT ratio with that seen after 40 min of stimulation with OAG (n = 16 cells). D, images show the effect of overexpression of STIM1 and Orai1 (S1/O1) on NFAT movement. E, time course of NFAT movement is compared between control cells (with endogenous CRAC channel activity) and cells transfected with STIM1 and Orai1 (n = 6–9 cells for each condition). F, histogram compares the rate of rise of the Ca2+ signal, obtained upon readmission of external Ca2+ to cells with depleted stores, for the two conditions shown (n = 16 for control and n = 11 for S1/O1). G, inhibition of Ca2+ extrusion with 1 mm La3+ increases the size and prolongs the time course of the Ca2+ rise to thapsigargin (Thap) in Ca2+-free external solution (n = 21 for 0Ca2+, n = 20 for 0Ca2+ + La3+, n = 21 for 2 Ca2+). In these experiments, we used the low affinity dye Fura-5F because Fura-2 would have been saturated by the larger Ca2+ signal. H, images show NFAT1 movement following stimulation with thapsigargin in Ca2+-free solution supplemented with La3+. I, aggregate data are compared for the conditions shown (n = 4 cells).
FIGURE 5.
FIGURE 5.
Quantification of the kinetics of NFAT1 activation and movement into and out of the nucleus. A, representative gel showing NFAT1 dephosphorylation (measured from the gel shift, as shown by the two arrows) following stimulation with thapsigargin. B, aggregate data from two independent gels is summarized. C, images show movement of NFAT out of nucleus after termination of Ca2+ entry together with block of calcineurin (0Ca2+ + cyclosporin A (CsA)). Cells were stimulated with thapsigargin (Thap) for 20 min in the presence of external Ca2+ before Ca2+ entry was terminated. D, graph plots the migration of NFAT1 out of the nucleus as a function of time in Ca2+-free solution (n = 5 cells). The upper dashed line shows the nuclear NFAT1-GFP fluorescence after 20 min stimulation with thapsigargin, and the lower dashed line indicates the absolute level of NFAT1-GFP fluorescence in the cytosol prior to stimulation (rest). The absolute levels of NFAT1-GFP (gray scale) were measured and not the ratio, because NFAT1 flux out of the nucleus was being evaluated, and error bars have been omitted for clarity. At 20 and 40 min for example, the absolute values were 161.5 ± 16.1 and 143.0 ± 11.7 (E). Schematic summarizes the kinetics (half-time (t½) of the various steps that regulate NFAT1 movement into and out of the nucleus.
FIGURE 6.
FIGURE 6.
Local Ca2+ entry evoked by an agonist drives NFAT1 movement. A, stimulation of Fura-5F-loaded cells with 120 nm LTC4 in the presence of external Ca2+ (black trace) or absence of external Ca2+ with 1 mm La3+ evokes Ca2+ oscillations of similar amplitude and frequency. B, images show distribution of NFAT1-GFP (left panels) and DAPI (right panels) for rest (nonstimulated), 0Ca2+ + La3+ (exposed to LTC4 for 10 min), and 2Ca2+ (LTC4 was applied for 10 min). C, aggregate data from several cells is shown. N/C, nuclear/cytoplasmic ratio. D, EGFP gene expression is compared between nonstimulated (control) cells and those exposed to 120 nm LTC4 (40 min). E, histogram compares the number of EGFP-positive cells for the different conditions. Data are the aggregate from six fields of view from three coverslips each.
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References

    1. Berridge M. J., Bootman M. D., Roderick H. L. (2003) Nat. Rev. Mol. Cell Biol. 4, 517–529 - PubMed
    1. Neher E. (1998) Neuron 20, 389–399 - PubMed
    1. Parekh A. B. (2008) J. Physiol. 586, 3043–3054 - PMC - PubMed
    1. Rizzuto R., Pozzan T. (2006) Physiol. Rev. 86, 369–408 - PubMed
    1. Dolmetsch R. E., Pajvani U., Fife K., Spotts J. M., Greenberg M. E. (2001) Science 294, 333–339 - PubMed

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