Cysteinyl leukotriene type I receptor desensitization sustains Ca2+-dependent gene expression

Abstract

Receptor desensitization is a universal mechanism to turn off a biological response; in this process, the ability of a physiological trigger to activate a cell is lost despite the continued presence of the stimulus. Receptor desensitization of G-protein-coupled receptors involves uncoupling of the receptor from its G-protein or second-messenger pathway followed by receptor internalization. G-protein-coupled cysteinyl leukotriene type I (CysLT1) receptors regulate immune-cell function and CysLT1 receptors are an established therapeutic target for allergies, including asthma. Desensitization of CysLT1 receptors arises predominantly from protein-kinase-C-dependent phosphorylation of three serine residues in the receptor carboxy terminus. Physiological concentrations of the receptor agonist leukotriene C(4) (LTC(4)) evoke repetitive cytoplasmic Ca(2+) oscillations, reflecting regenerative Ca(2+) release from stores, which is sustained by Ca(2+) entry through store-operated calcium-release-activated calcium (CRAC) channels. CRAC channels are tightly linked to expression of the transcription factor c-fos, a regulator of numerous genes important to cell growth and development. Here we show that abolishing leukotriene receptor desensitization suppresses agonist-driven gene expression in a rat cell line. Mechanistically, stimulation of non-desensitizing receptors evoked prolonged inositol-trisphosphate-mediated Ca(2+) release, which led to accelerated Ca(2+)-dependent slow inactivation of CRAC channels and a subsequent loss of excitation-transcription coupling. Hence, rather than serving to turn off a biological response, reversible desensitization of a Ca(2+) mobilizing receptor acts as an 'on' switch, sustaining long-term signalling in the immune system.

Figure 1. CysLT1 receptor-dependent c-fos expression requires PKC

a, Averaged Ca²⁺ signals to LTC₄ and thapsigargin are compared (>50 cells per graph). b, c-fos expression is compared between control (non-stimulated), 160 nM LTC₄- and 2 μM thapsigargin-stimulated cells. Stimulus was present for 8 minutes. c, Histograms show averaged responses from 3 independent experiments. LTC₄ and thapsigargin groups were different from control (p<0.001), but not from one another (p>0.3; Anova). d, Ca²⁺ entry rate was measured following readmission of Ca²⁺ to cells stimulated with LTC₄ or thapsigagin in Ca²⁺-free solution (* denotes p<0.01). e, Cells stained with antibody against c-fos protein. f, Aggregate data are compared (n>20 per bar). Thapsigargin and LTC₄ groups were different from control (p<0.001) but not from one another (p=0.11). g, G06983 (1 μM; 10 min pre-treatment) suppresses LTC₄-induced c-fos expression. h, Histogram comparing the effects of PKC blockers. LTC₄ control group (LTC₄ in absence of PKC block) was different from the other groups (p<0.01). There were no significant differences between the other groups. G0 is G06983. i, Single-cell Ca²⁺ signals to LTC₄ are compared for the conditions shown. j Averaged data is compared (> 45 cells for each condition). k, Histogram showing c-fos expression to thapsigargin in presence of PKC blockers. All thapsigargin-treated groups were significantly different from control (p<0.001) but were not significantly different from one another. l, Ca²⁺ signals to thapsigargin are unaffected by PKC block. m, Downregulation of PKC (PMA; 500 nM, 24 hours) reduces LTC₄- but not thapsigargin-induced c-fos expression (data from 4 independent experiments). All stimulated groups were significantly different from control (p<0.01). For LTC₄ the PMA group was different from the LTC₄ control (p<0.01). For thapsigargin, the PMA groups was not different from the thapsigargin control (p=0.07). n, PKC downregulation alters the LTC₄-evoked Ca²⁺ signal.

Figure 2. Gene expression to non-desensitizing CysLT1 receptors is rescued by preventing a cytoplasmic Ca²⁺ rise

a, Stimulation with LTC₄ in the presence of G06983 evokes a more sustained Ca²⁺ release response, and this leads to more extensive store depletion (measured through the extent of Ca²⁺ release evoked by 5 μM ionomycin). Both LTC₄ and ionomycin were applied in Ca²⁺-free external solution. Inset compares the kinetics of Ca²⁺ release. b, Cytosolic GFP-PH levels, a measure of InsP₃ levels, rise when CysLT1 receptors are stimulated in the presence of G06983. c, Upper panel, loading cells with the Ca²⁺ chelator EGTA prevents loss of gene expression to agonist when PKC is blocked. Lower panel, aggregate data from five independent gels are summarised. d as in c, but calphostin C was used to block PKC instead. Lower panel, aggregate data from three independent gels are summarised.

Figure 3. Ca²⁺-dependent slow inactivation underlies suppression of c-fos expression to non-desensitizing CysLT1 receptors

a, Ca²⁺-dependent fast inactivation is unaffected by non-desensitizing receptors (labelled –PKC). Cells were stimulated with LTC₄ (160 nM) prior to breaking in with a pipette solution containing thapsigargin and buffered Ca²⁺ (140 nM) and fast inactivation was measured within 60 seconds of break-in. b, dependence of Ca²⁺-dependent slow inactivation on patch pipette Ca²⁺ concentration. c, Stimulation of non-desensitizing receptors with LTC₄ prior to break-in significantly reduced the size of I_CRAC that developed in response to dialysis with thapsigargin in weak buffer (0.2 mM EGTA). d, as c, but cells were dialysed with a pipette solution containing strong Ca²⁺ buffer (10 mM EGTA, 140 nM free Ca²⁺). e, Store-operated Ca²⁺ entry recovers partially by increasing the time interval between Ca²⁺ release and subsequent Ca²⁺ entry. f, c-fos expression to non-desensitizing receptor stimulation is rescued partially when Ca²⁺ entry occurs several minutes after Ca²⁺ release has reached completion. g, c-fos expression in human nasal mast cells after CysLT1 receptor activation is suppressed by PKC inhibition. h, Aggregate data is compared (12-17 cells per bar; 3 patients each).

Figure 4

PKCα regulates CysLT1 receptor-driven c-fos transcription. a, Expression of PKCα, β and ζ (western blot) is shown in control cells and cells exposed to PMA for 24 hours. b, Quantification of data from 3 independent experiments as in a. c, Confocal microscopic images of PKC expression for the conditions shown. Cells were fixed before analysis. d, Quantification of images from experiments as in c. e, siRNA against PKCα or β significantly reduces corresponding protein expression. Left panel: DAPI staining of nuclei (left), GFP expression (indicating transfection; middle) and PKCα expression (right) after siRNA-mediated knockdown. Right panel, aggregate data from 4 experiments are depicted. Both siRNA groups were different from control (p<0.005). f, knockdown of PKCα, β and α + β on LTC₄-dependent c-fos expression. Data are compared with mock-transfected cells. For comparison, 24 hour exposure to PMA is included. All treated groups were significantly different from the LTC₄ control (black bar) group except siRNA β knockdown (p>0.1). α+β and 24 h PMA groups had p<0.01; α group had p<0.05.