CREB Is Activated by the SCF/KIT Axis in a Partially ERK-Dependent Manner and Orchestrates Survival and the Induction of Immediate Early Genes in Human Skin Mast Cells

Abstract

cAMP response element binding protein (CREB) functions as a prototypical stimulus-inducible transcription factor (TF) that initiates multiple cellular changes in response to activation. Despite pronounced expression in mast cells (MCs), CREB function is surprisingly ill-defined in the lineage. Skin MCs (skMCs) are critical effector cells in acute allergic and pseudo-allergic settings, and they contribute to various chronic dermatoses such as urticaria, atopic dermatitis, allergic contact dermatitis, psoriasis, prurigo, rosacea and others. Using MCs of skin origin, we demonstrate herein that CREB is rapidly phosphorylated on serine-133 upon SCF-mediated KIT dimerization. Phosphorylation initiated by the SCF/KIT axis required intrinsic KIT kinase activity and partially depended on ERK1/2, but not on other kinases such as p38, JNK, PI3K or PKA. CREB was constitutively nuclear, where phosphorylation occurred. Interestingly, ERK did not translocate to the nucleus upon SCF activation of skMCs, but a fraction was present in the nucleus at baseline, and phosphorylation was prompted in the cytoplasm and nucleus in situ. CREB was required for SCF-facilitated survival, as demonstrated with the CREB-selective inhibitor 666-15. Knock-down of CREB by RNA interference duplicated CREB's anti-apoptotic function. On comparison with other modules (PI3K, p38 and MEK/ERK), CREB was equal or more potent at survival promotion. SCF efficiently induces immediate early genes (IEGs) in skMCs (FOS, JUNB and NR4A2). We now demonstrate that CREB is an essential partaker in this induction. Collectively, the ancient TF CREB is a crucial component of skMCs, where it operates as an effector of the SCF/KIT axis, orchestrating IEG induction and lifespan.

Keywords: CREB; ERK1/2; KIT; SCF; apoptosis; mast cells; skin; survival.

Figure 1

CREB is phosphorylated upon SCF stimulation in a KIT kinase-dependent manner. (a) Skin-derived MCs were stimulated with SCF for the indicated times and Ser-133 phosphorylation of CREB was detected by immunoblot in whole-cell lysates. Upper panel: Image J based semi-quantification of the detected signal for pCREB normalized to the housekeeping protein Cyclophilin B (CycloB). Lower panel: Representative time-course. (b) Cells were pretreated with imatinib mesylate (KIT inh.) prior to stimulation with SCF for 15 min. Upper and lower panel as in (a). Each dot corresponds to one experiment (individual MC culture). * p < 0.05, ** p < 0.01.

Figure 2

SCF-elicited CREB phosphorylation occurs in an ERK1/2-dependent manner. (a) SkMCs were pretreated with inhibitors of ERK1/2 and PI3K and stimulated with SCF for 15 min; phosphorylation of CREB was detected by immunoblot in whole-cell lysates. Upper panel: Image J based semi-quantification of the detected signal for pCREB normalized to the housekeeping protein Cyclophilin B (CycloB). Lower panel: Representative time-course. (b) As in (a), but inhibitors of p38 and JNK were used instead. Each dot corresponds to one experiment (individual MC culture). ** p < 0.01, **** p < 0.0001.

Figure 3

CREB is constitutively located in the nucleus where phosphorylation occurs. MCs were treated with SCF for the times given, cytosolic and nuclear fractions were prepared separately and phosphorylation events were detected by immunoblot. Upper panel: CREB phosphorylated on Ser-133. Center: STAT5 phosphorylated on Tyr-694 given for comparison. Lower panel: β-actin (loading control). One of three experiments with comparable outcomes is shown. For total versus phosphorylated STAT5 and CREB, please refer to Figure S4.

Figure 4

ERK1/2 is constitutively present in the cytoplasm and nucleus; activation-dependent phosphorylation is not associated with translocation. MCs were treated with SCF for the times given, cytosolic and nuclear fractions were prepared separately, and phospho-ERK1/2 (pERK1/2), as well as total ERK1/2 (tERK1/2), were detected by immunoblot. β-actin served as the loading control. One of three experiments with identical outcomes is shown. The same membrane as in Figure 3 is shown.

Figure 5

CREB function is required for skMC survival—comparison with other signaling modules. (a) SkMCs were treated with different concentrations of the CREB inhibitor for 2 d. Cell numbers were determined and are expressed as % of the original numbers plated. (b) As in (a), but inhibitors of critical kinases (PI3K, p38 and ERK1/2) were used for comparison. (c,d) SkMCs were treated with the specified inhibitors for 2 d and viable versus non-viable/apoptotic cells were determined with the YoPro/PI technique. To one portion, fresh SCF was provided directly upon inhibitor pretreatment, the other part had SCF from the previous feeding only (3–4 d earlier). (c) Cumulative results. (d) Representative dot plots: upper panel with re-addition of fresh SCF, lower panel without. Each dot in (a–c) corresponds to an individual experiment. * p < 0.05 by the Kruskal–Wallis test (b) or by the Friedman test (c).

Figure 6

CREB is critically implicated in skMC survival—validation by RNA interference. (a) SkMCs were treated with control and selective siRNAs for 3 d. Cell numbers were determined and are expressed as % of the original numbers plated. (b,c) SkMCs were treated with control and selective siRNAs for 2 d (in the presence of SCF), then replated and kept for 2 more days with or without re-addition of fresh SCF. Viable versus non-viable/apoptotic cells were determined with the YoPro/PI technique. (b) Cumulative results of the two experiments run under two conditions each. (c) Dot plots of the two independent experiments. ** p < 0.01.

Figure 7

CREB is a critical intermediary in SCF-triggered induction of immediate-early genes but not MCL1. (a) Skin-derived MCs were pretreated with(out) the CREB inhibitor 666-15, then stimulated (or not) with SCF for 25 min. RT-qPCR was used to quantitate gene expression (normalized to several housekeeping genes). The fold-induction by SCF over control is depicted. Means ± SEM of eight experiments (separate cultures) for IEGs and four experiments for MCL1 are given. ** p < 0.01. (b) Skin-derived MCs were treated with CREB-selective versus control siRNA for 48 h, then stimulated as in (a) for immediate-early gene quantification. The knockdown efficiency is given on the left-hand side, which was determined in unstimulated cells.