CREB Is Indispensable to KIT Function in Human Skin Mast Cells-A Positive Feedback Loop between CREB and KIT Orchestrates Skin Mast Cell Fate

Abstract

Skin mast cells (MCs) are critical effector cells in acute allergic reactions, and they contribute to chronic dermatoses like urticaria and atopic and contact dermatitis. KIT represents the cells' crucial receptor tyrosine kinase, which orchestrates proliferation, survival, and functional programs throughout the lifespan. cAMP response element binding protein (CREB), an evolutionarily well-conserved transcription factor (TF), regulates multiple cellular programs, but its function in MCs is poorly understood. We recently reported that CREB is an effector of the SCF (Stem Cell Factor)/KIT axis. Here, we ask whether CREB may also act upstream of KIT to orchestrate its functioning. Primary human MCs were isolated from skin and cultured in SCF+IL-4 (Interleukin-4). Pharmacological inhibition (666-15) and RNA interference served to manipulate CREB function. We studied KIT expression using flow cytometry and RT-qPCR, KIT-mediated signaling using immunoblotting, and cell survival using scatterplot and caspase-3 activity. The proliferation and cycle phases were quantified following BrdU incorporation. Transient CREB perturbation resulted in reduced KIT expression. Conversely, microphthalmia transcription factor (MITF) was unnecessary for KIT maintenance. KIT attenuation secondary to CREB was associated with heavily impaired KIT functional outputs, like anti-apoptosis and cell cycle progression. Likewise, KIT-elicited phosphorylation of ERK1/2 (Extracellular Signal-Regulated Kinase 1/2), AKT, and STAT5 (Signal Transducer and Activator of Transcription) was substantially diminished upon CREB inhibition. Surprisingly, the longer-term interference of CREB led to complete cell elimination, in a way surpassing KIT inhibition. Collectively, we reveal CREB as non-redundant in MCs, with its absence being incompatible with skin MCs' existence. Since SCF/KIT regulates CREB activity and, vice versa, CREB is required for KIT function, a positive feedforward loop between these elements dictates skin MCs' fate.

Keywords: CREB; KIT; SCF; apoptosis; cell cycle; mast cells; proliferation; skin; survival; transcription factors.

Figure 1

KIT surface expression requires intact CREB activity. MCs were kept in the presence of CREBi or DMSO (ctrl) for the indicated periods, either without or with the addition of SCF. (a–d) Cells were harvested and incubated with an anti-CD117 antibody for the detection of KIT on the cell surface. The right panels represent the flow cytometry analysis, showing the distribution of KIT in the cell population. The left panels show the quantification of the mean fluorescence intensity relative to the vehicle control, set as 100%, (a) at 48 h, (b) at 48 h in the presence of SCF, (c) at 24 h, and (d) at 24 h in the presence of SCF. Shown are the means ± SEM of 4–6 independent experiments. (e) Top and bottom: cells were harvested and processed for RT-qPCR. The relative expression of KIT mRNA was normalized to that of the control. The mean ± SEM of 4–7 independent experiments are shown. * p < 0.05, ** p < 0.01; ns: not significant.

Figure 2

RNA interference with CREB reduces cell surface expression of KIT, while MITF has no significant effect. (a–c) MCs were transfected with either a control siRNA (ctrl: black) or an siRNA-targeting CREB (si-CREB: dark blue). Cells were kept in the presence of SCF during transfection to ensure survival under the minimal medium NOT AMOUNT! Minimal medium means it contains no serum, or growth factors (Accell) required for the procedure. Cells were harvested 48 h after the initial transfection. (a,c) RT-qPCR of CREB and KIT mRNAs, respectively. Shown are the means ± SEM of five independent experiments (ns: not significant). (b) The right panels are the representative flow cytometry analysis showing the distribution of KIT in the cell population (blue: control siRNA; red: siCREB). The left panels are the quantification of the mean fluorescence intensity relative to the control, set at 100%. Shown are the means ± SEM of five independent experiments. (d–f) Skin MCs were transiently transfected with either control siRNA (black: ctrl) or MITF-selective siRNA (red: si-MITF) as in (a–c). Cells were harvested after 72 h (due to the longer half-life of MITF). RT-qPCR and flow cytometry were performed as above. (d–f) are analogous to (a–c). Shown are the means ± SEM of 4–6 independent experiments. ** p < 0.01, *** p < 0.001, with one sample t-test; ns: not significant.

Figure 3

CREB is required for SCF-induced signaling through ERK, AKT, and STAT5. MCs were pretreated with CREBi (blue) or DMSO (green) for 1 day, washed extensively, and stimulated with SCF for the indicated periods in minutes. Cells were harvested and subjected to Western blotting analysis using antibodies against phospho-ERK1/2, phospho-AKT, and phospho-STAT5. An antibody against Cyclophilin B (Cycl B) was included as a loading control and was used for normalization. (a–c) Relative quantifications of the Western blot signals. Shown are the means ± SEM of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (d) Representative Western blot experiment, using the indicated antibodies.

Figure 4

The reduction in KIT following CREB inhibition is required for the induction of apoptosis. (a) MCs were deprived of SCF (ctr, black) or kept in the presence of SCF (yellow) for two days. Cells were harvested and the enzymatic activity of caspase-3 was measured. Shown are relative units that are the means of 4 independent experiments ± SEM. (b) MC cells were pretreated with either CREBi (pre-CREBi) or DMSO (pre-vehicle) for 1 day, washed extensively, and incubated either in the absence or the presence of SCF for two days, after which the caspase-3 activity was determined. Shown are relative units that are the means of at least 4 independent experiments ± SEM. (c) The 24 h supernatants of CREBi (SN_CREBi)- and vehicle (SN_vehicle)-treated cells (in the presence of SCF) were applied to MCs for two days, after which caspase-3 activity was measured. ctrl: control cells (left untreated). Shown are relative units that are the means ± SEM of 4 independent experiments. * p < 0.05. ns: not significant.

Figure 5

Reduced SCF-triggered proliferation as a result of impaired KIT expression due to CREB inhibition. MCs were treated with either CREBi (pre-CREBi) or DMSO (ctrl) for 24 h, washed extensively, and incubated in the presence of 100 ng/mL SCF and BrdU for 5 days. After application of the anti-BrdU antibody, total DNA was labeled with the 7-AAD dye and cells were analyzed using flow cytometry. (a) Effect of CREBi on the proportions of S-phase, G0/G1, and apoptotic cells. (b) Representative scatter plots showing the different cell cycle stages based on BrdU fluorescence against the total amount of DNA, either in the vehicle control (left panel) or in the presence of CREBi (right panel). (c) Representative histograms, displaying the extent of BrdU incorporation in the cell population upon CREBi treatment (right panel) or in the vehicle control (left panel). n = 5; * p < 0.05, ** p < 0.01.

Figure 6

CREBi eliminates skin MCs. MC cells were treated with either CREBi, KITi, PI3Ki or DMSO (ctrl), as indicated, and incubated in the presence of 100 ng/mL SCF and BrdU for a total of 5 days, as detailed in the Methods. Cells were harvested and analyzed using flow cytometry for the incorporation of BrdU and DNA quantity as shown in Figure 5. (a) Representative FSC/SSC scatter plots in the presence of the indicated inhibitors. (b) Representative scatter plots showing BrdU fluorescence against the total amount of DNA. (c) Representative histograms displaying the extent of BrdU incorporation into the cell population upon treatment with the inhibitors. (d,e) Effect of the indicated inhibitors on the proportions of identifiable cells and on the proportions of BrdU positive cells, respectively. n = 5; * p < 0.05, ** p < 0.003, **** p < 0.0001.