Blunted IgE-mediated activation of mast cells in mice lacking the serum- and glucocorticoid-inducible kinase SGK3

Abstract

Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 (sgk3^−/−) and their wild-type littermates (sgk3^+/+). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular Ca²⁺ and entry of extracellular Ca²⁺. Influx of extracellular Ca²⁺ but not Ca²⁺ release from intracellular stores was significantly blunted in sgk3^−/− BMMCs compared with sgk3^+/+ BMMCs. Antigen stimulation further led to a rapid increase of a K⁺-selective conductance in sgk3^+/+ BMMCs, an effect again blunted in sgk3^−/− BMMCs. In contrast, the Ca²⁺ ionophore ionomycin activated K⁺ currents to a similar extent in sgk3^−/− and in sgk3^+/+ BMMCs. β-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in sgk3^−/− BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in sgk3^−/− compared with sgk3^+/+ mice. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk3^−/− than in sgk3^+/+ mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function.

mast cells play a decisive role in IgE-dependent allergic reactions (45), including allergic rhinitis (65), asthma (18), anaphylactic shock, and delayed hypersensitivity reactions (5, 35, 78). Through cytokine release they regulate the function of other inflammatory cells, such as neutrophils and T cells (9, 35, 36, 59).

Mast cell function is governed by the activity of Ca²⁺ channels (20, 22, 25, 28, 29, 44, 57, 72), K⁺ channels (19, 20, 27, 55), and Cl⁻ channels (28, 29).

Mast cell ion channel activity (49) and function (2, 4, 38, 81) are dependent on phosphoinositol-3 (PI3) kinase. PI3 kinase-dependent signaling involves 3-phosphoinositide (PIP3)-dependent kinase PDK1, which phosphorylates and thus activates the protein kinase B (PKB) and serum- and glucocorticoid- inducible kinase (SGK) isoforms (1, 10, 47, 64). Recently, SGK1 has been demonstrated to play a critical role in mast cell activation (71).

The serum- and glucocorticoid-inducible kinase isoform SGK3 has originally been identified as a gene closely related to the serum- and glucocorticoid-inducible kinase SGK1 (48). SGK3 shares 80% of the amino acid sequence identity in its catalytic domain with SGK1 (48). SGK3 is identical to the cytokine-independent survival kinase CISK (52). The “SGK-like” gene (SGKL) in chromosome 8q12.3 (24) encodes a protein whose predicted amino acid sequence is virtually identical to that of human SGK3. SGK3 has been shown to be expressed in all tissues tested thus far (48). Unlike SGK1 (33, 50), SGK3 is not under transcriptional control of serum and glucocorticoids (50). While SGK1 is upregulated by cell stress and a wide variety of hormones and mediators, it is particularly important during stress conditions. SGK3, on the other hand, is constitutively expressed and serves basal functions (50). For instance, radiation leads to marked upregulation of SGK1 transcription (70) but decreases SGK3 transcript levels (76).

Neither knockout of SGK3 alone (58) nor of both SGK1 and SGK3 (39) leads to a severe phenotype, even though SGK1 (50, 70) and SGK3 (50, 79, 82) participate in the maintenance of cell survival. SGK3 further participates in the regulation of a variety of carriers and ion channels (50). Gene-targeted mice lacking functional SGK3 have a strikingly delayed hair growth (23, 56, 58, 60), which has been attributed to apoptosis of keratinocytes (3). Interestingly, SGK3 and Akt2 appear to have partially redundant roles in that the hair loss phenotype of mice deficient in both is markedly augmented than in mice lacking either one (56). Moreover, SGK3-deficient mice show decreased basal intestinal glucose transport (66) and a subtle decrease of locomotion (50).

In the present study, experiments were performed on gene-targeted mice lacking SGK3 (sgk3^−/−) and their wild-type littermates (sgk3^+/+) to elucidate whether mast cell ion channel regulation and degranulation involves SGK3.

MATERIALS AND METHODS

Mice.

All animal experiments were conducted according to the German law for the welfare of animals and were approved by local authorities.

The targeting strategy for disruption of the Sgk3 gene has been described earlier (58). To generate mice homozygous for the targeted allele, the resulting heterozygote (sgk3^+/−) males and females were interbred to yield SGK3-deficient mice (sgk3^−/−) and their wild-type littermates (sgk3^+/+).

Culture of bone marrow-derived mast cells.

Mast cells were isolated from femoral bone marrow of 6- to 8-wk-old naive sgk3^+/+ and sgk3^−/− mice and cultured for 4 wk in RPMI 1640 (Invitrogen Life Technologies) containing 10% FCS, 1% penicillin-streptomycin, 20 ng/ml IL-3 (R&D Systems), and 100 ng/ml of the c-kit ligand stem cell factor (PeproTech). Bone marrow mast cell (BMMC) maturation was confirmed by flow cytometry (FACSCalibur; BD Biosciences) using the following specific fluorescent-labeled Abs: PE-labeled anti-FcεRI (eBioscience), allophycocyanin-labeled anti-CD117 (BD Pharmingen), and FITC-labeled anti-CD34 (BD Pharmingen). Cells were kept in culture 4–6 wk before the experiments. For experiments, BMMCs were sensitized for 1 h with monoclonal mouse anti- dinitrophenyl (DNP) mouse IgE (anti-DNP IgE, 5–10 μg/ml per 1 × 10⁶ cells, clone SPE-7; Sigma-Aldrich) in culture medium and challenged with DNP-human serum albumin (DNP-HSA; 50 ng/ml; Sigma-Aldrich).

Determination of mast cell numbers in the ear conches.

Anesthetized mice were euthanized by cervical dislocation, and the skin was cleansed with 70% ethanol. Ear conches were cut off at the base, fixed in 4% paraformaldehyde overnight, and finally embedded in paraffin. Tissue sections (4-μm thick) taken from the middle of the conches were prepared, deparaffinized, and stained with toluidine blue. Mast cell numbers of 10 different areas on different slices per conch of 4 sgk3^+/+ and 3 sgk3^−/− mice were determined using a Zeiss Axiovert 200 microscope with a LD Achroplan ×20 lens in brightfield mode.

Patch clamp.

Patch-clamp experiments were performed at room temperature in voltage-clamp, fast-whole cell mode (41). BMMCs were continuously superfused by a flow system inserted into the dish. The bath was grounded via a bridge filled with NaCl-Ringer solution containing (in mM) 145 NaCl, 5 KCl, 2 MgCl₂, 1 CaCl₂, 10 glucose, and 10 HEPES/NaOH (pH 7.4, 300 mosM). Borosilicate glass pipettes (2–4 MΩ tip resistance; GC 150 TF-10, Harvard Apparatus, March-Hugstetten, Germany) manufactured by a microprocessor-driven DMZ puller (Zeitz, Augsburg, Germany) were used in combination with a MS314 electrical micromanipulator (MW, Märzhäuser, Wetzlar, Germany). The currents were recorded by an EPC-9 amplifier (HEKA, Lambrecht, Germany) using Pulse software (HEKA) and an ITC-16 Interface (Instrutech, Port Washington, NY). Whole cell currents were determined as 10 successive 200-ms square pulses from a −35 mV holding potential to potentials between −115 and +65 mV. The currents were recorded with an acquisition frequency of 10 and 3 kHz low-pass filtered.

The pipette solution contained (in mM) 140 K-gluconate, 5 KCl, 1.2 MgCl₂, 2 EGTA, 1.26 CaCl₂ (pCa 7), 2 Na₂ATP, and 10 HEPES/KOH (pH 7.2, 280 mosM) and was used in combination with NaCl-Ringer bath solution. Where indicated the antigen DNP-HSA, (50 ng/ml, Sigma-Aldrich, Germany) and the Ca²⁺ ionophore ionomycin (1 μM, Sigma-Aldrich) were added to the bath solution.

The offset potentials between both electrodes were zeroed before sealing. The potentials were corrected for liquid junction potentials as estimated according to Barry and Lynch (8). The original whole cell current traces are depicted without further filtering, and currents of the individual voltage square pulses are superimposed. The applied voltages refer to the cytoplasmic face of the membrane with respect to the extracellular space. The inward currents, defined as flow of positive charge from the extracellular to the cytoplasmic membrane face, are negative currents and depicted as downward deflections of the original current traces.

Intracellular calcium measurements.

Intracellular Ca²⁺ measurements were performed as described (69). Briefly, BMMCs were sensitized with IgE (10 μg/ml) for 1 h at 37°C and subsequently loaded with fura-2 AM (2 μM, Molecular Probes, Goettingen, Germany) for 20 min at 37°C. Fluorescence measurements were carried out with an inverted phase-contrast microscope (Axiovert 100, Zeiss, Oberkochen, Germany). Cells were excited alternatively at 340 and 380 nm, and the light was deflected by a dichroic mirror into either the objective (Fluar ×40/1.30 oil, Zeiss, Oberkochen) or a camera. Emitted fluorescence intensity was recorded at 505 nm and data acquisition was performed by using specialized computer software (Metafluor, Universal Imaging, Downingtown). Intracellular Ca²⁺ was measured before and following addition of DNP-HSA to IgE-sensitized BMMCs in the absence or presence of extracellular Ca²⁺.

As a measure for the increase of cytosolic Ca²⁺ activity, the slope and peak of the changes in the 340/380 nm ratio were calculated for each experiment. For intracellular calibration purposes, ionomycin (10 μM) was applied at the end of each experiment. Experiments were performed with Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1.2 MgSO₄, 2 CaCl₂, 2 Na₂HPO₄, 32 HEPES, and 5 glucose, pH 7.4. To reach nominally Ca²⁺-free conditions, experiments were performed using Ca²⁺-free Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1.2 MgSO₄, 2 Na₂HPO₄, 32 HEPES, 0.5 EGTA, and 5 glucose, pH 7.4.

Measurement of degranulation.

Mature BMMCs were seeded on 96-well plates in fresh medium with anti-DNP IgE antibody (5 μg/ml) for 1 h. Afterwards cells were washed in Tyrode salt solution (Sigma-Aldrich) and challenged with DNP-HSA (50 ng/ml). Twenty microliters of supernatant and 20 μl of 2 mM 4-nitrophenyl N-acetyl-β-d-glucosaminide (Sigma-Aldrich), diluted in 0.2 M citrate buffer, pH 4.5, were added to each well of the 96-well plate, and color was developed for 2 h at 37°C. The reaction was terminated with 1 M Tris buffer, pH 9.0, and the absorbance was measured at 405 nm in an ELISA microplate reader. The data are expressed as the percentage of the total release (Triton X-100 0.1%) and are corrected for spontaneous release.

Passive systemic anaphylaxis/antigen-induced anaphylaxis and serum histamine concentrations.

Mice were sensitized with 30 μg/250 μl anti-DNP IgE by intraperitoneal application. Five hours later, mice were challenged with either DNP-HSA (100 μg/200 μl) or PBS. Body temperature was monitored before and every minute after antigen challenge with an 8-Channel USB Thermometer (Tübingen, Germany) during the midportion of the light phase of the light cycle. Mice were placed with the tail raised, and the Vaseline-covered probe was inserted a standardized distance of 2 cm until a stable temperature reading was obtained. Baseline temperature was measured after mice were habituated to rectal probe insertion. Ambient room temperature was 23°C, and the animals were exposed to a 12-h light and 12-h dark cycle (7 am to 7 pm). Data are expressed as a change in body temperature following treatment (Δ°C). Histamine levels were analyzed in the blood of male sgk3^+/+ and sgk3^−/− mice 30 min after induction of an anaphylactic reaction. Histamine was measured by ELISA according to the instructions of the manufacturer (IBL-Hamburg, Hamburg, Germany).

Statistics.

Data are provided as means ± SE; n represents the number of animals/independent experiments. All data were tested for significance using Student's unpaired two-tailed t-test or ANOVA (Dunnets test), where applicable. P < 0.05 was considered to indicate statistical significance.

RESULTS

Cells were derived from the bone marrow (BMMCs) of SGK3 knockout mice (sgk3^−/−) and their wild-type littermates (sgk3^+/+), and expression of the mast cell surface markers CD117, CD34, and FcεRI (Fig. 1A) was determined. No significant difference in the abundance of any of the three markers for mast cell maturation was observed between BMMCs of the two genotypes (Fig. 1B).

Fig. 1.

Maturation of bone marrow mast cells (BMMCs) from sgk3^+/+ and sgk3^−/− mice. A: original dot plots of CD117-, CD34-, and FcεRI-positive BMMCs from sgk3^+/+ and sgk3^−/− mice. Numbers depict the percentage of cells in the respective quadrant, acquired within the mast cell gate. B: frequency of mast cells in primary culture. Mean percent (± SE; n = 6 individual BMMC cultures) of sgk3^+/+ (open bars) and sgk3^−/− (closed bars) BMMCs acquired within the mast cell gate. C: ear conche sections of sgk3^+/+ (top) and sgk3^−/− (bottom) mice stained with toluidine blue for mast cell detection (mast cells are indicated by black arrows). D: number of mast cells (±SE) in skin, analyzed by staining of ear conche sections with toluidine blue. Mean mast cell numbers of toluidine blue-positive cells in one area (×200 magnification) as calculated from 10 different areas on different slices per conch of 4 sgk3^+/+ (open bar) and 3 sgk3^−/− (closed bar) mice (P = 0.43, two-tailed unpaired t-test).

The number of mast cells in the skin, analyzed by staining of ear conch sections with toluidine blue (Fig. 1, C and D), was similar in sgk3^+/+ and sgk3^−/− mice (P = 0.43).

Stimulation with IgE and cognate antigen was followed by a sharp increase of cytosolic Ca²⁺ in sgk3^+/+ cells, an effect significantly blunted in sgk3^−/− cells (Fig. 2, A and B). Before addition of antigen the basal Ca²⁺ level was not different between sgk3^+/+ and sgk3^−/− BMMCs (fluorescence ratio: 1.40 ± 0.07 in sgk3^+/+ vs. 1.35 ± 0.14 in sgk3^−/− BMMCs). To further assess the effect of SGK3 on Ca²⁺ mobilization, the cells were sensitized with IgE and challenged with antigen in the absence of extracellular Ca²⁺ (Fig. 2C). As a result, in the nominal absence of extracellular Ca²⁺, the exposure to IgE and antigen was followed by a transient increase in intracellular Ca²⁺, an effect not significantly different between sgk3^+/+ and sgk3^−/− cells (Fig. 2D). Thus lack of SGK3 predominantly impaired the entry of extracellular Ca²⁺.

Fig. 2.

Antigen-induced Ca²⁺ entry into BMMCs from sgk3^+/+ and sgk3^−/− mice. A: representative original tracings showing the fura-2 fluorescence ratio of 340 over 380 nm in fura-2/AM-loaded BMMCs from sgk3^+/+ and sgk3^−/− mice before and following addition of antigen (Ag, 50 ng/ml). At the end of each experiment, ionomycin (10 μM) was added for calibration. For quantification of the Ca²⁺ entry into the BMMCs, the slope (Δratio/ms) and peak (Δratio) were calculated following addition of Ag as indicated in the figure. B: means (±SE) of the slope (left) and peak (right) of the fluorescence ratio change for sgk3^+/+ (n = 9, open bars) and sgk3^−/− (n = 8, closed bars) BMMCs following stimulation with Ag (50 ng/ml). *P < 0.05 and **P < 0.01, significant difference between both groups (two-tailed unpaired t-test). C: representative original tracings showing the ratio of 340/380 nm fura-2 fluorescence in fura-2-loaded sgk3^+/+ and sgk3^−/− BMMCs before and after addition of Ag (50 ng/ml) in the absence of extracellular Ca²⁺. To reach a Ca²⁺-free environment, EGTA (0.5 mM) was added to the Ca²⁺-free bath solution. D: means (±SE) of the slope (left) and peak value (right) of the fluorescence ratio change for sgk3^+/+ (n = 5, open bars) and sgk3^−/− (n = 5, closed bars) BMMCs upon stimulation with antigen in a Ca²⁺-free solution.

BMMCs are known to express Ca²⁺-activated K⁺ channels K_Ca3.1, which are important amplifiers of Ca²⁺ entry upon IgE-antigen-dependent stimulation (69). The K⁺ currents of BMMCs upon receptor stimulation were measured in patch-clamp experiments (Fig. 3). Addition of antigen to the bath solution resulted in a rapid increase of a K⁺-selective conductance. The current amplitude was growing after antigen application and reached its maximum in about 3 min. The maximal amplitude was significantly blunted in sgk3^−/− if compared with sgk3^+/+ cells (Fig. 3). However, when the cells were stimulated with a Ca²⁺ ionophore ionomycin, no difference in measured K⁺ currents was detected between the genotypes (Fig. 3C). Accordingly, the surface expression and maximal activity of the Ca²⁺-activated K⁺ channel did not differ between sgk3^+/+ and sgk3^−/− cells; i.e., SGK3 deficiency primarily decreased the Ag-stimulated Ca²⁺ entry.

Fig. 3.

Antigen-induced K⁺ currents are reduced in sgk3^−/− BMMCs. A: representative whole cell currents from sgk3^+/+ (left) and sgk3^−/− (right) BMMCs elicited by 200-ms pulses ranging from −115 to +65 mV in 20-mV increments from a holding potential of −35 mV. Currents were recorded in standard NaCl bath solution 3 min after stimulation with either Ag (50 ng/ml, top) or ionomycin (1 μM, bottom). The dotted line indicates the zero current value. B: mean current-voltage relationships (±SE, n = 7) in sgk3^+/+ (open symbols) and sgk3^−/− (closed symbols) BMMCs before (control, squares) and 3 min after stimulation with antigen (Ag, 50 ng/ml, triangles). C: mean whole cell conductance (± SE) of sgk3^+/+ (open bars) and sgk3^−/− (closed bars) BMMCs as recorded in B before (control) and after stimulation with either Ag (50 ng/ml) or ionomycin (1 μM). Data were calculated by linear regression between −55 and +5 mV. *P < 0.05, significant difference between sgk3^+/+ and sgk3^−/− cells (ANOVA); ###P < 0.001, significant difference from sgk3^+/+ cells under control conditions (ANOVA); §§§P < 0.001, significant difference from sgk3^−/− cells under control conditions (ANOVA).

Decreased Ca²⁺ entry in sgk3^−/− BMMCs could result in decreased antigen-induced mediator release. To determine whether SGK3 deficiency influences mast cell degranulation, the release of β-hexosaminidase was measured in sgk3^+/+ and sgk3^−/− cells. As shown in Fig. 4, β-hexosaminidase release was significantly reduced in sgk3^−/− BMMCs.

Fig. 4.

Degranulation of antigen-stimulated sgk3^−/− and sgk3^+/+ BMMCs. β-Hexosaminidase release from cultured sgk3^−/− BMMCs (closed bars) and their wild-type littermates sgk3^+/+ (open bars) stimulated for 15 min with 50 ng/ml antigen (±SE, n = 5 individual experiments). Release in the supernatant was calculated as percentage of total cellular (0.1% Triton X-100) β-hexosaminidase. The stimulated β-hexosaminidase release in each experiment was corrected for the spontaneous release. *Significant difference between genotypes (P < 0.05; two-tailed unpaired t-test).

To determine whether this defect in Ag-stimulated Ca²⁺ entry into sgk3^−/− BMMCs affects mast cell function in vivo, we tested sgk3^+/+ and sgk3^−/− mice for passive systemic anaphylaxis (Fig. 5). Mice were sensitized with anti-DNP IgE intraperitoneally, and after 5 h rest, they received DNP-HSA antigen or saline as a control by intraperitoneal injection, and body temperature was monitored over time. The measured drop in body temperature following antigen treatment was reduced in sgk3^−/− mice (Fig. 5, A and B). Serum histamine levels measured 30 min after induction of the anaphylactic reaction were significantly lower in sgk3^−/− than those levels in sgk3^+/+ mice (Fig. 5C), thus pointing to an impairment of sgk3-deficient mast cell function in vivo.

Fig. 5.

Systemic anaphylactic reaction in sgk3^+/+ and sgk3^−/− mice. A: changes in body temperature (Δ°C) of sgk3^−/− mice (n = 5, closed squares) and their wild-type littermates sgk3^+/+ (n = 6, open squares) following induction of anaphylaxis (±SE). Mice were given intraperitoneal anti- dinitrophenyl (DNP) IgE (30 μg) and challenged with 100 μg DNP- human serum albumin (HSA) after 5 h. B: arithmetic means (± SE) of maximal changes in body temperature (Δ°C) of sgk3^−/− mice (n = 5, closed bars) and their wild-type littermates sgk3^+/+ (n = 6, open bars) following induction of anaphylaxis. *Significant difference between genotypes (P < 0.05; two-tailed unpaired t-test). C: serum histamine levels 30 min after induction of anaphylaxis in sgk3^−/− mice (closed bar) and their wild-type sgk3^+/+ littermates (open bar) (n = 3). **Significant difference between genotypes (P < 0.01; two-tailed unpaired t-test).

DISCUSSION

The present study unravels a novel function of the serum- and glucocorticoid-inducible kinase SGK3. Specifically, Ca²⁺ entry, Ca²⁺-activated K⁺ channel activity, and degranulation are blunted in BMMCs from SGK3 knockout mice (sgk3^−/−) compared with BMMCs from their wild-type littermates (sgk3^+/+). Accordingly, sgk3^−/− mice appear to be more resistant to anaphylactic shock.

As shown earlier (6, 20, 22, 25, 28, 29, 44, 57, 72), Ca²⁺ entry through Ca²⁺ channels is critically important for the regulation of mast cell degranulation. The Ca²⁺ entry depends on the potential difference across the cell membrane (63) and is thus influenced by the activity of Ca²⁺-activated K⁺ channels (19, 20, 27, 55, 69).

The stimulation of K⁺ channels is blunted, but not completely inhibited, in the nominal absence of extracellular Ca²⁺ (71). Accordingly, the activation of K⁺ channels largely depends on extracellular Ca²⁺. However, the present data do not rule out additional mechanisms involved in the regulation of K⁺ channels.

In mast cells, PI3 kinase has been shown to regulate cell proliferation, adhesion, and migration, as well as antigen-IgE-induced degranulation and cytokine release (2). Moreover, PI3 kinase was suggested to target the TRPV2 Ca²⁺ channel in these cells (75). In the Xenopus oocyte heterologous expression system, SGK1 and SGK3 have previously been shown to increase the cell membrane abundance and activity of the Ca²⁺ channel TRPV5 (32) and TRPV6 (17). It is conceivable that SGK1 has a similar stimulating effect on TRPV2 and/or other Ca²⁺ channels important for Ca²⁺ entry into mast cells.

In the Xenopus oocyte system, SGK3 has been shown to stimulate the activity of several ion channels including the epithelial Na⁺ channel ENaC (34, 62), the renal and cochlear Cl⁻ channel complex ClC-Ka/barttin (30), the cell volume-regulated Cl⁻ channel ClC2 (61), the cardiac voltage-gated Na⁺ channel SCN5A (16), the cardiac K⁺ channels KCNE1/KCNQ1 (31) and HERG (54), the glutamate receptor GluR1 (73) as well as the voltage-gated K⁺ channels Kv1.3 (37, 42, 80), Kv1.5 (77), and Kv4.3 (7). SGK3 further stimulates the activity of a wide variety of transporters including the Na⁺-glucose cotransporter SGLT1 (26), the glutamine transporter SN1 (13), the glutamate transporters EAAT1 (12), EAAT2 (14), EAAT3 (67), and EAAT5 (15), the dicarboxylate cotransporter NaDC-1 (11), the creatine transporter CreaT (SLC6A8) (68), the myoinositol transporter SMIT (46), and the Na⁺-K⁺-ATPase activity (43). Both, SGK1 and SGK3 regulate the function of channels and transporters by influencing expression, trafficking, and degradation of the channel and transport proteins (50).

SGK3 has been shown in vitro to confer cell survival (52, 83), an effect that may be related to the effect of SGK3 on Kv1.3 channel activity. In human embryonic kidney cells and Jurkat lymphocytes, Kv1.3 is involved in the regulation of cell proliferation (37, 51) and apoptosis (40, 51, 74). The antiapoptotic effect may further be secondary to phosphorylation of forkhead transcription factors (21, 52, 83). Moreover, SGK3 has been shown to phosphorylate and thus inactivate Bad (52, 83). Phosphorylated Bad binds to the chaperone 14–3-3 and is thus prevented from traveling to the mitochondria, where it triggers apoptosis (53).

At first glance, it may be surprising that both the knockout of SGK1 (71) and knockout of SGK3 (this study) disrupt antigen-induced Ca²⁺ entry into and activation of mast cells. Accordingly, even though both kinases obviously serve similar functions in mast cells, they cannot fully replace each other and lack of one of the two kinases disrupts mast cell function. It must be kept in mind that SGK3 is constitutively expressed, while SGK1 is genomically upregulated following cell stress. Thus SGK3 may be required for the stimulation of channel or carrier expression in unstressed conditions; i.e., before antigen exposure, while SGK1 may be required for the full-blown entry of Ca²⁺ during stimulation.

In conclusion, SGK3 participates in antigen-stimulated Ca²⁺ entry and subsequent activation of Ca²⁺-activated K⁺ channels. The latter leads to augmentation of Ca²⁺ entry and thus similarly participates in the stimulation of mast cell degranulation. Those cellular effects are critical for anaphylactic response in vivo.

GRANTS

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, SFB 766) and NIH R01-DK56695 (to D. Pearce).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).