Transcription Factor NF-κB Regulates Expression of Pore-forming Ca2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca2+ Entry and Affect Cellular Functions*

The serum and glucocorticoid-inducible kinase SGK1 increases the activity of Orai1, the pore forming unit of store-operated Ca2+ entry, and thus influences Ca2+-dependent cellular functions such as migration. SGK1 further regulates transcription factor nuclear factor κB (NF-κB). This study explored whether SGK1 influences transcription of Orai1 and/or STIM1, the Orai1-activating Ca2+ sensor. Orai1 and STIM1 transcript levels were decreased in mast cells from SGK1 knock-out mice and increased in HEK293 cells transfected with active S422DSGK1 but not with inactive K127NSGK1 or in S422DSGK1-transfected cells treated with the NF-κB inhibitor Wogonin (100 μm). Treatment with the stem cell factor enhanced transcript levels of STIM1 and Orai1 in sgk1+/+ but not in sgk1−/− mast cells and not in sgk1+/+ cells treated with Wogonin. Orai1 and STIM1 transcript levels were further increased in sgk1+/+ and sgk1−/− mast cells by transfection with active NF-κB subunit p65 as well as in HEK293 cells by transfection with NF-κB subunits p65/p50 or p65/p52. They were decreased by silencing of NF-κB subunits p65, p50, or p52 or by NF-κB inhibitor Wogonin (100 μm). Luciferase assay and chromatin immunoprecipitation defined NF-κB-binding sites in promoter regions accounting for NF-κB sensitive genomic regulation of STIM1 and Orai1. Store-operated Ca2+ entry was similarly increased by overexpression of p65/p50 or p65/p52 and decreased by treatment with Wogonin. Transfection of HEK293 cells with p65/p50 or p65/p52 further augmented migration. The present observations reveal powerful genomic regulation of Orai1/STIM1 by SGK1-dependent NF-κB signaling.

Alterations of cytosolic Ca 2ϩ activity participate in the regulation of a wide variety of cellular functions including excitation-contraction coupling, exocytosis, migration, cell proliferation, and cell death (1)(2)(3)(4). Cytosolic Ca 2ϩ is increased by release of Ca 2ϩ from intracellular stores and/or Ca 2ϩ entry across the cell membrane (5). Ca 2ϩ release from intracellular stores results in the stimulation of Ca 2ϩ release-activated Ca 2ϩ channel (CRAC) 2 (6,7), which consists of the pore forming units Orai1, -2, and/or -3 (8 -10) and the endoplasmic reticulum-located regulatory subunit STIM1 or -2 (11)(12)(13). The stimulation of the channel leads to the inward current I CRAC and the store-operated Ca 2ϩ entry (SOCE).
Recent observations uncovered the powerful stimulation of I CRAC and SOCE by the serum and glucocorticoid-inducible kinase SGK1 (14), a kinase stimulated by growth factors and involved in stress response (15) and regulation of cell survival (16). SGK1 is partially effective through phosphorylation of the ubiquitin ligase Nedd4-2 (neuronal precursor cells expressed developmentally down-regulated). Nedd4-2 ubiquitinates Orai1, thus preparing the channel protein for degradation (14). The effect of Nedd4-2 on Orai1 parallels that of Nedd4-2 on the epithelial Na ϩ channel ENaC (16,17). The phosphorylation of Nedd4-2 leads to binding of the ubiquitin ligase to the protein 14-3-3, which prevents the interaction with the channel protein (18). Accordingly, SGK1 enhances Orai1 protein abundance in the cell membrane (14). STIM is similarly regulated by ubiquitination (19). However, the effect of SGK1 on Orai1 protein abundance is only in part explained by Nedd4-2-dependent protein degradation. Therefore, further experiments were performed to explore whether SGK1, in addition, stimulates Orai1 and/or STIM1 expression. As a matter of fact, RT-PCR revealed an increase of Orai1 and STIM1 transcript levels after expression of constitutively active SGK1. Thus, further experiments were performed to uncover the transcription factor involved. Previously, SGK1 has been shown to foster nuclear translocation and activation of nuclear factor B (NF-B) (20 -22). Accordingly, this study explored the putative involvement of NF-B subunits p65 (RELA), p50 (NFKB1), and p52 (NFKB2) in the regulation of Orai1 and STIM1 expression.
Site-directed Mutagenesis-To introduce an aspartic acid for a serine at position 276, QuikChange II site-directed mutagenesis kit (Stratagene, Agilent Technologies) was utilized using the following primer pair (5Ј-3Ј orientation, the mutation is in bold): forward (CAGCTACGGCGGCCTGATGATCGC-GAGCTCAGT) and reverse (ACTGAGCTCGCGATCAT-CAGGCCGCCGTAGCTG). Sequencing was performed to verify the mutation.
Primer pairs were designed binding to two different exons, creating a product of about 90 bp. Relative quantification of gene expression was performed using the 2 Ϫ⌬⌬ct method as described earlier (27).
Identification of Putative B-binding Sites-The genomic sequences of STIM1 and Orai1 promoter were checked for the 5Ј-GGGRNNYYCC-3Ј B-consensus sequence. DNA sequence of the promoter region (3000 bp upstream of transcription start) was used from ensemble.org, and each possible consensus sequence was tested by self-made sequence alignment.
Luciferase Assay-Sequences containing putative B-binding sites in the STIM1 and Orai1 promoter region were amplified by PCR from genomic template DNA. PCR was performed using Maxima TM Hot Start TaqDNA polymerase (Fermentas, Germany) with primers containing a KpnI site 5Ј and a HindIII site 3Ј: O1 forward (GTAGGTACCGGAAACAAAGCCAG-TAG), O1 reverse (CGTAAGCTTTTCCAGACCAGCCTA), O2 forward (CGTAGGTACCCCAGAGACTTCTTGGG), O2 reverse (GCTAAGCTTCAGGACGGCGAGG), S1 forward (CCTCGGTACCATCCATGTTGTAGCA), and S1 reverse (GCGAAAGCTTACGCTAAAATGGTGTCT). The separate STIM1/Orai1 promoter fragments were cloned into the pGL3-Luciferase Enhancer Vector (Promega). To ensure that the fragments were inserted in the correct orientation and to confirm the correctness of the sequence control restrictions and DNA sequencing was performed (Delphi Test).
Forty-eight hours after transfection, Dual-Luciferase Reporter Assays (Promega) were performed according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla activity. Renilla luciferase was constitutively expressed by the cotransfected vector pRL-TK and thus served as an internal control for the transfection rate.
Animals-Bone marrow was obtained from 6 -8-week-old female and male SGK1 knock-out (sgk1 Ϫ/Ϫ ) mice and their wild type (sgk1 ϩ/ϩ ) littermates. Generation, breeding, and genotyping of the mice has been described earlier (28). Animal experiments were conducted according to German law for the welfare of animals and were approved by local authorities.
Treatment of BMMCs-Mast cells were plated in 24-well plates (1 ϫ 10 6 cells/well). The cells were either treated with Wogonin (100 M) for 24 h or incubated in the medium deprived of growth factors (SCF, IL-3) in the presence or in the absence of Wogonin (100 M) for 20 h and after that treated with SCF (100 g/ml) in the presence or in the absence of Wogonin, respectively, for a further 4 h. Then the cells were collected and washed with PBS.
Immunofluorescence-HEK293 cells transfected with p65/ p50 or p65/p52, respectively, were cultured in 24-well plates with a coverslip inside, washed, and fixed with 4% paraformal-dehyde. For blocking, unspecific binding HEK293 cells were incubated with 5% normal goat serum, 1ϫ PBS, 0.1% Triton for 1 h at room temperature. Then the cells were exposed to rabbit anti-Orai1 (1:100, Millipore) or rabbit anti-STIM1 (1:100, Abnova) at 4°C overnight. The cells were rinsed three times with PBS and incubated with secondary FITC goat anti-rabbit antibody (1:1000; Invitrogen) or with Alexa Fluor 488-conjugated goat anti-mouse antibody (1:1000, Invitrogen) for 1.5 h at room temperature. After three washing steps the nuclei were stained with DRAQ-5 dye (1:1000; Biostatus) for 10 min at room temperature. The slides and coverslips were mounted with ProLong Gold antifade reagent (Invitrogen). Images were taken on a LSM 5 EXCITER confocal laser scanning microscope (Zeiss, Germany) with a water-immersion Plan-Neofluar 40ϫ/1.3 NA differential interference contrast and analyzed with the instrument's software.
Measurement of Intracellular Ca 2ϩ Concentration-Fura-2/AM fluorescence was utilized to determine intracellular Ca 2ϩ (29). Cells were excited alternately at 340 and 380 nm through an objective (Fluar 40ϫ/1.30 oil) built in an inverted phase-contrast microscope (Axiovert 100, Zeiss, Oberkochen, Germany). Emitted fluorescence intensity was recorded at 505 nm. Data were acquired using specialized computer software (Metafluor, Universal Imaging). Cytosolic Ca 2ϩ activity was estimated from the 340/380-nm ratio. HEK293 cells were loaded with Fura-2/AM (2.5 M, Molecular Probes) for 30 min at 37°C. SOCE was determined by extracellular Ca 2ϩ removal and subsequent Ca 2ϩ readdition in the presence of thapsigargin (1 M) (30). For quantification of the Ca 2ϩ entry, slope (⌬ ratio/s) and peak (⌬ ratio) were calculated after readdition of Ca 2ϩ .
Migration Assay-For migration assays, transwell inserts (BD Falcon 353097) and BD BioCoat TM Matrigel TM Invasion Chambers (BD Biosciences 354480) were used with a pore diameter size of 8 m. The transwells were placed in a 24-well cell culture plate containing cell culture medium (750 l). The upper chambers were filled with 500 l of cell culture medium containing HEK293 cells in a concentration of 5 ϫ 10 4 cells/ml. After an incubation time of 24 h at 37°C, migrated cells were analyzed by staining the cell nuclei with DAPI. Before that nonmigrated cells were removed by scrubbing with a cotton-tipped swab two times and washing with PBS. After a 15 min fixation in 4% paraformaldehyde, the membrane was removed with a scalpel. After removal from the inserts, membranes were mounted on slides with ProLong Gold antifade reagent (Invitrogen). To determine the total number of migrated cells, the slides were then viewed under the microscope, and the number of cells/ field in representative areas was counted. Experiments were performed in triplicate.
Statistics-Data are provided as the mean Ϯ S.E.; n represents the number of independent experiments. All data were tested for significance using Student's unpaired two-tailed t test or ANOVA (Tukey's test or Dunnett's test), where applicable. Results with p Ͻ 0.05 were considered statistically significant.

RESULTS
As illustrated in Fig. 1, A and B, transfection of HEK293 with the constitutively active mutant S422D SGK1 but not with the inactive mutant K127N SGK1 was followed by a significant increase of mRNA encoding STIM1 and Orai1. Accordingly, the transcript levels of both genes were enhanced by SGK1. Previous studies revealed that SGK1 activates NF-B (20 -22). Thus, S422D SGK1-transfected and non-transfected HEK293 cells were treated with the NF-B inhibitor Wogonin (100 M). The treatment significantly reduced the mRNA levels of STIM1 and Orai1 in both S422D SGK1-overexpressing and non-transfected HEK293 cells (Fig. 1, A and B). Transfection of HEK293 cells with S422D SGK1 but not with K127N SGK1 led to nuclear translocation of the NF-B subunit p65 (Fig. 1C). Again, the effect of S224D SGK could be reversed by treatment of the cells with Wogonin. For the positive control for p65 translocation, HEK293 cells were incubated with TNF␣ (100 ng/ml, 1 h). ⌻ elucidate whether the stimulating effect of SGK1 is apparent under endogenous levels of SGK1, the transcript levels of Orai1 and STIM1 were determined in BMMCs isolated from gene targeted mice lacking functional SGK1 (sgk1 Ϫ/Ϫ ) and their wild type (sgk1 ϩ/ϩ ) littermates. As shown in Fig. 2, the Orai1 and STIM1 transcript levels were both significantly higher in sgk1 ϩ/ϩ BMMCs compared with sgk1 Ϫ/Ϫ BMMCs. Stimulation of sgk1 ϩ/ϩ , but not of sgk1 Ϫ/Ϫ BMMCs, with SCF (20 ng/ml, 4 h) resulted in a translocation of the NF-B subunit p65 to the nucleus ( Fig. 2A) and accordingly led to significant increase of the STIM1 and Orai1 transcript levels in sgk1 ϩ/ϩ BMMCs but not in sgk1 Ϫ/Ϫ BMMCs (Fig. 2B), an effect abrogated by treat- ment of sgk1 ϩ/ϩ cells with Wogonin (Fig. 2B). Treatment of BMMCs with Wogonin alone tended to decrease Orai1 mRNA levels, an effect, however, not reaching statistical significance. To further evaluate whether NF-B-sensitive transcription of STIM1 and Orai1 was downstream of SGK1, BMMCs from sgk1 Ϫ/Ϫ and sgk1 ϩ/ϩ mice were transfected with a constitu-tively active mutant of p65 ( S276D p65). As shown in Fig. 2C, transfection with S276D p65 led to a significant increase in transcript levels of STIM1 and Orai1 in BMMCs from both sgk1 ϩ/ϩ and sgk Ϫ/Ϫ mice.
Further experiments explored whether transfection of HEK293 cells with NF-B influences the transcript levels of

. Effect of SGK1 and NF-B on STIM1 and Orai1 transcript levels in BMMCs.
A, confocal microscopy of p65 (red) translocation in BMMCs from genetargeted mice lacking functional SGK1 (sgk1 Ϫ/Ϫ ) and their wild type (sgk1 ϩ/ϩ ) littermates with or without (control) exposure to SCF (100 ng/ml) is shown. Nuclei were stained with DRAQ5 (blue). B, arithmetic means (ϮS.E., n ϭ 14) of relative mRNA levels of STIM1 (left panel) and Orai1 (right panel) in sgk1 ϩ/ϩ and sgk1 Ϫ/Ϫ BMMCs with or without exposure to SCF (100 ng/ml) and with or without the presence of Wogonin (100 M) are shown. Knock out of SGK1 was determined utilizing RT-PCR and agarose gel electrophoresis. ** (p Ͻ 0.01) and *** (p Ͻ 0.001) indicate statistically significant differences in one group, and # (p Ͻ 0.05) and ### (p Ͻ 0.001) indicate statistically significant differences between groups (ANOVA). C, arithmetic means (ϮS.E., n ϭ 6) of relative mRNA levels for STIM1 (left and middle left panel) and Orai1 (right and middle right panel) in BMMCs from sgk1 Ϫ/Ϫ and sgk1 ϩ/ϩ mice without (control) or with transfection with a constitutively active p65 mutant are shown. The success of transfection was determined utilizing RT-PCR and agarose gel electrophoresis. * (p Ͻ 0.05) and **(p Ͻ 0.01) indicate statistically significant differences (Student's unpaired t test). Fig. 3, transfection with the NF-B subunits p65/p50 or p65/p52 indeed significantly increased the STIM1 (Fig. 3A) and Orai1 (Fig. 3B) transcript levels. The success of transfection was verified by specific primer pairs for p65, p50, and p52 using RT-PCR (Fig. 3C). TBP was used as an internal control for RT-PCR. Further experiments explored whether STIM1 and Orai1 protein could also be increased by transfection with the NF-B subunits. As a result, fluorescence imaging (Fig. 4A) and Western blot analysis (Fig. 4B) revealed significant increases in STIM1 and Orai1 protein after transfection with p65/p50 or p65/p52. Tubulin was used as loading control for Western blot analysis. The success of transfection was controlled with specific antibodies against p65, p50, and p52, respectively.

STIM1 and Orai1. As shown in
In a next step, NF-B was silenced with siRNA in HEK293 cells. As illustrated in Fig. 5A, silencing of p65, p50, or p52 each led to a significant decrease of both STIM1 and Orai1 transcript levels. The success of the knockdown was verified by RT-PCR (Fig. 5B) and Western blotting (Fig. 5C). Silencing of NF-B subunits also decreased protein levels of STIM1 and Orai1 (Fig.  5D).
Analysis of the STIM1 and Orai1 promoter revealed putative NF-B-binding sites in the region 2980 bp upstream of STIM1 and 2420 bp upstream of Orai1 transcription start (Fig. 6A). We, therefore, explored the possibility of transcriptional activation of STIM1 and Orai1 by NF-B utilizing ChIP and luciferase assay. For STIM1 promoter (S1) and two regions of Orai1 promoter (O1 and O2), putative B-like binding sites were subcloned into pGL3-vector cotransfected with p65 and pRL-TK and analyzed for luciferase activity. For control of basal luciferase activity cells were transfected with a control plasmid (pcDNA3.1ϩ) instead of p65. Firefly luciferase activity was normalized to Renilla luciferase activity. As shown in Fig. 6B, cotransfection of pGL3-S1 with the integrated STIM1 promoter and p65 resulted in a 2.3 Ϯ 0.2 (n ϭ 14)-fold increase of luciferase activity. Cotransfection with pGL3-O2 (contains the promoter region O2 of Orai1) and p65 resulted in a 2.4 Ϯ 0.2 (n ϭ 21)-fold increase of luciferase activity. In contrast, cotransfection with pGL3-O1 containing the promoter region O1 of Orai1 and p65 did not activate luciferase activity. In ChIP experiments the binding of p65 to putative NF-B response elements was further investigated. This was achieved using anti-p65 (2 g, C20, Santa Cruz) or anti-rabbit IgG (1:1000, Abcam) antibodies to pull down p65-bound DNA extracted from HEK293 cells transiently transfected with p65. For comparison, the cells were transfected with a control plasmid. PCR indicated p65 binding to the STIM1 and Orai1 promoter sequence using primer pairs flanking the putative binding sites. As illustrated in Fig. 6C, we could observe pulldown of one putative binding site in the STIM1 promoter as well as in the Orai1 promoter (O2). There was no pulldown detectable in a second putative B-binding site in Orai1 promoter (O1) and when using rabbit IgG as negative control.
A further series of experiments explored whether NF-Bsensitive transcription of STIM1 and Orai1 influenced SOCE. To this end, the fura-2 fluorescence-ratio was determined by fluorescence spectrometry. SOCE was measured upon store depletion by inhibition of the vesicular Ca 2ϩ -ATPase with thapsigargin in HEK293 cells in the presence and absence of Wogonin (100 M). Stores were depleted by thapsigargin (1 M) first added to the Ca 2ϩ -free medium. Thapsigargin triggered release of Ca 2ϩ from intracellular stores, which was not modified by Wogonin treatment (Fig. 7A). Re-addition of Ca 2ϩ (1 mM) to the medium in the continued presence of thapsigargin led to an entry of Ca 2ϩ through plasma membrane Ca 2ϩ channels and to a subsequent increase of the fluorescence ratio (Fig. 7A). The fluorescence ⌬ratio (peak) and the slope of the ratio (⌬ratio/time) were analyzed upon Ca 2ϩ re-addition. Both, peak, and slope values of SOCE were significantly decreased by the NF-B inhibitor Wogonin (Fig. 7,  A-C). Conversely, both peak and slope were significantly increased after transfecting the cells with NF-B subunits p65/p50 or p65/p52 (Fig. 7, D-F). When p65/p50-and p65/ p52-transfected cells were treated with Wogonin, the stimulating effect of p65/p50 or p65/p52 overexpression was abrogated (Fig. 7, E and F). Finally, the impact of NF-Bsensitive transcriptional regulation of STIM1 and Orai1 on HEK293 cell migration was estimated in a transwell migration assay from translocation of cells from one chamber to another across a membrane with a pore diameter size of 8 m. As a result, migratory activity was significantly higher in  , lower panel). The experiment was completed three times. B, shown is a Western blot analysis of whole cell lysate in p65/p50-or p65/p52-transfected HEK293 cells showing STIM1 and Orai1 protein abundance. The blot was stripped and reprobed with tubulin to ensure equal loading and with p65, p50, and p52 to control transfection. Left, representative blot; middle, arithmetic means of STIM1/tubulin ratio (ϮS.E., n ϭ 6); right, arithmetic means of Orai1/tubulin ratio (ϮS.E., n ϭ 6). * (p Ͻ 0.05) and ** (p Ͻ 0.01) indicate statistically significant differences (Student's unpaired t test). p65/p50-and p65/p52-transfected HEK293 cells than in cells transfected with an empty vector (Fig. 8).

DISCUSSION
This study uncovers a novel element in the regulation of Orai1 and STIM1, the components that constitute the CRAC and contribute to the SOCE. We demonstrate that transfection of human embryonic kidney (HEK293) cells with constitutively active S422D SGK1, but not with inactive K127N SGK1, enhances the STIM1 and Orai1 transcript levels. SGK1 has previously been shown to activate the transcription factor NF-B (20 -22). Accordingly, treatment of S422D SGK1-transfected HEK293 cells with the NF-B inhibitor Wogonin strongly decreased STIM1 or Orai1 transcript levels. Transfection with S422D SGK1 but not with K127N SGK1 or with S422D SGK1 in the presence of Wogonin was further followed by nuclear translocation of p65 in HEK293 cells. STIM1 and Orai1 transcript levels were significantly lower in BMMCs from SGK1 knock-out (sgk1 Ϫ/Ϫ ) mice when compared with BMMCs from their corresponding wild type (sgk1 ϩ/ϩ ) littermates. Along those lines treatment with the stem cell factor SCF, a strong activator of the PI3K pathway (31), enhanced transcript levels of STIM1 and Orai1 in BMMCs from sgk1 ϩ/ϩ but not from sgk1 Ϫ/Ϫ mice. Consequently, active SGK1 is required for up-regulation of STIM1 and Orai1 transcription by growth factor stimulation of mast cells. Simultaneous treatment of sgk1 ϩ/ϩ BMMCs with SCF and the NF-B inhibitor Wogonin abolished the stimulating effect of SCF on STIM1 and Orai1 transcription. In addition, transfection of sgk1 Ϫ/Ϫ and sgk1 ϩ/ϩ mast cells with a constitutively active p65 mutant ( S276D p65) strongly increased transcript levels of STIM1 and Orai1.
According to this study the SGK1-dependent up-regulation of Orai1 and STIM1 transcription is mimicked in HEK293 cells by overexpressing the NF-B subunits p65/p50 or p65/p52 and reversed by silencing of the NF-B subunits p65, p50, and p52 and by treatment with the NF-B inhibitor Wogonin. Subsequently, overexpression of p65/p50 or p65/p52 also enhanced, whereas silencing of p65, p50, or p52 reduced, STIM1 and Orai1 protein abundance. SOCE was similarly up-regulated by overexpression of p65/p50 and p65/p52 and decreased by Wogonin treatment.
Application of the luciferase reporter assay allowed identifying putative binding regions of NF-B in the region upstream of STIM1 and Orai1. Two promoter regions, S1 and O2, were activated by p65 transfection. To distinguish between direct and indirect modes of binding, ChIP was performed to determine if any of these putative B-like binding sites exhibit bona fide p65 binding. Amplification of pulled down DNA with primers flanking B-like sites revealed binding of p65 to DNA for regions S1 and O2. Region O1 showed no binding of p65.
Thus, it appears safe to conclude that NF-B participates in the SGK1-dependent genomic regulation of STIM1 and Orai1.
NF-B-dependent STIM1 and Orai1 expression may be relevant for the regulation of migration (32), which can be stimulated by expression of Orai1, STIM1, and constitutively active SGK1 but not by coexpression of inactive SGK1 (14). Migration is stronger in HEK293 cells transfected with p65/p50 or p65/ FIGURE 6. NF-B-sensitive transcription of Orai1 and STIM1. A, analysis of the DNA sequence of 11p15.5 in the region adjacent to the STIM1 transcriptional start site (S1) and of 12q24.31 in the region adjacent to the Orai1 transcriptional start site (O1 and O2) revealed putative B-like binding sites (italic, underlined) is shown. For ChIP experiments DNA was amplified by PCR and subcloned into pGL3-vector for luciferase reporter assay using the primers depicted in bold. B, a luciferase reporter assay was applied to study the ability of NF-B to activate the target promoters of STIM1 and Orai1. HEK293 cells were transfected with luciferase reporter plasmids containing the putative B-binding sites and additionally transfected with p65 or control plasmid. The results are presented as -fold increase in the activity of luciferase activity (ϮS.E., n ϭ 11-21) compared with luciferase activity transfected with the control plasmid. * (p Ͻ 0.05) indicates statistically significant differences (ANOVA). C, binding of NF-B to DNA was explored utilizing ChIP of genomic DNA extracted from HEK293 cells transfected with p65 or a control plasmid. Protein-chromatin complexes were immunoprecipitated with anti-p65 or anti-rabbit IgG. The input served as a positive control, IgG as a negative control. The experiment was carried out five times. . E, shown are the arithmetic means (ϮS.E., n ϭ 3, each experiment 14 -33 cells) of peak (⌬ ratio) and slope (⌬ ratio/s) of fura-2 fluorescence increase after readmission of Ca 2ϩ in HEK293 cells transfected with control plasmid or p65/p50 with or without treatment with Wogonin (100 M). F, shown are the arithmetic means (ϮS.E., n ϭ 3, each experiment 14 -33 cells) of peak (⌬ ratio) and slope (⌬ ratio/s) of fura-2 fluorescence increase after readmission of Ca 2ϩ in HEK293 cells transfected with control plasmid or p65/p52 with or without treatment with Wogonin (100 M). * (p Ͻ 0.05), ** (p Ͻ 0.001), *** (p Ͻ 0.001) indicate statistically significant differences (ANOVA).
I CRAC activation is further controlled by Lyn and Syk kinases (66), which, however, other than SGK1, are not effective through regulation of STIM1 or Orai1 expression. In contrast to Lyn and Syk kinases, SGK1 plays a dual role in Orai1 regulation, i.e. inhibition of ubiquitination and stimulation of expression. Both effects increase Orai1 protein abundance. Unlike Lyn and Syk kinases, those effects of SGK1 are slow, rendering the respective cell more sensitive to immediate regulators of Orai1. In conclusion, NF-B is a powerful regulator of STIM1 and Orai1 expression and thus influences SOCE-dependent functions, including migration.