Glycogen Synthase Kinase 3β Activation Is a Prerequisite Signal for Production and Chemotaxis in Human Mast Cells

This information is current as Madeleine Rådinger, Hye Sun Kuehn, Mi-Sun Kim, Dean D. of October 2, 2021. Metcalfe and Alasdair M. Gilfillan J Immunol published online 11 December 2009 http://www.jimmunol.org/content/early/2009/12/11/jimmun ol.0902931 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 11, 2009, doi:10.4049/jimmunol.0902931 The Journal of Immunology

Glycogen Synthase Kinase 3b Activation Is a Prerequisite Signal for Cytokine Production and Chemotaxis in Human Mast Cells

Madeleine Ra˚dinger, Hye Sun Kuehn, Mi-Sun Kim, Dean D. Metcalfe, and Alasdair M. Gilfillan

In addition to regulating homeostasis, the activation of KIT following ligation by promotes a diversity of mast cell responses, including cytokine production and chemotaxis. Although we have previously defined a role for the mammalian target of rapamycin complex 1 in these responses, it is clear that other signals are also required for maximal KIT-dependent cytokine production and chemotaxis. In this study, we provide evidence to support a role for glycogen synthase kinase 3b (GSK3b) in such regulation in human mast cells (HuMCs). GSK3b was observed to be constitutively activated in HuMCs. This activity was inhibited by knockdown of GSK3b following transduction of these cells with GSK3b-targeted shRNA. This resulted in

a marked attenuation in the ability of KIT to promote chemotaxis and, in synergy with Fc«RI-mediated signaling, cytokine Downloaded from production. GSK3b regulated KIT-dependent mast cell responses independently of mammalian target of rapamycin. However, evidence from the knockdown studies suggested that GSK3b was required for activation of the MAPKs, p38, and JNK and downstream phosphorylation of the transcription factors, Jun and activating transcription factor 2, in addition to activation of the transcription factor NF-kB. These studies provide evidence for a novel prerequisite priming mechanism for KIT-dependent responses regulated by GSK3b in HuMCs. The Journal of Immunology, 2010, 184: 000–000. http://www.jimmunol.org/ ast cells are tissue-resident cells of hematopoietic origin naling events elicited by activated KIT, it is unclear how these that play a role in innate and acquired immune responses events subsequently differentially control the diverse group of M (1). Mast cell growth, development, and survival are responses mediated by KIT. In exploring this differential regula- driven by stem cell factor (SCF)-dependent activation of its receptor tion, we recently described, however, that the mammalian target of KIT (2). In addition to its role in mast cell homeostasis, however, rapamycin complex (mTORC)1 cascade, which is activated SCF can also regulate other mast cell functions. In this respect, SCF- downstream of PI3K following challenge of either mouse or hu- mediated activation of KIT potently induces mast cell chemotaxis man mast cells with SCF (12), contributes to the regulation of (3, 4) and adhesion to extracellular matrix (5), supporting a role for SCF-mediated chemotaxis and SCF/Ag-mediated cytokine pro- SCF in mast cell homing to their tissues of residence in vivo. Fur- duction (12). Nevertheless, because a substantial portion of these by guest on October 2, 2021 thermore, SCF-mediated KITactivation, particularly in conjunction responses remained following rapamycin-induced inhibition of with aggregation of the FcεRI, also promotes the generation of mTORC1 signaling, it was concluded that other signaling path- multiple and (5–7). ways apart from those regulated by mTORC1 participate in the KIT is member of the receptors with inherent regulation of SCF-mediated chemotaxis and transcriptional acti- tyrosine kinase activity family (8, 9). Dimerization of KIT, fol- vation, leading to cytokine and generation (12). lowing SCF binding, activates its inherent tyrosine kinase activity In this study, we present evidence to support a role for glycogen resulting in phosphorylation of specific tyrosine residues in the synthase kinase 3b (GSK3b) in such regulation. GSK3b is cytoplasmic tail of KIT allowing recruitment of critical adaptor a ubiquitously expressed serine/threonine kinase, which has been and signaling molecules (10). These receptor–proximal events reported to play a role in the regulation of diverse cellular re- lead to the initiation of multiple downstream signaling process, sponses including cell growth, tumorigenesis, cell migration, and eventually culminating in transcriptional regulation (8, 9, 11). cytokine generation (13–15). However, it is not fully understood Despite a comprehensive understanding of these immediate sig- how GSK3b regulates these responses. In studies that use knockdown of GSK3b expression, we now demonstrate that GSK3b activation is a prerequisite signal for SCF-mediated che- Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Dis- motaxis and SCF/Ag-mediated cytokine generation. Thus, this eases, National Institutes of Health, Bethesda, MD 20892 may constitute a novel priming mechanism for specific mast cell Received for publication September 3, 2009. Accepted for publication November 5, 2009. responses. The regulation of the chemotactic response by GSK3b This work was supported by the Division of Intramural Research, National Institute appears dependent on its modulation of JNK and p38-dependent of Allergy and Infectious Diseases, National Institutes of Health. M.R. is grateful for pathways, whereas the regulation of cytokine generation by support from the Swedish Heart-Lung Foundation. GSK3b may be explained by the differential regulation of tran- Address correspondence and reprint requests to Dr. Alasdair M. Gilfillan, Laboratory scriptional control downstream of JNK and p38. of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11C206, 10 Center Drive, MSC 1881, Be- thesda, MD 20892-1881. E-mail address: agilfi[email protected] Materials and Methods The online version of this article contains supplemental material. Mast

Abbreviations used in this paper: ATF2, activating transcription factor 2; GS, glycogen + synthase; GSK3b, glycogen synthase kinase 3b; HuMC, human mast cell; mTORC, Primary human mast cells (HuMCs) were derived from CD34 peripheral mammalian target of rapamycin complex; MKK, map kinase kinase; p, phospho; rHu, blood progenitor cells (16) obtained from normal volunteers following recombinant human; SA, streptavidin; SCF, stem cell factor; sh, short hairpin. informed consent under a protocol approved by the National Institutes of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902931 2 ROLE OF GSK3b IN MAST CELL FUNCTION

Health internal review board. The cells were developed in StemPro-34 Biosciences, Fredrick, MD). expression was analyzed using real-time culture medium containing StemPro-34 supplement (Invitrogen, Carlsbad, PCR on an ABI7500 SDS system (Applied Biosystems, Foster City, CA). CA), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ A common cytokine PCR array was purchased from SA Biosciences, and ml), recombinant human (rHu) IL-3 (30 ng/ml, first week only), rHuIL-6 real-time PCR was performed according to manufacturer’s instructions. (100 ng/ml), and rHuSCF (100 ng/ml) (PeproTech, Rocky Hill, NJ). Ex- All reactions (two different HuMCs donors) were performed in triplicate periments were conducted 7–9 wk after the initiation of HuMC cultures. for 40 cycles. The relative fold expression levels of cytokines was calcu- lated as follows: for each sample the threshold cycle (Ct) was determined Lentivirus short hairpin RNA transfection of 293T cells and and normalized to the average of five different housekeeping in the transduction of HuMCs KIT (DCt). The DCt of treated or untreated cells was then subtracted from untreated control shRNA-transduced cells (DDCt) and the relative fold The following GSK3b-targeted short hairpin (sh)RNAs were purchased DD expression was calculated using the equation 2 Ct. from Sigma-Aldrich (St. Louis, MO): CCGGGTGTGGATCAGTTGGTA- GAAACTCGAGTTTCTACCAACTGATCCACACTTTTT (TRCN00000 Cytokine quantitation 10552) (construct A); CCGGGACACTAAAGTGATTGGAAATCTCGA- GATTTCCAATCACTTTAGTGTCTTTTTG (TRCN0000040002) (cons- Cell-free supernatants from activated cells were harvested and cytokine truct B); CCGGCCACTGATTATACCTCTAGTACTCGAGTACTAGAGG- content was measured by using DuoSet ELISA System (R&D Systems, TATAATCAGTGGTTTTTG (TRCN0000039998) (construct C); CCGGC- Minneapolis, MN). CCAAACTACACAGAATTTAACTCGAGTTAAATTCTGTGTAGTTTG- GGTTTTTG (TRCN0000039999) (construct D); CCGGGCAGGACAAGA- Chemotaxis GATTTAAGAACTCGAGTTCTTAAATCTCTTGTCCTGCTTTTTG (TR Chemotaxis assays were performed using Transwell polycarbonate CN000040000) (construct E); andCCGGCAACAAGATGAAGAGC- 3 5 ACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT (SHC002) membranes (8-mm pore size) (Corning, Corning, NY). HuMCs (1 10 (control nontarget control vector).The packaging vector (MissionLentiviral /well) were incubated in cytokine-free StemPro medium for 4 h and then resuspended in cytokine-free StemPro medium containing 0.5% BSA. packaging mix [Sigma-Aldrich]), the pLKO1 transfer vectors with GSK3b Downloaded from m shRNA, or control shRNA (3.4 mg) were cotransfected into 293T packaging The cell suspension (100 l) was placed in the upper chamber and 6 m cells (4 3 10 cells) with FuGENE6 transfection reagent (Roche, Indian- preincubated in the bottom chamber containing 600 l cytokine-free apolis, IN) in Opti-MEM medium. The transfected 293T cells were grown in StemPro medium for 30 min at 37˚C. After 30 min, the inserts were replaced into the bottom chambers with or without SCF (30 ng/ml). After DMEM containing FBS (10%), L-glutamine (4 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml). Following 16–19 h of transfection, medium 4 h, the migrated cells were collected in the bottom chamber and counted was removed and replaced with fresh DMEM. After 62–65 h of transfection, under microscopy. virus was collected by centrifugation (25,000 rpm, 1 h 40 min, 4˚C), then the

Immunoblotting http://www.jimmunol.org/ resulting pellet resuspended in 3 ml prewarmed complete StemPro medium. Transduction of HuMCs was conducted by transfering the 3 ml resuspended Cell lysates were prepared as described previously (18). Aliquots of lysates virus to a T75 culture flask containing 3–4 3 106 HuMCs in 15 ml complete were loaded onto a 4–12% NuPage BisTris gel (Invitrogen) and following StemPro medium. Two days postinfection, the medium was changed to vi- electrophoresis, were transferred onto nitrocellulose membranes. rus-free complete StemPro medium, and antibiotic selection was initiated The proteins were probed with following phospho (p)-specific Abs from (0.2 mg/ml puromycin; Sigma-Aldrich). Experiments were conducted on day Technology (Beverley, MA): p-AKT (S473), p-GSK3b 7 posttransduction. Cytospins of HuMCs transduced with shControl or (S9), p-JNK (T183 and Y185), p-map kinase kinase (MKK) 3/6 (S189 and shGSK3b were stained with toluidine blue as described previously (17). The S207), p-p38 (T180), p-GS (S641), p-mTOR (S2448), p-p70 S6K (T389), cells treated with GSK3b-targeted shRNA are hereafter termed GSK3b p-4E-BP1 (T37 and T46), phospho-activating transcription factor 2 (ATF2) knockdown HuMCs. (T71), p-NFkB (S536), p-NFkB (S276), total JNK, total p38, and total NF-

« kBp65. p-cJun (S73) was from Upstate Biotechnology (Lake Placid, NY), by guest on October 2, 2021 Flow cytometric analysis for Fc RI and KIT surface and p-GSK3a/b (Y279/Y216) was from Invitrogen. Total Syk, total KIT, expression total Lyn, and total cJun Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoreactive proteins were visualized by probing with Cells were incubated in cytokine-free media for 4 h and washed twice in HRP-conjugated secondary Abs and then by ECL (PerkinElmer, Wellesley, PBS containing 0.1% BSA. Cells were then stained with CD117-PE (BD MA). Quantitation of changes in protein phosphorylation were performed Biosciences, San Jose, CA) or PE-conjugated isotype control (BD Bio- using a Quantity One scanner (Bio-Rad, Hercules, CA). sciences) and FcεRI-allophycocyanin (eBioscience, San Diego, CA) or allophycocyanin-conjugated isotype control (eBioscience) for 1 h on ice. Statistical analysis Cell fluorescence was analyzed (10,000 events) on a gated forward scatter and side light scatter area previously determined as mast cell- Data are represented as the mean 6 SE. The statistical analyses were specific using a FACSCalibur flow cytometer (BD Biosciences) and as- performed by unpaired Student’s t test. Differences were considered sig- sociated CellQuest software. nificant when p , 0.05. The n values represent experiments from multiple preparations. Cell activation HuMCs were incubated overnight in complete Stem Pro medium containing Results human myeloma IgE (100 ng/ml; Calbiochem, La Jolla, CA), biotinylated Expression and knockdown of GSK3b in human mast cells within the National Institute of Allergy and Infectious Diseases Core Fa- cility. The cells were then starved in cytokine-free StemPro medium for 4 h Disruption of the GSK-3b gene in mice leads to an embryonically (for chemotaxis assay and corresponding cell lysate preparations), then the lethal phenotype (19), therefore, to explore the role of GSK3b in cells were activated by the addition of SCF (30 ng/ml). For cytokine release studies, HuMCs (1 3 106/ml) were sensitized in complete StemPro culture human mast cell function, we elected to use a gene knockdown medium overnight and the next day washed in culture medium and triggered approach. To achieve this, HuMCs were stably transduced with concurrently via KIT with SCF (30 ng/ml) and via the FcεRI with strepta- GSK3b-targeted shRNA, using a lentivirus system. Five (A–E) vidin (SA) (100 ng/ml) for 6 h. In some experiments, cells were pretreated different constructs were examined for their ability to selectively with the mTOR inhibitor, rapamycin (100 nM) or the PI3K inhibitor, wort- knockdown GSK3b expression. As a control, we used a scrambled maninn (100 nM), (Calbiochem) for 20 min prior to activation. shRNA construct purchased from Sigma-Aldrich. The level of ex- Real-time PCR analysis pression of GSK3b in the cells treated with the control-scrambled HuMCs (2–3 3 106/sample) were sensitized overnight with biotinylated shRNA was not substantially different from that observed in un- human IgE (100 ng/ml) in complete StemPro medium. The following day, treated cells (data not shown). Of the shRNAs targeting GSK3b, cells were washed with the same medium three times to remove excess four decreased GSK3b levels to varying degrees within the cells IgE, then the cells were stimulated with SA (100 ng/ml) and SCF (30 ng/ following transduction. Of these, two (A and B) were selected for ml) for 4 h. Total RNA was isolated from each preparation using the RNeasy Mini Kit (Qiagen, Valencia, CA). One microgram of total cellular further characterization based on their differential abilities to reduce RNA was treated for genomic DNA contamination and reverse transcribed GSK3b expression in HuMCs (A: 41 6 3%; B: 69 6 4% reduction) using SA Biosciences Reverse Transcription reagents and oligo(dT) (SA (Fig. 1A). These constructs had little effect on the expression of The Journal of Immunology 3

other molecules examined, including phospholipase Cg1, Lyn, Syk, phosphorylation of this residue. Similarly, the GSK3b substrate, KIT, and NF-kΒ (Fig. 1B). In addition, no marked differences were GS, was constitutively phosphorylated at S641 (Fig. 2A,2C). seen in surface expression of KIT and FcεRI (Fig. 1C) in cells However, in contrast to the phosphorylation of GSK3b, this transduced with GSK3b-targeted shRNA (86.7 6 1.7% double- phosphorylation was slightly elevated in cells activated through positive cells compared with the control cells [91.6 6 2.9% double- KIT and FcεRI. Surprisingly, in light of this observation, we also positive cells, n = 3]). There was also little change in the gross observed that the phosphorylation of the inhibitory S9 residue of morphology of the cells treated with the GSK3b-targeted shRNA GSK3b was enhanced in the activated HuMCs (Fig. 2A,2D). compared with control cells (Fig. 1D) or nontransduced cells (data Similar responses to the above were observed in HuMCs activated not shown). In subsequent studies, responses observed in GSK3b via either FcεRI or KIT alone (data not shown). knockdown HuMCs are compared with those observed in HuMCs As expected, because of the effective reduction in total GSK3b incubated with scrambled control shRNA. protein in the GSK3b knockdown HuMCs, the phosphorylation of GSK3b-Y216 and GSK3b-S9 was substantially reduced in both Knockdown of GSK3b phosphorylation in quiescent and quiescent and activated HuMCs (Fig. 2). The phosphorylation of GS activated mast cells at S641, however, was also reduced in these cells. In contrast, and by Optimal cytokine generation in HuMCs requires that the cells be means of a negative control, we observed no decrease in the SCF/ concurrently activated through KIT and the FcεRI (6, 20). We SA-dependent phosphorylation of AKT, a surrogate marker for therefore examined GSK3b phosphorylation under these con- PI3K activation, in these cells (Fig. 2A,2E). Taken together, these ditions following GSK3b knockdown. GSK3b activity is regu- data suggest that, in HuMCs, GSK3b is active under resting con- ditions and that, upon cell activation through FcεRI and KIT, this lated by the phosphorylation status of Y216 and S9. Y216 is Downloaded from reported to be constitutively phosphorylated in resting cells thus permits an increase in phosphorylation of the GSK3b substrate GS. maintaining GSK3b in an active state (14, 21, 22). However, the These responses can be effectively reduced in the HuMCs following ability of GSK3b to phosphorylate its specific substrates, for ex- shRNA-induced GSK3b knockdown. ample, glycogen synthase (GS), requires priming of the substrate Effect of shRNA-induced knockdown of GSK3b on mast cell by means of prior phosphorylation by an additional kinase (23, cytokine production 24). In resting HuMCs, we similarly observed constitutive phos- http://www.jimmunol.org/ phorylation of Y216 (Fig. 2A,2B). When the cells were activated Having successfully established knockdown of GSK3b activity in through KIT and FcεRI, we observed no consistent increase in the HuMCs, we next investigated the role of GSK3b in mast cell cytokine generation. For these studies, the cells were again cos- timulated through KIT, via SCF, and the FcεRI, through sensi- tizing with biotinylated IgE, and challenging with SA as the response to either stimulus alone is not marked (20). To initially screen the effect of GSK3b knockdown on multiple cytokines, cells were stimulated for an optimal period of 4 h, based on previous kinetic studies (25), then cytokine was by guest on October 2, 2021 determined by a commercially available array. In these studies, we observed a marked increase in mRNA’s for multiple cytokines, including those for GM-CSF, IL-8, and IL-13 (Fig. 3A–C and Supplemental Material). These responses were markedly attenu- ated in GSK3b knockdown cells compared with the control cells (Fig. 3A–C). On the basis of this initial screen, we examined the amounts of GM-CSF and IL-8 protein present in the supernatants of control and GSK3b knockdown HuMCs challenged with SCF in the presence of SA. As demonstrated in Fig. 3D and 3E, there was a marked reduction in SCF/SA-induced generation of GM- CSF and IL-8 in the GSK3b knockdown HuMCs compared with controls. The relative inhibition produced by the two constructs (Fig. 3D,3E) correlated with their relative abilities to knockdown GSK3b protein levels (Fig. 1A). This close correlation was further illustrated by plotting the relative expression of GSK3b protein levels to that of IL-8 production (Fig. 3F). Effect of shRNA-induced knockdown of GSK3b on mast cell chemotaxis In addition to stimulating cytokine generation in mast cells, SCF is FIGURE 1. shRNA-mediated knockdown of GSK3b in quiescent a potent chemotactic agent for HuMCs (4). To thus explore HuMCs. HuMCs transduced with scrambled shRNA (shContr) or shRNA whether SCF-mediated chemotaxis was similarly dependent on for GSK3b (two different constructs; shGSK3b-A and shGSK3b-B). GSK3b, we next examined the relative ability of HuMCs to mi- Whole-cell extracts were prepared and immunoblotted with anti-GSK3b, grate in response to SCF following GSK3b knockdown. From Fig. anti-phospholipase Cg (anti-PLCg ), anti-Lyn, anti-Syk, anti-KIT, and 1 1 3G, it can be seen that GSK3b knockdown HuMCs displayed anti–NF-kB Abs (A and B). FcεRI and KIT surface expression on HuMCs transduced with scrambled shRNA (shContr) or shRNA for GSK3b a reduced capacity to migrate toward SCF compared with control (shGSK3b) analyzed by flow cytometry (C). HuMCs transduced with cells. The extent of inhibition of migration again correlated to the scrambled shRNA (shContr) or shRNA for GSK3b (shGSK3b) stained extent of knockdown of GSK3b observed in these studies, with with toluidine blue (D; original magnifications 340 [upper panels] and GSK3b shRNA B producing a greater knockdown of GSK3b and 3100 [lower panels]) (n =6inA and n = 2–3 in B–D). migration than that produced by GSK3b shRNA A (Fig. 3G). 4 ROLE OF GSK3b IN MAST CELL FUNCTION

FIGURE 2. Knockdown of GSK3b phosphorylation in quiescent and activated HuMCs. HuMCs, transduced with scrambled shRNA (shContr) or shRNA for GSK3b (shGSK3b-B), were sensitized overnight and then stimulated with SA (100 ng/ml) and SCF (30 ng/ml) for 2 min as described in Materials and Methods. Whole- cell extracts were prepared and immunoblotted with anti–p-GSK3b(Y216), anti–p-GS (p-GS(S641)), anti– Downloaded from p-GSK3b(S9), or anti–p-AKT(S473) Abs (A). Protein loading of the samples was normalized by probing for Syk. Data were generated by scanning the blots from three to five independent experiments, and normalized to the response obtained at 2 min with SCF/SA stimu- lation in shContr-transduced HuMCs (B–E)(n = 3–5, ppp , 0.001 and pppp , 0.0001, by Student’s t test). http://www.jimmunol.org/ by guest on October 2, 2021

GSK3b regulates mast cell responses independently of mTOR cells compared with controls (Fig. 4A–E). Furthermore, as discussed We next examined how GSK3b may regulate mast cell cytokine earlier, there was also no difference in the resting or SCF/SA in- production and chemotaxis. GSK3b has been reported to phos- duced, mTORC2-dependent phosphorylation of AKT(S473) in the phorylate the mTOR regulator, tuberin (TSC2), in HEK293T cells GSK3b knockdown HuMCs when compared with control cells (Fig. (26), thereby influencing the activation of the mTORC1 and 2A,2E). Similarly, the mTORC1 inhibitor, rapamycin, failed to mTORC2 cascades. Our previous studies in HuMCs demonstrated block the phosphorylation of GSK3b (S9, Y216) (Fig. 4F) despite that the mTORC1 cascade, downstream of PI3K, contributes to a marked inhibition of GSK3b (S9 only) by the PI3K inhibitor SCF/SA-mediated cytokine production and SCF-mediated chemo- wortmannin. Taken together, these data support the conclusion that taxis in human mast cells (12). The mTORC1 cascade is initiated by GSK3b regulates SCF/SA-induced mast cell responses in- the PI3K-dependent activation of AKT, which phosphorylates and dependently of both the mTORC1 and mTORC2 cascades. We inactivates TSC1 and TSC2 (tuberin), negative regulators of mTOR therefore next examined whether GSK3b regulated human mast cell activation. This results in the sequential phosphorylation and acti- cytokine production and chemotaxis by an alternative mechanism. vation of mTOR and its downstream substrates: the transcriptional b regulators p70 ribosomal S6 kinase (p70S6K) and eukaryotic ini- Regulation of the MAPK activation by GSK3 in human mast tiation factor 4E binding protein 1 (4E-BP1) (27, 28). In contrast, the cells mTORC2 cascade leads to the phosphorylation (S473) and activa- The MAPKs, p38, and JNK, regulate transcriptional activation tion of AKT (29–31). We therefore investigated whether the reduced pathways that contribute to mast cell function, including cytokine cytokine production in the GSK3b knockdown HuMCs may be generation (32). Thus, we next investigated whether the MAPK a consequence of inhibition of the mTORC1- or mTORC2-regulated cascade represented a key intermediary step in the regulation of signaling cascades. GSK3b-regulated mast cell cytokine production. As can be seen As before, GSK3b knockdown significantly reduced GSK3b in Fig. 5A, in contrast to the lack of effect on the mTOR cascades, expression in HuMCs (Fig. 4A,4B). However, there were minimal the increase in both JNK (Fig. 5A,5B) and p-38 (Fig. 5A,5C) differences in the basal and SCF/SA-induced phosphorylation of phosphorylation in response to SCF/SA was reduced in the mTOR, p70S6K, and 4E-BP1 in the GSK3b knockdown HuMCs GSK3b knockdown HuMCs compared with the control treated The Journal of Immunology 5

FIGURE 3. GSK3b regulates SCF/SA-mediated GM-CSF, IL-8 production, and SCF-mediated chemotaxis in HuMCs. HuMCs transduced with scrambled shRNA (shControl) or shRNA for GSK3b (shGSK3b-B) were sensitized overnight and then stimulated with SA (100 ng/ml) and SCF (30 ng/ml) for 4 h. Total RNAwas isolated from the indicated samples, reverse transcribed, and quan- tified by real-time PCR. Relative expression of GM-SCF, IL-8, and IL-13 mRNAwas calculated as described in Materials and Methods (A–C). Data are shown from two independent experiments. HuMCs, transduced with scrambled shRNA (shContr) or shRNA for GSK3b (two different constructs; construct A or B), were sensitized overnight and then stimulated with SA (100 ng/ml) and SCF (30 ng/ml) for 6 h (D and E). Supernatants were collected, and ELISA was performed for hu- man GM-SCF and IL-8 (D and E)(n = 6–9; pp , Downloaded from 0.05, ppp , 0.001, and pppp , 0.0001, by Stu- dent’s t test). The degree of GSK3b knockdown inversely correlates with SCF/SA-mediated IL-8 release (F)(n = 6, Spearman correlation test). The diamond symbol represents the means, and shown SEs, of the values obtained from control cells (n =

6). These data were not included in the statistical http://www.jimmunol.org/ analysis of the correlation. HuMCs, transduced with scrambled shRNA (shContr) or shRNA for GSK3b (two different constructs; construct A or B), were starved for 4 h and then stimulated with SCF (30 ng/ml) for 4 h in a Transwell chemotaxis assay (G)(n = 3–5; ppp , 0.001, by Student’s t test). by guest on October 2, 2021 cells. There was no effect on total p38 or JNK protein levels under be phosphorylated in mast cells, may contribute to mast cell cytokine these conditions (Fig. 5A). The SCF/SA-induced phosphorylation production and other transcriptional-dependent processes. We of MKK3/6, upstream of p38, was however also markedly reduced therefore examined whether GSK3b regulated the phosphorylation in the GSK3b knockdown-HuMCs (Fig 5A,5D). of Jun, ATF2, and NF-kB. As can be seen in Fig. 6, the phosphor- Because the MAPKs JNK and p38 have also been shown to ylation of the AP-1 transcription factors c-Jun (Fig. 6A,6B) and regulate SCF/KIT-induced mast cell migration (33–35), we next ATF2 (Fig. 6A,6C) in response to SCF/SA stimulation was greatly determined the phosphorylation status of these proteins under the reduced in GSK3b knockdown HuMCs compared with control- conditions used for the chemotaxis studies, i.e., stimulation with treated cells. Furthermore, the phosphorylation of the p65 NF-kB SCF after a 4-h period of starvation in SCF-depleted media. As subunit was also significantly reduced in the GSK3b knockdown shown in Fig. 5E–I, SCF-mediated MKK3/6, JNK, and p38 acti- cells compared with control-treated cells (Fig. 6A,6D). Taken to- vation, but not total p38 or JNK protein, were significantly reduced gether, these data support the conclusion that GSK3b activation is in the GSK3b knockdown cells, compared with control-treated cells a prerequisite signal for the MAPK-dependent activation of c-Jun under these conditions. In contrast, SCF-induced AKT activation and ATF2and also NF-kB p65 activity, which in turn regulates SCF/ was unaffected compared with control treated cells (Fig. 5E,5I). SA-induced cytokine production. From these data, we conclude that GSK3b regulates the activation of the MAPKs p38 and JNK, thus providing an explanation for the reduced SCF-induced chemotaxis observed in the GSK3b knock- Discussion down HuMCs. Furthermore, GSK3b-dependent, p38- and JNK- In this study, we present evidence that supports a prerequisite role mediated transcription activation would also provide an explanation for GSK3b in KIT-mediated mast cell chemotaxis and KIT/FcεRI- for the reduced SCF/SA-induced cytokine production of served in mediated enhanced gene expression leading to cytokine pro- the GSK3b knockdown HuMCs. We thus next examined the SCF/ duction in HuMCs. As reported in other cell types (14, 21, 22), in SA-induced phosphorylation of specific transcription factors in the quiescent HuMCs, GSK3b was determined to be constitutively GSK3b knockdown HuMCs. activated. This conclusion was supported by the observed basal phosphorylation of the activating tyrosine residue (Y216) in b Transcription factors regulation by GSK3 GSK3b, the phosphorylation of its substrate GS at S641, and the The MAPK pathway leads to the phosphorylation and, thus, regu- reduction of these phosphorylation states in the GSK3b knock- lation of multiple transcription factors including Jun and ATF2. down cells (Fig 2). Although we did not observe a consistent in- These and potentially other transcription factors, which are known to crease in phosphorylation of GSK3b at the Y216 position in 6 ROLE OF GSK3b IN MAST CELL FUNCTION

FIGURE 4. GSK3b regulates SCF/SA-mediated mast cell responses independently of mTOR. HuMCs, transduced with scrambled shRNA (shContr) or shRNA for GSK3b (shGSK3b-B), were sensitized overnight and then stimulated with SA (100 ng/ml) and SCF (30 ng/ml) for 10 min as described in Ma- terials and Methods. Whole-cell extracts were pre- pared and immunoblotted with anti-GSK3b, anti–p- mTOR(S2448), anti–p-p70S6K(T389), and anti–p- 4E-BP1(T37, T46) Abs (A). Data in B–E were gen- erated by scanning the blots from three to five in- dependent experiments and normalized to the Downloaded from response obtained at 10 min with SCF/SA stimulation (n = 3–5, ppp , 0.001 for comparison with SCF/SA response in shContr-transduced HuMCs by Student’s t test). In F, HuMCs were pretreated with rapamycin (100 nM) or wortmaninn (100 nM) 20 min prior stimulation with SCF or SA at the time indicated. http://www.jimmunol.org/ Whole-cell extracts were prepared and immuno- blotted with anti–p-AKT(S473), anti–p-GSK3b (Y216), anti–p-GSK3b(S9), or anti–p-GS(S641) Abs. Protein loading of the samples was normalized by probing for Syk. by guest on October 2, 2021

response to SCF and/or SA, under the conditions used to examine may occur latently to the constitutive activation. We have pre- chemotaxis and cytokine production, there was an apparent in- viously demonstrated that PI3K, and signals dependent on PI3K crease in the phosphorylation of GS at S641 under these con- activity, are delayed responses compared with other signals initi- ditions, which was reduced in the GSK3b knockdown. The ated upon FcεRI or KIT activation (12). Thus, it is likely that any constitutive Y216 phosphorylation of GSK3b may be due to the response due to downregulating GSK3b activity would be chro- cells being maintained in SCF. Indeed, when the cells were starved nologically secondary to those regulated by GSK3b activation. of SCF for a prolonged period of time (overnight) prior to stim- Nevertheless, these data do suggest that the ability of GSK3b to ulation, we were able to observe an SCF-dependent increase in the phosphorylate its substrates may depend on the net balance be- phosphorylation of this residue. Thus, the phosphorylation of this tween positive and negative regulation of GSK3b activity. residue may be directly dependent on KIT. Regardless, our results The marked reduction in the ability of SCF/SA to enhance IL-8, suggest that triggering of mast cells through KIT and/or FcεRI IL-13, and GM-CSF mRNA levels and IL-8 and GM-CSF secre- facilitates the ability of GSK3b to phosphorylate its substrate(s) tion, associated with the diminution of GSK3b activity in the without necessarily increasing its constitutive activity, a potential GSK3b knockdown HuMCs (Fig 3F), strongly supports a re- mechanism of action that is elaborated upon below. quirement for GSK3b activity in the regulation of KIT/FcεRI- In addition to the phosphorylation of (Y216) in GSK3b,how- mediated cytokine production. This conclusion is further sup- ever, we observed that the inhibitory S9 residue GSK3b was also ported by the close statistical correlation between the degree of phosphorylated in a PI3K-dependent manner following SCF/ SA GSK3b knockdown and IL-8 secretion. Similarly, the close cor- challenge (Figs. 2D,4F). This phenomenon has also been reported relation between GSK3b knockdown and reduction in SCF- in monocytes, dendritic cells, and T cells, following exposure to induced chemotaxis in the GSK3b knockdown HuMCs also pro- TLR2, TLR4, TLR5, and TLR9 agonists, Escherichia coli, and vides evidence for a prerequisite role for GSK3b in the SCF-in- viral respectively (36–38). In our study, however, the duced chemotactic response. observed increased phosphorylation of GS, at least at early time There are conflicting reports regarding the role of GSK3b in points, would suggest that downregulation of GSK3b activation cytokine production in other cells of hematopoietic lineage. The Journal of Immunology 7

FIGURE 5. GSK3b regulates SCF/SA- and SCF-mediated MAPK activity in HuMCs. HuMCs, transduced with scrambled shRNA (shContr) or shRNA for GSK3b (shGSK3b-B), were sensitized overnight and then stimulated with SA (100 ng/ml) and SCF (30 ng/ml) (A) or starved for 4 h then stimulated with SCF (30 ng/ml) (E) for 2 min as described in Materials and Methods. Whole-cell extracts were prepared and im- munoblotted (A and E) with anti–p-JNK (T183, Y185), total JNK, anti–p-MKK3/6 (S189, S207), anti–p-p38 (T180), total p-38, or p-AKT (S473) Abs. Protein loading of the samples was normalized by probing for Syk (A and E). Data in B–D and F–I were gen- Downloaded from erated by scanning the blots in three to five independent experiments and normalized to the response obtained with SCF/SA stimu- lation (B–D), or SCF response (F–I), in shContr-transduced HuMCs (n = 3–5; pp , 0.05, ppp , 0.001 and pppp , 0.0001, by http://www.jimmunol.org/ Student’s t test).

Treatment of monocytes with GSK3b inhibitors such as LiCl and/ GSK3b inhibitors were observed to enhance viral peptide-induced or SB216763, or with GSK3b-targeted small interfering RNA, has IL-2 production, whereas overexpression of GSK3b in T cells by guest on October 2, 2021 been reported to inhibit TLR2-, 4-, 5-, and 9-dependent release of downregulated the response (38). This apparent dichotomy in the IL-1b, IL-6, TNF-a, IL-12, and IFN-g but to enhance TLR- GSK3b-dependent regulation of cytokine generation in the vari- dependent production of IL-10 (36). GSK3b inhibitors were also ous cell types may reflect the potential for GSK3b to both nega- reported to inhibit E. coli-induced IL-12, IL-6, and TNF-a, but not tively and positively regulate transcriptional signaling pathways IL-10, release from dendritic cells (37). In contrast, in T cells, for cytokine production. Indeed, it is possible that, in addition to

FIGURE 6. GSK3b regulates SCF/SA-mediated AP1 transcription factors and NF-kB activity in HuMCs. HuMCs, transduced with scrambled shRNA (shContr) or shRNA for GSK3b (shGSK3b- B), were sensitized overnight and then stimulated with SA (100 ng/ml) and SCF (30 ng/ml) for 30 min as described in materials and methods. Whole-cell extracts were prepared and immunoblotted with anti–p-cJun (S73), total cJun, anti–p-ATF2(T71), or anti–p-NF-kB(S536) Abs (A). Protein loading of the samples was normalized by probing for Syk (A). Data in B–D were generated by scanning in three to four independent experiments and normalizing to the responses obtained with SA/SCF stimulation in shContr-transduced HuMCs (pp , 0.05, ppp , 0.001, and pppp , 0.0001, by Student’s t test). 8 ROLE OF GSK3b IN MAST CELL FUNCTION regulating positive signals, negative signaling pathways may also previously described to regulate mast cell chemotaxis (33–35). be regulated by GSK3b in mast cells. In this respect, it has been Therefore, the reduced SCF/SA-induced cytokine production and suggested that the ability of AKT to enhance cytokine generation SCF-induced chemotaxis observed in the GSK3b knockdown through NF-AT activation in mouse mast cells may be due to HuMCs may be explained by defective JNK and p38 signaling in downregulation of GSK3b activity (39). Whether this may also be these cells (Fig. 5A–D). true for HuMCs is unclear from the current study; however, the How GSK3b may act as a prerequisite signal for the regulation of induced phosphorylation of the inhibitory GSK3b S9 residue in these pathways may be explained by the unique manner in which response to SCF/SA in HuMCs was reduced by the PI3K inhibitor GSKb phosphorylates its substrates. As discussed, GSK3b sub- wortmannin (Fig. 4F). strates require prior phosphorylation by a secondary kinase at amino There has emerged no common mechanistic explanation as to acids 4–5 COOH-termini to the GSK3b phosphorylation sites for how GSK3b may be exerting its regulatory influence on cytokine optimal GSK3b-mediated phosphorylation. Thus, although GSK3b generation and other processes in hematopoietic cells. As we have is active in resting conditions, it cannot optimally phosphorylate its previously demonstrated that the mTORC1 cascade contributes to substrates, until upon FcεRI or KIT kinase activation, the GSK3b KIT/FcεRI-mediated cytokine production and KIT-mediated mast substrates become phosphorylated as a consequence of the activa- cell chemotaxis (12), and as GSK3b has been proposed to regulate tion of one of the kinases downstream of these receptors. This would the mTOR pathway through phosphorylation of tuberin (26), the then allow GSK3b to optimally phosphorylate its target signaling scenario existed that, in the HuMCs, GSK3b may be acting via proteins and hence transduce the signals required for gene expres- regulation of mTOR pathways. However, the observations that the sion leading to cytokine production and the processes required for KIT/FcεRI-mediated phosphorylation of components of the chemotaxis (Fig. 7). Of potential relevance may be the presence of mTORC1 and mTORC2 cascades was not reduced in the GSK3b two highly conserved SxxxS/T sequences in MKK3 and MKK6, Downloaded from knockdown HuMCs (Fig. 4), argues against this possibility. It has which are responsible for the phosphorylation and activation of p38, been proposed, that the contrasting roles for GSK3b in TLR cy- and in MKK4 and MKK7, which are responsible for the phos- tokine production in monocytes may be explained by opposing phorylation and activation of JNK. Multiple such sequences are also regulation of the transcription factors CREB and NF-kB through found in MEK kinase 1 and MEK kinase 4, upstream kinases of competition for binding to a common coactivator protein CREB MKK4 and MKK7. Thus, phosphorylation of these sites by GSK3b binding protein (36). According to this model, GSK3b inhibition following initial phosphorylation by a “priming” serine/threonine http://www.jimmunol.org/ would increase CREB activation allowing CREB to compete with kinase may provide a mechanism by which constitutive activation of the p65 subunit of NF-kB for binding to CREB binding protein. In GSK3b may regulate the activation of p38 and JNK and subsequent our present study, however, although SCF/SA-induced phosphor- downstream transcription factors. In support of this conclusion, we ylation of the p65 subunit of NF-ĸB was observed to be signifi- observed that SCF and SA/SCF-mediated MKK3/6 phosphorylation cantly reduced in the GSK3b knockdown HuMCs (Fig. 6 A,6D), was markedly reduced in the GSK3b knockdown HuMCs (Fig. 5). we did not consistently observe an increase in CREB activity in In summary, in this study, we have presented evidence to support these cells (data not shown). Regardless, these data are in agree- the conclusion that GSK3b is a prerequisite signal for KIT- ment with other studies showing that GSK3b is required for NF- mediated chemotaxis and KIT/FcεRI-mediated cytokine pro- by guest on October 2, 2021 kB activation (19). duction in human mast cells. The regulation of cytokine genera- The most remarkable defects that we observed, however, in the tion by GSK3b could be explained by the differential regulation of GSK3b knockdown HuMCs were in the p38 and JNK MAPK transcriptional control downstream of JNK and p38, as well as pathways and, particularly, in the respective downstream tran- transcriptional control of NF-kB p65 subunit, whereas the regu- scription factors ATF2 and c-Jun. JNK activity has previously lation of the chemotactic response by GSK3b may be explained been shown to regulate cytokine production mediated by AP1 by its modulation of JNK- and p38-dependent pathways. As with transcription factors in both mouse bone marrow-derived mast other cells types, it is, however, possible that as-yet undefined cells (32) and HuMCs (6) Similarly, both JNK and p38 have been inhibitory pathways both regulating GSK3b activation and

FIGURE 7. Potential model by which constitutively activated GSK3b may regulate HuMC cytokine pro- duction and chemotaxis. Under resting conditions, GSK3b is constitutively active, because of phosphor- ylation of the Y216 residue, but is unable to optimally phosphorylate its substrates as they require initial phosphorylation by a “priming” kinase to allow these reactions to take place. Upon SCF/SA challenge, GSK3b substrates are phosphorylated by priming kinases, thus allowing GSK3b to phosphorylate and activate these substrates. This leads to activation of the JNK and p38 MAPK pathways, thereby initiation of to chemotaxis, and downstream transcription factors and NF-kB leading to cytokine production. GSK3b activity is terminated upon phosphorylation at the S9 position as a consequence of activation of PI3K. On the basis of other reports, it is possible that GSK3b may also reg- ulate an inhibitory pathway for NF-AT activation (39), leading to cytokine production. 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SABiosciences PCR Array Catalog #: PAHS-021 SA+ SCF 4h stimulaton AVG(Ct) Position Unigene Refseq Symbol Description shContr shContr-4h shGSKβ A01 Hs.1274 NM_006129 BMP1 Bone morphogenetic protein 1 25.25 25.69 26.75 A02 Hs.73853 NM_001200 BMP2 Bone morphogenetic protein 2 36.25 34.87 36.11 A03 Hs.387411 NM_001201 BMP3 Bone morphogenetic protein 3 (osteogenic) A04 Hs.68879 NM_130851 BMP4 Bone morphogenetic protein 4 33.27 34.49 34.92 A05 Hs.296648 NM_021073 BMP5 Bone morphogenetic protein 5 A06 Hs.285671 NM_001718 BMP6 Bone morphogenetic protein 6 29.57 31.27 32.99 A07 Hs.473163 NM_001719 BMP7 Bone morphogenetic protein 7 (osteogenic protein 1) 36.23 A08 Hs.664022 NM_001720 BMP8B Bone morphogenetic protein 8b (osteogenic protein 2) 24.07 24.66 25.81 A09 Hs.591402 NM_000757 CSF1 Colony stimulating factor 1 (macrophage) 24.61 24.43 25.63 A10 Hs.1349 NM_000758 CSF2 Colony stimulating factor 2 (granulocyte-macrophage) 29.59 25.31 36.29 A11 Hs.473877 NM_058186 FAM3B Family with sequence similarity 3, member B 33.85 34.32 A12 Hs.2007 NM_000639 FASLG (TNF superfamily, member 6) 29.93 29.28 31.97 B01 Hs.11392 NM_004469 FIGF C-fos induced growth factor 31.92 32.64 33.51 B02 Hs.2171 NM_004962 GDF10 Growth differentiation factor 10 33.44 B03 Hs.643604 NM_005811 GDF11 Growth differentiation factor 11 23.90 23.63 26.18 B04 Hs.279463 NM_016204 GDF2 Growth differentiation factor 2 34.69 B05 Hs.86232 NM_020634 GDF3 Growth differentiation factor 3 24.96 25.43 29.32 B06 Hs.1573 NM_000557 GDF5 Growth differentiation factor 5 34.01 B07 Hs.41565 NM_005259 MSTN Myostatin 34.45 36.88 35.43 B08 Hs.25022 NM_005260 GDF9 Growth differentiation factor 9 29.51 30.68 31.83 B09 Hs.37026 NM_024013 IFNA1 , alpha 1 B10 Hs.211575 NM_000605 IFNA2 Interferon, alpha 2 37.45 B11 Hs.1510 NM_021068 IFNA4 Interferon, alpha 4 B12 Hs.37113 NM_002169 IFNA5 Interferon, alpha 5 35.16 38.61 C01 Hs.73890 NM_002170 IFNA8 Interferon, alpha 8 C02 Hs.93177 NM_002176 IFNB1 Interferon, beta 1, fibroblast 35.80 36.77 C03 Hs.856 NM_000619 IFNG Interferon, gamma 36.33 36.12 C04 Hs.591083 NM_020124 IFNK Interferon, kappa 36.21 33.74 35.81 C05 Hs.193717 NM_000572 IL10 20.49 21.32 25.36 C06 Hs.467304 NM_000641 IL11 C07 Hs.673 NM_000882 IL12A Interleukin 12A 28.68 29.04 30.40 C08 Hs.674 NM_002187 IL12B Interleukin 12B 38.02 C09 Hs.845 NM_002188 IL13 32.54 30.41 37.36 C10 Hs.699306 NM_175852 TXLNA Taxilin alpha 22.41 22.51 24.15 C11 Hs.654378 NM_000585 IL15 36.38 35.17 34.20 C12 Hs.459095 NM_004513 IL16 ( chemoattractant factor) 23.55 24.10 25.60 D01 Hs.41724 NM_002190 IL17A Interleukin 17A D02 Hs.156979 NM_014443 IL17B Interleukin 17B 38.35 39.39 D03 Hs.278911 NM_013278 IL17C Interleukin 17C 32.08 32.40 34.22 D04 Hs.302036 NM_022789 IL25 D05 Hs.83077 NM_001562 IL18 (interferon-gamma-inducing factor) 20.69 21.38 23.58 D06 Hs.661017 NM_013371 IL19 Interleukin 19 27.09 26.92 29.47 D07 Hs.1722 NM_000575 IL1A Interleukin 1, alpha 24.26 24.91 29.53 D08 Hs.126256 NM_000576 IL1B Interleukin 1, beta 29.21 30.39 32.07 D09 Hs.306974 NM_173161 IL1F10 Interleukin 1 family, member 10 (theta) D10 Hs.516301 NM_012275 IL1F5 Interleukin 1 family, member 5 (delta) D11 Hs.278910 NM_014440 IL1F6 Interleukin 1 family, member 6 (epsilon) D12 Hs.166371 NM_173205 IL1F7 Interleukin 1 family, member 7 (zeta) 33.26 32.97 38.34 E01 Hs.278909 NM_173178 IL1F8 Interleukin 1 family, member 8 (eta) E02 Hs.211238 NM_019618 IL1F9 Interleukin 1 family, member 9 E03 Hs.89679 NM_000586 IL2 E04 Hs.272373 NM_018724 IL20 32.74 32.56 34.63 E05 Hs.567559 NM_021803 IL21 E06 Hs.287369 NM_020525 IL22 36.03 35.46 34.74 E07 Hs.411311 NM_006850 IL24 29.30 29.49 32.13 E08 Hs.694 NM_000588 IL3 (colony-stimulating factor, multiple) 35.04 30.69 E09 Hs.73917 NM_000589 IL4 33.37 35.13 36.57 E10 Hs.2247 NM_000879 IL5 Interleukin 5 (colony-stimulating factor, eosinophil) 33.28 32.50 34.28 E11 Hs.654458 NM_000600 IL6 (interferon, beta 2) 36.10 37.95 E12 Hs.591873 NM_000880 IL7 28.98 30.25 32.22 F01 Hs.624 NM_000584 IL8 29.35 27.13 33.85 F02 Hs.960 NM_000590 IL9 F03 Hs.407506 NM_002191 INHA Inhibin, alpha 35.82 F04 Hs.583348 NM_002192 INHBA Inhibin, beta A 30.09 29.86 30.82 F05 Hs.520187 NM_003240 LEFTY2 Left-right determination factor 2 37.36 36.90 37.43 F06 Hs.36 NM_000595 LTA alpha (TNF superfamily, member 1) 28.24 29.08 29.93 F07 Hs.376208 NM_002341 LTB (TNF superfamily, member 3) 27.39 28.92 29.29 F08 Hs.370414 NM_018055 NODAL Nodal homolog (mouse) 33.44 32.95 35.62 F09 Hs.707991 NM_002607 PDGFA Platelet-derived growth factor alpha polypeptide 23.95 23.83 25.47 F10 Hs.170009 NM_003236 TGFA Transforming growth factor, alpha 21.51 22.08 24.87 F11 Hs.645227 NM_000660 TGFB1 Transforming growth factor, beta 1 19.89 19.77 21.95 F12 Hs.133379 NM_003238 TGFB2 Transforming growth factor, beta 2 30.58 32.09 32.52 G01 Hs.592317 NM_003239 TGFB3 Transforming growth factor, beta 3 27.37 27.55 28.78 G02 Hs.241570 NM_000594 TNF (TNF superfamily, member 2) 26.94 27.66 28.63 G03 Hs.81791 NM_002546 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b G04 Hs.478275 NM_003810 TNFSF10 Tumor necrosis factor (ligand) superfamily, member 10 18.80 19.31 19.19 G05 Hs.333791 NM_003701 TNFSF11 Tumor necrosis factor (ligand) superfamily, member 11 29.71 30.01 34.45 G06 Hs.415839 NM_003809 TNFSF12 Tumor necrosis factor (ligand) superfamily, member 12 23.84 24.58 27.07 G07 Hs.54673 NM_003808 TNFSF13 Tumor necrosis factor (ligand) superfamily, member 13 26.26 27.03 28.66 G08 Hs.525157 NM_006573 TNFSF13B Tumor necrosis factor (ligand) superfamily, member 13b 29.68 30.28 32.57 G09 Hs.129708 NM_003807 TNFSF14 Tumor necrosis factor (ligand) superfamily, member 14 30.38 29.83 32.77 G10 Hs.181097 NM_003326 TNFSF4 Tumor necrosis factor (ligand) superfamily, member 4 24.88 26.31 30.07 G11 Hs.501497 NM_001252 CD70 CD70 molecule 30.79 31.06 31.97 G12 Hs.654445 NM_001244 TNFSF8 Tumor necrosis factor (ligand) superfamily, member 8 35.83 36.19 39.37 H01(HKG) Hs.534255 NM_004048 B2M Beta-2-microglobulin 15.87 16.11 17.75 H02(HKG) Hs.412707 NM_000194 HPRT1 Hypoxanthine phosphoribosyltransferase 1 21.22 21.69 24.44 H03(HKG) Hs.523185 NM_012423 RPL13A Ribosomal protein L13a 18.17 18.49 19.57 H04(HKG) Hs.544577 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 16.85 17.21 18.60 H05(HKG) Hs.520640 NM_001101 ACTB Actin, beta 15.79 16.39 18.44 H06 N/A SA_00105 HGDC Human Genomic DNA Contamination H07 N/A SA_00104 RTC Reverse Transcription Control 21.63 22.04 22.33 H08 N/A SA_00104 RTC Reverse Transcription Control 21.58 22.01 22.38 H09 N/A SA_00104 RTC Reverse Transcription Control 21.63 22.00 22.52 H10 N/A SA_00103 PPC Positive PCR Control 18.11 18.46 18.39 H11 N/A SA_00103 PPC Positive PCR Control 18.52 18.88 18.85 H12 N/A SA_00103 PPC Positive PCR Control 18.28 18.49 18.43 SA+ SCF 4h stimulaton AVG Delta(Ct) Fold Change (Ct(sample) - Ave Ct(HKG)) (comparing to shCont w/o stimulation) shGSKβ-4h shContr shContr-4h shGSKβ shGSKβ-4h shContr shContr-4hshGSKβ shGSKβ-4h 27.73 7.67 7.71 6.99 6.99 1.00 0.97 1.60 1.61 38.14 17.42 16.89 15.24 14.26 1.00 1.44 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 33.33 15.69 16.51 15.16 12.59 1.00 0.56 1.44 8.57 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 34.64 11.99 13.29 13.23 13.90 1.00 0.41 0.42 0.27 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 26.77 6.49 6.68 6.05 6.03 1.00 0.88 1.36 1.38 26.54 7.03 6.45 5.87 5.80 1.00 1.49 2.24 2.34 32.94 12.01 7.33 15.24 12.20 1.00 25.63 0.11 0.87 16.27 16.34 15.24 14.26 1.00 0.95 2.04 4.02 33.35 12.35 11.30 12.21 12.61 1.00 2.07 1.10 0.83 33.85 14.34 14.66 13.75 13.11 1.00 0.80 1.50 2.34 17.42 15.46 15.24 14.26 1.00 3.89 4.54 8.94 27.10 6.32 5.65 6.41 6.36 1.00 1.59 0.93 0.97 17.11 17.02 15.24 14.26 1.00 1.06 3.65 7.20 30.89 7.38 7.45 9.56 10.14 1.00 0.95 0.22 0.15 16.43 17.02 15.24 14.26 1.00 0.67 2.29 4.51 37.03 16.87 17.02 15.24 14.26 1.00 0.90 3.09 6.09 32.41 11.93 12.70 12.07 11.67 1.00 0.59 0.91 1.20 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 39.10 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 15.76 15.24 14.26 1.00 3.17 4.54 8.94 27.42 2.91 3.34 5.60 6.68 1.00 0.74 0.16 0.07 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 31.96 11.10 11.06 10.63 11.21 1.00 1.03 1.38 0.92 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 36.69 14.96 12.43 15.24 14.26 1.00 5.75 0.82 1.62 24.72 4.83 4.53 4.39 3.98 1.00 1.23 1.36 1.81 17.42 17.02 14.44 14.26 1.00 1.32 7.87 8.94 26.02 5.97 6.12 5.84 5.28 1.00 0.90 1.10 1.62 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 35.57 14.50 14.42 14.46 14.26 1.00 1.06 1.03 1.18 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 24.79 3.11 3.40 3.82 4.05 1.00 0.82 0.61 0.52 30.24 9.51 8.94 9.70 9.50 1.00 1.49 0.87 1.01 31.26 6.68 6.93 9.77 10.52 1.00 0.84 0.12 0.07 35.90 11.63 12.41 12.31 14.26 1.00 0.58 0.63 0.16 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 34.00 15.68 14.99 15.24 13.26 1.00 1.61 1.35 5.33 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 15.16 14.58 14.87 14.26 1.00 1.49 1.22 1.87 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 36.40 17.42 17.02 14.98 14.26 1.00 1.32 5.43 8.94 32.46 11.72 11.51 12.36 11.72 1.00 1.16 0.64 1.00 17.42 12.71 15.24 14.26 1.00 26.10 4.54 8.94 15.79 17.02 15.24 14.26 1.00 0.43 1.47 2.90 39.15 15.70 14.52 14.52 14.26 1.00 2.25 2.27 2.71 36.11 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 34.08 11.39 12.27 12.46 13.34 1.00 0.54 0.48 0.26 32.94 11.77 9.15 14.09 12.20 1.00 6.13 0.20 0.74 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 34.63 17.42 17.02 15.24 13.89 1.00 1.32 4.54 11.53 32.15 12.50 11.88 11.06 11.41 1.00 1.54 2.73 2.14 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 30.05 10.66 11.11 10.17 9.30 1.00 0.73 1.40 2.55 30.01 9.81 10.94 9.53 9.27 1.00 0.46 1.22 1.46 37.41 15.86 14.97 15.24 14.26 1.00 1.86 1.54 3.04 25.42 6.37 5.85 5.71 4.68 1.00 1.44 1.59 3.23 25.23 3.93 4.10 5.11 4.49 1.00 0.89 0.44 0.68 22.01 2.31 1.79 2.19 1.27 1.00 1.43 1.09 2.06 32.84 13.00 14.11 12.76 12.10 1.00 0.46 1.18 1.86 29.05 9.79 9.57 9.02 8.31 1.00 1.16 1.71 2.79 29.29 9.36 9.68 8.87 8.55 1.00 0.81 1.41 1.76 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 19.66 1.22 1.33 -0.57 -1.08 1.00 0.93 3.47 4.94 37.05 12.13 12.03 14.69 14.26 1.00 1.07 0.17 0.23 27.86 6.26 6.60 7.31 7.12 1.00 0.79 0.48 0.55 29.67 8.68 9.05 8.90 8.92 1.00 0.77 0.86 0.84 34.03 12.10 12.30 12.81 13.29 1.00 0.87 0.61 0.44 32.14 12.80 11.85 13.01 11.39 1.00 1.93 0.87 2.65 31.94 7.30 8.33 10.30 11.20 1.00 0.49 0.12 0.07 32.06 13.21 13.08 12.21 11.32 1.00 1.10 2.00 3.72 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 18.55 -1.71 -1.87 -2.01 -2.19 1.00 1.11 1.23 1.40 25.67 3.64 3.71 4.68 4.93 1.00 0.95 0.49 0.41 20.13 0.59 0.51 -0.19 -0.61 1.00 1.06 1.71 2.30 19.28 -0.73 -0.77 -1.17 -1.46 1.00 1.02 1.35 1.66 20.08 -1.79 -1.59 -1.32 -0.66 1.00 0.87 0.72 0.46 17.42 17.02 15.24 14.26 1.00 1.32 4.54 8.94 22.57 4.05 4.06 2.57 1.83 1.00 1.00 2.80 4.68 22.62 4.00 4.03 2.62 1.88 1.00 0.98 2.61 4.37 22.60 4.05 4.02 2.76 1.86 1.00 1.02 2.45 4.55 19.26 0.53 0.48 -1.37 -1.48 1.00 1.03 3.73 4.03 19.71 0.94 0.90 -0.91 -1.03 1.00 1.02 3.60 3.91 18.22 0.70 0.51 -1.33 -2.52 1.00 1.15 4.10 9.38