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Akt-dependent cytokine production in mast cells.

Cross-linking of FcεRI induces the activation of three protein tyrosine kinases, Lyn, Syk, and Bruton's tyrosine kinase (Btk), leading to the secretion of a panel of proinflammatory mediators from mast cells. This study showed phosphorylation at Ser-473 and enzymatic activation of Akt/protein kinase B, the crucial survival kinase, upon FcεRI stimulation in mouse mast cells. Phosphorylation of Akt is regulated positively by Btk and Syk and negatively by Lyn. Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-α promoters. Transcription from the nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1) sites within these promoters is under the control of Akt activity. Accordingly, the signaling pathway involving IκB-α, a cytoplasmic protein that binds NF-κB and inhibits its nuclear translocation, appears to be regulated by Akt in mast cells. Catalytic activity of glycogen synthase kinase (GSK)-3β, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt. Importantly, Akt regulates the production and secretion of IL-2 and TNF-α in FcεRI-stimulated mast cells. Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-κB, NF-AT, and AP-1 that contributes to the production of cytokines.

In this study, we found that Akt is activated upon crosslinking of Fc ⑀ RI in mast cells. Using primary cultured mast cells and mast cell lines deficient in PTK critical for mast cell activation, we investigated the requirements for Akt activation. Downstream of Akt activation, the signaling pathways to transcriptional activation of cytokine genes were analyzed. Importantly, we revealed that Akt is critically involved in cytokine production induced by Fc ⑀ RI stimulation.
Rat basophilic leukemia (RBL)-2H3 is a rat mast cell line used extensively for studies on Fc ⑀ RI signal transduction. A Syk-deficient RBL-2H3 variant and its syk cDNA-transfected cell line (63) were provided by Dr. Reuben P. Siraganian (National Institutes of Health, Bethesda, MD).
Retroviral Transfection. Hemagglutinin (HA)-tagged Akt cDNAs were recloned into the EcoRI and XhoI sites of the retroviral vector pMX-puro (38). These plasmids were transfected into BOSC-23 packaging cells with Lipofectamine (Life Technologies) to generate recombinant retroviruses. BMMCs were infected with the retroviruses in the presence of 10 g/ml poly-brene, and drug selection was started 48 h after infection. Mass populations of puromycin-resistant cells were grown and then cultured in the absence of selection drug for 48 h before Fc ⑀ RI stimulation.
Measurements of Secreted Cytokines. For cytokine measurements, mast cells were stimulated in BMMC medium instead of Tyrode buffer. TNF-␣ and IL-2 secreted into the culture medium for 20 h were measured by ELISA kits (Endogen).
Immunoblotting and Immunoprecipitation. Cells were lysed in ice-cold 1% NP-40-containing lysis buffer (20 mM Tris-HCl [pH 8.0], 0.15 M NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 25 M p-nitrophenyl pЈ-guanidinobenzoate, 1 M pepstatin, and 0.1% sodium azide) immediately after stimulation. Lysates were centrifuged in an Eppendorf microcentrifuge at 4ЊC for 10 min. Protein concentrations were measured using D C protein assay reagents (Bio-Rad Laboratories). Cleared lysates were either directly analyzed by SDS-PAGE or immunoprecipitated before SDS-PAGE analysis. For immunoprecipitation, lysates were incubated on ice with an appropriate antibody for 2-4 h, and immune complexes were recovered by brief centrifugation after another 30-min incubation with Pansorbin for rabbit polyclonal antibodies or anti-mouse immunoglobulinconjugated agarose (Sigma-Aldrich) for mouse mAbs. Immune complexes were washed in lysis buffer four times before SDS-PAGE analysis. Proteins separated by SDS-PAGE were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (NEN Life Science Products). Membranes were blocked, then incubated consecutively with primary antibody and horseradish peroxidase-conjugated secondary antibody, and immunoreactive proteins were visualized by enhanced chemiluminescence reagents (NEN Life Science Products).

Results
Akt Activation by Fc⑀RI Cross-Linking in Mast Cells. Because Akt is activated by numerous stimuli, we investigated whether Fc⑀RI cross-linking induces Akt activation. In situ Akt activity was monitored before and after cell stimulation by immunoblotting with a phosphospecific antibody to the phosphorylated Ser-473 of Akt. Ser-473 phosphorylation is Cells were lysed, and lysates were analyzed by SDS-PAGE followed by immunoblotting with antiphospho-Akt antibody that specifically recognizes the phosphorylated Ser-473 residue and its flanking sequence. The same blot was reprobed with anti-Akt antibody (left). Shown is the result representative of at least five independent experiments. Cell lysates were immunoprecipitated with anti-Akt, and immune complexes were subjected to kinase assays using histone H2B as an exogenous substrate (right). The kinase result is representative of three similar experiments. Stim., stimulation. (B) IgE-sensitized BMMCs were pretreated with the indicated concentrations of genistein, wortmannin, or LY294002 for 15 min before antigen (100 ng/ml DNP-HSA) stimulation (Stim.) for 10 min. Cell lysates were analyzed for Ser-473 phosphorylation as above.
crucial for Akt activation. Antigen treatment of IgE-primed BMMCs caused a remarkable phosphorylation of Ser-473 at its peak ‫01-3ف‬ min after antigen stimulation (Fig. 1 A,  left). Enzymatic activation of Akt in a time course similar to that of Ser-473 phosphorylation was shown in in vitro kinase assays on anti-Akt immunoprecipitates using histone H2B as an exogenous substrate (Fig. 1 A, right). Dependence of Fc⑀RI-induced Akt activation on PTK was revealed by pretreatment of BMMCs with genistein, similar to BCR-induced Akt activation (64,72,73). PI3K inhibitors wortmannin and LY294002 also blocked Akt activation very efficiently, confirming the PI3K dependence of Akt activation (Fig. 1 B). These results are consistent with PI3K activation induced by the engagement of Fc⑀RI and other related receptors that induces activation of several PTKs as well (74)(75)(76)(77).
Hyperphosphorylation of Akt in Lyn-deficient Cells and Reduced Akt Phosphorylation in Btk-and Syk-deficient Cells. PTK dependence of Akt phosphorylation induced by Fc⑀RI cross-linking was further studied using BMMCs derived from lyn Ϫ/Ϫ and btk Ϫ/Ϫ mice and a Syk-deficient variant of RBL-2H3 rat mast cells. Strikingly, lyn Ϫ/Ϫ cells showed a severalfold more robust and prolonged Akt Ser-473 phosphorylation than wt cells (Fig. 2 A). In contrast, btk Ϫ/Ϫ mast cells exhibited a reduced phosphorylation of Akt compared to wt cells (Fig. 2 B). btk Ϫ/Ϫ lyn Ϫ/Ϫ double-mutant cells showed a prolonged, but not stronger, phosphorylation compared with wt cells (Fig. 2 A), indicating that the enhanced Akt phosphorylation in lyn Ϫ/Ϫ cells is dependent on Btk. On the other hand, wt RBL-2H3 cells exhibited high basal levels of Akt Ser-473 phosphorylation and further induction upon Fc⑀RI stimulation (Fig. 2 C), consistent with the presence of active mutations of c-Kit, the receptor for stem cell factor (SCF), in this and other mast cell lines (78). But basal and induced levels of Akt phosphorylation were significantly reduced in Syk-deficient RBL-2H3 cells and enhanced in Syk-deficient cells transfected with wt syk cDNA (Fig. 2 C). These results demonstrate that Akt activity is regulated positively by Syk and Btk and negatively by Lyn in mast cells.
Regulation of IL-2 and TNF-␣ Promoters by Akt. One of the cardinal features of Fc⑀RI-induced mast cell activation is the production and secretion of various cytokines including IL-2 and TNF-␣, at least partly through the transcriptional activation of these cytokine genes. Our recent studies indicate that this aspect of mast cell activation is exaggerated in lyn Ϫ/Ϫ cells (79), and Btk is required for optimal production of these cytokines (80). Given the Akt hyperphosphorylation in lyn Ϫ/Ϫ cells and the Akt hypophosphorylation in btk Ϫ/Ϫ cells, we examined whether Akt activity is involved in cytokine gene expression. To this end, we cotransfected BMMCs with wt or DN Akt cDNA expression vectors together with IL-2/luc or TNF-␣/luc reporter plasmids (Fig. 3). Overexpression of wt Akt enhanced twofold induction of IL-2 promoter-driven luciferase expression over the vector-transfected cells upon Fc⑀RI cross-linking. Fc⑀RI-induced transcriptional activation was almost abrogated by two different DN Akt mutants. Similar results were obtained with TNF-␣/luc. Given these results, together with higher transcriptional activity of IL-2 and TNF-␣ promoters (Figure 8 in reference 79) and higher production of these cytokines ( Figure  7 in reference 79) in lyn Ϫ/Ϫ mast cells compared with wt  cells, higher cytokine production in Fc⑀RI-stimulated lyn Ϫ/Ϫ cells may be accounted for at least partly by the higher activation levels of Akt via the transcriptional regulation. Indeed, overexpression of wt Akt in lyn Ϫ/Ϫ cells enhanced the transcriptional activity of IL-2 and TNF-␣ promoters more than that in wt cells, and DN Akt inhibited it (data not shown).
Transcription Factor NF-B Is under the Control of Akt. Expression of IL-2 and TNF-␣ genes requires several transcription factors, including NF-B, AP-1, and NF-AT (65). Therefore, we examined whether the activity of these transcription factors is regulated by Akt in mast cells. First, the luciferase gene under the control of multiple copies of the NF-B site (Ϫ206 to Ϫ202, eight times) of the murine IL-2 promoter was cotransfected into BMMCs with wt, E40K, or DN Akt (Fig. 4 A). wt Akt transfection doubled NF-B-dependent transcription before stimulation, whereas Fc⑀RI stimulation did not enhance it any further. Constitutively active E40K mutant transfectants exhibited a further enhancement in basal and induced levels of NF-B activity. Importantly, these activities were strongly inhibited by DN Akt. Because of the possible general negative effect of DN Akt expression, we evaluated whether DN Akt induces apoptosis in BMMCs. Because the efficiency of transient transfection used in the above experiments was too low (Ͻ20%), to address this issue, we made stable transfectants using wt or DN Akt retroviral constructs. Puromycinresistant transfectants were stained with annexin V (to detect early apoptotic cells) and propidium iodide (to detect late apoptotic or dead cells), followed by FACS ® analysis. The results indicated no enhanced apoptosis in DN Akttransfected cells (data not shown; see Fig. 6 A for Akt expression). Therefore, these results indicate that Akt positively regulates the NF-B activity.
IB regulates NF-B by sequestering NF-B in the cytoplasm, and phosphorylation by IKK and subsequent degradation of IB releases this inhibition (60). Therefore, we examined whether DN IB-␣ (IB␣M) and DN IKK␣ (K44M) affect NF-B/luc reporter activity. As shown in Fig. 4 B, transcriptional activation of NF-B was strongly inhibited by DN IB-␣ or DN IKK␣. In keeping with these results and the enhanced Akt phosphorylation in lyn Ϫ/Ϫ cells, phosphorylation of Ser-32 in IB-␣ (by IKK) was severalfold higher in Fc⑀RI-stimulated lyn Ϫ/Ϫ mast cells than that in wt cells (Fig. 4 C). These data are also consistent with a recent study that demonstrated that Akt can directly phosphorylate IKK␣ at Ser-32 (81).
Transcription Factors NF-AT and AP-1 Are also Regulated by Akt. Luciferase constructs driven by multiple copies of other cis-elements of the IL-2 promoter, i.e., NF-AT (Ϫ290 to Ϫ261, seven times) and AP-1 (65), were also tested by cotransfection with wt or DN Akt (Fig. 5, A and  B). NF-AT/luc activity was significantly enhanced in Fc⑀RI-stimulated, wt Akt-transfected cells compared with Fc⑀RI-stimulated, vector-transfected cells. Importantly, these activities were inhibited by two DN Akt (K179M and AAA) mutants, indicating a role for Akt in signal transduction leading to activation of this transcription factor in mast cells. AP-1/luc activity was also affected significantly, albeit to a lesser extent, by wt or DN Akt expression.
JNK is involved in the regulation of both NF-AT and AP-1 activities (41,65). Therefore, effects of Akt on JNK activity were examined with wt BMMCs stably transfected with wt or DN Akt (Fig. 5 C). Fc⑀RI-induced JNK1 activation was not significantly affected by wt or DN Akt. Similar to JNK activity, Fc⑀RI-induced ERK activation, as measured by phosphorylation at the activation-loop Thr-202 and Tyr-204 residues of ERK1 and ERK2, was barely affected by wt and DN Akt.
These data suggest that the effect of Akt on NF-AT and AP-1 activities is not through the regulation of JNK. GSK-3 controls the nuclear export of NF-AT (71). Phosphorylation at Ser-9 of GSK-3␤ by Akt inhibits its catalytic activity and blocks the nuclear export of NF-AT. Therefore, we compared the phosphorylation status of Ser-9 of GSK-3␤ between wt and lyn Ϫ/Ϫ BMMCs. As shown in Fig. 5 D, Ser-9 phosphorylation in GSK-3␤ was higher at ‫03-01ف‬ min after Fc⑀RI stimulation in lyn Ϫ/Ϫ cells than in wt counterparts. A slower mobility of GSK-3␣ on SDS gels was observed at ‫03-01ف‬ min in wt cells, and this mobility shift was more pronounced in lyn Ϫ/Ϫ cells than in wt cells. These data are consistent with the notion that Akt-mediated NF-AT activation in Fc⑀RI-stimulated mast cells is through the phosphorylation and inhibition of GSK-3 by Akt. To further examine whether GSK-3 is involved in the regulation of NF-AT activity, wt BMMC was transiently cotransfected with DN GSK-3␤ and NF-AT/luc plasmids. DN GSK-3␤ expression enhanced the Fc⑀RI-induced activation of NF-AT-driven transcription (Fig. 5 A). Expression of DN GSK-3␤ also enhanced the Fc⑀RI-induced activation of AP-1-driven transcription (Fig. 5 B). Given the lack of the effect of Akt on the activity of JNK and ERK, Akt-mediated AP-1 activation may also be through GSK-3, as GSK-3 is known to inhibit AP-1 activity by phosphorylating Jun (82).
Akt Regulates Cytokine Production in Mast Cells. All the above data suggest that cytokine production may be regulated by Akt through the regulation of multiple transcription factors including NF-B, NF-AT, and AP-1. To di-rectly test this possibility, we mused puromycin-resistant, stable transfectants of wt BMMCs by retroviral infection with empty vector, HA-tagged wt, or K179M Akt viruses. Transfected Akt was expressed at approximately two-to threefold more than the endogenous Akt level (Fig. 6 A). Akt Ser-473 phosphorylation induced by Fc⑀RI stimulation in wt Akt-transfected cells was higher than that in control vector-transfected cells, whereas it was less pronounced in K179M Akt-transfected cells (Fig. 6 A). More than 98% of these trasnfected cells were viable in culture medium containing IL-3 and SCF. Upon Fc⑀RI stimulation, wt Akt transfectants secreted more IL-2 and TNF-␣ than vector-transfected cells, whereas K179M Akt transfectants secreted less than vector-transfectants (Fig. 6 B). lyn Ϫ/Ϫ BMMCs transfected with wt or K179M Akt viruses exhibited similar patterns of IL-2 and TNF-␣ production (data not shown). Taken together, these data indicate that Akt positively regulates IL-2 and TNF-␣ production and secretion in mast cells.

Discussion
This study indicates that Akt is activated by Fc⑀RI stimulation in mast cells. Extracellular stimuli that induce Akt activation include growth factors, cytokines, and antigen Stim., stimulation. (C) wt BMMCs stably transfected with an empty vector, wt Akt or K179M Akt, were IgE sensitized and stimulated with antigen. Cell lysates were either directly analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-ERK (for ERK assays) or immunoprecipitated with anti-JNK1 antibody before being subjected to kinase assays using GST-c-Jun as an exogenous substrate (for JNK1 assays). Equal expression of ERK1, ERK2, JNK1, and JNK2 was confirmed by probing immunoblots of total cell lysates with appropriate antibodies. Stim., stimulation. (D) wt and lyn Ϫ/Ϫ BMMCs were stimulated with IgE and antigen. Cell lysates were analyzed by immunoblotting with phosphospecific anti-GSK-3␤ (pSer9) antibody. The same blot was then reprobed with anti-GSK-3␣/␤ antibody. Stim., stimulation.
receptors. Most, if not all, of these stimuli promote cell survival and proliferation. Indeed, Fc⑀RI cross-linking and stimulation with SCF and IL-3, two major mast cell growth factors, exhibited these properties, i.e., survival/ proliferation and Akt activation ( Fig. 1; reference 83; data on Akt phosphorylation by IL-3 and SCF not shown). Although mechanisms by which Akt activation contributes to cell proliferation are not fully understood, some of the known Akt targets are involved in cell survival. A proapoptotic member of the Bcl-2 family BAD is phosphorylated at Ser-136 by Akt (67,84,85). The phosphorylated BAD becomes sequestered by 14-3-3 proteins and cannot heterodimerize with and inhibit the survival activity of the proteins Bcl-2 or Bcl-X L . However, some cytokine-mediated cell survival and Akt activation are not correlated with BAD phosphorylation (86)(87)(88). Caspase-9, an initiator caspase of apoptosis, is another target of Akt, and phosphorylation of caspase-9 at Ser-196 by Akt inhibits its protease activity (89). Another antiapoptotic mechanism by Akt is phosphorylation of forkhead family transcription factors. Phosphorylation of multiple sites in FKHRL1, AFX, and FKHR1 transcription factors results in inhibiting their transcriptional activity (90)(91)(92). FKHRL1 phosphorylated by Akt is bound to 14-3-3 proteins and retained in the cytoplasm, and is prevented from activating the gene(s) in-volved in apoptosis (90). It remains to be explored whether any of these known antiapoptotic mechanisms contribute to the mast cell survival/proliferation induced by Fc⑀RI stimulation.
A hallmark of Fc⑀RI-induced mast cell activation is the production and secretion of various cytokines including IL-1, IL-2, IL-3, IL-4, IL-6, IL-9, IL-13, GM-CSF, TNF-␣, etc. Among this expanding list of cytokines, TNF-␣ is known to play a critical role in late phase reactions of hypersensitivity (19). Potential mechanisms for allergic inflammation may include the antiapoptotic effect of TNF-␣ on monocytes (93). Production of cytokines such as IL-2 and TNF-␣ is regulated at several steps: gene transcription, mRNA stability, translation, and posttranslational modification. One of the critical regulatory steps is transcription. As shown for other cell types, numerous cis-transcriptional elements that are binding sites for transcription factors are involved in transcriptional activation of the IL-2 gene in Fc⑀RI-stimulated mast cells (65). This study provides evidence that Akt regulates transcriptional activity of NF-B, NF-AT, and AP-1 that is critical for the expression of the IL-2 and TNF-␣ genes. Phosphorylation of the inhibitor IB-␣ seems to be under the control of Akt as a signaling intermediary from Akt to NF-B, because phosphorylation of IB-␣ at Ser-32 (the phosphorylation site by IKK) was enhanced in lyn Ϫ/Ϫ mast cells in which Akt is hyperactivated. Because DN IB-␣ and DN IKK␣ inhibit Fc⑀RI-induced NF-B activity, Akt may phosphorylate and activate IKK, and IKK in turn phosphorylates and promotes the degradation of IB. Then free from IB, NF-B can translocate to the nucleus. During the revision of this manuscript, two groups showed that Btk regulates NF-B in B cells (94,95), in apparent agreement with our results in mast cells. As several other kinases such as Cot, MEKK1-3, and NF-Binducing kinase were shown to be capable of phosphorylating IKK, it will be interesting to determine whether Akt directly phosphorylates IKK␣ in mast cells, as shown for TNF-and platelet-derived growth factor (PDGF)-stimulated cells (81,96). GSK-3 phosphorylation by Akt may be involved in NF-AT and AP-1 activation in mast cells. Akt can phosphorylate and inhibit GSK-3 (71). Consistent with this established fact, phosphorylation of GSK-3␤ at Ser-9 was enhanced in lyn Ϫ/Ϫ mast cells, which exhibit hyperactive Akt upon Fc⑀RI stimulation. GSK-3 in turn is known to phosphorylate NF-AT and regulate the nuclear exit of this transcription factor. GSK-3 is also known to phosphorylate Jun proteins to inhibit AP-1 activity (82). Although Akt overexpression exhibited little effect on ERK and JNK activity, it significantly affected the activities of NF-AT and AP-1 (Fig. 5). Therefore, Akt-dependent phosphorylation of GSK-3 may regulate NF-AT and AP-1 without substantially affecting the canonical MAPK activation pathways, i.e., the Raf-1/MEK/ERK and MEKK/MAPK kinase 4 (or MAPK kinase 7)/JNK pathways.
In summary, we provide the first evidence for the involvement of Akt in Fc⑀RI-induced production of IL-2 and TNF-␣. Production of these cytokines is critical to late phase reactions of IgE-dependent hypersensitivity. Although Akt itself may not be a target for pharmaceutical interference to control allergic reactions and immunological diseases, our findings of Akt as a novel component of the signaling pathways leading to cytokine production have provided new insight into the exquisite networks of signaling molecules for mast cell activation.