IL-3-Induced Immediate Expression of c-fos and c-jun Is Modulated by the IKK2-JNK Axis

Interleukin (IL)-3 is a pleiotropic cytokine that regulates the survival, proliferation, and differentiation of hematopoietic cells. The binding of IL-3 to its receptor activates intracellular signaling, inducing transcription of immediate early genes (IEGs) such as c-fos, c-jun, and c-myc; however, transcriptional regulation under IL-3 signaling is not fully understood. This study assessed the role of the inhibitor of nuclear factor-κB kinases (IKKs) in inducing IL-3-mediated expression of IEGs. We show that IKK1 and IKK2 are required for the IL-3-induced immediate expression of c-fos and c-jun in murine hematopoietic Ba/F3 cells. Although IKK2 is well-known for its pivotal role as a regulator of the canonical nuclear factor-κB (NF-κB) pathway, activation of IKKs did not induce the nuclear translocation of the NF-κB transcription factor. We further revealed the important role of IKK2 in the activation of c-Jun N-terminal kinase (JNK), which mediates the IL-3-induced expression of c-fos and c-jun. These findings indicate that the IKK2-JNK axis modulates the IL-3-induced expression of IEGs in a canonical NF-κB-independent manner.


Introduction
Interleukin (IL)-3, mainly produced by activated T cells and mast cells, is a key cytokine that regulates the survival, proliferation, and differentiation of hematopoietic cells [1,2]. Although IL-3 is not essential for steady-state hematopoiesis, it stimulates the proliferation and differentiation of myeloid cells and modulates the cellular functions of multiple immune cell types in response to infection or inflammation [3][4][5][6][7]. In addition to its role in immune responses, IL-3 is involved in malignant hematopoiesis. The levels of expression of the IL-3 receptor α (IL-3Rα) subunit are elevated in the progenitor and stem cells of patients with several hematological malignancies, e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and basic plasmacytoid dendritic cell neoplasm [8][9][10][11][12]. Indeed, its levels of expression strongly correlate with a reduced survival rate in patients with AML [12,13], suggesting that targeting IL-3 signaling might be a potential therapeutic strategy for AML. These findings indicate the need to identify and validate mediators of IL-3 signaling.
IL-3 belongs to the β common (βc) cytokine family, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. The βc family functions through heterodimeric receptors composed of a cytokine-specific α chain and a shared βc chain. The receptors of the βc cytokine family lack intrinsic kinase activity, but the βc chain associates with Janus kinases (JAKs) at the membrane-proximal cytoplasmic domain [14]. The binding of IL-3 to IL-3Rα triggers the assembly of the receptor complex to activate JAKs, thereby promoting multiple signaling cascades. Major downstream signaling pathways associated with βc receptor cytokine signaling include the JAK-signal transducer and activator of transcription (STAT), the phosphoinositide 3-kinase (PI3K), and the mitogen-associated protein kinase (MAPK) pathways [15,16]. These signaling pathways are associated with various cellular responses, including cell survival, proliferation, and differentiation. IL-3 (IL-3 depletion medium), and stimulated by adding 10 ng/mL IL-3 or 20 ng/mL tumor necrosis factor (TNF)-α (410MT025/CF, R&D Systems, Minneapolis, MN, USA). Cells were collected by centrifugation and subjected to subsequent experiments. For inhibitor experiments, cells were incubated for 6 h in an IL-3 depletion medium containing the indicated concentrations of IKK-16 (13313, Cayman, Ann Arbor, MI, USA), JNK-IN-8 (HY-13319, Med Chem Express, Monmouth Junction, NJ, USA), or DMSO. For IL-3 neutralization experiments, 0.4 µg/mL IL-3 was incubated with 10 µg/mL anti-mouse IL-3 neutralizing antibody (Ab) (503901, BioLegend, San Diego, CA, USA) or control IgG (sc-2026, Santa Cruz Biotechnology, Dallas, TX, USA) at 37 • C in RPMI 1640 for 6 h, and then added to IL-3-starved cells. The final concentration of IL-3 was 10 ng/mL.
To generate IKK1 or IKK2 single KO clones, 5 × 10 6 parental Ba/F3 cells were electroporated with 120 µg of a Cas9 expression plasmid (41815, Addgene), 120 µg of a sgRNA expression plasmid targeting IKK1 or IKK2, and 1 µg of a plasmid encoding a puromycin resistant gene using the Gene Pulser Xcell (1652660J1, Bio-Rad, Richmond, CA, USA). For IKK1 and IKK2 double KO (DKO) clones, IKK1 KO Ba/F3 clones were electroporated with the IKK2 targeting sgRNA expression plasmid together with the Cas9 expression plasmid and the plasmid encoding the puromycin resistant gene. One day after electroporation, 1.0 µg/mL puromycin was added, and cells were cultured for 2 days. The cells were subsequently cultured in a puromycin-free complete medium, and each cell was isolated by limiting dilution. After the expansion of each cell, the knock out of each target gene was confirmed by genotyping PCR followed by Sanger sequencing and immunoblot analysis.

RNA Extraction, Reverse Transcription (RT), and Quantitative PCR
Total RNA was extracted with Isogen II (317-07363, Nippon Gene, Tokyo, Japan) and reverse transcribed using ReverTra Ace qPCR RT Master Mix with a genomic DNA remover (FSQ-301, Toyobo, Osaka, Japan) in accordance with the manufacturer's protocols. The complementary DNA was subjected to quantitative real-time PCR using THUNDERBIRD SYBR qPCR Mix (QPS-101, Toyobo) and the primers shown in Table S1. The level of expression of each mRNA was normalized relative to that of 18S rRNA.

Plasmid Construction and Retroviral Transduction
The plasmid encoding FLAG-tagged IKK1 was purchased from Addgene (15467) [39]. To construct the retroviral plasmid encoding FLAG-IKK1, the coding sequence of FLAG-IKK1 was amplified by PCR using mIKK1-InFusion-F and mIKK1-InFusion-R primers (Table S1). The PCR product was purified and subjected to In-Fusion cloning (639648, Takara Bio, Shiga, Japan) with the pMSCVpuro vector (634401, Takara Bio) that had been digested with Bgl II and EcoR I. The sequence of the cloned fragment was confirmed by Sanger sequencing. To construct the retroviral plasmid encoding 3 × FLAG-BAP, the coding sequence of 3 × FLAG-BAP was cleaved by SnaB I and Sma I from the p3 × FLAG-CMV-7-BAP plasmid (C7472, Sigma-Aldrich, St. Louis, MO, USA) and ligated into the pMSCVpuro vector that had been cleaved by Hpa I.
For retrovirus production, the packaging cell line PLAT-E was transfected with 10 µg of the retroviral plasmids in a 10 cm culture dish using Lipofectamine 3000 (L3000015, Thermo Fisher Scientific). After incubation for 48 h, the viral supernatant (10 mL) was collected and filtered through a 0.45 µm pore size membrane. IKK1-deficient Ba/F3 cells were cultured with the viral media supplemented with 10 µg/mL polybrene and 1 ng/mL IL-3 for 5 h, and then an equal volume of complete medium was added. Following incubation overnight, the viral media were replaced with a complete medium containing 1.0 µg/mL puromycin to select and expand infected cells.

Cell Lysis, Immunoprecipitation, and Immunoblot Analyses
Cells were washed with PBS and incubated on ice for 30 min in lysis buffer (20 mM Tris-HCl, pH 8.0, 420 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM Na 3 VO 4 , 1 mM Na 4 P 2 O 7 , cOmplete protease inhibitor cocktail without EDTA (11697498001, Roche, Basel, Switzerland). After centrifugation at 20,000× g for 10 min at 4 • C, the supernatants were collected as whole-cell extracts. Cytosol and nuclear extracts were prepared with NE-PER Nuclear and Cytoplasmic Extraction reagents (78833, Thermo Fisher Scientific) according to the manufacturer's protocol with slight modifications, namely, additional incubation with CER II for 2 min at the cytoplasmic protein extraction step and an additional wash of nuclei with CER I before the nuclear protein extraction step. Protein concentrations were determined using the Bradford method with Protein Assay Dye Reagent Concentrate (500-0006, Bio-Rad).

Statistical Analyses
Differences between the two groups were assessed by the Mann-Whitney U test. Relative expression levels in multiple groups were compared by Kruskal-Wallis one-way analysis of variance followed by the Steel-Dwass multiple-comparisons test using R software (v3.5.3, R Foundation for Statistical Computing, Vienna, Austria) [40].

Suppression of IL-3-Mediated Expression of IEGs by an IKK Inhibitor
To determine whether IKK1 and IKK2 are involved in the IL-3-induced expression of IEGs, the effects of IKK-16, a selective inhibitor of IKK1 and IKK2 [41], on IL-3-induced expression of IEGs were tested. IL-3-dependent cell lines, Ba/F3, and 32D were utilized in this study. These cell lines have been extensively studied as models to investigate IL-3 signaling because of their steady expression of the IL-3 receptor and signaling molecules. Ba/F3 or 32D cells were starved of IL-3 for 6 h in the presence of IKK-16 or DMSO (negative control). Upon IL-3 stimulation, expression of c-fos, c-jun, and c-myc was induced rapidly in the absence of IKK-16 in Ba/F3 cells ( Figure 1A). By contrast, IKK-16 (0.25 µM) strongly suppressed the IL-3-induced expression of c-fos and c-jun but not c-myc ( Figure 1A). Similarly, IL-3-induced expression of c-fos was suppressed by 0.25 µM IKK-16 in 32D cells; however, that of c-myc was not ( Figure 1B). In 32D cells, c-jun expression was not detectable [42]. Lower concentrations of IKK-16 (≤0.25 µM) also suppressed c-fos and c-jun (in Ba/F3 cells), but not c-myc, expression 30 min after IL-3 stimulation. By contrast, higher concentrations of IKK-16 (≥0.5 µM) decreased the expression of all these genes ( Figure 1C and Figure S1). These results suggest that IKK-16 suppresses two distinct signaling pathways at different concentrations, one that induces c-fos/c-jun and another that induces c-myc. Cell-free assays have shown that IKK-16 inhibits kinase activities of IKK1 and IKK2 at different concentrations (IC 50 : 0.2 and 0.04 µM, respectively) [41]. Therefore, the different sensitivities of the c-fos/c-jun and c-myc pathways to suppression by IKK-16 may be due to the differential contributions of IKK1 and IKK2 to the mechanisms of induction of these IEGs, or to an inherent off-target effect of IKK-16. We hereafter used Ba/F3 cells expressing c-fos, c-jun, and c-myc to investigate the roles of IKKs in the IL-3-mediated expression of different IEGs.

KO of IKKs Suppressed IL-3-Mediated Expression of IEGs
To identify the IKK isoforms responsible for IL-3-mediated expression of IEGs, IKK1 KO, IKK2 KO, and IKK1/2 DKO cells were generated by genome editing using the CRISPR/Cas9 system. The expression plasmids for sgRNAs that target nucleotide sequences encoding the N-terminus of the IKK1 or IKK2 protein were co-transfected, along with a Cas9 expression plasmid to induce frameshift mutations. The absence of IKK1 and/or IKK2 proteins was confirmed by immunoblot analysis (Figure 2A).
Assessments of the levels of expression of c-fos, c-jun, and c-myc mRNAs after IL-3 stimulation showed that KO of IKK1 and/or IKK2 reproducibly led to a reduction in the level of c-fos mRNA when compared with parental cells, suggesting that IKK1 and IKK2 are functionally important for IL-3-induced expression of the c-fos gene ( Figure 2B and Figure S2A,B). In addition, KO of IKK1 alone led to a reduction in expression of c-jun mRNA, whereas only one IKK2 KO clone showed a reduction in expression of c-jun mRNA compared with parental cells ( Figure 2C). Further analysis using two additional IKK2 KO clones showed similar results ( Figure S2B). Although we cannot exclude the possibility that the variation in c-jun mRNA expression in IKK2 KO clones is due to clonal variation, compensatory mechanisms, such as functional redundancy, could mask the loss-of-function phenotypes of some clones. Together with evidence showing that c-jun expression was suppressed by a low concentration of IKK-16 ( Figure 1A,C), these findings suggest that IKK2 could be involved in the induction of c-jun transcription. In contrast to c-fos and c-jun mRNAs, the expression of c-myc mRNA was not affected by either IKK1 or IKK2 KO ( Figure 2D). A slight reduction in c-myc mRNA expression was observed in IKK1/2 DKO cells, suggesting a functional redundancy between IKK1 and IKK2 in the mechanism of c-myc expression ( Figure 2D).
In summary, these findings showed that (1) IKK1 is required for IL-3-induced immediate expression of c-fos and c-jun, (2) IKK2 is also required for expression of c-fos and c-jun, although a marked difference in clones was observed, and (3) both IKK1 and IKK2 could be marginally involved in the expression of c-myc. The finding that Ab neutralization of IL-3 suppressed expression of IEGs ( Figure S3) suggests that their expression was induced by IL-3 associated signaling, not by potentially contaminating lipopolysaccharide (LPS) or other Toll-like receptor ligands. Furthermore, these results of loss-of-function experiments

KO of IKKs Suppressed IL-3-Mediated Expression of IEGs
To identify the IKK isoforms responsible for IL-3-mediated expression of IEGs, IKK1 KO, IKK2 KO, and IKK1/2 DKO cells were generated by genome editing using the CRISPR/Cas9 system. The expression plasmids for sgRNAs that target nucleotide sequences encoding the N-terminus of the IKK1 or IKK2 protein were co-transfected, along with a Cas9 expression plasmid to induce frameshift mutations. The absence of IKK1 and/or IKK2 proteins was confirmed by immunoblot analysis (Figure 2A). Assessments of the levels of expression of c-fos, c-jun, and c-myc mRNAs after IL-3 stimulation showed that KO of IKK1 and/or IKK2 reproducibly led to a reduction in the level of c-fos mRNA when compared with parental cells, suggesting that IKK1 and IKK2 are functionally important for IL-3-induced expression of the c-fos gene ( Figures 2B and  S2A,B). In addition, KO of IKK1 alone led to a reduction in expression of c-jun mRNA, whereas only one IKK2 KO clone showed a reduction in expression of c-jun mRNA compared with parental cells ( Figure 2C). Further analysis using two additional IKK2 KO clones showed similar results ( Figure S2B). Although we cannot exclude the possibility that the variation in c-jun mRNA expression in IKK2 KO clones is due to clonal variation, compensatory mechanisms, such as functional redundancy, could mask the loss-of-function phenotypes of some clones. Together with evidence showing that c-jun expression was suppressed by a low concentration of IKK-16 ( Figure 1A,C), these findings suggest that IKK2 could be involved in the induction of c-jun transcription. In contrast to c-fos and c-jun mRNAs, the expression of c-myc mRNA was not affected by either IKK1 or IKK2 KO ( Figure 2D). A slight reduction in c-myc mRNA expression was observed in IKK1/2 DKO cells, suggesting a functional redundancy between IKK1 and IKK2 in the mechanism of cmyc expression ( Figure 2D).
In summary, these findings showed that (1) IKK1 is required for IL-3-induced immediate expression of c-fos and c-jun, (2) IKK2 is also required for expression of c-fos and c-

Regulation of IL-3-Mediated Expression of IEGs by Activated IKKs in a Canonical NF-κB-Independent Manner
IKK2 is the main regulator of the canonical NF-κB pathway and phosphorylated (activated) following the exposure of cells to inducing agents, such as TNF-α and LPS. This, in turn, induces the degradation of IκB and translocation of NF-κB transcription factors from the cytoplasm to the nucleus, leading to the activation of the expression of NF-κB target genes [28,43]. In addition, IKK-dependent but canonical NF-κB-independent induction of gene expression has been reported [44,45]. This study, therefore, evaluated whether IL-3 induction of IKK-dependent expression of IEGs involves canonical NF-κB signaling. To confirm that IKK1 and IKK2 were activated by IL-3, lysates were prepared from IL-3-stimulated Ba/F3 cells, and phosphorylation of IKK1 and IKK2 was evaluated by immunoblot analysis. Consistent with previous findings [32,33], phosphorylation of IKKs was induced within 5 min after IL-3 stimulation ( Figure 3A). Although several additional bands were detected, the specificity of the phospho-IKK1 and phospho-IKK2 bands was confirmed using the lysates of IKK KO cells ( Figure 3B). Signals of phosphorylated IKK1 were weak; therefore, phosphorylation of IKK1 in response to IL-3 was further confirmed by immunoprecipitation followed by immunoblot analysis. To this end, FLAG-tagged IKK1 (FLAG-IKK1) was expressed in IKK1 KO Ba/F3 cells. After IL-3 stimulation, FLAG-IKK1 was immunoprecipitated with anti-FLAG Ab. Immunoprecipitated FLAG-IKK1 was phosphorylated 5 min after the stimulation ( Figure S4A). In addition to FLAG-IKK1, endogenous IKK2 that co-immunoprecipitated with FLAG-IKK1 was also phosphorylated after the stimulation ( Figure S4A), which is consistent with the results presented in Figure 3A,B. Ab neutralization of IL-3 reduced the phosphorylation of IKK1 and IKK2 ( Figure S4B), indicating that the signaling cascade evoked by IL-3 is responsible for IKK1 and IKK2 activation. Notably, FLAG-IKK1 restored the expression levels of c-fos and c-jun in IKK1 KO cells (Figure S5), confirming that IKK1 is involved in the expression of IEGs.
We next investigated if the canonical NF-κB pathway was activated during IL-3 signaling. IκB-α protein levels in whole-cell extracts were analyzed by immunoblot analysis. TNF-α stimulation, used as a positive control, obviously decreased IκB-α protein levels (especially at 15 min), whereas IL-3 stimulation did not ( Figure 3C). We also evaluated the nuclear translocation of the transcription activator p65, a member of the family of canonical NF-κB transcription factors. Following IL-3 stimulation, the cytosolic and nucleic fractions were separated, and p65 was detected by immunoblot analysis. Although TNF-α stimulation increased the amounts of p65 in nuclear extracts, IL-3 stimulation did not ( Figure 3D). The constant protein levels of IκB-α and p65 in the nucleus after IL-3 stimulation were consistent with the previous results showing that GM-CSF, which binds to a βc receptor subunit, does not induce phosphorylation and degradation of IκB and nuclear translocation of p65 [46]. These findings suggested that IL-3 signaling immediately activates IKK1 and IKK2 but does not induce the subsequent degradation of IκB and nuclear translocation of p65, a characteristic feature of the canonical NF-κB pathway. Thus, IL-3-mediated expression of IEGs is regulated by activated IKKs in a canonical NF-κB-independent manner.

JNK Mediates c-fos and c-jun Expression Downstream of IKK2
To characterize the IEG expression mechanism controlled by IKKs, we investigated the signaling cascade downstream of IKKs. IKKs mediate the activation of the JNK pathway responsible for IL-3-induced proliferation [33]. The JNK pathway regulates the transcription of c-fos and c-jun in response to various stimuli [26]. Based on these previous findings, we hypothesized that (1) IKKs activated by IL-3 stimulation phosphorylate and thereby activate JNK, and (2) activated JNK induces expression of c-fos and c-jun. To test these hypotheses, we evaluated the phosphorylation levels of JNK using parental and IKK KO Ba/F3 cells. In parental Ba/F3 cells, the phosphorylation level of JNK was increased within 10 min after IL-3 stimulation ( Figure 4A). In IKK1/2 DKO cells, the phosphorylation level of JNK was attenuated compared with that in parental cells 10 min after IL-3 stimulation, despite these cells having similar JNK expression levels ( Figure 4B). Further analysis using IKK1 KO and IKK2 KO cells showed that IKK2 KO, but not IKK1 KO, reduced JNK phosphorylation ( Figure 4C,D). These data demonstrate that IKK2 is relevant for JNK activation.
To confirm the regulation of c-fos and c-jun expression by JNK during IL-3 signaling, Ba/F3 cells were treated with the selective JNK inhibitor JNK-IN-8 [47]. Treatment with a series of different concentrations of JNK-IN-8 decreased c-fos and c-jun expression levels in a dose-dependent manner, but c-myc expression levels remained constant 20 min after IL-3 stimulation ( Figure S6). Treatment with a low dose (0.5 µM) of JNK-IN-8 partially or remarkably reduced IL-3-induced c-fos and c-jun expression (20 min, Figure 4E). By contrast, in the presence of the inhibitor, c-myc expression was unchanged or slightly augmented after IL-3 stimulation. These results suggest that JNK mediates the induction of c-fos and c-jun expression in an IKK2-dependent manner during IL-3 signaling, which is consistent with our hypothesis.  We next investigated if the canonical NF-κB pathway was activated during IL-3 signaling. IκB-α protein levels in whole-cell extracts were analyzed by immunoblot analysis. TNF-α stimulation, used as a positive control, obviously decreased IκB-α protein levels (especially at 15 min), whereas IL-3 stimulation did not ( Figure 3C). We also evaluated the nuclear translocation of the transcription activator p65, a member of the family of canonical NF-κB transcription factors. Following IL-3 stimulation, the cytosolic and nucleic frac- . IL-3-induced IKK activation was not associated with the degradation of IκB-α followed by nuclear translocation of p65. (A) Phosphorylation of IKKs after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 for the indicated times, and wholecell lysates were immunoblotted using the indicated Abs. Long and short exposures to detect phosphorylated IKK1 and IKK2 are shown. An arrowhead indicates the band corresponding to phosphorylated IKK1 at 5 min after IL-3 stimulation. A dagger denotes the detection of IKK2 by potential cross-reactivity of the anti-IKK1 Ab. (B) Phosphorylation of IKKs in IKK KO cells after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 parental (WT), IKK1 KO, IKK2 KO, and IKK1/2 DKO cells were stimulated with IL-3 for 5 min. Immunoblot analysis was performed with whole-cell lysates using the indicated Abs. Arrowheads indicate the bands corresponding to phosphorylated IKK1. (C) Degradation of IκB-α by TNF-α but not by IL-3 stimulation. Following IL-3 deprivation, Ba/F3 cells were stimulated with IL-3 or TNF-α for the indicated durations, and whole-cell lysates were immunoblotted using the indicated Abs. (D) Induction of nuclear translocation of p65 by TNF-α but not by IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 or TNF-α for the indicated times and immunoblotted with Abs to the cytosol marker α-tubulin and the nuclear marker fibrillarin. Long and short exposures to detect p65 are shown.   Figure 4E). By con-

Discussion
IL-3 triggers multiple signaling cascades that modulate cellular responses, including cell survival, proliferation, and differentiation. However, the mechanisms by which IL-3 regulates multiple signals resulting in distinct outcomes have remained elusive. The Srcfamily kinase-and calcium-dependent activation of IKKs by IL-3 has been found to prevent apoptosis and mediate mitogen signaling for cell proliferation, suggesting that IKKs are important mediators of IL-3 signaling [32,33]. The present study focused on the role of IKKs in the IL-3-induced expression of IEGs. IKKs immediately activated by IL-3 signaling were found to modulate the expression of IEGs, especially c-fos and c-jun, in a canonical NF-κB-independent manner. In addition, IKK2 mediated the activation of JNK, resulting in c-fos and c-jun expression ( Figure 5). While the expression mechanism downstream of IKK1 remains elusive, our findings also indicate that IKK1 is involved in IEG expression. The transcription factors encoded by these genes are immediately expressed in response to IL-3 stimulation and modulate transcription cascades. Thus, the regulation of such early response genes by IKKs could be one aspect of the molecular mechanisms underlying late phases of IL-3-induced cellular activities.
stream of IKK1 remains elusive, our findings also indicate that IKK1 is involv expression. The transcription factors encoded by these genes are immediately in response to IL-3 stimulation and modulate transcription cascades. Thus, the r of such early response genes by IKKs could be one aspect of the molecular me underlying late phases of IL-3-induced cellular activities.
Although IKK2 is the main regulator of canonical NF-κB signaling, activatio by IL-3 did not induce degradation of IκB and nuclear translocation of p65. The members of the βc cytokine family to activate canonical NF-κB signaling is unc trophoretic mobility shift assays have shown that GM-CSF or IL-3 stimulation the DNA-binding activity of NF-κB [46,48,49]. Although IL-3 was found to indu clear translocation of p65 in mouse embryonic fibroblasts [49], the nuclear tra of p65 and the degradation of IκB were not detected in bone marrow mast cel mune cell lines [33,46]. Despite the inconsistency among experiment assays and our findings indicate that IKKs can mediate IL-3 signaling in a canonical NF-κB-i ent manner. IKK1 has been found to regulate a non-canonical NF-κB pathway th the nuclear translocation of the RelB/p52 NF-κB complex [29]. In contrast to the tivation of canonical NF-κB signaling, activation of non-canonical NF-κB signali due to the requirement for de novo protein synthesis [50,51]. Because the transc IEGs occurs within 20 min of stimulation, non-canonical NF-κB signaling was related to IL-3-induced IEGs transcription. Although IKK2 is the main regulator of canonical NF-κB signaling, activation of IKKs by IL-3 did not induce degradation of IκB and nuclear translocation of p65. The ability of members of the βc cytokine family to activate canonical NF-κB signaling is unclear. Electrophoretic mobility shift assays have shown that GM-CSF or IL-3 stimulation enhanced the DNA-binding activity of NF-κB [46,48,49]. Although IL-3 was found to induce the nuclear translocation of p65 in mouse embryonic fibroblasts [49], the nuclear translocation of p65 and the degradation of IκB were not detected in bone marrow mast cells and immune cell lines [33,46]. Despite the inconsistency among experiment assays and cell types, our findings indicate that IKKs can mediate IL-3 signaling in a canonical NF-κB-independent manner. IKK1 has been found to regulate a non-canonical NF-κB pathway that induces the nuclear translocation of the RelB/p52 NF-κB complex [29]. In contrast to the rapid activation of canonical NF-κB signaling, activation of non-canonical NF-κB signaling is slow due to the requirement for de novo protein synthesis [50,51]. Because the transcription of IEGs occurs within 20 min of stimulation, non-canonical NF-κB signaling was likely unrelated to IL-3-induced IEGs transcription.
A JNK inhibitor abrogated c-jun expression and decreased c-fos expression ( Figure 4E). In general, c-jun expression is primarily regulated by JNK through direct phosphorylation and transcriptional activation of c-Jun-ATF2 heterodimers [26]. Therefore, IL-3-induced c-jun expression is mediated by the same JNK-dependent pathway. Accumulated evidence indicates that phosphorylation of Elk-1 by ERK plays a pivotal role in the ternary complex formation of Elk-1 with SRF, thereby enhancing c-fos transcription [52]. Indeed, IL-3-dependent activation of ERK enhances the transcriptional ability of Elk-1 [53]. Therefore, ERK-Elk-1 signaling may be involved in IL-3-induced c-fos expression. On the other hand, JNK can also phosphorylate (activate) Elk-1 in the presence of extracellular stimulation [54,55]. The JNK inhibitor partially suppressed IL-3-induced c-fos expression ( Figure 4E); therefore, JNK may mediate this expression via phosphorylation of Elk-1 to some extent.
IKK1 phosphorylates various substrates involved in transcription mechanisms. For example, IKK1 phosphorylates histone tails, resulting in chromatin remodeling to recruit transcription factors, followed by the onset of transcription [56,57]. Optimal transcription of c-fos and some IEGs in response to epidermal growth factor requires IKK1 to regulate promoter-specific histone H3 phosphorylation independently of IκB degradation in mouse embryonic fibroblasts [44]. These findings suggest that IKK1 activated by IL-3 could lead to the phosphorylation of histone tails, initiating the transcription of c-fos and c-jun. Other examples of transcriptional regulation by IKK1 are phosphorylation of transcription factors and cofactors, e.g., AP-1, Myc, and CBP, to fine-tune signals for proper gene expression through NF-κB-dependent and -independent mechanisms [30,31,58]. Therefore, known or unknown substrates of IKK1 could regulate the transcription of IEGs in response to IL-3.
IL-3 supports the differentiation of hematopoietic progenitor cells and stimulates various myeloid cells. In addition, IL-3 plays a role in the development and functions of lymphocytes. IL-3 induces the expansion of stimulated B cells [59] and immunoglobulin secretion [60]. Activated T helper cells and regulatory T cells express functional IL-3Rα and exhibit IL-3-induced activation of the JAK-STAT pathway [61,62]. However, little is known about intracellular signaling and the roles of IL-3 in lymphocytes. It will be interesting to study this and to investigate the difference in IL-3 signaling between myeloid cells and lymphocytes. This study utilized IL-3-dependent cell lines, Ba/F3 and 32D; however, there is a general concern about their phenotypic differences from primary cells, similar to other cell lines. Further studies are needed to elucidate the roles of IKK-mediated IL-3 signaling and the underlying mechanisms in more physiological conditions.
The present study provides evidence that IKK1 and IKK2 regulate IL-3-induced expression of IEGs in a canonical NF-κB-independent manner and that JNK is a key mediator downstream of IKK2. In response to IL-3 stimulation, JNK plays a role in cell survival through the inactivation of proapoptotic signaling [63]. Therefore, the IKK2-JNK axis could be responsible for cell survival via IEG expression. These findings may help clarify the molecular mechanisms of cell survival and proliferation induced by IL-3 and those underlying IL-3-mediated malignant hematopoiesis.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11091451/s1, Table S1: Primers used in this study. Figure S1: Expression levels of c-fos and c-myc 0 and 30 min after IL-3 stimulation in 32D cells in the presence of IKK-16. Figure S2: Effects of IKK2 KO on c-fos and c-jun expression. Figure S3: IL-3-dependent induction of IEGs transcription. Figure S4: IL-3-induced phosphorylation of IKK1 and IKK2. Figure S5: Recovery of c-fos and c-jun expression in IKK1 KO cells by exogenous expression of FLAG-IKK1.