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A. Tobio et al.
KIT tyrosine kinase activity is required for IL-6 production in mast cells
Ligand-activated KIT signaling induces IL-6 production in mast cells and enhances IgE-receptor-driven IL-6 pro- duction.20 Indeed, stimulation of KIT by SCF in the LAD2 cell line, which expresses normal KIT, induced IL-6 release, albeit the amounts were quantitatively limited (Figure 3A). HMC-1.1 showed higher production of IL-6 than LAD2 cells, particularly when stimulated with SCF (Figure 3A). However, not only was IL-6 production approximately 100-fold higher in unstimulated HMC-1.2 than in LAD2 or HMC-1.1 cells, but ligand-induced stim- ulation of KIT did not cause any further IL-6 secretion by HMC-1.2 cells (Figure 3A). These data are consistent with the conclusion that ligand-independent signals induced by oncogenic KIT activity, particularly those from D816V- KIT, are more effective in inducing IL-6 secretion than lig- and-activated KIT signals.
We next blocked D816V-KIT activity in HMC-1.2 cells with dasatinib, a tyrosine kinase inhibitor that effectively
AB
suppresses the activity of the active, open conformation of D816V-KIT. Dasatinib markedly reduced IL-6 mRNA lev- els (Figure 3B) and IL-6 secretion (Figure 3C), while con- centrations of the tyrosine kinase inhibitor imatinib that are ineffective in blocking D816V-KIT, did not alter IL-6 mRNA levels (Figure 3B), further suggesting an involve- ment of D816V-KIT signaling. Inhibition by gefitinib of the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor that can drive IL-6 production in trans- formed cells,21 had no significant effects on IL-6 mRNA levels in HMC-1.2 cells (Figure 3B). Similar to HMC-1.2 cells, dasatinib effectively inhibited IL-6 release in P815 murine mastocytoma cells (Figure 3D). In contrast, persist- ent IL-6 production in HMC-1.2 cells did not appear to be mediated via feed-forward loops of activation by other receptors as those reported for the IL-6 receptor (IL-6R),22 sphingosine-1-phosphate receptors23 or the TGFβ recep- tor24 in other neoplastic cells, since it was not altered by specific blockage of these receptors (Online Supplementary Figure S2A-C). Overall, these data suggest that signals
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Figure 3. D816V-KIT oncogenic activity drives ligand-independent IL-6 induction. (A) IL-6 released to the extracellular media was measured in LAD2, HMC-1.1 and HMC-1.2 cells incubated for 48 hours (h) (37C, 5%CO2) in serum-free medium in the presence or absence of 100 ng/mL stem cell factor (SCF). (B) IL-6 mRNA levels were measured in HMC-1.2 after 2 h incubation in serum-free medium in the presence or absence of the KIT inhibitors, dasatinib and imatinib, or the EGFR inhibitor gefitinib at the indicated concentrations. Relative expression of IL-6 mRNA was obtained by normalizing to the expression of GAPDH using the DCt method and the results are expressed as fold change compared to untreated cells. The effect of the KIT inhibitor dasatinib (0.5 mM) on the secretion of IL-6 by HMC-1.2 (C) or by P815 mast cells (D) was determined after 6 h incubation in serum free medium. All data are the mean±Standard Error of Mean of three independent experiments done in triplicates. SCF: stem cell factor. *P=0.01, **P<0.01 and ****P<0.0001.
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haematologica | 2020; 105(1)