In this study, we have sought to examine the concept that concurrent inhibition of Kit- and FcεRI-mediated signaling may provide a coordinated suppression of mast cell activation. Of the known Kit inhibitors, the most widely investigated is imatinib. This agent has been described as a relatively selective inhibitor that, besides Kit, affects the activity of Abelson cytoplasmic tyrosine kinase and the platelet-derived growth factor receptor (Heinrich et al., 2000
; Scheinfeld, 2006
). It has been reported that imatinib decreases allergic peribronchial eosinophil accumulation, airway hyperreactivity, and cytokine levels in vivo
(Berlin and Lukacs, 2005
; Berlin et al., 2006
), although in mast cell explant in culture anti-IgE-mediated histamine release was unaffected (Beck et al., 2004
). However, as with other compounds which target Kit, imatinib has not been reported to inhibit any of the kinases involved in the FcεRI signaling pathway. In contrast, as reported here, hypothemycin not only blocks Kit- but also FcεRI-mediated signaling and therefore appeared to be an attractive compound to test the efficacy of co-inhibition of Kit- and FcεRI-mediated responses.
The ability of hypothemycin to inhibit Kit kinase activation was confirmed by the inhibition of the SCF-induced autophosphorylation of critical tyrosine residues within the cytosolic domain of Kit (). As these serve as docking sites for SH2 domain-containing signaling molecules which mediate the downstream signaling of Kit, SCF-mediated signaling events including Btk phosphorylation, AKT phosphorylation, and PLCγ1
phosphorylation leading to calcium mobilization were also inhbited (). The inhibition of these signalling events account for the ability of hypothemycin to effectively inhibit SCF-mediated mast cell adhesion (), chemotaxis (data not shown) and, as with imatinib, the ability of SCF to potentiate antigen-mediated degranulation () and cytokine production (). Unlike imatinib, however, hypothemycin also blocked the ability of antigen to induce both degranulation and cytokine production, thus demonstrating the ability of hypothemycin to inhibit the FcεRI-, as well in addition to the Kit-mediated signaling pathway. As hypothemycin was relatively ineffective at inhibiting Src kinases and Syk when screened against a panel of kinases (Schirmer et al., 2006
), these data, and our observations that hypothemycin does not block antigen-mediated phosphorylation of Src kinases and LAT (), indicated that the target of hypothemycin in the FcεRI-dependent signaling cascade was downstream from these initial signaling events and, by inference, FcεRI aggregation and phosphorylation. Moreover, as hypothemycin failed to inhibit AKT phosphorylation (), it appears that the target is also downstream of PI3K.
As antigen-mediated Btk phosphorylation, as well as downstream signalling events, were markedly suppressed (), hypothemycin most likely inhibits FcεRI-mediated degranulation at the level of Btk activation. However, resting Btk kinase activity was not blocked in an in vitro
kinase assay (Schirmer et al., 2006
), indicating that Btk is not directly inhibited by hypothemycin. Instead, hypothemycin may inhibit a subsidiary cryptic event associated with the phosphorylation and activation of Btk downstream of the activation of Lyn, Syk, and PI3K. Regardless, the data clearly indicate that Btk phosphorylation is the first recognizable step in the FcεRI-mediated signaling cascade inhibited by hypothemycin ().
Proposed sites of action of hypothemycin on SCF- and antigen-mediated mast cell activation
Data obtained with mast cells derived from Btk-/-
mice have demonstrated that Btk is a critical enzyme for optimal phosphorylation of PLCγ1
calcium mobilization, degranulation, and cytokine production in mast cells (Hata et al., 1998
; Iwaki et al., 2005
). We have, furthermore, previously demonstrated that Btk is also important for the ability of SCF to potentiate antigen-dependent mast cell degranulation (Iwaki et al., 2005
). This may be partially explained by Btk providing a mechanism for amplification of the PLCγ1
-dependent calcium signal (Iwaki et al., 2005
). Thus, the observed ability of hypothemycin to attenuate the antigen induced increase in PLCγ1
phosphorylation, calcium mobilization, and subsequently, degranulation, is entirely consistent with our conclusion that hypothemycin in blocking the FcεRI-mediated signaling cascade at the level of Btk. The suppression of cytokine production by hypothemycin could also be attributable to inhibition of Btk. Indeed, it has been previously demonstrated that antigen-mediated cytokine production is defective in Btk-/-
BMMCs (Hata et al., 1998
). However it is possible that hypothemycin may also be blocking cytokine production by inhibiting the activation of MAP kinases. Regardless, the net outcome of either or both of these mechanisms would account for the observed inhibition of elevated phosphorylation of transcription factors in response to antigen and SCF ().
The ability of hypothemycin to inhibit the synergistic release of mast cell activation in culture translated into a significant inhibition of the antigen-induced passive cutaneous anaphylaxis response in mice (), demonstrating its efficacy against a mast cell-driven allergic reaction in vivo. The residual response observed in these studies, may however reflect the need to optimize the bioavailability and dosage regimen of potential inhibitory molecules such as hypothemycin. Future studies to expand the therapeutic repertoire for a coordinated approach targeting Kit and FcεRI-mediated signaling would require testing of hypothemycin in a more complex model, for example in a mouse asthma mouse of allergic airway inflammation. This would provide information on how hypothemycin would affect a more complex allergic reaction involving multiple cell types. However, the further properties of hypothemycin to inhibit SCF dependent mast cell adhesion and chemotaxis would suggest that, in addition to inhibiting mast cell degranulation, hypothemycin may also prevent mast cell infiltration into sites of inflammation.
In summary, the results presented in this study provide proof of principle for the concept of concurrent inhibition of Kit- and FcεRI-mediated signaling as an approach for inhibition of mast cell-driven allergic reactions. To test this concept, we have utilized a molecule, hypothemycin, which inhibits both responses, with the aim of providing a basis for further investigation of this concept and potentially the development and optimization of novel compounds which may have a similar mode of action. Although hypothemycin irreversibly inhibits a specific subset of protein kinases, these properties may have inherent therapeutic advantages especially when used topically in disorders such as atopic asthma and dermatitis. As noted here, the ability to suppress both Kit and FcεRI-mediated signals at multiple levels would allow maximum suppression of antigen-induced release of inflammatory mediators and possibly other physiological functions such as migration and maturation of mast cells. An alternative approach however could be the concurrent administration of separate compounds targeting Kit- and FcεRI-specific signaling cascades. This latter approach may help to minimize potential issues with specificity of targeting.