p66Shc Is a Negative Regulator of FcεRI-Dependent Signaling in Mast Cells

Abstract

Aggregation of FcεRI on mast cells activates signaling pathways, resulting in degranulation and cytokine release. Release of mast cell-derived inflammatory mediators is tightly regulated by the interplay of positive and negative signals largely orchestrated by adapter proteins. Among these, the Shc family adapter p52Shc, which couples immunoreceptors to Ras activation, positively regulates FcεRI-dependent signaling. Conversely, p66Shc was shown to uncouple the TCR for the Ras–MAPK pathway and prime T cells to undergo apoptotic death. Loss of p66Shc in mice results in breaking of immunologic tolerance and development of lupus-like autoimmune disease, which includes alopecia among its pathological manifestations. The presence of numerous activated mast cells in alopecic skin areas suggests a role for this adapter in mast cells. In this study, we addressed the involvement of p66Shc in FcεRI-dependent mast cell activation. We showed that p66Shc is expressed in mast cells and that mast cells from p66Shc−/− mice exhibit enhanced responses following Ag stimulation of FcεRI. Furthermore, using RBL-2H3 cell transfectants, we showed that aggregation of FcεRI resulted in the recruitment of a p66Shc–SHIP1 complex to linker for activation of T cells. Collectively, our data identified p66Shc as a negative regulator of mast cell activation.

p66Shc regulates vesicle-mediated secretion in mast cells by affecting F-actin dynamics

“…BMMCs with IgE in the absence of antigen, which we have shown previously to reduce spontaneous degranulation to control levels [22], resulted in the recovery of a continous F-actin ring, similar to that observed in resting control cells (Fig. 2A).…”

“…This suggests that p66Shc participates in mast cell homeostasis by controlling the constitutive release of mediators through piecemeal degranulation. Hence, the enlarged granules, documented in mast cells lacking p66Shc, using toluidine blue staining, which does not allow partially empty discrimination from dense-core granules, are likely to reflect the increase in the number of partially empty granules rather than an increase in mediator contents, as supported by the comparable cellular levels of ␤-hexosaminidase in BMMCs from control and p66Shc Ϫ/Ϫ mice [22].…”

“…We have shown recently that p66Shc Ϫ/Ϫ BMMCs display enlarged toluidine blue-positive granules and substantial spontaneous degranulation compared with control BMMCs, indicating that p66Shc limits constitutive secretion in mast cells [22]. To gain further insight into the role of p66Shc in the process of constitutive secretion, we analyzed mast cell granules in WT and p66Shc Ϫ/Ϫ BMMCs by electron microscopy.…”

“…Lysis, immunoprecipitation, F-actin skeleton fractionation, and immunoblot assays Cells (2ϫ10 6 /sample), left untreated or stimulated as described above in Cell activation, were lysed, as described elsewhere [22]. Alternatively, postnuclear supernatants from 50 ϫ 10 6 cells/sample were immunoprecipitated using rabbit anti-SHIP1 or anti-collagen homology 2 polyclonal antibodies and protein A-Sepharose (Amersham Biosciences, Buckinghamshire, UK).…”

“…BMMCs exhibit increased responses, such as ␤-hexosaminidase and cytokine release, in the absence of stimulation and following Fc⑀RI engagement [22].…”