TSC1 deficiency promotes IL-3 depletion-induced mast cell apoptosis via ROS. (A) Increase of mitochondrial contents in TSC1KO BMMCs. Relative mitochondrial to nuclear DNA contents were analyzed by qRT-PCR using specific primers for 12S rRNA for mtDNA and 18S rRNA for nuclear DNA. (B) Increased ROS levels in TSC1KO BMMCs. ROS were analyzed by flow cytometry using DHE in the presence or absence of IL-3. (C) Hypersensitivity of TSC1KO BMMCs to H2O2-induced apoptosis. BMMCs were treated with H2O2 (100μM) in the presence of IL-3 for 24 hours, and then stained with annexin V. (D) Effect of ROS scavenger and pan-caspase inhibitor on IL-3 withdrawal-induced cell death in TSC1-deficient mast cells. TSC1KO BMMCs were cultured in IL-3–depleted media with the supplement of NAC (2mM), Nec1 (50μM), and zVAD (50μM). Annexin V–positive cells was measured by flow cytometry (mean ± SEM; *P < .05). (E) Increased caspase-3 cleavage in TSC1KO BMMCs. Cell lysates were subject to immunoblot using anti–caspase-3 antibody 24 hours after IL-3 withdrawal with or without NAC (2mM). p35 indicates procaspase-3; p19, partly processed caspase 3 fragment; and p17, fully cleaved, active form of caspase 3. Data shown are representative of 2 to 3 experiments. (F) Model for TSC1 function in mast cells. TSC1 inhibits mTORC1 as well as Erk1/2 and PKCδ but promotes mTORC2 activation in mast cells. Enhanced mTORC1, Erk1/2, and PKCδ activities in the absence of TSC1 may enhance mast cell cytokine production. However, TSC1 deficiency impairs mast cell degranulation. Furthermore, TSC1 inhibits p53/miR-34a and decreases the copy number of mitochondria, leading to increased Bcl-2 expression and decreased ROS levels to promote mast cell survival.
