Transgenic Expression of Dominant-Negative Fas-Associated Death Domain Protein in β Cells Protects against Fas Ligand-Induced Apoptosis and Reduces Spontaneous Diabetes in Nonobese Diabetic Mice

Abstract

In type 1 diabetes, many effector mechanisms damage the β cell, a key one being perforin/granzyme B production by CD8+ T cells. The death receptor pathway has also been implicated in β cell death, and we have therefore generated NOD mice that express a dominant-negative form of the Fas-associated death domain protein (FADD) adaptor to block death receptor signaling in β cells. Islets developed normally in these animals, indicating that FADD is not necessary for β cell development as it is for vasculogenesis. β cells from the transgenic mice were resistant to killing via the Fas pathway in vitro. In vivo, a reduced incidence of diabetes was found in mice with higher levels of dominant-negative FADD expression. This molecule also blocked signals from the IL-1R in culture, protecting isolated islets from the toxic effects of cytokines and also marginally reducing the levels of Fas up-regulation. These data support a role for death receptors in β cell destruction in NOD mice, but blocking the perforin/granzyme pathway would also be necessary for dominant-negative FADD to have a beneficial clinical effect.

Proapoptotic BH3-Only Protein Bid Is Essential For Death Receptor–Induced Apoptosis of Pancreatic β-Cells

“…The importance of the perforin pathway is probably best documented by the finding that loss of perforin greatly reduces the incidence of diabetes in NOD mice (43). Experiments with transgenic NOD mice expressing dnFADD in their ␤-cells indicated that death receptor signaling plays a partial role in diabetes development (20). Antigen-specific autoreactive cytotoxic T-cells kill ␤-cells in vitro using the Fas/FasL pathway in the absence of perforin (24,44), indicating that this mechanism may still contribute to diabetes development.…”

“…It appears likely that these death stimuli activate other BH3-only proteins, such as Bim in the case of ER stress (41) or Puma in the case of DNA damage (42). Since ␤-cell killing induced by IFN-␥ plus IL-1␤, which requires NO production, cannot be inhibited by either loss of Bid, caspase blockade, or Bcl-2 overexpression (20), it appears likely that this death occurs through a process independent of this pathway.…”

“…Biddeficient (bid Ϫ/Ϫ ) mice were generated on an inbred C57BL/6 background using C57BL/6-derived embryonic stem cells (17). Homozygous H-2 bm1 RIPBcl-2 and NOD.RIP-Bcl-2 transgenic mice (backcrossed Ն10 generations), which express human Bcl-2 in ␤-cells under control of the rat insulin promoter, have previously been described (19,20). The generation of bax Ϫ/Ϫ (21), bak Ϫ/Ϫ (22) and bak Ϫ/Ϫ bax ϩ/Ϫ (23) mice has previously been described, and those used here had been backcrossed for Ͼ10 generations onto a C57BL/6 background.…”

“…Intracellular insulin staining. The expression of insulin in islet ␤-cells was determined as previously described (20). Freshly isolated islets were trypsinized, fixed, and permeabilized.…”

Mediators and mechanisms of pancreatic beta-cell death in type 1 diabetes

“…Activated caspase-8 subsequently cleaves the effector caspase-3 and/or activates the mitochondrial death pathway via cleavage of the BH3 protein Bid. The role of Fas in T-cell mediated beta-cell destruction in vivo has remained controversial for a long time, but targeted overexpression of a dominant negative form of Fas (FADD) in beta-cells delays the onset of diabetes in NOD mice, implicating a role for Fas in the early stages of autoimmune beta-cell destruction (24,29,30).…”