RESUMO
Tumor necrosis factor-alpha (TNF) activates caspase-8 to cleave effector caspases or Bid, resulting in type-1 or type-2 apoptosis, respectively. We show here that TNF also induces caspase-8-dependent C-terminal cleavage of the retinoblastoma protein (Rb). Interestingly, fibroblasts from Rb(MI/MI) mice, in which the C-terminal caspase cleavage site is mutated, exhibit a defect in Bid cleavage despite caspase-8 activation. Recent results suggest that TNF receptor endocytosis is required for the activation of caspase-8. Consistent with this notion, inhibition of V-ATPase, which plays an essential role in acidification and degradation of endosomes, specifically restores Bid cleavage in Rb(MI/MI) cells. Inhibition of V-ATPase sensitizes Rb(MI/MI) but not wild-type fibroblasts to TNF-induced apoptosis and stimulates inflammation-associated colonic apoptosis in Rb(MI/MI) but not wild-type mice. These results suggest that Rb cleavage is required for Bid cleavage in TNF-induced type-2 apoptosis, and this requirement can be supplanted by the inhibition of V-ATPase.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/metabolismo , Proteína do Retinoblastoma/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Sítios de Ligação , Caspase 8/metabolismo , Citocromos c/metabolismo , Fibroblastos/metabolismo , Camundongos , Estrutura Terciária de Proteína , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Frações Subcelulares/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The retinoblastoma tumour suppressor protein RB is cleaved by caspases during apoptosis. Here we have mutated the caspase cleavage site in the carboxy terminus of the murine Rb protein in the mouse germ line to create the Rb-MI allele. After endotoxic shock, expression of Rb-MI inhibits apoptosis in the intestines, but not in the spleen, and promotes the survival of male mice. Fibroblasts expressing Rb-MI protein are protected from apoptosis induced by the tumour-necrosis factor-alpha type I receptor (TNFRI) but remain sensitive to cell death induced by DNA damage. Correspondingly, the release of cytochrome c and the activation of caspase-3 induced by TNFRI, but not by DNA damage, are defective in cells expressing Rb-MI. Our results highlight the importance of Rb cleavage in TNFRI-induced apoptosis.