RESUMEN
Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.
Asunto(s)
Apoptosis , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Ratones , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/metabolismo , Multimerización de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND AND AIMS: Tumor necrosis factor (TNF) is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, because TNF does not kill normal IECs. Here, we investigated how chronic nuclear factor (NF)- κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice. METHODS: Human IBD specimens were stained for p65 and cleaved caspase-3. C57BL/6 mice with constitutively active IKKß in IEC (Ikkß(EE)IEC), Ripk1D138N/D138N knockin mice, and Ripk3-/- mice were injected with TNF or lipopolysaccharide. Enteroids were also isolated from these mice and challenged with TNF with or without RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole. Ripoptosome-mediated caspase-8 activation was assessed by immunoprecipitation. RESULTS: NF-κB activation in human IBD correlated with appearance of cleaved caspase-3. Congruently, unlike normal mouse IECs that are TNF-resistant, IECs in Ikkß(EE)IEC mice and enteroids were susceptible to TNF-dependent apoptosis, which depended on the protein kinase function of RIPK1. Constitutively active IKKß facilitated ripoptosome formation, a RIPK1 signaling complex that mediates caspase-8 activation by TNF. Butylated hydroxyanisole treatment and RIPK1 inhibitors attenuated TNF-induced and ripoptosome-mediated caspase-8 activation and IEC death in vitro and in vivo. CONCLUSIONS: Contrary to common expectations, chronic NF-κB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase inhibitors selectively inhibited TNF destructive properties while preserving its survival and proliferative properties, which do not require RIPK1 kinase activity. RIPK1 kinase inhibition could be a potential treatment for IBD.