Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice

Abstract

A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappaB (NF-kappaB) activity and tumor necrosis factor (TNF)-mediated programmed cell death (PCD). TNF dramatically increases A20 messenger RNA expression in all tissues. Mice deficient for A20 develop severe inflammation and cachexia, are hypersensitive to both lipopolysaccharide and TNF, and die prematurely. A20-deficient cells fail to terminate TNF-induced NF-kappaB responses. These cells are also more susceptible than control cells to undergo TNF-mediated PCD. Thus, A20 is critical for limiting inflammation by terminating TNF-induced NF-kappaB responses in vivo.

Fig. 1

Generation and histology of A20^–/– mice. (A) Gross appearance of 4-week-old A20^+/+ and A20^–/– mice. (B) Gross appearance of A20^+/+ and A20^–/– livers. Note pale acellular regions of A20^–/– livers. (C) Gross appearance of A20^+/+ and A20^–/– kidneys. Note atrophied kidney in A20^–/– mouse. (D) Flow cytometric analyses of TNF expression in A20^+/+ and A20^–/– splenocytes stimulated with LPS. (E) Hematoxylin- and eosin-stained (H and E) sections from A20^+/+ and A20^–/– livers. Note inflammation and hepatocyte loss in A20^–/– livers. (F) H and E kidney sections. Note interstitial nephritis, glomerular dilatation, and cortical tubular atrophy in small A20^–/– kidney. (G) H and E colonic sections. Note colitis, including lamina propria inflammation, crypt abscess, and branching epithelial crypts in A20^–/– colon. (H) H and E joint and bone sections. Note bone marrow replacement with inflammatory cells, thinned trabecular bone, and destructive arthritis in A20^–/– bone and joint. (I) H and E skin sections. Note thickened epidermis and dermis, and loss of hair follicles and fat in A20^–/– skin. (J) H and E kidney sections from A20^+/– RAG-1^–/– (left panel) and A20^–/– RAG-1^–/– (right panel) mice. Note normal appearance of A20^+/– RAG-1^–/– kidney. Note interstitial nephritis, glomerular dilatation, and cortical tubular atrophy in A20^–/– RAG-1^–/– kidney (right panel), comparable to A20^–/– RAG-1^+/+ kidney [right panel in (F)].

Fig. 2

Sensitivity of A20^–/– thymocytes and MEFs to TNF-mediated PCD. (A) Northern analysis of A20 mRNA expression in tissues from TNF-injected normal mice (LIV, liver; KID, kidney; SPL, spleen; THY, thymus; COL, colon; LN, lymph node). Comparable RNA loading and integrity was confirmed by ethidium staining of 28S rRNA. (B) Survival of A20^–/– (solid bars) and A20^+/– (hatched bars) thymocytes from 2- to 3-week-old mice 5 hours after in vitro treatment with the indicated agents. TNF was used at 10 ng/ml, and cycloheximide (CHX) was used at 10 μg/ml in all experiments. Asterisk indicates P < 0.001 by Tukey's test. (C) Western analyses of IκBα [Santa Cruz Biotechnology (SCB)], phospho-SAPK/JNK [New England Biolabs (NEB)], SAPK/JNK (NEB), Bcl-x (Transduction Labs), and Bcl-2 (Pharmingen) proteins in lysates from TNF-treated thymocytes. (D) Northern analysis of A20 mRNA expression in TNF-treated A20^+/+ MEFs. (E) Survival of A20^–/– (solid bars) and A20^+/– (hatched bars) MEFs 10 hours after in vitro treatment with the indicated agents. (F) Western analyses of TRAF2 (MedBiol Labs), cIAP-1 (Trevigen), phospho-SAPK/JNK, and SAPK/JNK proteins in lysates from TNF-treated MEFs.

Fig. 3

Prolonged NF-κB responses to TNF in A20^–/– MEFs. Electrophoretic mobility-shift assay (EMSA), Western, and Northern blot analyses of A20^+/+ and A20^–/– MEFs treated repeatedly with TNF and harvested at the indicated time points. (A) EMSA analyses of NF-κB activity, using an NF-κB consensus oligonucleotide (SCB). (B) Western blot analysis of IκBα expression. (C) Northern blot analyses of IκBα and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA expression in MEFs. (D) Western blot analyses of IκBα and phospho-IκBα expression after proteasome inhibition. (E) IKK kinase assay of TNF-treated MEFs. Total cell lysates from repeatedly TNF-treated MEFs were immunoprecipitated with an anti-IKKγ antibody (SCB), and kinase activity was assessed using a GST-IκBα (1-54) substrate (upper panels). Comparable IKKβ protein in immunoprecipitated samples confirmed by Western analysis (lower panels). (F) Western analysis of IκBα expression in IL-1β–treated MEFs.