Impaired regulation of NF-κB and increased susceptibility to colitis-associated tumorigenesis in CYLD-deficient mice
Jun Zhang, Brigid Stirling, Stephane T. Temmerman, Chi A. Ma, Ivan J. Fuss, Jonathan M.J. Derry, Ashish Jain
Jun Zhang, Brigid Stirling, Stephane T. Temmerman, Chi A. Ma, Ivan J. Fuss, Jonathan M.J. Derry, Ashish Jain
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Research Article Oncology

Impaired regulation of NF-κB and increased susceptibility to colitis-associated tumorigenesis in CYLD-deficient mice

Abstract

Cylindromatosis (CYLD) is a deubiquitinating enzyme that is altered in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. However, the regulatory function of CYLD remains unsettled. Here we show that the development of B cells, T cells, and myeloid cells was unaffected in CYLD-deficient mice, but that the activation of these cells with mediators of innate and adaptive immunity resulted in enhanced NF-κB and JNK activity associated with increased TNF receptor–associated factor 2 (TRAF2) and NF-κB essential modulator (NEMO) ubiquitination. CYLD-deficient mice were more susceptible to induced colonic inflammation and showed a dramatic increase in the incidence of tumors compared with controls in a colitis-associated cancer model. These results suggest that CYLD limits inflammation and tumorigenesis by regulating ubiquitination in vivo.

Authors

Jun Zhang, Brigid Stirling, Stephane T. Temmerman, Chi A. Ma, Ivan J. Fuss, Jonathan M.J. Derry, Ashish Jain

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Figure 3

Comparison of triggered NF-κB activation and cytokine production in Cyld–/– and wild-type cells.

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Comparison of triggered NF-κB activation and cytokine production in Cyld...
(A) Peritoneal macrophages were stimulated with TNF-α (500 U/ml), CD40 agonist Ab (1 μg/ml), LPS (2.5 μg/ml), or Pam3CSK4 (150 ng/ml). Cellular extracts were prepared from cells stimulated for the indicated intervals and analyzed by EMSA for NF-κB binding activity. Quantification of NF-κB activity is represented as fold change compared with unstimulated cells (set at 1.0). (B) Peritoneal macrophages were stimulated with LPS (2.5 μg/ml), Pam3CSK4 (150 ng/ml), or CD40 agonist Ab (5 μg/ml) for 24 hours. IFN-γ (10 ng/ml) was added to all stimulation conditions, and the production of IL-6 and TNF-α into culture supernatants was assessed by ELISA. Med, medium. *P < 0.005, Cyld–/– versus wild-type. (C) CYLD overexpression suppresses NF-κB activation by CD40, EDAR, and RANK, but not by TNF-α. 293M cells were transfected with an NF-κB reporter construct either in combination with a CYLD-bearing plasmid or with empty vector as a control. Cells were stimulated with TNF-α, and luciferase activity was measured. To activate other TNFR pathways, vectors bearing CD40, EDAR, and RANK were cotransfected into 293M cells. This led to receptor overexpression and ligand-independent NF-κB activation caused by spontaneous receptor trimerization. Quantitation of NF-κB activity in the presence of CYLD is indicated as fold suppression. *P < 0.005, Cyld–/– versus vector.