Short form FLICE-inhibitory protein promotes TNFα-induced necroptosis in fibroblasts derived from CFLARs transgenic mice

Biochem Biophys Res Commun. 2016 Nov 4;480(1):23-28. doi: 10.1016/j.bbrc.2016.10.015. Epub 2016 Oct 6.

Abstract

Cellular FLICE-inhibitory protein (cFLIP) is a catalytically inactive homolog of the initiator caspase, caspase 8 and blocks apoptosis through binding to caspase 8. Human CFLAR gene encodes two proteins, a long form cFLIP (cFLIPL) and a short form cFLIP (cFLIPs) due to an alternative splicing. Recent studies have shown that expression of cFLIPs, but not cFLIPL promotes programmed necrosis (also referred to as necroptosis) in an immortalized human keratinocyte cell line, HaCaT. Here, we found that expression of cFLIPs similarly promoted necroptosis in immortalized fibroblasts. To further expand this observation and exclude the possibility that immortalization process of keratinocytes or fibroblasts might affect the phenotype induced by cFLIPs expression, we generated human CFLARs transgenic (Tg) mice. Primary fibroblasts derived from CFLARs Tg mice were increased in susceptibility to TNFα-induced necroptosis, but not apoptosis compared to wild-type (WT) fibroblasts. Moreover, hallmarks of necroptosis, such as phosphorylation of receptor-interacting protein kinase (RIPK)1 and RIPK3, and oligomer formation of mixed lineage kinase domain-like (MLKL) were robustly induced in CFLARs Tg fibroblasts compared to wild-type fibroblasts following TNFα stimulation. Thus, cFLIPs-dependent promotion of necroptosis is not unique to immortalized keratinocytes or fibroblasts, but also to generalized to primary fibroblasts.

Keywords: Apoptosis; Necroptosis; RIPK1; TNFα; cFLIP.

MeSH terms

  • Animals
  • CASP8 and FADD-Like Apoptosis Regulating Protein / genetics*
  • CASP8 and FADD-Like Apoptosis Regulating Protein / metabolism
  • Cell Death / drug effects
  • Cell Line, Transformed
  • Cells, Cultured
  • Dipeptides / pharmacology
  • Fibroblasts / metabolism*
  • Fibroblasts / pathology
  • Humans
  • Indoles / pharmacology
  • Mice, Transgenic
  • Necrosis / genetics
  • Oligopeptides / pharmacology
  • Phosphorylation
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism
  • Tumor Necrosis Factor-alpha / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology*

Substances

  • CASP8 and FADD-Like Apoptosis Regulating Protein
  • CFLAR protein, human
  • Dipeptides
  • Indoles
  • Oligopeptides
  • Tumor Necrosis Factor-alpha
  • benzyloxycarbonyl-valyl-alanyl-aspartic acid
  • birinapant
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk1 protein, mouse
  • Ripk3 protein, mouse