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Alternative splicing generates a constitutively active Fn14 isoform that signals independently of TWEAK

ABSTRACT: Fibroblast growth factor-inducible 14 (Fn14) is a tumour necrosis factor (TNF) receptor family member and therapeutic target in cancer and inflammatory disease. However, clinical strategies aimed at blocking its activation by preventing engagement with its ligand TNF-like weak inducer of apoptosis (TWEAK) have demonstrated limited efficacy, suggesting alternative mechanisms of Fn14 activation. Here, we identified Fn14β, a novel Fn14 isoform, encoded by the alternatively spliced transcript TNFRSF12A-202. Transcriptomic analyses showed that TNFRSF12A-202 is expressed across human tissues and liver disease contexts, localises to fibrotic regions in vivo, and is associated with reduced overall survival in hepatocellular carcinoma. Structural modelling predicted that Fn14β lacks the extracellular TWEAK-binding domain while retaining the intracellular TRAF-binding signalling domain. CRISPR/Cas9-mediated selective ablation of the canonical Fn14 isoform demonstrated that endogenous Fn14β is expressed and sufficient to drive downstream transcriptional programmes shared with canonical Fn14a. Functional analyses further showed that Fn14β activates NF-κB signalling and promotes proliferation independently of TWEAK. Together, these findings identified Fn14β as a novel constitutively active, TWEAK-independent Fn14 isoform and provided a mechanistic basis for ligand-independent Fn14 signalling, with implications for therapeutic strategies targeting the TWEAK/Fn14 pathway.

ORGANISM(S): Homo sapiens

PROVIDER: GSE335579 | GEO | 2026/06/17

REPOSITORIES: GEO

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Project description:To test the efficacy of TNFR-Fc and anti-TWEAK mAb treatment alone and in combination Tumor necrosis factor (TNF)-alpha is a major effector in various inflammatory conditions. TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that promotes inflammatory tissue damage through its receptor, FGF-inducible molecule 14 (Fn14). Since both TWEAK and TNF-alpha have been shown to mediate pathological responses through inter-dependent or independent pathways by in vitro, the potential interplay of these pathways was investigated in a mouse colitis model. Acute colitis was induced by rectal injection of trinitrobenzene sulfonic acid (TNBS), with administration of control IgG, TNF receptor (TNFR)-Ig chimeric protein, anti-TWEAK monoclonal antibody, or the combination of TNFR-Ig and anti-TWEAK antibody. On day 4, disease severity was evaluated and gene expression profiling was analyzed using whole colon tissue. Levels of transcript of TWEAK, Fn14 and NF-kB-related molecules were measured in purified colon epithelial cells (ECs). NF-kB activation was investigated with Western blot and immunohistochemical analysis. As a result, activation of the canonical, but not noncanonical NF-kB pathway was the hallmark of inflammatory responses in this model. Inflammation induced upregulation of Fn14 only in ECs but not in other cell types. Combination treatment of TNFR-Ig and anti-TWEAK antibody synergistically reduced disease severity in comparison with the control antibody or single agent treatment. Gene expression profile of the colon indicated downregulation of canonical NF-kB pathway with combination treatment. In conclusion, synergistic activation of canonical NF-kB by TWEAK and TNF-alpha is critical for the induction of inflammatory tissue damage in acute inflammation. TNBS colitis was induced by intrarectal administration of a 2 % solution of TNBS in phosphate-buffered saline (PBS): ethanol (1:1). For acute inflammatory responses, 70 M-NM-<g/g body weight of TNBS was given on day 0 and animals sacrificed on day 4. One hour prior to administration of TNBS, groups of mice were injected i.p. with the control IgG2a mAb (anti-human CD20) (10 mg/kg), TNFR-Ig (0.3 mg/kg), anti-TWEAK (mP2D10, 10 mg/kg), the combination of TNFR-Fc (0.3 mg/kg) and anti-TWEAK mP2D10 (10 mg/kg), or were untreated. For single agent and combination treatments groups, 0.3 mg/kg TNFR-Ig was employed, since 1 mg/kg of TNFR-Ig markedly ameliorated TNBS colitis but 0.3 mg/kg was much less effective as monitored by the effect on colon length and body weight. Colon tissue was rolled and snap-frozen in liquid nitrogen. Specimens for RNA extraction were cut from the frozen rolled colon. A set of experiments using 5-8 mice for each experimental group was performed twice, labelled 7 and 10 in the CEL files. Total number of mice for each experimental condition was as follows; untreated TNBS colitis group, 8; control antibody, 15; TNFR-Ig, 12; anti-TWEAK mAb, 14; combination of TNFR-Ig and anti-TWEAK mAb, 15.

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Alternative splicing generates a constitutively active Fn14 isoform that signals independently of TWEAK

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