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TWEAK-independent Fn14 self-association and NF-κB activation is mediated by the C-terminal region of the Fn14 cytoplasmic domain.

Brown SA, Cheng E, Williams MS, Winkles JA - PLoS ONE (2013)

Bottom Line: Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions.Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122.These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

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Endogenously expressed Fn14 has slower electrophoretic mobility when analyzed under non-reducing conditions.Cell lysates were prepared from the indicated cell lines and equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-Fn14 antibody (from CST) or an anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).
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pone-0065248-g006: Endogenously expressed Fn14 has slower electrophoretic mobility when analyzed under non-reducing conditions.Cell lysates were prepared from the indicated cell lines and equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-Fn14 antibody (from CST) or an anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).

Mentions: We noted that in our ligand blot experiments, in which cell lysate samples are run under non-reducing conditions (no DTT in loading buffer, sample is not heated), the overexpressed Fn14-FL protein had slower electrophoretic mobility when compared to its mobility under normal (reducing) SDS-PAGE conditions (Fig. 2B). Therefore, we examined whether endogenously-expressed Fn14 also had different electrophoretic mobility under these two conditions. Lysates were prepared from three different cancer cell lines, protein was subjected to SDS-PAGE under either reducing or non-reducing conditions, and Western blot analysis was performed. In all three cell lines, Fn14 migrated as a single species with an apparent molecular mass of ∼17-kDa under reducing conditions (Fig. 6). In comparison, under non-reducing conditions, the major immunoreactive Fn14 species detected in the cell lines was ∼29-kDa, with two lower molecular mass species of ∼26- and ∼17-kDa also present in the A375 cell lysate. These results indicate that endogenous Fn14 could be forming a disulfide-linked dimer, either in the cell itself or after cell harvest during the cell lysis procedure.


TWEAK-independent Fn14 self-association and NF-κB activation is mediated by the C-terminal region of the Fn14 cytoplasmic domain.

Brown SA, Cheng E, Williams MS, Winkles JA - PLoS ONE (2013)

Endogenously expressed Fn14 has slower electrophoretic mobility when analyzed under non-reducing conditions.Cell lysates were prepared from the indicated cell lines and equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-Fn14 antibody (from CST) or an anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672086&req=5

pone-0065248-g006: Endogenously expressed Fn14 has slower electrophoretic mobility when analyzed under non-reducing conditions.Cell lysates were prepared from the indicated cell lines and equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-Fn14 antibody (from CST) or an anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).
Mentions: We noted that in our ligand blot experiments, in which cell lysate samples are run under non-reducing conditions (no DTT in loading buffer, sample is not heated), the overexpressed Fn14-FL protein had slower electrophoretic mobility when compared to its mobility under normal (reducing) SDS-PAGE conditions (Fig. 2B). Therefore, we examined whether endogenously-expressed Fn14 also had different electrophoretic mobility under these two conditions. Lysates were prepared from three different cancer cell lines, protein was subjected to SDS-PAGE under either reducing or non-reducing conditions, and Western blot analysis was performed. In all three cell lines, Fn14 migrated as a single species with an apparent molecular mass of ∼17-kDa under reducing conditions (Fig. 6). In comparison, under non-reducing conditions, the major immunoreactive Fn14 species detected in the cell lines was ∼29-kDa, with two lower molecular mass species of ∼26- and ∼17-kDa also present in the A375 cell lysate. These results indicate that endogenous Fn14 could be forming a disulfide-linked dimer, either in the cell itself or after cell harvest during the cell lysis procedure.

Bottom Line: Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions.Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122.These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory and pro-angiogenic cytokine implicated in physiological tissue regeneration and wound repair. TWEAK binds to a 102-amino acid type I transmembrane cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK:Fn14 engagement activates several intracellular signaling cascades, including the NF-κB pathway, and sustained Fn14 signaling has been implicated in the pathogenesis of chronic inflammatory diseases and cancer. Although several groups are developing TWEAK- or Fn14-targeted agents for therapeutic use, much more basic science research is required before we fully understand the TWEAK/Fn14 signaling axis. For example, we and others have proposed that TWEAK-independent Fn14 signaling may occur in cells when Fn14 levels are highly elevated, but this idea has never been tested directly. In this report, we first demonstrate TWEAK-independent Fn14 signaling by showing that an Fn14 deletion mutant that is unable to bind TWEAK can activate the NF-κB pathway in transfected cells. We then show that ectopically-expressed, cell surface-localized Fn14 can self-associate into Fn14 dimers, and we show that Fn14 self-association is mediated by an 18-aa region within the Fn14 cytoplasmic domain. Endogenously-expressed Fn14 as well as ectopically-overexpressed Fn14 could also be detected in dimeric form when cell lysates were subjected to SDS-PAGE under non-reducing conditions. Additional experiments revealed that Fn14 dimerization occurs during cell lysis via formation of an intermolecular disulfide bond at cysteine residue 122. These findings provide insight into the Fn14 signaling mechanism and may aid current studies to develop therapeutic agents targeting this small cell surface receptor.

Show MeSH
Related in: MedlinePlus