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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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Related in: MedlinePlus

Cloning of a human Fn14 mRNA predicted to encode an Fn14 protein missing most of the extracellular domain.(A) Schematic representation of human Fn14 gene organization (via UCSC Genome Browser). The four Fn14 exons are numbered and boxed and the intron sizes are indicated in nucleotides (nt) at the top. The Fn14 amino acid (aa) numbers (1–129) and mature mRNA nucleotide (nt) numbers (1–1033) are provided above or below each exon, respectively. The positions of the two oligonucleotide primers used for RT-PCR analysis are indicated with arrows. (B) RNA was isolated from MDA-MB-231 and U87 cells and RT-PCR was performed using an exon 1 sense primer and an exon 4 antisense primer. PCR was also performed with this primer pair in the absence of cDNA (NT, no template). Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The positions of DNA size markers (M) are shown on the left (in base pairs). The two PCR products that were isolated and sequenced are indicated on the right as a and b. (C) Predicted amino acid sequence of PCR amplification products a and b. The last six amino acids of the signal peptide are indicated with an arrow, the Fn14 extracellular domain is bracketed with asterisks and the six cysteine residues found in this domain are in red. The Fn14 transmembrane domain is overlined and the cytoplasmic tail is underlined. (D) The Fn14 mRNA translation initiation codon and selected codons surrounding Fn14 introns 1 and 2 are shown for the Fn14 full-length (Fn14-FL) and Fn14 extracellular domain deletion (Fn14-ΔEC) mRNAs. The predicted Fn14-FL and Fn14-ΔEC amino acid sequence is shown below the nucleotide sequence. Abbreviation: I, intron. (E) Schematic representation of the Fn14-FL and Fn14-ΔEC proteins showing structural domains in relation to their exon coding regions. Amino acid numbers corresponding to the beginning of each protein domain and the C-terminal amino acid are indicated at the top of each diagram. The region of the extracellular domain that is missing in Fn14-ΔEC is shown in black. Abbreviations: SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CYT, cytoplasmic tail.
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pone-0065248-g001: Cloning of a human Fn14 mRNA predicted to encode an Fn14 protein missing most of the extracellular domain.(A) Schematic representation of human Fn14 gene organization (via UCSC Genome Browser). The four Fn14 exons are numbered and boxed and the intron sizes are indicated in nucleotides (nt) at the top. The Fn14 amino acid (aa) numbers (1–129) and mature mRNA nucleotide (nt) numbers (1–1033) are provided above or below each exon, respectively. The positions of the two oligonucleotide primers used for RT-PCR analysis are indicated with arrows. (B) RNA was isolated from MDA-MB-231 and U87 cells and RT-PCR was performed using an exon 1 sense primer and an exon 4 antisense primer. PCR was also performed with this primer pair in the absence of cDNA (NT, no template). Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The positions of DNA size markers (M) are shown on the left (in base pairs). The two PCR products that were isolated and sequenced are indicated on the right as a and b. (C) Predicted amino acid sequence of PCR amplification products a and b. The last six amino acids of the signal peptide are indicated with an arrow, the Fn14 extracellular domain is bracketed with asterisks and the six cysteine residues found in this domain are in red. The Fn14 transmembrane domain is overlined and the cytoplasmic tail is underlined. (D) The Fn14 mRNA translation initiation codon and selected codons surrounding Fn14 introns 1 and 2 are shown for the Fn14 full-length (Fn14-FL) and Fn14 extracellular domain deletion (Fn14-ΔEC) mRNAs. The predicted Fn14-FL and Fn14-ΔEC amino acid sequence is shown below the nucleotide sequence. Abbreviation: I, intron. (E) Schematic representation of the Fn14-FL and Fn14-ΔEC proteins showing structural domains in relation to their exon coding regions. Amino acid numbers corresponding to the beginning of each protein domain and the C-terminal amino acid are indicated at the top of each diagram. The region of the extracellular domain that is missing in Fn14-ΔEC is shown in black. Abbreviations: SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CYT, cytoplasmic tail.

Mentions: The original goal of this study was to determine whether Fn14 protein-positive cancer cells expressed more than one Fn14 mRNA species. To test this, we performed RT-PCR analysis using RNA isolated from human MDA-MB-231 breast cancer cells and U87 glioma cells and exon 1- or exon 4-targeted oligonucleotide primers (Fig. 1A). Two major amplification products of 310- and 205-bp were detected (Fig. 1B). The U87 cell mRNA-derived DNA products were isolated, cloned and sequenced. The product A nucleotide sequence was 100% identical to the corresponding region of the human Fn14 cDNA sequence that we previously reported (GenBank Accession Number AF191148) [2]. This cDNA encodes full-length (129-aa) Fn14 (referred to here as Fn14-FL). The product B nucleotide sequence was 100% identical to the corresponding region of an unpublished human Fn14 cDNA sequence that was deposited in GenBank by S. Tanaka in 1999 (GenBank Accession Number AB035481). In comparison to product A, the product B sequence has a 105-bp deletion, and is predicted to encode a shorter Fn14 isoform missing an internal 35-aa region within the 53-aa extracellular domain (amino acids 33–67; we refer to this protein here as Fn14 extracellular domain deletion (Fn14-ΔEC) (Fig. 1C). Analysis of the human Fn14 exon sequences revealed that exon 2 is 105-bp in length and encodes these same 35 amino acids, suggesting that this exon is a cassette-type alternative exon (reviewed in [40]). Indeed, further analysis of the Fn14 gene exon/intron junction sequences is consistent with the proposal that alternative splicing of the Fn14 primary transcript via exon 2 skipping would generate the Fn14-ΔEC mRNA and protein represented by product B (Fig. 1D). The structural domains of the Fn14-FL and Fn14-ΔEC proteins are shown in reference to the Fn14 coding exons in Fig. 1E. We have been unable to detect an Fn14 protein species in MDA-MB-231 or U87 cells that we can conclusively identify as the Fn14-ΔEC protein by Western blotting using an antibody recognizing the Fn14 cytoplasmic tail (data not shown). Thus, this smaller Fn14 isoform may either not be expressed by these cells at all, or alternatively, it could be expressed at relatively low levels and not detected by the antibody we utilized.


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)

Cloning of a human Fn14 mRNA predicted to encode an Fn14 protein missing most of the extracellular domain.(A) Schematic representation of human Fn14 gene organization (via UCSC Genome Browser). The four Fn14 exons are numbered and boxed and the intron sizes are indicated in nucleotides (nt) at the top. The Fn14 amino acid (aa) numbers (1–129) and mature mRNA nucleotide (nt) numbers (1–1033) are provided above or below each exon, respectively. The positions of the two oligonucleotide primers used for RT-PCR analysis are indicated with arrows. (B) RNA was isolated from MDA-MB-231 and U87 cells and RT-PCR was performed using an exon 1 sense primer and an exon 4 antisense primer. PCR was also performed with this primer pair in the absence of cDNA (NT, no template). Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The positions of DNA size markers (M) are shown on the left (in base pairs). The two PCR products that were isolated and sequenced are indicated on the right as a and b. (C) Predicted amino acid sequence of PCR amplification products a and b. The last six amino acids of the signal peptide are indicated with an arrow, the Fn14 extracellular domain is bracketed with asterisks and the six cysteine residues found in this domain are in red. The Fn14 transmembrane domain is overlined and the cytoplasmic tail is underlined. (D) The Fn14 mRNA translation initiation codon and selected codons surrounding Fn14 introns 1 and 2 are shown for the Fn14 full-length (Fn14-FL) and Fn14 extracellular domain deletion (Fn14-ΔEC) mRNAs. The predicted Fn14-FL and Fn14-ΔEC amino acid sequence is shown below the nucleotide sequence. Abbreviation: I, intron. (E) Schematic representation of the Fn14-FL and Fn14-ΔEC proteins showing structural domains in relation to their exon coding regions. Amino acid numbers corresponding to the beginning of each protein domain and the C-terminal amino acid are indicated at the top of each diagram. The region of the extracellular domain that is missing in Fn14-ΔEC is shown in black. Abbreviations: SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CYT, cytoplasmic tail.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672086&req=5

pone-0065248-g001: Cloning of a human Fn14 mRNA predicted to encode an Fn14 protein missing most of the extracellular domain.(A) Schematic representation of human Fn14 gene organization (via UCSC Genome Browser). The four Fn14 exons are numbered and boxed and the intron sizes are indicated in nucleotides (nt) at the top. The Fn14 amino acid (aa) numbers (1–129) and mature mRNA nucleotide (nt) numbers (1–1033) are provided above or below each exon, respectively. The positions of the two oligonucleotide primers used for RT-PCR analysis are indicated with arrows. (B) RNA was isolated from MDA-MB-231 and U87 cells and RT-PCR was performed using an exon 1 sense primer and an exon 4 antisense primer. PCR was also performed with this primer pair in the absence of cDNA (NT, no template). Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The positions of DNA size markers (M) are shown on the left (in base pairs). The two PCR products that were isolated and sequenced are indicated on the right as a and b. (C) Predicted amino acid sequence of PCR amplification products a and b. The last six amino acids of the signal peptide are indicated with an arrow, the Fn14 extracellular domain is bracketed with asterisks and the six cysteine residues found in this domain are in red. The Fn14 transmembrane domain is overlined and the cytoplasmic tail is underlined. (D) The Fn14 mRNA translation initiation codon and selected codons surrounding Fn14 introns 1 and 2 are shown for the Fn14 full-length (Fn14-FL) and Fn14 extracellular domain deletion (Fn14-ΔEC) mRNAs. The predicted Fn14-FL and Fn14-ΔEC amino acid sequence is shown below the nucleotide sequence. Abbreviation: I, intron. (E) Schematic representation of the Fn14-FL and Fn14-ΔEC proteins showing structural domains in relation to their exon coding regions. Amino acid numbers corresponding to the beginning of each protein domain and the C-terminal amino acid are indicated at the top of each diagram. The region of the extracellular domain that is missing in Fn14-ΔEC is shown in black. Abbreviations: SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CYT, cytoplasmic tail.
Mentions: The original goal of this study was to determine whether Fn14 protein-positive cancer cells expressed more than one Fn14 mRNA species. To test this, we performed RT-PCR analysis using RNA isolated from human MDA-MB-231 breast cancer cells and U87 glioma cells and exon 1- or exon 4-targeted oligonucleotide primers (Fig. 1A). Two major amplification products of 310- and 205-bp were detected (Fig. 1B). The U87 cell mRNA-derived DNA products were isolated, cloned and sequenced. The product A nucleotide sequence was 100% identical to the corresponding region of the human Fn14 cDNA sequence that we previously reported (GenBank Accession Number AF191148) [2]. This cDNA encodes full-length (129-aa) Fn14 (referred to here as Fn14-FL). The product B nucleotide sequence was 100% identical to the corresponding region of an unpublished human Fn14 cDNA sequence that was deposited in GenBank by S. Tanaka in 1999 (GenBank Accession Number AB035481). In comparison to product A, the product B sequence has a 105-bp deletion, and is predicted to encode a shorter Fn14 isoform missing an internal 35-aa region within the 53-aa extracellular domain (amino acids 33–67; we refer to this protein here as Fn14 extracellular domain deletion (Fn14-ΔEC) (Fig. 1C). Analysis of the human Fn14 exon sequences revealed that exon 2 is 105-bp in length and encodes these same 35 amino acids, suggesting that this exon is a cassette-type alternative exon (reviewed in [40]). Indeed, further analysis of the Fn14 gene exon/intron junction sequences is consistent with the proposal that alternative splicing of the Fn14 primary transcript via exon 2 skipping would generate the Fn14-ΔEC mRNA and protein represented by product B (Fig. 1D). The structural domains of the Fn14-FL and Fn14-ΔEC proteins are shown in reference to the Fn14 coding exons in Fig. 1E. We have been unable to detect an Fn14 protein species in MDA-MB-231 or U87 cells that we can conclusively identify as the Fn14-ΔEC protein by Western blotting using an antibody recognizing the Fn14 cytoplasmic tail (data not shown). Thus, this smaller Fn14 isoform may either not be expressed by these cells at all, or alternatively, it could be expressed at relatively low levels and not detected by the antibody we utilized.

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