TWEAK mediates signal transduction and differentiation of RAW264.7 cells in the absence of Fn14/TweakR. Evidence for a second TWEAK receptor

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor family that is implicated in apoptosis, proliferation, migration, and inflammation. We describe our findings showing that TWEAK mediated the differentiation of RAW264.7 (RAW) monocyte/macrophage cells into multinuclear, functional osteoclasts. The effect of TWEAK was direct and not mediated by the receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL) as shown by the use of TWEAK- or RANKL-neutralizing antibodies and by osteoprotegerin, a decoy receptor for RANKL. Recently, fibroblast growth factor-inducible 14 (Fn14) was suggested to be a receptor for TWEAK. We show that the Fn14/TWEAK receptor (TweakR) was not responsible for the osteoclastic effect of TWEAK on RAW cells. Flow cytometry analysis did not reveal the expression of Fn14/TweakR on RAW cells. Moreover, Fn14/TweakR-neutralizing antibodies did not block TWEAK-induced RAW cell differentiation into osteoclasts. This indicated that a second TweakR, TweakR2, exists on RAW cells and is responsible for mediating TWEAK-induced differentiation. We next compared the signaling pathways that are activated by the two receptors. TWEAK binding to TweakR2 activated the NF-kappa B, mitogen-activated protein kinase and c-Jun N-terminal kinase signaling cascades in RAW cells. In contrast, TWEAK binding to Fn14/TweakR activated the NF-kappa B and c-Jun N-terminal kinase pathways but induced only a weak activation of MAPK in HT-29 human colon adenocarcinoma cells expressing endogenous Fn14/TweakR. We propose that the biological effects of TWEAK are mediated by binding to one of at least two distinct receptors that induce differential activation of downstream signaling pathways.     

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-kappaB (NF-kappaB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-kappaB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast- and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.     

Functional Expression of TWEAK and the Receptor Fn14 in Human Malignant Ovarian Tumors: Possible Implication for Ovarian Tumor Intervention

The aim of this current study was to investigate the expression of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) in human malignant ovarian tumors, and test TWEAK’s potential role on tumor progression in cell models in-vitro. Using immunohistochemistry (IHC), we found that TWEAK and its receptor Fn14 were expressed in human malignant ovarian tumors, but not in normal ovarian tissues or in borderline/benign epithelial ovarian tumors. High levels of TWEAK expression was detected in the majority of malignant tumors (36 out of 41, 87.80%). Similarly, 35 out of 41 (85.37%) malignant ovarian tumors were Fn14 positive. In these malignant ovarian tumors, however, TWEAK/Fn14 expression was not corrected with patients’ clinical subtype/stages or pathological features. In vitro, we demonstrated that TWEAK only inhibited ovarian cancer HO-8910PM cell proliferation in combination with tumor necrosis factor-α (TNF-α), whereas either TWEAK or TNF-α alone didn’t affect HO-8910PM cell growth. TWEAK promoted TNF-α production in cultured THP-1 macrophages. Meanwhile, conditioned media from TWEAK-activated macrophages inhibited cultured HO-8910PM cell proliferation and invasion. Further, TWEAK increased monocyte chemoattractant protein-1 (MCP-1) production in cultured HO-8910PM cells to possibly recruit macrophages. Our results suggest that TWEAK/Fn14, by activating macrophages, could be ovarian tumor suppressors. The unique expression of TWEAK/Fn14 in malignant tumors indicates that it might be detected as a malignant ovarian tumor marker.     

Studies of binding of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to fibroblast growth factor inducible 14 (Fn14)

To perform highly sensitive cellular binding studies with TNF-like weak inducer of apoptosis (TWEAK), we developed a bioluminescent variant of soluble TWEAK (GpL-FLAG-TNC-TWEAK) by fusing it genetically to the C terminus of the luciferase of Gaussia princeps (GpL). Equilibrium binding studies on human (HT1080 and HT29) and murine (Renca and B16) cell lines at 37 °C revealed high affinities of human TWEAK from 53 to 112 pm. The dissociation rate constant of the TWEAK-Fn14 interaction was between 0.48×10(-3) s(-1) (HT29) and 0.58×10(-3) s(-1) (HT1080) for the human molecules, and the association rate constant obtained was 3.3×10(6) m(-1) s(-1) for both cell lines. It has been shown previously that oligomerization of soluble TWEAK trimers results in enhanced Fn14-mediated activation of the classical NFκB pathway. Binding studies with GpL-FLAG-TNC-TWEAK trimers oligomerized by help of a FLAG tag-specific antibody gave no evidence for a major increase in Fn14 occupancy by oligomerized ligand despite strongly enhanced induction of the NFκB target IL8. Thus, aggregated complexes of soluble TWEAK and Fn14 have a higher intrinsic activity to stimulate the classical NFκB pathway and qualitatively differ from isolated trimeric TWEAK-Fn14 complexes. Furthermore, determination of IL8 induction as a function of occupied activated receptors revealed that the intrinsic capability of TNFR1 to stimulate the classical NFκB pathway and IL8 production was ～100-fold higher than Fn14. Thus, although ～25 activated TNFR1 trimers were sufficient to trigger half-maximal IL8 production, more than 2500 cell-bound oligomerized TWEAK trimers were required to elicit a similar response.     

Development of an Fn14 agonistic antibody as an anti-tumor agent

TNF-like weak inducer of apoptosis (TWEAK), a tumor necrosis factor (TNF) family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is upregulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231) and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect or tumor killing activity caused by antibody-dependent cell-mediated cytotoxicity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.Key words: TWEAK, Fn14, monoclonal antibody, agonist, xenograft, apoptosis     

A novel llama antibody targeting Fn14 exhibits anti-metastatic activity in vivo

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (5B6, 18D1, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 5B6, efficiently blocked TNF-like weak inducer of apoptosis (TWEAK) binding at low concentrations (0.2–2 µg/ml). Oligomerization and Fcγ receptor (FcγR) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 18D1 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RENCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEAK-Fn14 interaction in some cases.     

A previously unrecognized protein-protein interaction between TWEAK and CD163: potential biological implications

TWEAK (TNF-like weak inducer of apoptosis) is a TNF superfamily member implicated in several mechanisms. Although fibroblast growth factor inducible 14 (Fn14)/TweakR has been reported as its receptor, an as yet unrecognized surface molecule(s) might modulate TWEAK function(s). Thus, we set out to identify TWEAK-binding proteins by screening a combinatorial peptide library. Cyclic peptides containing a consensus motif (WXDDG) bound to TWEAK specifically. These peptides were similar to CD163, a scavenger receptor cysteine-rich domain family member, restricted to the monocyte/macrophage lineage and responsible for the uptake of circulating haptoglobin-hemoglobin (Hp-Hb) complexes. Sequence profile analysis suggested that TWEAK mimicked the CD163 natural ligand (Hp-Hb). Consistently, we show dose-dependent TWEAK binding to CD163 and blockade by an anti-CD163 Ab. In a competition assay, both soluble CD163 and Fn14/TweakR were able to compete off TWEAK binding to coated Fn14/TweakR or CD163, respectively. Flow-cytometry and immunofluorescence assays showed that human monocytes (Fn14/TweakR negative and CD163 positive) bind TWEAK, thus blocking the recognition of CD163 and reducing the activation mediated by a specific mAb in these cells. We demonstrate that monocytes can sequester TWEAK from supernatants, thus preventing tumor cell apoptosis; this effect was reverted by preincubation with the peptide mimicking CD163 or with a mAb anti-CD163, indicating specificity. Finally, we show that recombinant human TWEAK binding to CD163-transfected Chinese hamster ovary cells is inhibited by the presence of either unlabeled TWEAK or the Hp-Hb complex. Together, these data are consistent with the hypothesis that CD163 either acts as a TWEAK scavenger in pathological conditions or serves as an alternate receptor for TWEAK in cells lacking Fn14/TweakR.     

Development of an Fn14 agonistic antibody as an anti-tumor agent

TWEAK, a TNF family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is up-regulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling, and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231), and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect and/or ADCC mediated tumor killing activity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.     

Characterization of murine TWEAK and its receptor (Fn14) by monoclonal antibodies

In the present study, we characterized murine TWEAK and its receptor (Fn14) by generating cDNA transfectants and specific monoclonal antibodies (mAbs). Recombinant murine TWEAK bound to murine Fn14-transfected L5178Y (mFn14/L5178Y) cells and induced cell death. Some anti-human Fn14 mAbs we previously generated strongly cross-reacted with murine Fn14 and induced cell death in mFn14/L5178Y cells. Murine TWEAK-transfected L5178Y cells expressed murine TWEAK on cell surface and secreted functional TWEAK, which were detected by a newly generated anti-murine TWEAK mAb (MTW-1). Although thioglycolate-elicited murine peritoneal macrophages did not express a detectable level of TWEAK on their surface, they secreted functional TWEAK that was cytotoxic against mFn14/L5178Y cells and neutralized by MTW-1. The anti-murine TWEAK and Fn14 mAbs will be useful for further investigating the physiological and pathological functions of TWEAK and Fn14.     

TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease

First described as a weak apoptosis inducer, the TNF superfamily ligand TWEAK has since emerged as a cytokine that regulates multiple cellular responses, including proinflammatory activity, angiogenesis and cell proliferation, suggesting roles in inflammation and cancer. More recently TWEAK's ability to regulate progenitor cell fate was elucidated. Experiments using genetic overexpression and pathway inhibition or deficiency in mice indicate that TWEAK coordinates inflammatory and progenitor cell responses in settings of acute injury through its highly inducible receptor, FGF-inducible molecule 14 (Fn14), establishing the pathway's physiological role in facilitating acute tissue repair. In contrast, in chronic inflammatory disease models characterized by persistent TWEAK/Fn14 activation, TWEAK functions as a novel pathogenic mediator by amplifying inflammation, promoting tissue damage and potentially impeding endogenous repair mechanisms. Herein we aim not only to review the multifaceted functions of this emerging pathway, but also propose a conceptual framework for TWEAK/Fn14 pathway function in health and disease, supported by studies employing TWEAK and Fn14 deficient mice and anti-TWEAK blocking mAbs in acute injury and inflammatory disease settings. In addition to a perspective of the biology, we discuss potential therapeutic strategies targeting this pathway for the treatment of tissue injury, chronic inflammatory diseases and cancer.     

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

Introduction

TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.

Methods

TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.

Results

TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38⁺ plasma cells and with CD22⁺ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22⁺ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1⁺ osteoblasts.

Conclusions

The expression of TWEAK by CD22⁺ B cells and CD38⁺ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.     

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.     

The fibroblast growth factor-inducible 14 receptor is highly expressed in HER2-positive breast tumors and regulates breast cancer cell invasive capacity

Genomic characterization is beginning to define a molecular taxonomy for breast cancer; however, the molecular basis of invasion and metastasis remains poorly understood. We report a pivotal role for the fibroblast growth factor-inducible 14 (Fn14) receptor in this process. We examined whether Fn14 and its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) were expressed in breast tumors and whether deregulation of Fn14 levels affected malignant behavior of breast cancer cell lines. Analysis of TWEAK and Fn14 in publicly available gene expression data indicated that high Fn14 expression levels significantly correlated with several poor prognostic indicators (P < 0.05). Fn14 expression was highest in the HER2-positive/estrogen receptor-negative (HER2(+)/ER(-)) intrinsic subtype (P = 0.0008). An association between Fn14 and HER2 expression in breast tumors was confirmed by immunohistochemistry. Fn14 levels were elevated in invasive, ER(-) breast cancer cell lines. Overexpression of Fn14 in weakly invasive MCF7 and T47D cells resulted in a marked induction of invasion and activation of nuclear factor-kappaB (NF-kappaB) signaling. Ectopic expression of Fn14tCT, a Fn14 deletion mutant that cannot activate NF-kappaB signaling, was not able to induce invasion. Moreover, ectopic expression of Fn14tCT in highly invasive MDA-MB-231 cells reduced their invasive capability. RNA interference-mediated inhibition of Fn14 expression in both MDA-MB-231 and MDA-MB-436 cells reduced invasion. Expression profiling of the Fn14-depleted cells revealed deregulation of NF-kappaB activity. Our findings support a role for Fn14-mediated NF-kappaB pathway activation in breast tumor invasion and metastasis.     

Structural Basis and Targeting of the Interaction between Fibroblast Growth Factor-inducible 14 and Tumor Necrosis Factor-like Weak Inducer of Apoptosis

Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr¹⁷⁶, but not Trp²³¹, resulted in the loss of TWEAK binding to Fn14 substantiating Tyr¹⁷⁶ as the anchoring residue. Importantly, mutation of TWEAK at Tyr¹⁷⁶ did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.     

Expression of TWEAK and its receptor Fn14 in human subcutaneous adipose tissue. Relationship with other inflammatory cytokines in obesity

TWEAK, a cytokine of the TNF family, has been found to be expressed under different inflammatory conditions but no data is available concerning the expression of this cytokine and its receptor (Fn14) in human obesity. In the present work we have evaluated the expression of many pro-inflammatory TNF system cytokines (TNF-alpha, TWEAK and their respective receptors, TNFR1, TNFR2 and Fn14) in human adipose tissue of 84 subjects some with different degree of obesity and type 2 diabetes, and its relation with inflammation by also measuring the expression of macrophage marker CD68. We detected expression of TWEAK and Fn14 in isolated mature adipocytes and in the stromovascular fraction. Additionally, we found that LPS upregulates the expression of both genes on THP-1 human monocytic cell line. TWEAK was expressed in adipose tissue of all studied subjects with no differences between obesity group, and was associated with Fn14 expression in morbid obese, mainly in women with type 2 diabetes. The data obtained here also showed that TNF-alpha and TNFR2 mRNAs were significantly more expressed in subcutaneous adipose tissue of subjects with morbid obesity compared to obese and non-obese subjects. In contrast, TNFR1 gene expression was negatively associated with BMI. Our results suggest that the expression of TNF-derived pro-inflammatory cytokines are increased in severe obesity, where macrophage infiltrate could modulate the inflammatory environment through activation of its receptors.     

Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death

TWEAK, a TNF family member, is produced by IFN-gamma-stimulated monocytes and induces multiple pathways of cell death, including caspase-dependent apoptosis, cathepsin B-dependent necrosis, and endogenous TNF-alpha-mediated cell death, in a cell type-specific manner. However, the TWEAK receptor(s) that mediates these multiple death pathways remains to be identified. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which was responsible for TWEAK-induced proliferation of endothelial cells and angiogenesis. Because Fn14 lacks the cytoplasmic death domain, it remains unclear whether Fn14 can also mediate the TWEAK-induced cell death. In this study, we demonstrated that TWEAK could induce apoptotic cell death in Fn14 transfectants. A pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, rather sensitized the Fn14 transfectants to TWEAK-induced cell death by necrosis via reactive oxygen intermediates and cathepsin B-dependent pathway. By using newly generated agonistic anti-Fn14 mAbs, we also observed that Fn14 is constitutively expressed on the cell surface of all TWEAK-sensitive tumor cell lines, and can transmit the multiple death signals. Moreover, an anti-Fn14 mAb that blocks TWEAK-Fn14 interaction could totally abrogate TWEAK binding and TWEAK-induced cell death in all TWEAK-sensitive tumor cell lines. These results revealed that the multiple pathways of TWEAK-induced cell death are solely mediated by Fn14.     

Solution structure of the cysteine‐rich domain in Fn14, a member of the tumor necrosis factor receptor superfamily

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF‐like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor‐ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand‐binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine‐rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28‐Ala70) by heteronuclear NMR, with a ¹³C‐/¹⁵N‐labeled sample. The tertiary structure of the CRD comprises a β‐sheet with two strands, followed by a 3₁₀ helix and a C‐terminal α‐helix, and is stabilized by three disulfide bonds connecting Cys36‐Cys49, Cys52‐Cys67, and Cys55‐Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1‐C2 module type), but not to the CRD of B‐cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1‐D2 module type). This is the first structural report about the A1‐C2 type CRD that could bind to the known target.     

TWEAK/Fn14 interaction stimulates human bronchial epithelial cells to produce IL-8 and GM-CSF

TNF-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) family, is a multifunctional cytokine that regulates cellular proliferation, angiogenesis, inflammation, and apoptosis. In this study, we investigated the effect of TWEAK on human bronchial epithelial cells. A human bronchial epithelial cell line, BEAS2B, expressed a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14), and produced IL-8 and GM-CSF upon TWEAK stimulation in a dose-dependent manner, which was abrogated by anti-Fn14 blocking antibody. TWEAK induced phosphorylation of IkappaBalpha and BAY11-7082, a selective inhibitor of IkappaBalpha phosphorylation, inhibited the TWEAK-induced IL-8 and GM-CSF production by BEAS2B cells. Moreover, primary cultured human bronchial epithelial cells also expressed Fn14 and produced IL-8 and GM-CSF upon TWEAK stimulation. Collectively, TWEAK stimulated human bronchial epithelial cells to produce IL-8 and GM-CSF through Fn14. Because IL-8 and GM-CSF are associated with inflammatory conditions, these results suggest that TWEAK/Fn14 interaction may play some roles in airway inflammatory responses.     

The role of TWEAK/Fn14 in cardiac remodeling

The pathophysiological basis of heart failure is cardiac remodeling, a process that comprises structural and functional changes including cardiomyocyte proliferation, hypertrophy, necrosis, apoptosis, autophagy, interstitial fibrosis, contractile dysfunction and ventricular dilatation. Accumulating evidence demonstrate that tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is involved in the process by binding its receptor fibroblast growth factor-inducible molecule 14 (Fn14). In this review, we will discuss the potential role of the TWEAK/Fn14 axis in cardiac remodeling, elucidate its possible mechanisms and explore new therapeutic targets for heart failure.