Sharron A.N. Brown

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway.

TWEAK Is an Endothelial Cell Growth and Chemotactic Factor That Also Potentiates FGF-2 and VEGF-A Mitogenic Activity

Objective—TWEAK, a member of the tumor necrosis factor superfamily, binds to the Fn14 receptor and stimulates angiogenesis in vivo. In this study, we investigated Fn14 gene expression in human endothelial cells (ECs) and examined the effect of TWEAK, added either alone or in combination with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor-A (VEGF-A), on EC proliferation, migration, and survival in vitro. We also determined whether a soluble Fn14-Fc fusion protein could inhibit TWEAK biologic activity on ECs and investigated TWEAK signal transduction in ECs. Methods and Results—We found that both FGF-2 and VEGF-A could induce Fn14 mRNA expression in ECs. TWEAK was a mitogen for ECs, and this proliferative activity could be inhibited by an Fn14-Fc decoy receptor. Furthermore, TWEAK treatment activated several intracellular signaling pathways in ECs and potentiated FGF-2– and VEGF-A–stimulated EC proliferation. TWEAK also had EC chemotactic activity, but it did not promote EC survival. Conclusions—These results indicate that TWEAK is an EC growth and migration factor but not a survival factor. TWEAK can also enhance both FGF-2 and VEGF-A mitogenic activity on ECs. Thus, TWEAK may act alone as well as in combination with FGF-2 or VEGF-A to regulate pathological angiogenesis.

Soluble Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Overexpression in HEK293 Cells Promotes Tumor Growth and Angiogenesis in Athymic Nude Mice

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of structurally related cytokines. TWEAK acts on responsive cells via binding to a cell surface receptor named Fn14. Recent studies have demonstrated that TWEAK can stimulate numerous cellular responses including cell proliferation, migration, and proinflammatory molecule production. It has also been reported that TWEAK can stimulate blood vessel formation in the rat cornea angiogenesis assay, but it is presently unknown whether this cytokine could play a role in the pathological angiogenesis associated with human diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. In the present study we investigated whether TWEAK was expressed in human tumors and whether it could promote tumor growth and angiogenesis in vivo. TWEAK mRNA expression was detected in many tumor types by cDNA array hybridization analysis, and TWEAK protein expression was confirmed in human colon cancer tissue by immunohistochemistry. As an initial approach to address whether TWEAK might act as a tumor angiogenesis factor, we established several human embryonic kidney cell lines that constitutively secrete a soluble TWEAK protein and examined their growth properties in vitro and in vivo. We found that although TWEAK-overexpressing cells do not have a growth advantage in vitro, they form larger and more highly vascularized tumors in athymic mice when compared with control, vector-transfected cells. This result suggests that the TWEAK-Fn14 signaling system may be a potential regulator of human tumorigenesis.

Full-length, Membrane-anchored TWEAK Can Function as a Juxtacrine Signaling Molecule and Activate the NF-κB Pathway

Tumor necrosis factor (TNF) family members are initially synthesized as type II transmembrane proteins, but some of these proteins are substrates for proteolytic enzymes that generate soluble cytokines with biological activity. TWEAK (TNF-like weak inducer of apoptosis), a member of the TNF family, is a multifunctional cytokine that acts via binding to a cell surface receptor named Fn14 (fibroblast growth factor-inducible 14). Studies conducted to date indicate that TWEAK-producing cells can co-express both membrane-anchored and soluble TWEAK isoforms, but there is little information on TWEAK proteolytic processing. Also, it is presently unclear whether membrane-anchored TWEAK, like soluble TWEAK, is biologically active. Here we show that full-length human TWEAK is processed intracellularly by the serine protease furin and identify TWEAK amino acid residues 90–93 as the predominant furin recognition site. In addition, we report that full-length, membrane-anchored TWEAK can bind the Fn14 receptor on neighboring cells and activate the NF-κB signaling pathway. Thus, TWEAK can act in a juxtacrine manner to initiate cellular responses, and this property may be important for TWEAK function during physiological wound repair and disease pathogenesis.

Progressive Ankylosis (Ank) Protein Is Expressed by Neurons and Ank Immunohistochemical Reactivity Is Increased by Limbic Seizures

Ank is a 492-amino acid multipass transmembrane protein involved in the regulation of extracellular inorganic pyrophosphate levels and the control of tissue calcification. Previous Northern blot hybridization experiments revealed that Ank mRNA was expressed in the brain, but there have been no reports describing the anatomical sites or specific cell types in the brain that express Ank protein. In this study, we demonstrate that Ank is expressed primarily in human brain neurons, with the highest levels of expression observed in the thalamus, the III and V cortical layers, the Purkinje cells of the cerebellum, clusters of cells in the dorsal portion of the pons and midbrain, and neurons of the anterior horn of the spinal cord. In primary mouse neuronal cell cultures, Ank is detected on both the cell body and on cell extensions, mainly dendrites. In the rat brain, Ank mRNA is expressed at relatively high levels in the thalamus, midbrain, and spinal cord, and the Ank protein expression pattern is similar to that observed in the human brain. Finally, we observed a significant increase in Ank immunoreactivity in the rat amygdala, the CA-2 and CA-3 layers of the hippocampus, and the cerebral cortex after the induction of seizure activity. Ank regulation of ATP and/or inorganic pyrophosphate release from neurons may function to modulate the membrane excitability and cell death associated with seizure activity.

DEVELOPMENT OF A SPECIFIC RADIOIMMUNO ASSAY FOR E DOMAIN CONTAINING FORMS OF INSULIN-LIKE GROWTH FACTOR II

The analysis of cDNA clones for human (1,2) and rat (3) insulin-like growth factor-II (IGF-II)1 has led to the prediction that the processed forms of the growth factors, i.e. Mr = 7422 (67 amino acids) for human IGF-II are synthesized as precursors with an extension of 89 amino acids at the carboxyl terminus. This extension is termed the E domain. Moses et al (4) identified two precursor forms of rat IGF-II (originally designated multiplication-stimulating activity) in the conditioned medium of Buffalo rat liver, i.e. BRL-3A, cells with appMrs = 16,270 (MSA-1) and 8,700 (MSA-II). Human serum, spinal fluid and tissue extracts also contain high Mr forms of IGF-II (5-8). Zumstein et al., (5) have purified a Mr = 10,000 variant form of IGF-II from serum that contained Cys-Gly-Asp for Ser33 in the C domain and an E domain extension of 21- amino acids. This 10 kDa IGF-II may be similar or identical to the “big IGF-II” that was reported to be present in human serum and in spinal fluid (6). We have isolated a still larger form of IGF-II from normal human serum (7). N-terminal amino acid sequence analysis through the first 28 residues and RRAs using rat placental membranes established it as a form of IGF-II. As evidenced from its mobility during SDS-PAGE, it has an appMr = 15,000. Very recently, Hudgins et al., (8) established that normal human serum contains several forms of precursor IGF- II with acidic isoelectric points, i.e. pI’s. The mass and acidic nature of one of these molecules with an apparent Mr= 15,000 was contributed, in part, by polysaccharides and sialic acids, respectively (8). These results may explain the observations of several investigators that extracts from the tissues and serum of patients with malignant tumors contain a broad size range of IGF-II (2,9,10).

TWEAK activation of the non-canonical NF-κB signaling pathway differentially regulates melanoma and prostate cancer cell invasion

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that binds with high affinity to a plasma membrane-anchored receptor named Fn14. Both TWEAK and Fn14 expression has been detected in human cancer tissue, and studies have shown that TWEAK/Fn14 signaling can promote either “pro-cancer” or “anti-cancer” cellular effects in vitro, depending on the cancer cell line under investigation. In this study, we engineered murine B16 melanoma cells to secrete high levels of soluble TWEAK and examined their properties. TWEAK production by B16 cells preferentially activated the non-canonical NF-κB signaling pathway and increased the expression of several previously described TWEAK-inducible genes, including Fn14. TWEAK overexpression in B16 cells inhibited both cell growth and invasion in vitro. The TWEAK-mediated reduction in B16 cell invasive capacity was dependent on activation of the non-canonical NF-κB signaling pathway. Finally, we found that this same signaling pathway was also important for TWEAK-stimulated human DU145 prostate cancer cell invasion. Therefore, even though TWEAK:Fn14 binding activates non-canonical NF-κB signaling in both melanoma and prostate cancer cells, this shared cellular response can trigger a very different downstream outcome (inhibition or stimulation of cell invasiveness, respectively).