TWEAK

TWEAK Jeffrey L. Browning1,*, Christine Ambrose2 and Catherine Hession2 1

Cell Biology, Inflammation and Immunology, Biogen, 12 Cambridge Center, Cambridge, MA 02142, USA 2 Molecular Genetics, Biogen, 12 Cambridge Center, Cambridge, MA 02142, USA * corresponding author tel: 617-679-3312, fax: 617-679-2304, e-mail: [email protected] DOI: 10.1006/rwcy.2000.05009.

SUMMARY TWEAK is a ligand in the TNF family that could potentially serve as a secreted mediator.

BACKGROUND

Discovery Mouse TWEAK was identified in 1996 as a novel ligand in the TNF family by Yves Chicheportiche during an examination of potential erythropoietinrelated transcripts (Chicheportiche et al., 1997). A partial human homolog was identified in the EST database and, using this sequence, a full-length human cDNA was cloned from a tonsil library. Following on the observation that TWEAK can induce cell death only in the presence of IFN or weakly in a very limited set of cells, the designation TWEAK was given, reflecting the TNF family relationship and the weak death.

Alternative names

The N short terminus is hydrophilic and very basic, probably indicative of a `stop transfer' type of function. This region is followed by a 27 amino acid hydrophobic transmembrane domain, a `stalk' region of about 55±60 amino acids, and lastly the receptorbinding domain of about 150 amino acids. There is conservation of the structural motifs required to form a sheet structure that forms the receptor-binding domain and also serves to trimerize the molecule. This conservation indicates that TWEAK is, in all likelihood, a trimer of folded sheets as defined for TNF, LT, and CD40L.

Main activities and pathophysiological roles Currently the actual function of this protein remains unknown. The protein is readily secreted from transfected cell lines, suggesting the possibility of a secreted cytokine, although the in vivo relevance of such facile in vitro cleavage events is unclear.

GENE AND GENE REGULATION

The gene has no other names except a possible designation as the Apo-3 ligand (Marsters et al., 1998).

Accession numbers

Structure

Human cDNA: AF030099 Mouse: AF030100

The protein consists of 249 amino acids although there could be possible usage of further inframe upstream start sites. TWEAK was readily identified as a member of the TNF family of ligands due to the presence of a classical type II membrane sequence.

Chromosome location The gene for TWEAK is located on human chromosome 17p13 by radiation hybrid mapping.

486 Jeffrey L. Browning, Christine Ambrose and Catherine Hession The gene has six exons and lies within 1 kb of the gene for APRIL on the telomeric end. The genomic organization of TWEAK closely resembles that of APRIL. Mouse chromosome 11 is syntenic with this region.

Relevant linkages The p53 tumor suppresser gene lies within 70 kb of the TWEAK/APRIL locus (J. Tschopp, Lausanne group, personal communication).

Regulatory sites and corresponding transcription factors Both the mouse and human cDNAs have a conserved AU-rich RNA-destabilizing element roughly 20 bp 50 to the polyadenylation signal. This motif can shorten the RNA half-life and is often observed in proteins involved in inflammatory events.

Cells and tissues that express the gene By northern analysis, a 1.4 kb human TWEAK mRNA was found to be expressed more highly in the mature secondary lymphoid organs than in primary Figure 1

organs. The RNA was also abundant in heart, pancreas, colon, small intestine, lung, brain, ovary, and prostate. The least amount, yet readily detectable, was observed in kidney, testis, and liver. A roughly similar pattern of high-level expression has been observed in many organs, including lung, heart, liver, and kidney. Among transformed cell lines, nonhematopoietic cells exhibited high-level expression (Chicheportiche et al., 1997) while several hematopoietically derived tumors showed low level expression. U937 cells expressed TWEAK RNA as well as primary resident and elicited peritoneal murine macrophages. The relatively ubiquitous nature of this expression pattern is unusual for the TNF family of ligands and is most similar to that of TRAIL. The expression pattern suggests that control is at the translation/posttranslational level. The underlying mechanism of control for these types of ligands appears different from the induced expression of other TNF family members seen in inflammatory settings or upon cell activation.

PROTEIN

Sequence See Figure 1. The mouse and human genes are closely related with 93% identity in the receptor-binding domain. This level of identity is among the highest seen in the TNF family of ligands, suggestive of a highly conserved function.

Comparison of human and mouse TWEAK. Nonidentical residues are starred.

COMPARISON OF HUMAN AND MOUSE TWEAK human mouse human mouse human mouse human mouse human mouse

1 60 MAARRSQRRRGRRGEPGTALLVPLALGLGLALACLGLLLAVVSLGSRASLSAQEPAQEEL MAARRSQRRRGRRGEPGTALLAPLVLSLGLALACLGLLLVVVSLGSWATLSAQEPSQEEL * * * * * * * 61 120 VAEEDQDPSELNPQTEESQDPAPFLNRLVRPRRSAPKGRKTRARRAIAAHYEVHPRPGQD TAEDRREPPELNPQTEESQDVVPFLEQLVRPRRSAPKGRKARPRRAIAAHYEVHPRPGQD * **** * ** ** * * 121 180 GAQAGVDGTVSGWEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQVHFDEGKAVYLKLD GAQAGVDGTVSGWEETKINSSSPLRYDRQIGEFTVIRAGLYYLYCQVHFDEGKAVYLKLD ** * * * 181 240 LLVDGVLALRCLEEFSATAASSLGPQLRLCQVSGLLALRPGSSLRIRTLPWAHLKAAPFL LLVNGVLALRCLEEFSATAASSPGPQLRLCQVSGLLPLRPGSSLRIRTLPWAHLKAAPFL * * * 241 249 TYFGLFQVH TYFGLFQVH

TWEAK

Description of protein When engineered as a soluble form, the protein behaves as a compact trimer similar to other soluble ligands in the family. Based on the nature of the conserved sequence homology, it is believed to be a classical TNF family structure of parallel sheets. When the full-length protein and/or various secretion constructs were expressed in a number of protein expression systems including yeast, mammalian, or insect cell, extensive cleavage in the stalk region occurred, leading to proteolyzed forms in the cell supernatants. As a type II membrane protein, it is likely that the protein is synthesized as a membrane protein and then subsequently cleaved in the stalk region either on the outer surface of the cell or while in transit in the endoplasmic reticulum. During expression of recombinant TWEAK, cleavages were observed between Arg92 and Arg93, leading to a 17± 18 kDa secreted form. Proteolysis within the multibasic stretch of RPRR has been observed for other ligands such as APRIL, and this site resembles the furin recognition sequence. TNF cleavage is a regulated event, while in the case of LT, the membrane intermediate is basically undetectable even though the protein clearly has a type II sequence. To address the issue of secretion, secretion or retention from HEK 293 cells transfected with TNF, LT, LT/ , CD40L, and TWEAK was assessed with the expectation that LT and TNF were classic secreted proteins and LT/ and CD40L were fundamentally cell surface proteins. A potential hallmark of a secreted protein may be the presence within the cell lysates of cleaved ligand forms. LT is only found in a cleaved form while TNF is intermediate with both cleaved and full-length forms being observed. Many cleaved forms of TWEAK were seen in the cell and roughly 20±40% of the synthesized TWEAK was secreted. Whether TWEAK is actually secreted in vivo is unclear and the HEK 293 cells may simply express proteases that readily cleave the basic motifs found in the TWEAK stalk. Such behavior has been observed for other ligands such as APRIL. The protein is very basic and sticks to most gel exclusion chromatography matrices under low salt conditions.

Important homologies Typically, ligands in the TNF family are poorly homologous and any homology is limited to those regions critical to forming the scaffold sheet structure. Notably, TWEAK is somewhat related to

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APRIL in agreement with their genomic clustering and similar exon/intron structures.

Posttranslational modifications The recombinant protein can be glycosylated at position 139 and, depending on the expression system, we have observed heterogeneous occupation of this site leading to microheterogeneity in the preparations.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce In a limited metabolic labeling study, the natural protein was observed in U937 cells, wherein about 50% was cleaved, yet none was observed to be secreted (Browning, unpublished).

RECEPTOR UTILIZATION Initial surveys analyzing the ability of recombinant FLAG-labeled TWEAK to bind to most of the known TNF family receptors failed to define a receptor (J. Browning, C. Ambrose and J. Tschopp, unpublished). Receptor-binding studies have shown that TWEAK can bind to a number of both cultured cell lines and primary cells. For example the HT29 colon carcinoma line binds TWEAK well and bound ligand can be competed off with non-FLAG or myc labeled TWEAK, indicating specificity. Binding saturates at 2±10 nM as expected for receptor/ligand interactions in this family. One report described an interaction between TWEAK and death receptor-3 (DR3/TRAMP, WSL, LARD) of the TNF family of receptors (Marsters et al., 1998). In this study, soluble recombinant TWEAK was found to associate with soluble DR3 receptor, indicating some interaction between the two proteins. The pattern of cell types that can undergo apoptosis in response to TWEAK exposure is not very consistent with the activation of a powerful death domain-containing receptor. For example, very few cell types are sensitive to TWEAK and the pattern is vaguely reminiscent of cells that respond to LT R activation. For this reason, it is likely that additional TWEAK receptors exist.

488 Jeffrey L. Browning, Christine Ambrose and Catherine Hession

IN VITRO ACTIVITIES

In vitro findings TWEAK, like TNF, LT, LT/ , TRAIL, and FasL can induce the death of HT29 cells when cultured in the presence of IFN (Chicheportiche et al., 1997). The death induced by TWEAK appears to be apoptotic and occurs quickly, in contrast to LT/ triggered death which requires several days. Very few cell lines will undergo death in response to TWEAK in this fashion and this `weak' death resembles that induced by several other TNF family receptors lacking canonical death domain in the intracellular portion. This weak death has been reported for the putative TWEAK receptor, lymphotoxin R, CD30, CD27, and TNFR75 activation (Declerqc et al., 1998) and is in contrast to the strong apoptotic death that accompanies TNFR55, Fas, DR3, and several of the TRAIL receptors. Some receptors can also trigger cell death when overexpressed, although no natural counterpart has been observed, e.g. CD40 and the p75 NGF-R. On this basis, one would predict that the TWEAK receptor will lack a canonical death domain. TWEAK can effectively induce the proliferation of endothelial cells when they are grown in minimal medium (Lynch et al., 1998) and specifically TWEAK could substitute for EGF and hydrocortisone in the growth media. Another newly defined TNF family member called TL-1 or VEGI, which is expressed in endothelial cells, can inhibit endothelial cell proliferation (Zhai et al., 1999; Yue et al., 1999). These observations suggest that TNF family members may be instrumental in controlling survival and/or growth of the vasculature, which is not surprising given that the original tumor-necrosing activity of TNF stemmed largely from its ability to induce the death of neovasculature in tumors. Whether TWEAK plays a role in these processes will require more study. On the other hand, TNF can stimulate fibroblast and

thymocyte proliferation, and the in vivo significance of these in vitro activities still remains unclear more than a decade after they were first observed. Chemokine secretion may underlie many of the actions of the TNF family members and TWEAK as well as TNF and LT/ can induce IL-8 secretion from some epithelial tumor lines (Chicheportiche et al., 1997). The biological significance of this event is unclear and may simply reflect NFB activation following receptor occupancy, as is the case for many TNF family ligands.

References Chicheportiche, Y., Bourdon, P. R., Xu, H., Hsu, Y.-M., Scott, H., Hession, C., Garcia, I., and Browning, J. L. (1997). TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis. J. Biol. Chem. 272, 32401±32410. Declerqc, W., Denecker, G., Fiers, W., and Vandenabeele, P. (1998). Cooperation of both TNF receptors in inducing apoptosis: Involvement of the TNF-receptor-associated factor binding domain of TNF receptor 75. J. Immunol. 161, 390±399. Lynch, C., Chen, Y.-W., and Wiley, S. (1998). TWEAK induces proliferation in endothelial cells and substitutes for EGF and hydrocortisone. J. Interferon Cytokine Res. 18, A46. Marsters, S. A., Sheridan, J. P., Pitti, R. M., Brush, J., Goddard, A., and Ashkenazi, A. (1998). Identification of a ligand for the death-domain containing receptor Apo3. Curr. Biol. 8, 525±528. Zhai, Y., Ni, J., Jiang, G.-W., Lu, J., Xing, L., Lincoln, C., Carter, K. C., Janat, F., Kozak, D., Xu, S., Rojas, L., Aggarwal, B. B., Ruben, S., Li, L.-Y., Gentz, R., and Yu, G. L. (1999). VEGI, a novel cytokine of the TNF family is an angiogenesis inhibitor that suppresses the growth of colon carcinomas in vivo. FASEB J. 13, 181±189. Yue, T.-L., Ni, J., Romanic, A. M., Gu, J.-L., Keller,P., Wang, C., Kumar, S., Yu, G.-L., Hart, T. K., Wang, X., Xia, Z., DeWolf, W. E., and Feuerstein, G. Z. (1999). TL-1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial cells. J. Biol. Chem. 274, 1479±1486.