A Novel p75TNF Receptor Isoform Mediating NFκB Activation

We report the identification of a novel p75TNF receptor isoform termed icp75TNFR, which is generated by the use of an alternative transcriptional start site within the p75TNFRgene and characterized by regulated intracellular expression. The icp75TNFR protein has an apparent molecular mass of ∼50 kDa and is recognized by antibodies generated against the transmembrane form of p75TNFR. The icp75TNFR binds the tumor necrosis factor(TNF) and mediates intracellular signaling. Overexpression of the icp75TNFR cDNA results in TNF-induced activation of NFκB in a TNF receptor-associated factor 2 (TRAF2)-dependent manner. Thus, our results provide an example for intracellular cytokine receptor activation.

TNF 1 is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation and cytotoxicity in a variety of different cell types. Two distinct receptor molecules with an apparent molecular mass of 55 kDa (p55TNFR, TNFR type 1) and 75 kDa (p75TNFR, TNFR type 2) have been identified, and their corresponding cDNAs have been cloned (1)(2)(3)(4). The p55TNFR is expressed rather constitutively on a broad spectrum of different cell types and has been shown to mediate most of the commonly known biological effects of TNF (5,6). In contrast, expression of the p75TNFR seems to be modulated by various stimuli, and there are only a few cellular responses that can be attributed exclusively to signaling via the p75TNFR, e.g. proliferation of NK cells (7), B cells (8), thymocytes (9), and mature T cells (10), and GM-CSF secretion of T lymphocytes (11). Moreover, the p75TNFR has been shown to be preferentially activated by membrane-bound TNF (12). Although the intracellular receptor domains show only little similarity, they share activities like NFB activation. Although p55TNFR is capable of mediating these effects when expressed at physiologically relevant levels, induction of NFB via the p75TNFR alone is observed only in cells overexpressing this receptor subtype (13,14).
In our report we describe the identification of a novel p75TNFR isoform, termed icp75TNFR, generated by the use of an additional transcriptional start site. The elucidated open reading frame of icp75TNFR revealed that the leader sequence in exon 1 of p75TNFR is replaced by an alternative exon. Immunohistochemical staining indicated intracellular expression of the icp75TNFR protein. We further present evidence that expression of icp75TNFR induces NFB activation in TNF-stimulated cells, thus providing an example for intracellular cytokine receptor activation.
Immunofluorescence Microscopy-HeLa cells seeded on glass slides were transfected with expression plasmids using LipofectAMINE (Life Technologies Inc.) according to the manufacturer's instructions. Nuclei of cells were stained with Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) at 37°C for 10 min. To visualize mitochondria cells were incubated with MitoTracker Red CMXRos (Molecular Probes) for 30 min at 37°C. Cells were fixed with 2% paraformaldehyde for 10 min, permeabilized with acetone (-20°C) for 15 min, and incubated in blocking solution (3% bovine serum albumin in PBS) for 2 h at 4°C. Primary anti-Myc antibody (2 g/ml in PBS containing 2% bovine serum albumin, Invitrogen) was added, and cells were incubated at 4°C overnight. A secondary FITC-conjugated rabbit anti-mouse IgG (Dako, Hamburg, Germany) was added for 1 h at room temperature.
Colocalization of the Myc-tagged icp75TNFR protein with mitochondria was studied with the exhaustive photon reassignment (EPR) method.
To examine TNF binding of cells overexpressing p75TNFR or icp75TNFR, HeLa cells were seeded on glass slides and transfected with the corresponding expression plasmids as described above. Twenty-four h later, cells were washed with PBS and incubated with biotinylated TNF (100 ng/ml in PBS; R&D Systems, Wiesbaden, Germany) for 2 h on ice and then washed again with PBS and incubated at 37°C under standard culture conditions. After 2 h, cells were fixed, permeabilized, and blocked as described before. Primary anti-Myc antibody (2 g/ml (Invitrogen) in PBS containing 2% bovine serum albumin) was added, and cells were incubated at 4°C overnight. The following day, cells were incubated with FITC-conjugated rabbit anti-mouse IgG (Dako) and Cy3-conjugated streptavidin (Dako) for 1 h at room temperature. Transient Transfection and Reporter Assays-Murine L929 cells stably expressing p75TNFR (L929 p75TNFR), icp75TNFR (L929 icp75TNFR), icp75TNFR ⌬TNF (L929 icp75TNFR ⌬TNF) or mocktransfected cells (L929 pcDNA3.1) were seeded at a density of 1 ϫ 10 5 . Cells were transiently transfected with an NFB-dependent luciferase reporter plasmid or cotransfected with the NFB-dependent luciferase reporter plasmid and the expression plasmids encoding icp75TNFR ⌬TRAF or icp75TNFR ⌬TNF deletion mutants using DOTAP (Roche Diagnostics) according to the manufacturer's instructions. Sixteen h after transfection cells were incubated with medium with or without 20 ng/ml mouse TNF for 6 h, and luciferase activity was assayed using the Luciferase Assay System (Promega) according to the manufacturer's directions. Each transfection was done in triplicate and repeated at least three times.

RESULTS
While studying the transcriptional regulation of the human p75TNFR it was realized that this promoter sequence lacks a consensus TATA element within the first few hundred base pairs proximal to the translational start site ATG, whereas several TATA elements are located further upstream of the translational start site. To determine whether these TATA elements are active transcriptional initiation sites we performed primer extension analysis using RNA derived from THP-1 cells. Two oligonucleotides complementary to the nucleotides ϩ16 to Ϫ19 in the cDNA and 5Ј-UTR (primer 2) and complementary to the nucleotides Ϫ795 to Ϫ835 in the 5Ј-flanking region (primer 1) were used as primers (Fig. 1, bottom). The primer extension analysis identified two transcriptional start sites within the 5Ј-flanking region of the p75TNFR gene. One of them is located at position Ϫ47 (TSI) relative to the translational start site, whereas a second transcriptional start site can be localized at position Ϫ870 (TSII) relative to the translational start site (Fig. 1, top). We previously reported about two functional major start sites of transcription in the promoter of the mouse p75TNFR gene (15).
To identify the corresponding cDNA complementary to the mRNA originating from TSII we performed RT-PCR using a 5Ј-primer located at position Ϫ858 to Ϫ826 and a 3Ј-primer located at the 3Ј-end of the p75TNFR cDNA ( Fig. 2A). Three independent cDNA clones were isolated and revealed sequence identity. Primary sequence analysis indicated that transcriptional initiation at TSII within the 5Ј-flanking region of the p75TNFR gene results in a novel p75TNFR cDNA. Comparison of the two p75TNFR cDNAs indicated sequence identity in the extracellular (ED), transmembrane (TM) and intracellular domain (ID). In contrast, the new p75TNFR cDNA isoform lacks the 5Ј-UTR and the first exon of p75TNFR ( Fig. 2A). Alignment with the genomic sequence further shows that the mRNA transcript initiated at TSII generates a splice donor site at position Ϫ600, which fuses the newly transcribed exon 1a to the splice acceptor site at the 5Ј-end of exon 2 of the p75TNFR transcript. The fusion of this new exon 1a generates an open reading frame (ORF 1a) by positioning the ATG at Ϫ681 in frame with exon 2 of the p75TNFR transcript as a potential translational initiation codon (Fig. 2B). Thus, the ORF 1a of the new p75TNFR transcript generates a cDNA that encodes for a mature p75TNFR preceded by a stretch of 27 amino acids encoded by exon 1a (Fig. 2C). To address the question whether exon 1a encodes a putative signal sequence, we compared the predicted amino acid sequences of the p75TNFR cDNA (amino acids 1-26) and the new p75TNFR cDNA (amino acids 1-27) using two different signal peptide prediction methods (SignalP V1.1 and PSORT II, data not shown). Although the first 26 amino acids of p75TNFR were identified as a signal peptide capable of directing a nascent protein to the cell surface, no similarity to any known targeting sequence can be found within the first 27 amino acids of the new p75TNFR.
To determine the expression pattern of icp75TNFR mRNA, we performed RT-PCR analysis using a primer specific for the 5Ј-end of icp75TNFR cDNA and a 3Ј-primer annealing in the second exon of the icp75TNFR cDNA resulting in a 360-base pair PCR product. From various cell lines cDNA was prepared and the PCR products were verified by hybridization with a probe specific for the 5Ј-untranslated region of the icp75TNFR cDNA. Whereas no icp75TNFR mRNA could be detected in HEK 293 and HepG2 cells, HeLa, Kym-1, THP-1, as well as HMEC cells expressed the icp75TNFR mRNA (Fig. 2D). Furthermore, stimulation of HUVEC and U937 cells with lipopolysaccharide resulted in an up-regulation of icp75TNFR mRNA suggesting that the icp75TNFR expression is regulated by proinflammatory stimuli (Fig. 2E). The same cell lines that were positive for the icp75TNFR were also found positive for p75TNFR mRNA expression in parallel experiments (data not shown). Compared with the p75TNFR mRNA expression level, the relative abundance of the icp75TNFR mRNA seemed much lower in all cell lines and primary cells we have tested so far.
Transient expression of the icp75TNFR cDNA with a Cterminal V5 His-tag in HeLa cells followed by purification and Western blot analysis using an antibody to V5 resulted in detection of a single protein band with an apparent molecular mass of ϳ50 kDa (Fig. 3A, lane 2), whereas no protein expression was detected in wild-type Hela cells (Fig. 3A, lane 1). Thus, the identified ORF 1a generated by the use of TSII and subsequent alternative splicing of the p75TNFR mRNA resulted in the icp75TNFR protein with an expected molecular mass. The elucidated molecular mass of icp75TNFR is very close to the calculated value of 48.5 kDa. In contrast, it has been shown in previous studies that the expression of the p75TNFR cDNA results in a protein signal ϳ75 kDa using Western blot analysis.
HeLa cells were transiently transfected with Myc-tagged icp75TNFR cDNA and incubated with biotinylated human TNF followed by simultaneous staining with FITC-coupled anti-Myc antibody and Cy3-coupled streptavidin (Fig. 3B). In contrast to the staining of TNF bound to endogenous TNFRs in wild-type HeLa cells, which is visible as clustering in close proximity to the nucleus, the staining pattern of HeLa cells overexpressing icp75TNFR protein was characterized by a dispersed intracellular receptor staining that was colocalized with biotinylated TNF. This result indicates that the icp75TNFR protein is capable of binding TNF.
To further characterize the icp75TNFR protein, immunohistochemical staining of transiently transfected mouse L929 cells was performed using different mAbs as well as a polyclonal antiserum raised against the human p75TNFR protein. We observed positive staining in L929 cells transfected with either the p75TNFR or icp75TNFR cDNA indicating that the icp75TNFR is indistinguishable from p75TNFR by the use of a polyclonal anti-human p75TNFR serum and by all mAbs we have tested so far (Fig. 3C). No difference was observed in fluorescence-activated cell sorter staining experiments when U937 cells were either nontreated for staining of membrane p75TNFR or when the cell membrane was perforated for intracellular staining (data not shown). In both cases the staining of the p75TNFR protein was clearly enhanced after lipopolysaccharide stimulation.
To investigate the subcellular localization of the icp75TNFR protein we performed immunohistochemical staining of transiently transfected HeLa cells expressing either of the two p75TNFR cDNA isoforms fused to a C-terminal Myc-tag. Staining with an anti-Myc antibody revealed that the expression of p75TNFR can easily be recognized on the cell surface of the transiently transfected HeLa cells (Fig. 4A). In contrast, the expression of icp75TNFR was localized predominantly in the intracellular compartment (Fig. 4B), whereas no specific staining was detected in cells transfected with expression plasmid alone (Fig. 4C).
The result of an EPR analysis, which provides a computer-based digital-confocal imaging, confirmed intracellular expression of the icp75TNFR protein characterized by a diffuse intracellular staining (Fig. 4D). Colocalization of icp75TNFR with a mitochondrial marker was recognized only to a small extent.
Because there have been several reports in the past correlating the expression level of p75TNFR with NFB activation (16) we asked whether the expression of icp75TNFR would influence the TNF-induced activation of NFB in L929 cells. L929 cells stably transfected with the cDNA of p75TNFR or icp75TNFR were tested for their ability to activate a NFB-de-pendent reporter gene. Although expression of the p75TNFR protein in L929 cells resulted in only marginal activation of NFB after treatment with TNF compared with a control transfection, a significant increase in NFB activation was detected in L929 cells transfected with the cDNA for the icp75TNFR (Fig. 5A). In contrast L929 cells expressing icp75TNFR protein lacking the TNF binding domain are no longer capable of inducing NFB after TNF treatment (Fig.  5B). This result indicates that ligand binding by the icp75TNFR protein is necessary for signal transduction and NFB activation.
It has been shown by mutational analysis within the cytoplasmic domain of p75TNFR that a C-terminal region responsible for the binding of TNF receptor-associated factor 2 (TRAF2) is indispensable for signal transduction and NFB activation. Coexpression of a p75TNFR mutant, which lacks the TRAF2 domain, resulted in a dominant-negative effect implicating that this domain is essential for the response (17). Accordingly, we tested whether the TRAF2 domain of icp75TNFR is also involved in signal transduction of the icp75TNFR. In fact, coexpression of icp75TNFR lacking the TRAF2 binding domain in the corresponding stably transfected L929 cells resulted in inhibition of TNF-induced NFB activation (Fig. 5C). This observation suggests that, also for the intracellularly expressed form of icp75TNFR, the TRAF2 domain is critical for signal transduction. In contrast, coexpres- HeLa cells were transiently transfected with an expression plasmid containing the V5 His-tagged icp75TNFR cDNA. Twenty-four h after transfection cells were lysed and the icp75TNFR V5 His fusion protein was purified as described under "Experimental Procedures." An aliquot of the purified protein was separated by SDS-polyacrylamide gel electrophoresis and detected by Western blotting using an anti-V5 antibody. B, TNF binding capacity of icp75TNFR. HeLa cells were seeded on glass slides and transiently transfected with the expression plasmid encoding Myc-tagged icp75TNFR cDNA. Twenty-four h later, cells were incubated with biotinylated human TNF as described under "Experimental Procedures" and stained with an anti-Myc antibody and a secondary FITC-conjugated antibody (top) as well as with Cy3-conjugated streptavidin (bottom). Magnification factor of each panel is 1000ϫ. C, recognition of icp75TNFR protein by different anti-p75TNFR antibodies. L929 cells were seeded on glass slides and transiently transfected with expression plasmids encoding Myc-tagged p75TNFR or icp75TNFR cDNA. Twenty-four h later, cells were stained with the indicated anti-p75TNFR antibodies as described under "Experimental Procedures." sion of icp75TNFR lacking the TNF binding domain did not reduce TNF-induced NFB activation (Fig. 5C). This supports the result of our previous experiment (Fig. 5B) indicating that intracellular signaling by icp75TNFR seems to be ligand-dependent because coexpression of icp75TNFR ⌬TNF, which is incapable of ligand-dependent oligomerization of its TRAF2 binding domains, did not interfere with TNF-induced NFB activation. DISCUSSION TNF exerts pleiotropic activities affecting proliferation, differentiation, or function in a wide variety of cell types (18). In the past two distinct receptors for TNF, referred here to as p55TNFR and p75TNFR, have been characterized (19). Our present data identify a novel human p75TNFR mRNA isoform termed icp75TNFR, which originates from an additional transcriptional start site in the 5Ј-flanking region of the p75TNFR gene. The deduced ORF is characterized by sequence identity concerning the extracellular, transmembrane, and intracellular receptor domains, but differs in the N-terminal sequence as well as in the 5Ј-untranslated region. Replacement of the leader sequence encoded by exon 1 by a novel sequence that does not contain characteristics for directing the protein into a specific cellular compartment suggests that the icp75 protein may not be transported to the cell membrane by the regular endosomal secretion pathway. The presence of the transmembrane domain in the icp75TNFR suggests that rather the protein remains inserted into a intracellular perinuclear membrane and not expressed in the cytoplasma. Our results indicate that the icp75TNFR protein is indistinguishable from p75TNFR using different mAbs as well as a polyclonal antiserum raised against human p75TNFR. This fact represents a major problem in the attempt to specifically detect endogenous icp75TNFR protein in cells that produce the membrane form of the p75TNFR at the same time. Immunohistochemical analysis revealed that the localization of the novel icp75TNFR is characterized by a diffuse intracellular staining pattern. Further FIG. 5. TNF-mediated NFB activation in L929 cells expressing different p75TNFR variants. A, enhanced NFB activation in L929 expressing icp75TNFR. L929 cells were stably transfected with expression plasmids coding for p75TNFR or icp75TNFR, respectively, or were mock-transfected. Stable transfectants were assayed for NFB activation by transient transfection of a NFB luciferase reporter plasmid. Sixteen h after transfection, cells were stimulated with 20 ng/ml mouse TNF (hatched bars) or cultured in TNF-free medium for another 6 h (white bars). Luciferase activity was determined as described under "Experimental Procedures." B, impaired NFB activation in L929 cells expressing an icp75TNFR deletion mutant lacking the TNF binding domain. Stable transfectants were assayed as described above in A. C, different effects on NFB activation by overexpression of icp75TNFR deletion mutants lacking the TRAF2 binding domain or the TNF binding domain. L929 cells stably expressing icp75TNFR were cotransfected with a NFB reporter plasmid and the expression plasmids for the icp75TNFR proteins without the TRAF2 binding domain (icp75 ⌬TRAF) or without the TNF binding domain (icp75 ⌬TNF). Sixteen h later, cells were stimulated with 20 ng/ml mouse TNF or cultured in TNF-free medium for another 6 h. Luciferase activity was determined as described under "Experimental Procedures." HeLa cells transfected with p75TNFR demonstrate a cell membrane staining, whereas cells transfected with icp75TNFR show a diffuse intracellular staining. D, EPR analysis of the subcellular distribution of icp75TNFR. HeLa cells were transiently transfected with the Myctagged icp75TNFR expression plasmid. Cells were incubated with the mitochondria marker MitoTracker Red CMXRos for 30 min. Myctagged icp75TNFR was detected with a FITC-conjugated antibody as described above. A diffuse intracellular staining of icp75TNFR was confirmed using the EPR method as described under "Experimental Procedures." Colocalization of icp75TNFR (green) with the mitochondrial marker (red) can be seen only to a small extent using combined immunofluorescence (yellow). evidence for intracellular expression of icp75TNFR was given by the observation that the expressed protein is detected with an apparent molecular mass of ϳ50 kDa, which is close to the calculated value of the primary amino acid sequence (48.5 kDa). This suggests that no posttranslational modifications, e.g. glycosylation, occur by which the transmembrane form of the p75TNFR is characterized. Interestingly, Ledgerwood et al. (20) described a 60-kDa protein localized in the inner mitochondrial membrane of umbilical vein endothelial cells, which was recognized by a specific mAb against human p75TNFR and was also capable of binding TNF. Our data show that the icp75TNFR protein was recognized by all mAbs raised against human p75TNFR that we have tested so far including the one used by Ledgerwood et al. EPR analysis showed that in addition to diffuse intracellular staining a small amount of icp75TNFR protein possibly was colocalized with a mitochondrial marker. The difference in the staining pattern may be due to overexpression of icp75TNFR in our experiments. Specific staining of endogenous icp75TNFR protein by cytochemistry or fluorescence-activated cell sorter analysis was not possible so far because no reagents are available that can distinguish between the membrane form of the p75TNFR and the icp75TNFR. More detailed localization studies have to be done to dissect the expression pattern of two p75TNFR isoforms.
Overexpression of icp75TNFR induced significant NFB activation in L929 cells after TNF stimulation suggesting that this receptor subtype is capable of triggering signaling events in a TNF-dependent manner. L929 cells stably transfected with the p75TNFR cDNA encoding the transmembrane receptor subtype as confirmed by immunocytochemical staining and RT-PCR analysis show only a marginal TNF-induced increase in NFB-dependent reporter gene activation compared with the control cells. It has been shown previously that p75TNFR is capable of inducing NFB activation and that for this signal transduction the TRAF2 binding domain is indispensable (17). This also seems to be the case for NFB activation by the icp75TNFR protein arguing for a direct involvement in TNFmediated signaling. It remains to be analyzed whether TNF has to be internalized by the transmembrane receptors as described earlier (19), by a suggested TNFR-independent mechanism (20), and/or whether endogenously produced TNF is responsible for activation of icp75TNFR followed by intracellular signaling. In addition, the functional consequences of such intracellular TNF signaling via the icp75TNFR is currently under investigation.