From the Department of Pathology/Tumor Immunology, University of Regensburg, D-93042 Regensburg, Germany
Received for publication, February 12, 2001, and in revised form, March 1, 2001
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ABSTRACT |
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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 p75TNFR
gene 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 NF TNF1 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 (1-4). 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 NF 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 NF Reagents, Cell Lines--
The rat anti-human p75TNFR antibody
had been purchased from Genzyme (Genzyme, Cambridge, MA). The
polyclonal antiserum M80 as well as the monoclonal antibodies (mAbs)
80M2, 80A5, and MR2-1 had been kindly provided by M. Grell (Stuttgart,
Germany). Mouse L929 cells (fibrosarcoma cells) and the human cell
lines HEK 293, HepG2, KYM-1, THP1, U937, and HeLa (ATCC, American Type
Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies Inc.) and 0.05 mg/ml gentamicin (PAA
Laboratories Linz, Austria). HUVECs and HMEC were kindly provided by G. Eisner (Regensburg, Germany).
Primer Extension Analysis--
Total RNA was isolated
from THP-1 cells, and primer extension analysis was performed as
described previously (15) using the following primers: Primer 1, 5'-CGGCCTGTTTAGACTCTAGAGCCAGACCACCTGGGTCTGG-3'; Primer 2, 5'-GGCGACGGGCGCCATGGGTGCGGGCGGGGTCCGG-3').
PCR amplification was performed using sense and antisense
primers for icp75TNFR (5'-GGGAAGCTTGACAACATGGCGAAACCCCAT-3'
and 5'-GGGTCTAGAGGTTAACTGGGCTTCATCCCAGCATCAGGC-3').
p75TNFR expression plasmids (p75TNFR,
icp75TNFR, icp75TNFR Protein Expression and Western Blot Analysis--
Twenty-four h
after transfection, cells were lysed in 1 ml of lysis buffer (1%
Nonidet P-40, 0.1% Triton X-100, 0.1% SDS, 150 mM NaCl,
50 mM Tris-HCl, pH 8.0, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride)
for 30 min on ice. Supernatants were incubated overnight at 4 °C
with 50 µl of NTA-agarose (Qiagen), washed twice with 1 ml of
denaturing wash buffer (8 M urea, 0.1 M
Na2HPO4, 0.01 M Tris base, pH 5.9). Eluted proteins were subjected to SDS polyacrylamide electrophoresis and blotted. Immunoblot analysis was performed with a horseradish peroxidase coupled-anti-V5 epitope antibody (Invitrogen) using the
Enhanced Chemiluminescence (ECL) Western Blot Detection System (Energene, Regensburg, Germany).
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 µg/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 µg/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
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 +16 to
B in a TNF
receptor-associated factor 2 (TRAF2)-dependent manner.
Thus, our results provide an example for intracellular cytokine
receptor activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation. Although p55TNFR is capable of
mediating these effects when expressed at physiologically relevant
levels, induction of NF
B via the p75TNFR alone is observed only in
cells overexpressing this receptor subtype (13, 14).
B activation in TNF-stimulated cells, thus
providing an example for intracellular cytokine receptor activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TRAF, and
icp75TNFR
TNF) were constructed by inserting
the corresponding cDNAs into pcDNA 3.1Hygro (Invitrogen,
Groningen, The Netherlands). All expression constructs have been
verified by sequencing.
TNF (L929 icp75TNFR
TNF) or mock-transfected
cells (L929 pcDNA3.1) were seeded at a density of 1 × 105. Cells were transiently transfected with an
NF
B-dependent luciferase reporter plasmid or
cotransfected with the NF
B-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
19 in the cDNA and 5'-UTR (primer 2) and complementary to the
nucleotides
795 to
835 in the 5'-flanking region (primer 1) were
used as primers (Fig. 1,
bottom). The primer extension analysis identified two
transcriptional start sites within the 5'-flanking region of the
p75TNFR gene. One of them is located at position
47
(TSI) relative to the translational start site, whereas a second
transcriptional start site can be localized at position
870 (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).
View larger version (50K):
[in a new window]
Fig. 1.
Analysis of the p75TNFR
transcriptional start sites. RNA derived from THP1 cells was
hybridized with either primer 1 or 2, and reverse transcription
products were analyzed on a 10% sequencing gel along with a sequencing
reaction serving as a size marker. The size of the reaction products
are indicated on the left, and the positions of the
corresponding transcriptional start sites are indicated on the
right. Lane 1, product of the primer extension
reaction performed with primer 1; lane 2, product of the
primer extension reaction performed with primer 2. The annealing
positions of primers 1 and 2 within the RNA template are given
below. bp, base pair.
To identify the corresponding cDNA complementary to the mRNA
originating from TSII we performed RT-PCR using a 5'-primer located at
position 858 to
826 and a 3'-primer located at the 3'-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 5'-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 5'-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
600, which fuses the newly
transcribed exon 1a to the splice acceptor site at the 5'-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
681 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.
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To determine the expression pattern of icp75TNFR mRNA, we performed RT-PCR analysis using a primer specific for the 5'-end of icp75TNFR cDNA and a 3'-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 5'-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
C-terminal 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 ~50
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 ~75 kDa using Western blot analysis.
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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).
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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 NF
B in L929 cells. L929 cells stably transfected with
the cDNA of p75TNFR or icp75TNFR were tested for their ability to
activate a NF
B-dependent reporter gene. Although
expression of the p75TNFR protein in L929 cells resulted in only
marginal activation of NF
B after treatment with TNF compared with a
control transfection, a significant increase in NF
B 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 NF
B after TNF treatment (Fig.
5B). This result indicates that ligand binding by the
icp75TNFR protein is necessary for signal transduction and NF
B
activation.
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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 NF
B 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, coexpression of icp75TNFR lacking
the TNF binding domain did not reduce TNF-induced NF
B 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 NF
B activation.
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DISCUSSION |
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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 5'-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 5'-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 evidence for intracellular expression of icp75TNFR was given by the observation that the expressed protein is detected with an apparent molecular mass of ~50 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
NF
B-dependent reporter gene activation compared with the
control cells. It has been shown previously that p75TNFR is capable of
inducing NF
B activation and that for this signal transduction the
TRAF2 binding domain is indispensable (17). This also seems to be the
case for NF
B activation by the icp75TNFR protein arguing for a
direct involvement in TNF-mediated 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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Matthias Grell for the anti-p75TNFR antibodies and helpful discussions.
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FOOTNOTES |
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* This work was supported by Grant HE 3116 1-1 from the Deutsche Forschungsgemeinschaft (to T. H. and D. N. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. Pathology/Tumor
Immunology, Univ. of Regensburg, F.-J.-Strauss-Allee, D-93042 Regensburg, Germany. Tel.: 49-941-944-6622; Fax: 49-941-944-6602; E-mail: daniela.maennel@klinik.uni-regensburg.de.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M101336200
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ABBREVIATIONS |
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The abbreviations used are: TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; mAb(s), monoclonal antibody(ies); PCR, polymerase chain reaction; PBS, phosphate-buffered solution; FITC, fluorescein isothiocyanate; EPR, exhaustive photon reassignment; ORF, open reading frame; RT, reverse transcription; TRAF2, TNF receptor-associated factor 2.
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