Section of Nephrology and Department of Internal Medicine, Yale University School of Medicine and the Veterans Affairs Connecticut Healthcare Systems, New Haven, Connecticut 06520
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ABSTRACT |
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Transforming growth factor-1 (TGF-
1) has been implicated
to play an important role both in the process of normal development and
in the pathogenesis of a wide variety of disease processes, including
those of the kidney. TGF-
1 regulates diverse cellular functions via
a heteromeric signaling complex of two transmembrane serine/threonine
kinase receptors (types I and II). Several distinct type I receptors
have been described and are thought to determine specificity of the
TGF-
response and confer multifunctionality. This report reveals the
cloning of a novel, naturally occurring soluble form of TGF-
type I
receptor, designated sT
R-I, from a rat kidney cDNA library. In vivo
expression of a mRNA transcript encoding the sT
R-I, which lacks the
transmembrane and cytoplasmic domains, is confirmed by RT-PCR followed
by Southern blot analysis and by RNase protection assay. The sT
R-I
mRNA abundance is greater in the neonatal rat kidney compared with the
adult rat kidney. Furthermore, sT
R-I is a functional protein capable
of binding TGF-
1 ligands in the presence of a TGF-
type II
receptor on the cell surface, as determined by affinity cross-linking
with 125I-labeled TGF-
1.
Studies using p3TP-Lux reporter construct reveal that this novel
protein may function as a potentiator of TGF-
signaling. The
discovery of a sT
R-I provides an additional level of complexity to
the TGF-
receptor system.
variant activin receptor-like kinase-5; alternative splicing; signaling; renal development; transforming growth factor-1
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INTRODUCTION |
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TRANSFORMING GROWTH factor-1 (TGF-
1) is a
multifunctional cytokine that regulates diverse cellular functions
including cell proliferation and differentiation, as well as
extracellular matrix protein synthesis. TGF-
1 has been implicated in
both the process of normal development and morphogenesis (19) and in
the pathogenesis of a wide variety of disease processes, including
tissue fibrosis, inflammation, and tumorigenesis (16). In embryonic
kidney tissues, both temporally and spatially regulated differential
expression patterns of TGF-
and its receptors have been
demonstrated, suggesting an important role for TGF-
in renal
development (6, 19).
TGF-1 exerts its multiple biological actions by the interaction with
two transmembrane serine/threonine kinases (types I and II), which are
coexpressed by most cells (3). Molecular cloning has revealed that a
distinct type II receptor exists for TGF-
1 and for members of the
TGF-
superfamily, including activin and bone morphogenetic protein
(BMP), and that the type II receptor is capable of binding its
respective ligands directly and interacting with different type I
receptors (2, 10, 15). To date, at least six distinct type I receptors
of the TGF-
superfamily, named activin receptor-like kinases (ALK),
have been cloned. ALK-5 has been identified as the predominant TGF-
type I receptor (T
R-I) in most cell types and has been shown to
mediate TGF-
signaling (12). ALK-1 and ALK-2 are thought to be
activin type I receptors, but both have also been demonstrated to bind
TGF-
(1, 9). ALK-4 also appears to be an activin type I receptor
(21). ALK-3 and ALK-6 are thought to be type I receptors for BMP (22).
Features common to all type I receptors include a cysteine-rich
extracellular domain and a single transmembrane-spanning domain.
Furthermore, besides the characteristic cytoplasmic serine/threonine
kinase domain, type I receptors have a region, between the
transmembrane and kinase domains, containing a conserved TSGSGSG motif
denoted the GS domain. Mutational analyses have revealed
that phosphorylation of serine and threonine residues in the GS domain
of T
R-I by the TGF-
type II receptor (T
R-II) is essential for
TGF-
signaling (20, 25).
TR-I is thought to determine the specificity of the cellular
response to TGF-
1, whereas T
R-II determines the ligand
specificity. T
R-I alone is unable to bind TGF-
1, on the basis of
125I-labeled TGF-
1
cross-linking studies, and T
R-II is unable to signal without T
R-I
(27). Thus interaction of T
R-II with different type I receptors may
be a mechanism that confers multifunctionality of TGF-
1. This report
shows that there are variant forms of the T
R-I. Three unique cDNA
clones encoding multiple receptor forms of ALK-5 have been isolated
from a neonatal rat kidney cDNA library. Two of the rat cDNA clones
encode membrane-spanning receptors that differ in the COOH-terminal
region of the extracellular domain. The third clone encodes a
previously undescribed soluble form of T
R-I (sT
R-I). The present
studies clearly demonstrate, for the first time, the existence of a
naturally occurring sT
R-I mRNA expressed in greater abundance in the
neonatal rat kidney compared with the adult rat kidney. Furthermore,
the sT
R-I is a functional protein capable of binding TGF-
1
ligands in the presence of T
R-II. This novel protein may function as
a potentiator of TGF-
signaling.
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METHODS |
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Cloning and sequencing of rat TR-I
cDNA clones.
A 333-bp cDNA probe for ALK-5 (12) was generated by RT-PCR of total RNA
from rat kidneys using sense primer A
(nt 88-117 of human ALK-5,
5'-GGGGCGACGGCGTTACAGTGTTTCTGCCAC-3') and antisense primer B (nt 391-420 of human
ALK-5, 5'-TGAGATGCAGACGAAGCACACTGGTCCAGC-3'). Using this
PCR-amplified cDNA probe, a rat neonatal kidney cDNA library was
screened by methods previously described (6). Three positive clones
were identified. Partially overlapping nested deletion clones were
prepared from the cDNAs with the Erase-a-Base kit (Promega), and then
the sense and antisense strands were sequenced using the dideoxy chain
termination method with 35S-dATP
and Sequenase version 2.0 (Amersham).
RT-PCR. Total RNA (3 µg) from Sprague-Dawley rat neonatal and adult kidneys (Charles River, Wilmington, MA) was reverse-transcribed using M-MLV reverse transcriptase (GIBCO BRL), and the first-strand cDNA was used as a template for amplification. Each template cDNA was combined with 1 µM of each set of PCR primers, 200 µM dNTPs, and 2 U of AmpliTaq DNA polymerase in 1× PCR buffer (Perkin-Elmer); placed in the GeneAmp PCR System 9600 (Perkin-Elmer), and heated to 95°C for 1 min. This was followed by 30 cycles consisting of denaturing for 15 s at 95°C, annealing for 15 s at 50°C, and extending for 15 s at 72°C. The sequences of primers A and B were as described above; the sequence of primer C (nt 480-505 of rat cDNA clone 29-1, 3'-untranslated region) was 5'-GAGTAGCCAGGAGCCTGACTCCTGGG-3'. All experiments were performed at least three times, using RNA isolated from at least three separate neonatal rats and three adult rats.
Southern blot analysis.
After RT-PCR, the amplified products were separated on a 1.5% agarose
gel, incubated for 15 min in 0.25 N HCl, placed in denaturing solution
(0.5 N NaOH and 1.5 M NaCl), and finally placed in neutralizing solution (1.5 M NaCl and 0.5 M Tris · HCl, pH 7.4)
and then transferred and UV-linked to a nylon membrane (Nytran,
Schleicher & Schuell). The blot was prehybridized and then hybridized
in the presence of a random primer
[32P]dCTP-labeled cDNA
probe, as recommended by the manufacturer (GIBCO BRL). The two TR-I
cDNA probes were Hind
III/Sph I and Sph I fragments of clone 29-1 cDNA in
pSPORT1 (GIBCO BRL), produced by restriction digests with the
respective enzymes.
Solution hybridization/RNase
protection.
RNase protection analysis was done using the RPA II kit (Ambion)
according to the manufacturer's instructions. The
32P-labeled antisense riboprobe
was prepared from the linearized plasmid containing the
Hind
III/Sma I fragment of clone 29-1 cDNA using Sp6 RNA polymerase, yielding a probe 337 nt long. One, ten, and
twenty micrograms of total RNA from neonatal and adult rat kidneys, or
10 µg of total RNA from wild-type and transfected COS-7 cells, were
hybridized with the 32P-labeled
probe for 16-18 h at 42°C, then digested with RNase A/T1 and
resolved on a 6% acrylamide/7.7 M urea sequencing gel. A sample of
32P-labeled, 1-kb ladder DNA was
loaded in an adjacent lane as the molecular size marker. The -actin
mRNA was used as an internal standard to determine equivalent amounts
of RNA used in parallel reactions. All experiments were repeated at
least three times, using RNA isolated from at least three different
neonatal and adult rats.
Cell culture and stable transfection of COS-7
cells.
To generate clones that stably expressed TR-II and sT
R-I, COS-7
cells (ATCC CRL-1651) were transfected using Lipofectin (GIBCO BRL) as
follows. Cells grown to ~40% confluency on six-well plates were
incubated in OPTI-MEM (GIBCO BRL) in the presence of 1-5 µg
full-length, wild-type T
R-II cDNA (5) or sT
R-I cDNA ligated in
pcDNA3 (Invitrogen) and 5-10 µl Lipofectin suspension for 5 h at
37°C in a 5% CO2 atmosphere.
Control cells were incubated with pcDNA3 vector (without T
R-II or
sT
R-I) and Lipofectin. After a 5-h incubation, DMEM containing 20%
fetal bovine serum (FBS) was added to make a final concentration of
10% FBS. After 48 h, the medium was changed to 10% FBS in
DMEM and incubated for another 24 h. To select for stable
transfectants, cells were treated with 400 µg/ml Geneticin (GIBCO
BRL) in DMEM containing 10% FBS, and the medium was changed every
2-3 days. Clones emerging ~10 days after lipofection were
subcloned using ring cylinders, expanded, and maintained in DMEM
containing 10% FBS, 200 µg/ml Geneticin, 5 U/ml
penicillin, and 5 µg/ml streptomycin. Four independent, stably
transfected clones containing T
R-II or cotransfected with T
R-II
and sT
R-I, and three clones containing empty vector were expanded.
Nontransfected cells similarly treated with Lipofectin served as
additional controls.
Covalent labeling of TGF-
receptors on cell surface.
Cells on 100-mm plates were affinity labeled with 400 pM
125I-TGF-
1 (Amersham) in the
presence or absence of 100 nM unlabeled TGF-
1 (Collaborative
Biomedical Products) and covalently cross-linked using disuccinimidyl
suberate (Pierce), as previously described (5). The cells were
subsequently lysed with 100 µl of 1% Triton X-100, 10 mM Tris 7.4, 1 mM EDTA, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, and 10 µg/ml pepstatin. Sample loading buffer
(sucrose, 0.01% bromophenol blue, 2%
-mercaptoethanol, and 5 mM
EDTA) was added (1:1, vol/vol) and boiled for 5 min, followed by 12%
SDS-PAGE. A sample of rainbow-colored protein molecular weight markers
(Amersham) was loaded in an adjacent lane. The gel was stained with
Coomassie brilliant blue (Bio-Rad) to allow visualization of
equivalence in protein loading and was destained before autoradiography.
Luciferase assay.
Luciferase reporter plasmid, p3TP-Lux (kindly provided by J. Massagué), was used to assay TGF-1-induced transcription of plasminogen activator inhibitor-1 (PAI-1). The p3TP-Lux contains elements from the PAI-1 promoter and drives the expression of a
luciferase reporter gene in response to TGF-
1 (26). COS-7 cells were
transiently transfected with p3TP-Lux and the indicated receptor
constructs, or with empty vector pcDNA3 (see Fig. 5). A truncated
T
R-I construct (T
R-IM)
that lacks the cytoplasmic GS and kinase domains but contains the
transmembrane and extracellular domains was generated by PCR using rat
T
R-I cDNA (6) as a template, using a similar strategy as previously
described for the kinase-deleted T
R-II construct
(T
R-IIM) (5), and cloned in
pcDNA3. Primer sequences used to amplify the
T
R-IM construct were as
follows: sense primer,
5'-AC
CCATGGAGGCGGCGTCGGCTGCTTT-3'; antisense primer,
5'-GC
GCGCCTATGGCACGCGGTGGTGAATGACA-3'. They contained the sequences for the restriction enzymes
Kpn I and
Xba I, respectively (underlined), for
directional cloning, and a stop codon in the antisense primer. Control
reporter vector pRL-CMV (Promega) was included in all transfections as
an internal control to normalize transfection efficiency. Forty-eight
hours after transfection using Lipofectin, cells were incubated for 20 h in the absence or presence of TGF-
1 (1 ng/ml). Luciferase activities in cell lysates were measured using the dual-Luciferase reporter assay system (Promega) in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). All experiments were performed at least three times.
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RESULTS |
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Identification of a soluble
TR-I.
A 333-bp rat cDNA fragment obtained by RT-PCR, containing nucleotides
of the extracellular region and homologous to the human ALK-5 cDNA
(12), was used as a probe to screen a neonatal rat kidney library to
identify cDNAs encoding variant forms of T
R-I that differ in their
extracellular ligand binding domains. A schematic representation of the
three cDNA clones isolated is shown in Fig. 1A. All
three rat cDNA clones were identical in the extracellular ligand
binding domain with the exception of the COOH-terminal region. Clone
12-1 was found to be more than 95% homologous with the human ALK-5 at
the amino acid level. In clone 21-1, an insert of 12 nt corresponding
to amino acid residues GPFS was noted just upstream to the
transmembrane domain. The remainder of the sequence was identical to
clone 12-1, including the single hydrophobic transmembrane spanning
domain. Interestingly, in clone 29-1, the divergent region in the
extracellular domain also differed. It contained an insert of 15 nt,
corresponding to amino acid residues GKLLY, followed by an in-frame
stop codon. The remainder of the sequence downstream from the stop
codon contained no homologous regions with the other two clones, and no
sequence that would predict a transmembrane or a serine/threonine
kinase domain was found in clone 29-1. The nucleotide sequence,
including the flanking untranslated regions, and the deduced amino acid
sequence are shown in Fig. 1B. Like
the membrane-anchored form of ALK-5, clone 29-1 contained an
NH2-terminal signal peptide
domain. Based on the nucleotide sequence of clone 29-1, the deduced
amino acid sequence predicted a soluble form of T
R-I.
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In vivo expression of sTR-I
mRNA.
To determine whether mRNA corresponding to the sT
R-I is endogenously
expressed in vivo, RT-PCR followed by Southern blot analysis were
performed with rat neonatal and adult kidney tissue samples. As
illustrated in Fig. 2,
A and
B
(top), primer pairs A and C should only
amplify sequences homologous to the soluble form of ALK-5, a predicted
product 430 bp in length. Primers A and B are expected to amplify
sequences corresponding to the membrane-anchored form of ALK-5, with a
predicted product 333 bp long. As controls, 29-1 and 21-1 cDNAs were
used as templates. Primers A and
C would only amplify the 430-bp
product with clone 29-1 and not clone 21-1. Conversely,
primers A and
B would amplify the 333 bp with clone
21-1 cDNA and not clone 29-1.
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Overexpression of sTR-I in COS-7
cells.
COS-7 cells were stably transfected with a construct containing the
coding sequence for the rat sT
R-I ligated in pcDNA3, either alone or
cotransfected with a full-length, wild-type rat T
R-II cDNA.
Wild-type COS-7 cells and cells transfected with empty vector pcDNA3
were used as controls. mRNA expression of the sT
R-I construct was
confirmed by RNase protection assay using the same riboprobe
complementary to clone 29-1 from Hind
III to Sma I sites, which contained
268 nt of the coding sequence of sT
R-I. As expected, no protected
fragments were observed in control wild-type COS-7 cells (data not
shown) and in cells transfected with empty vector alone (Fig. 3,
lane 9). The predicted 268-bp protected fragments were observed in COS-7 cells transfected with sT
R-I construct alone (Fig. 3, lane
10) and in cells cotransfected with sT
R-I and
T
R-II constructs (Fig. 3, lane
11).
sTR-I binds TGF-
1
in the presence of T
R-II.
Cell-surface ligand binding is demonstrated by affinity cross-linking
with 125I-labeled TGF-
1. Two
distinct bands, approximately 97 and 70 kDa in size, corresponding to
T
R-II and T
R-I, respectively, are detected in wild-type COS-7
cells (data not shown) and in cells transfected with empty vector (Fig.
4, lane
2). In COS-7 cells transfected with full-length
T
R-II cDNA, a more intensely labeled 97-kDa band is observed (Fig.
4, lane 4). Because T
R-I alone
did not exhibit significant binding of TGF-
1 ligand when assessed by
cross-linking analysis (27), COS-7 cells were cotransfected with
full-length T
R-II and sT
R-I cDNAs. In these cells, additional specifically labeled bands of lower molecular weight are seen (Fig. 4,
lanes 6 and
8), corresponding to sT
R-I.
Preincubation with unlabeled TGF-
1 demonstrates specificity of
ligand binding. Based on the deduced amino acid sequence of clone 29-1, the sT
R-I protein has a predicted molecular mass of ~12 kDa. It
also contains a potential N-glycosylation site, as indicated in Fig.
1B. Hence, the 45-kDa bands observed
by affinity cross-linking are interpreted to represent glycosylated
sT
R-I protein cross-linked to TGF-
dimer. Evidence for
glycosylation of the ALK-5 protein, which also contains the same
potential site for N-glycosylation in the extracellular domain, has
been previously shown by enzymatic deglycosylation of the affinity
cross-linked complexes using endoglycosidase F (12).
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TGF-1 signaling.
To explore the potential function of sT
R-I, the effects of its
expression on TGF-
1 signaling were determined using the luciferase reporter plasmid, p3TP-Lux (Fig. 5).
Transient transfection of p3TP-Lux and empty vector pcDNA3 (as
controls) into COS-7 cells resulted in the induction of luciferase
activity by exogenous TGF-
1, presumably due to endogenous expression
of TGF-
receptors in these cells. Remarkably, in cells transfected
with sT
R-I and p3TP-Lux, further enhancement of TGF-
1 signaling
was observed, with consistently higher levels of TGF-
1-induced
luciferase activity observed in cells expressing sT
R-I compared with
pcDNA3 controls. In cells transfected with full-length T
R-II and
p3TP-Lux, increased basal luciferase activity was observed, which
increased little with exogenous TGF-
1 addition. Similarly, in cells
cotransfected with full-length T
R-II and sT
R-I, and p3TP-Lux,
basal luciferase activity was increased, but in the presence of
exogenous TGF-
1, further induction of luciferase activity above
basal levels was noted. TGF-
1 responsiveness was completely
abrogated when signaling was blocked by transfection of a dominant
negative mutant T
R-IM or
T
R-IIM.
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DISCUSSION |
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TGF-1 exerts diverse biological activities through the interaction
with a heteromeric signaling complex consisting of T
R-I and T
R-II
(28). Given that T
R-II is unable to mediate TGF-
1 signaling
without T
R-I, the specificity of cellular response to TGF-
1 is
thought to be determined by T
R-I. One plausible mechanism whereby
different sets of cellular responses may be elicited by a single
regulatory molecule is the interaction of the type II receptor with
different type I receptors, or ALKs, to form the heteromeric signaling
complex. Although ALK-5, ALK-1, and ALK-2 have all been demonstrated to
complex with ligand-bound T
R-II, ALK-5 is the predominant T
R-I in
most cell types (12).
Alternatively, diversity of TGF- actions may be mediated by T
R-II
interacting with variant forms of the signaling T
R-I subtype. This
study reports the identification of three variant forms of ALK-5. Two
of the cDNA clones encoded membrane-anchored forms of T
R-I but
differed in the COOH-terminal region of the extracellular
ligand-binding domain. One clone contained an in-frame insertion of
four amino acid residues, GPFS, not present in the second clone. At
precisely the same position in the extracellular domain, where the
nucleotide sequences diverged in the two membrane-anchored forms of
ALK-5, the third cDNA clone contained an insert of five amino acid
residues, GKLLY, followed by an in-frame stop codon, and lacked the
entire transmembrane and cytoplasmic serine/threonine domains. Based on
the nucleotide sequence of this clone, the deduced amino acid sequence
predicted a secreted form of ALK-5, named sT
R-I. In vivo expression
of the sT
R-I mRNA was demonstrated in the neonatal and adult rat
kidneys by RT-PCR followed by Southern analysis (Fig. 2), and confirmed
by RNase protection assay (Fig. 3).
To date, a naturally occurring soluble form of TGF- receptor had not
previously been reported. Curiously, similar variant receptor forms,
including a naturally occurring soluble form, have been described for
another cytokine, fibroblast growth factor (FGF) (8). The FGF receptor
1 (FGFR-1) gene contains three alternative exons encoding the
COOH-terminal portion of the extracellular region (13). One of these
alternative exons includes a stop codon and therefore encodes a
secreted form of FGFR-1, and the other two exons encode
membrane-anchored forms of FGFR-1 with the characteristic intracellular
tyrosine kinase domain. As with the FGFR-1, the variant forms of
T
R-I described here may arise from three alternatively spliced exons
at the position in the extracellular domain where the nucleotide
sequences diverge. The published human T
R-I gene consists of nine
exons, and alignment with the nucleotide sequences for the three
variant forms of ALK-5 described in this report reveals that the point
of sequence divergence occurs precisely between exons 2 and 3, located
just upstream of the transmembrane domain (24). Thus it is likely that
the three variant receptor forms of ALK-5 are produced by major
alternative splicing. Moreover, given the high degree of nucleotide
sequence homology between the human and rat T
R-I, indicating that
the gene is highly conserved during evolution, it is likely that
similar alternatively spliced forms of ALK-5 also occur in humans.
The significance for the existence of the variant forms of TR-I is
yet unknown but may represent an endogenous mechanism to carefully and
precisely control and modulate the activity of TGF-
. Given that the
multiple receptor forms vary in their extracellular ligand-binding
domain, a potential physiological function may be to confer specificity
for binding TGF-
ligands. Alternative exons conferring
ligand-binding specificity by altering ligand affinity have been
demonstrated for the FGF receptors. For instance, an alternatively
spliced variant of FGFR-2, named keratinocyte growth factor (KGF)
receptor, bound acidic FGF with higher affinity than basic FGF (17), in
contrast to the bek form of FGFR-2, which bound both basic
and acidic FGF with high affinity (7). Moreover, a secreted form of
FGFR-1 protein, when expressed in Chinese hamster ovary cells,
oligomerized on ligand binding and preferentially bound basic FGF over
acidic FGF (8).
In the case of TGF- receptors, although T
R-II is required for
initial binding of TGF-
1 and thus is said to possess ligand-binding specificity (27), additional ligand specificity may be determined by
the particular form of T
R-I involved in the heteromeric signaling complex with T
R-II, perhaps by differing affinities for the three isoforms of TGF-
. During embryonic development, all three isoforms, TGF-
1, -
2, and -
3, have been shown to be temporally and
spatially regulated and differentially expressed in embryonic tissues
including the kidneys (19). Both T
R-I and T
R-II have also been
shown to be temporally and spatially regulated and differentially
expressed in developing kidneys (6). It is possible that each of the multiple forms of TGF-
receptors may possess specific roles and patterns of expression during embryonic development and may mediate different functions. Interestingly, the sT
R-I mRNA expression was
observed to be greater in abundance in the rat neonatal kidneys compared with the adult kidneys, similarly to a previously reported (6)
developmental study of T
R-I mRNA abundance, thus implicating a role
for sT
R-I in renal development.
Soluble receptors may potentially act as either antagonists or
potentiators of cytokine activities. Inhibitory actions by soluble
receptors, presumably through competition with membrane-anchored receptors for ligand binding, have been demonstrated for soluble receptors of interleukin (IL)-4, platelet-derived growth factor (PDGF)-, and vascular endothelial cell growth factor (VEGF) (14, 18,
23). In contrast, potentiating action of IL-6 by its
soluble receptor, IL-6sR, to markedly induce collagenase 3 expression in osteoblasts has also been demonstrated (11).
In this report, the functional role of sTR-I in TGF-
signaling
was explored using a TGF-
-inducible luciferase reporter assay.
First, the ability of sT
R-I to complex with ligand-bound T
R-II on
the cell surface was demonstrated by affinity cross-linking with
125I-labeled TGF-
1 in COS-7
cells expressing both the sT
R-I and T
R-II. Next, the effects of
its expression on TGF-
1 signaling were determined using the
luciferase reporter plasmid, p3TP-Lux, to ascertain TGF-
1-induced
transcriptional activation of the PAI-1 promoter. Transient
transfection of sT
R-I with p3TP-Lux in COS-7 cells resulted in
higher luciferase activity in response to exogenous TGF-
1 compared
with control COS-7 cells transfected with empty vector pcDNA3 and
p3TP-Lux. This increased TGF-
1 responsiveness demonstrates the
ability of sT
R-I to potentiate signaling by the endogenously
expressed wild-type TGF-
receptors. To determine whether TGF-
1
responsiveness could be further enhanced by increased levels of
T
R-II and ligand binding, the effects on TGF-
1 signaling were
assayed on transfection of T
R-II and in combination with sT
R-I.
Transient transfection of wild-type T
R-II in COS-7 cells resulted in
higher basal luciferase activity, indicating that high-level expression
of the receptors can initiate TGF-
signaling, due to either the
presence of endogenous TGF-
1 or the inherent affinity of the
receptors to interact and cooperate with each other to signal in a
ligand-independent manner. Transfection and overexpression of wild-type
T
R-I have previously been shown to exhibit ligand-independent
activation (4). The inability of exogenous TGF-
1 to further increase
luciferase activity in T
R-II-transfected cells suggests probable
saturation of the signaling receptors with the ligand. However,
exogenous TGF-
1 was able to induce increased luciferase activity
upon expression of sT
R-I, by cotransfection of T
R-II with
sT
R-I, but not above the levels observed in the sT
R-I-transfected
cells. Taken together, these data suggest that the signaling T
R-I
may be expressed in limiting levels and that the sT
R-I functions as
a potentiator but not as a direct signaling receptor, as it lacks the
cytoplasmic domain. Next, the effects of sT
R-I were compared with
those of another truncated form, T
R-IM, which is membrane
anchored but lacks the cytoplasmic GS and kinase domains and inhibits
TGF-
1 signaling. As predicted, complete inhibition of
TGF-
1-induced luciferase activity was observed in cells expressing
the truncated receptors, T
R-IM. Thus the sT
R-I actions clearly differ from those of the
kinase-deleted mutant T
R-IM,
which inhibits TGF-
1 signaling, presumably by functioning in a
dominant negative fashion to compete with membrane-anchored T
R-I for
complexing with the ligand-bound T
R-II. Similarly, transfection of a
dominant negative T
R-IIM also
inhibited TGF-
signaling.
The stimulatory action exhibited by a soluble type I receptor for
TGF- is rather intriguing. In contrast to IL-6sR, which binds IL-6
directly to potentiate its action, sT
R-I and T
R-I require the
presence of T
R-II for ligand binding. Thus the soluble form of
T
R-I presumably functions as an agonist to augment TGF-
1 actions
through an entirely novel mechanism of action not yet understood. The
secreted sT
R-I might serve as a chaperone, for instance, by
recognizing and interacting with ligand-bound T
R-II to stabilize the
ligand-bound complex and prevent its dissociation and degradation, and
may cooperate to facilitate the recruitment of the membrane-anchored
T
R-I to form the signaling heteromeric complex. The soluble form of
receptor may then dissociate and allow the membrane-anchored T
R-I to
form a more stable complex with ligand-bound T
R-II to allow
transphosphorylation and initiation of the signaling cascade. The
concept of agonist activity of a natural sT
R-I to potentiate
TGF-
1 actions has important clinical implications for its potential
role in a wide range of biological processes, such as growth and
development, wound repair, and pathogenesis of fibrotic diseases. The
present studies demonstrate upregulation of sT
R-I expression in the
developing kidney, potentially to enhance TGF-
signaling and thereby
amplify TGF-
actions during renal development.
In summary, three variant forms of TR-I were isolated from a rat
neonatal kidney library, and data presented in this report demonstrate
for the first time the existence of a naturally occurring variant
T
R-I transcript whose nucleotide sequence and deduced amino acid
sequence predict a soluble form of ALK-5. Physiological functions of
these variant receptor forms are not yet known, but their variable in
vivo expression may represent a mechanism conferring multiple cellular
responses to TGF-
1. Moreover, the biological importance of a soluble
T
R-I is yet to be determined, but the findings here suggest that it
may serve as a natural potentiator of TGF-
signaling. The molecular
cloning and identification of a naturally occurring sT
R-I adds a new
level of complexity to our present knowledge of the TGF-
receptor
system and will facilitate further investigations to expand our
understanding of the mechanism of TGF-
1 actions.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Physician Scientist Award 5-K12-DK-0129809, a Grant-in-Aid (96015510) from the American Heart Association, and a U.S. Department of Veterans Affairs Career Development Award.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: M. E. Choi, Section of Nephrology, Yale University School of Medicine, 333 Cedar St., 2073 LMP, New Haven, CT 06520-8029.
Received 18 August 1998; accepted in final form 8 October 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Attisano, L.,
J. Cárcamo,
F. Ventura,
F. M. B. Weis,
J. Massagué,
and
J. L. Wrana.
Identification of human activin and TGF- type I receptors that form heteromeric kinase complexes with type II receptors.
Cell
75:
671-680,
1993[Medline].
2.
Attisano, L.,
J. L. Wrana,
S. Cheifetz,
and
J. Massagué.
Novel activin receptors: distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors.
Cell
68:
97-108,
1992[Medline].
3.
Cárcamo, J.,
A. Zentella,
and
J. Massagué.
Disruption of TGF- signaling by a mutation that prevents transphosphorylation within the receptor complex.
Mol. Cell. Biol.
15:
1573-1581,
1995[Abstract].
4.
Chen, F.,
and
R. A. Weinberg.
Biochemical evidence for the autophosphorylation and transphosphorylation of transforming growth factor receptor kinases.
Proc. Natl. Acad. Sci. USA
92:
1565-1569,
1995[Abstract].
5.
Choi, M. E.,
and
B. J. Ballermann.
Inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth factor- receptors.
J. Biol. Chem.
270:
21144-21150,
1995
6.
Choi, M. E.,
A. Liu,
and
B. J. Ballermann.
Differential expression of transforming growth factor- receptors in rat kidney development.
Am. J. Physiol.
273 (Renal Physiol. 42):
F386-F395,
1997
7.
Dionne, C. A.,
G. Crumley,
F. Bellot,
J. M. Kaplow,
G. Searfoss,
M. Ruta,
W. H. Burgess,
M. Jaye,
and
J. Schlessinger.
Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors.
EMBO J.
9:
2685-2692,
1990[Abstract].
8.
Duan, D.-S.,
S. Werner,
and
L. T. Williams.
A naturally ocurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF.
J. Biol. Chem.
267:
16076-16080,
1992
9.
Ebner, R.,
R.-H. Chen,
L. Shum,
S. Lawler,
T. F. Zioncheck,
A. Lee,
A. R. Lopez,
and
R. Derynck.
Cloning of a type I TGF- receptor and its effect on TGF-
binding to the type II receptor.
Science
260:
1344-1348,
1993[Medline].
10.
Estevez, M.,
L. Attisano,
J. L. Wrana,
P. S. Albert,
J. Massagué,
and
D. L. Riddle.
The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans larva development.
Nature
365:
644-649,
1993[Medline].
11.
Franchimont, N.,
S. Rydziel,
A. M. Delany,
and
E. Canalis.
Interleukin-6 and its soluble receptor cause a marked induction of collagenase 3 expression in rat osteoclasts cultures.
J. Biol. Chem.
272:
12144-12150,
1997
12.
Franzén, P.,
P. ten Dijke,
H. Ichijo,
H. Yamashita,
P. Schulz,
C. H. Heldin,
and
K. Miyazono.
Cloning of a TGF type I receptor that forms a heteromeric complex with the TGF
type II receptor.
Cell
75:
681-692,
1993[Medline].
13.
Johnson, D. E.,
J. Lu,
H. Chen,
S. Werner,
and
L. T. Williams.
The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain.
Mol. Cell. Biol.
11:
4627-4634,
1991[Medline].
14.
Kendall, R. L.,
and
K. A. Thomas.
Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.
Proc. Natl. Acad. Sci. USA
90:
10705-10709,
1993[Abstract].
15.
Lin, H. Y.,
W.-F. Wang,
E. Ng-Eaton,
R. A. Weinberg,
and
H. F. Lodish.
Expression cloning of the TGF- type II receptor, a functional transmembrane serine/threonine kinase.
Cell
68:
775-785,
1992[Medline].
16.
Markowitz, S. D.,
and
A. B. Roberts.
Tumor suppressor activity of the TGF- pathway in human cancers.
Cytokine Growth Factor Rev.
7:
93-102,
1996[Medline].
17.
Miki, T.,
T. P. Fleming,
D. P. Bottaro,
J. S. Rubin,
D. Ron,
and
S. A. Aaronson.
Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop.
Science
251:
72-75,
1991[Medline].
18.
Mosley, B.,
M. P. Beckmann,
C. J. March,
R. L. Idzerda,
S. D. Gimpel,
T. VandenBos,
D. Friend,
A. Alpert,
D. Anderson,
J. Jackson,
J. M. Wignall,
C. Smith,
B. Gallis,
J. E. Sims,
D. Urdal,
M. B. Widmer,
D. Cosman,
and
L. S. Park.
The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms.
Cell
59:
335-348,
1989[Medline].
19.
Roelen, B. A.,
H. Y. Lin,
V. Knezevic,
E. Freund,
and
C. L. Mummery.
Expression of TGF-s and their receptors during implantation and organogenesis of the mouse embryo.
Dev. Biol.
166:
716-728,
1994[Medline].
20.
Saitoh, M.,
H. Nishitoh,
T. Amagasa,
K. Miyazono,
M. Takagi,
and
H. Ichijo.
Identification of important regions in the cytoplasmic juxtamembrane domain of type I receptor that separate signaling pathways of transforming growth factor-.
J. Biol. Chem.
271:
2769-2775,
1996
21.
Ten Dijke, P.,
H. Yamashita,
H. Ichijo,
P. Franzen,
M. Laiho,
K. Miyazono,
and
C. H. Heldin.
Characterization of type I receptors for transforming growth factor-beta and activin.
Science
264:
101-104,
1994[Medline].
22.
Ten Dijke, P.,
H. Yamashita,
T. K. Sampath,
A. H. Reddi,
M. Estevez,
D. L. Riddle,
H. Ichijo,
C. H. Heldin,
and
K. Miyazono.
Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4.
J. Biol. Chem.
269:
16985-16988,
1994
23.
Tiesman, J.,
and
C. E. Hart.
Identification of a soluble receptor for platelet-derived growth factor in cell-conditioned medium and human plasma.
J. Biol. Chem.
268:
9621-9628,
1993
24.
Vellucci, V. F.,
and
M. Reiss.
Cloning and genomic organization of the human transforming growth factor- type I receptor gene.
Genomics
46:
278-283,
1997[Medline].
25.
Weis-Garcia, F.,
and
J. Massagué.
Complementation between kinase-defective and activation-defective TGF- receptors reveals a novel form of receptor cooperativity essential for signaling.
EMBO J.
15:
276-289,
1996[Abstract].
26.
Wrana, J. L.,
L. Attisano,
J. Cárcamo,
A. Zentella,
J. Doody,
M. Laiho,
X. F. Wang,
and
J. Massagué.
TGF- signals through a heteromeric protein kinase receptor complex.
Cell
71:
1003-1014,
1992[Medline].
27.
Wrana, J. L.,
L. Attisano,
R. Wieser,
F. Ventura,
and
J. Massagué.
Mechanism of activation of the TGF- receptor.
Nature
370:
341-347,
1994[Medline].
28.
Yamashita, H.,
P. ten Dijke,
P. Franzén,
K. Miyazono,
and
C.-H. Heldin.
Formation of hetero-oligomeric complexes of type I and type II receptors for transforming growth factor-.
J. Biol. Chem.
269:
20172-20178,
1994