(Received for publication, November 28, 1994)
From the
Although transforming growth factor- (TGF
) is
implicated in differentiation and disease, proof of in vivo function requires specific inhibitors of the TGF
cascade.
TGF
binds a family of type I and type II receptors (T
RI,
T
RII), containing a cytoplasmic serine/threonine kinase domain. We
previously reported that kinase-deficient T
RII (
kT
RII)
blocks TGF
-dependent transcription in cardiac myocytes. It is
controversial whether both receptors are needed in all cells for gene
regulation by TGF
or whether they mediate distinct subsets of
TGF
-dependent events. To resolve this uncertainty,
TGF
-dependent transcription was investigated in cardiac myocytes versus mink lung epithelial cells. 1)
kT
RII inhibits
induction of a TGF
-responsive reporter gene, in both cell
backgrounds. 2) Charged-to-alanine mutations of key residues of the
T
RII kinase, including consensus ATP binding and amino acid
recognition motifs, are competent for binding but not transcriptional
activation. Each inactive receptor inhibits TGF
-dependent
transcription in both cell types. 3) Kinase-deficient T
RI
(
kT
RI) likewise impairs TGF
-dependent transcription,
less completely than
kT
RII; kinase-deficient activin type I
receptor has no effect. 4) TGF
-binding proteins in cardiac cells
and Mv1Lu cells are comparable by affinity labeling and
immunoprecipitation; however, Mv1Lu cells express up to 3-fold higher
levels of T
RII and T
RI. Thus, the model inferred from
TGF
-resistant cell lines (that T
RII and T
RI are
necessary in tandem for the TGF
-signaling complex to regulate
transcription) is valid for cardiac myocytes, the cell type most
prominently affected in TGF
-deficient animals.
Cytokines of the type transforming growth factor
(TGF
) (
)superfamily participate in remarkably diverse
aspects of development, including pattern formation, organogenesis, and
tissue-specific transcription, as well as in human disease(1) .
For the cardiovascular system, TGF
has been implicated in cardiac
myogenesis itself, formation of the heart valves, protection of
contractility from depressant effects of interleukin 1
,
atherosclerosis in apolipoprotein(a) transgenic mice, and activation of
a ``fetal'' program of transcription during work-induced
hypertrophy (2, 3, 4, 5, 6, 7) .
Despite multiple roles for TGF
anticipated from embryonal stem
cells and other model systems, both embryonic development and perinatal
survival are normal in mice homozygous for a null mutation of
TGF
1(8, 9) . While, a priori, such a
discrepancy might be explained by functional redundancy among the three
mammalian isoforms of TGF
, a more direct explanation possibly is
furnished by the unexpected finding that maternal TGF
crosses the
placenta and also can be conferred by breast milk(10) . The
TGF
1-null offspring of heterozygous mothers, which initially are
normal, soon develop a multifocal inflammatory response primarily
affecting the heart and lungs(8, 9) , with enhanced
expression of major histocompatibility antigens, resembling autoimmune
disease(11) . By contrast, the homozygous offspring of a mother
homozygous for the null mutation die uniformly in the first day of
life, with gross abnormalities of only ventricular myocardium and the
atrioventricular valves(10) . Thus, the heart is reported to be
the predominant, if not the exclusive, target organ affected by
TGF
1 deficiency in the embryo and newborn.
Affinity labeling
with radioiodinated TGF has identified three major TGF
receptors in most mammalian lineages, designated type III (betaglycan;
mass 200-400 kDa), type II (T
RII; mass 70-80 kDa), and
type I (T
RI; mass 53 kDa); however, additional membrane proteins,
of less certain function, also bind TGF
in various cell
types(12, 13) . Betaglycan, a membrane-spanning
proteoglycan with an extremely short cytoplasmic domain, is absent from
certain TGF
-responsive cells and is thus dispensable for TGF
signal generation. Soluble fragments of betaglycan have been identified
that interfere with access of TGF
to the signaling receptors,
whereas membrane-anchored betaglycan enhances the action of TGF
via increased binding of TGF
, particularly TGF
2, to the type
II and type I receptors(14, 15) . In the presence of
ligand, betaglycan associates with the type I and type II receptors,
forming a ternary complex(14) . However, homodimers of
betaglycan are the predominant type III receptor complex present on the
cell surface in both the absence and presence of TGF
(16) .
TRII, first identified by expression cloning and subsequently
isolated in several species, is a transmembrane protein characterized
by a single transmembrane segment and an intracellular domain
comprising a highly conserved serine/threonine kinase, the hallmark of
this emerging superfamily of receptors for TGF
, activins, bone
morphogenetic proteins, and related
cytokines(17, 18, 19, 20) . The
protein kinase domain itself is characterized by two inserts,
positioned between subdomains VIa and VIb (insert I) and between
subdomains X and XI (insert II), while carboxyl-terminal to the kinase
domain is a serine/threonine-rich cytoplasmic tail. The ligand-binding
domain is abundant in cysteine residues, and an especially conserved
motif, the Cys box, is found in all members of the receptor
serine/threonine kinase superfamily. A distinguishable subfamily of
type I receptors has been isolated, based upon sequence homologies
within the kinase domains, which show high homology to each other
(60-90% amino acid sequence identity) but lesser homology to the
kinase domains of the known type II receptors
(30-40%)(21, 22, 23, 24) .
Features common to the type I receptors include the spacing of
extracellular cysteine residues, and a glycine- and serine-rich motif
amino-terminal to the kinase domain.
One distinction between the
cloned type I and type II receptors is the inability of type I
receptors to bind TGF in the absence of type II receptors, a
feature predicted from mutant cells unresponsive to TGF
(25) . Conversely, the type II receptor is fully sufficient for
ligand binding, yet not for signal transduction. Antibodies directed
against the type II receptor coprecipitate type I receptor from cells
treated with TGF
, suggesting that type I and type II receptors
form a heteromeric complex in the presence of ligand(18) .
Hence, the genetic evidence from TGF
-resistant cell lines implies
that only a heteromeric complex comprising type I and type II receptor
in tandem is competent to support TGF
signal
transduction(18) . Although TGF
-related growth factors
bind with promiscuity to a variety of type I receptors under the
conditions of forced expression in COS cells, physical association of
the respective endogenous type I and type II receptors exhibits greater
specificity, as does the ability of type I receptors to support
biological
responses(26, 27, 28, 29) . Of six
type I receptors, only one (ALK-5/R4) has been proven to rescue gene
induction by TGF
when introduced into type I receptor-deficient
cells(26, 30) . Thus, ALK-5/R4 is a functional type I
receptor for TGF
. Activity of the T
RII kinase is constitutive
even in the absence of TGF
, and ligand binding is understood to
initiate the TGF
signal transduction cascade through the physical
recruitment of T
RI into a receptor heterodimer and, consequently,
the asymmetric phosphorylation of T
RI by T
RII(31) .
We demonstrated previously that a truncation of TRII, lacking
the serine/threonine kinase domain (
kT
RII), introduced into
cardiac muscle cells by transient transfection, is sufficient to
suppress TGF
-dependent activation of the skeletal
-actin
promoter, by all three isoforms of the peptide, and to block
down-regulation of the
-myosin heavy chain promoter(32) .
Thus, by itself, the truncated type II receptor is sufficient to block
both positive and negative control of transcription by TGF
, as
predicted from models requiring the action of T
RII and T
RI in
concert(18, 27, 31, 33) . This model
for signal generation via a heteromeric complex of T
RI and
T
RII is disputed, however. Differing conclusions for a
dominant-negative type II receptor have been drawn in Mv1Lu mink lung
epithelial cells, used extensively for the analysis of TGF
signaling mechanisms: a related kinase-defective truncation of the
human type II receptor failed to suppress three representative
TGF
-dependent genes, including plasminogen activator inhibitor-1
(PAI-1), yet was capable of disrupting growth inhibition by
TGF
(34) . These results lend support to an alternative
model-that inactivation of the type II receptor exclusively impairs a
pathway important for growth control, whereas transcriptional control
might be mediated by the type I receptor alone.
To address these
uncertainties regarding the comparative role of TRI and T
RII
in gene regulation, in the present study we have developed three
independent lines of evidence. First, we transfected Mv1Lu cells with
our kinase-defective truncation of the type II TGF
receptor,
together with a TGF
-inducible reporter construct derived from the
PAI-1 promoter. In concordance with our prior observations using
ventricular myocytes, expression of
kT
RII inhibited induction
of the reporter gene by TGF
in the Mv1Lu cell background. Second,
charged-to-alanine point mutations were engineered at conserved
residues within subdomains II, VI-B, and kinase insert II of the
T
RII kinase, which are associated with ATP binding, amino acid
recognition, and protein kinase activity. Each substitution failed to
rescue receptor-deficient cells and was fully as active as
kT
RII for inhibiting TGF
-dependent gene induction both
in normal Mv1Lu cells and in cardiac myocytes. Third, we compared the
ability of a kinase-deficient truncation of the type I receptor
(
kT
RI) to likewise block TGF
signaling events that
mediate gene activation. Overexpression of
kT
RI in both
cardiac myocytes and mink lung cells blocked gene induction by
TGF
, although less effectively than did
kT
RII. Although
ALK-2, a type I receptor for activin, can bind TGF
both in its
full-length and truncated form(35) , neither full-length ALK-2
nor a corresponding truncation of ALK-2 interfered with
TGF
-dependent transcription. Thus, our data concur with the model
inferred from TGF
receptor-deficient cells, that the function of
type I and type II receptors is necessary in tandem for the
TGF
-signaling complex to regulate gene transcription.
Figure 1:
Structure of the TRII
mutations. Subdomains denote the consensus subdomains
conserved within the serine/threonine and tyrosine kinase
superfamilies(39) . The schematic representation of T
RII
indicates the extracellular cysteines as verticallines at the left of the figure and the transmembrane domain as
a solidbar. The whitebar denotes
subdomains II-XI of the cytoplasmic serine/threonine kinase
domain; the hatchedbar indicates the amino-terminal
remnant of the kinase domain retained in
kT
RII; graybars indicate kinase inserts 1 and 2. Sense and antisense primers used in conjunction with the mutagenic
oligonucleotides are illustrated below. See ``Experimental
Procedures'' and Table 1for
details.
For the purpose of subcloning mutagenized sequences
of TRII into the wild-type receptor background, full-length
T
RII was first subcloned into pGem4Z. Site-directed
charged-to-alanine mutations of the human T
RII cDNA were generated
by PCR, using H2-3FF (17) as the template. To generate
each point mutation of the T
RII kinase domain, two PCR reactions
were performed using overlapping primer pairs: e.g. primer 1
or 2, respectively, with the antisense or sense primers for E272A and
K277A (Fig. 1; Table 1). The resulting PCR products for
each primer pair were gel-purified, pooled, and subjected to another
round of PCR amplification using primers 1 and 2. The final product was
subcloned using the HpaI-SmaI (K277A) or HpaI-BglII (E272A) sites of human T
RII.
For
all other point mutations, a similar three-step PCR mutagenesis
protocol was employed. Primers 3 and 4, respectively, were used in
conjunction with antisense or sense primers for H362A, K372A/H377A,
R378A/D379A, K381A, and D397A; secondary amplification was performed
using primers 3 and 4, and the final product was subcloned into the SmaI-AccI sites of TRII-pGem4Z. Primers 3 and 6
were utilized for amplification of D446A. Primers 5 and 6 were used,
respectively, with the antisense or sense primer for R497/H507 and
R528A; secondary amplification was performed using primers 5 and 6, and
the final product was subcloned into AccI-HindIII
sites of T
RII-pGem4Z. The truncation of the carboxyl-terminal
serine-rich tail was generated by amplifying H2-3FF using primers
1 and
tail (Table 1) and was subcloned as a BglII-HindIII fragment into the BglII-HindIII sites of wild-type T
RII. Reaction
conditions were 1 min each at 94, 55, and 72 °C for 12 cycles. All
constructs were authenticated by dideoxy sequencing (U. S. Biochemical
Corp., Sequenase version 1) and were subcloned for expression in
eukaryotic cells into the vector pcDNA-1 (Invitrogen; Fig. 3) or
pSV-Sport-1 (Life Technologies, Inc.; Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8).
Transfections in each experiment were balanced for promoter and total
DNA content using the corresponding empty vector control, and all
comparisons between receptor constructs used the identical promoter for
each.
Figure 3:
The truncated type II receptor,
kT
RII, is a dominant inhibitor of TGF
-dependent
transcription in mink lung epithelial cells. A, Mv1Lu cells
transfected with
kT
RII and full-length T
RII cDNA in the
amounts indicated were analyzed for the activity of p3TP-lux and
CMV-
-galactosidase reporter genes. Results (mean ± S.E.)
are shown for vehicle-treated (
) and TGF
1-treated cells
(
). Levels of luciferase expression, corrected for transfection
efficiency, are expressed relative to the vehicle-treated,
vector-transfected cells. B, Mv1Lu cells were transfected with
a fixed amount (10 µg) of
kT
RII (
) or the vector
(&cjs2113;), together with decreasing amounts (7.5, 5, 2.5, or 1
µg) of the p3TP-lux reporter gene; the ratio of expression vector
to p3TP-lux was thus 1.3, 2, 4, or 10, respectively. Cells were
incubated for 16 h in the absence(-) or presence (+) of
TGF
1 and were assayed for luciferase and
-galactosidase
activity. Results (mean values ± S.E.) are expressed relative to
p3TP-lux expression in vehicle-treated, vector transfected
cells.
Figure 4:
TGF receptors in cardiac myocyte and
Mv1Lu cell cultures. A, cardiac myocytes, Mv1Lu
cells, and the receptor-deficient derivatives shown were
affinity-labeled with 100 pM
I-TGF
1
± 60 nM unlabeled TGF
1. B, Mv1Lu cells
and cardiac myocytes were affinity-labeled with
I-TGF
1, and the cell lysates were subjected to
immunoprecipitation with polyclonal antibody directed against T
RII (left) or T
RI (right). For reference, lysate not
subjected to immunoprecipitation is shown at the left of panelB. Immunoprecipitation with antibody directed
against T
RI was performed in the absence or presence, as
indicated, of synthetic T
RI peptide. Specificity of
immunoprecipitation with antibody directed against T
RII was
corroborated, analogously, using synthetic T
RII peptide, in
additional experiments not illustrated
here.
Figure 5:
Dominant-negative activity of point
mutations of the kinase domain of TRII. Cardiac myocytes (A), Mv1Lu cells (B), and T
RII-deficient DR-26
cells (C) were transfected with vector,
tail, or the
missense mutations indicated. Cell lysates were analyzed for the
activity of skeletal
-actin- (A) or p3TP-luciferase (B and C) and CMV-
-galatosidase reporter genes.
Results are shown for cells cultured in the absence (
) and
presence (
) of 1 ng/ml TGF
1. Luciferase activity (mean
± S.E.) is expressed relative to that of the p3TP-lux activity
in vehicle, vector-transfected cells.
Figure 6:
Cell surface expression of point mutations
of TRII. COS-1 cells were transfected with the indicated
charged-to-alanine substitutions of T
RII, wild-type T
RII, or
the vector alone, and were then affinity-labeled with 100 pM
I-TGF
1 in the absence(-) or presence
(+) of 60 nM unlabeled TGF
1 as competitor. For
comparison as a positive control, Mv1Lu cells were included. Cell
lysates were subjected to gel electrophoresis and
autoradiography.
Figure 7:
A deletion of the TGF type I receptor
kinase specifically inhibits TGF
-dependent transcription. Mv1Lu
cells were transfected with vector,
kT
RII,
kT
RI,
wild-type ActRI, or
kActRI. Cell lysates were analyzed for the
activity of p3TP-lux and CMV-
-galatosidase reporter genes. Results
are shown for cells cultured in the absence (
) and presence
(
) of 1 ng/ml TGF
1. Luciferase activity (mean ± S.E.)
is expressed relative to that in vehicle, vector-transfected
cells.
Figure 8:
Truncated type II receptor inhibits
TGF-dependent transcription at least as effectively as the
truncated type I receptor. Mv1Lu cells (A) and cardiac
myocytes (B) were transfected with
kT
RII, (
,
),
kT
RI (
,
), or
kT
RII +
kT
RI (
,
) expression vectors at the
concentrations shown, in the absence (
,
,
) or
presence (
,
,
) of 1 ng/ml TGF
1. For
cotransfection of
kT
RII +
kT
RI, the ordinate
indicates the sum of the two receptor plasmids (e.g.4
µg denotes 2 µg of each). Cell lysates were analyzed for
the activity of p3TP- (A) or skeletal
-actin-luciferase (B) and CMV-
-galatosidase reporter genes. Mean luciferase
activity is expressed relative to that in vehicle-treated,
vector-transfected cells.
A kinase-deficient truncation of the TGF type I receptor,
kT
RI, was constructed using PCR primers
kT
RI
and
kT
RI
(Table 1) and human
ALK-5-pSV7D (26) as the template. Primers upstream of the start
codon were problematic, given the unusually high GC content in this
region. Therefore, a sense-strand primer was used, positioned 135
nucleotides 3` to the start site. The resulting fragment was subcloned
into the EcoRI-HindIII sites of pSV-Sport-1. The
missing 5` end was rescued by subcloning an EcoRI/XbaI fragment of ALK-5-pSV7D into the EcoRI-XbaI sites of
kT
RI-pSV-Sport-1. The
truncated, kinase-deficient activin type I receptor,
kActRI, was
produced by subcloning a 1.0-kilobase pair EcoRI/BglII fragment of SKR1/ALK-2 (22) into
the EcoRI and BglII sites of
kT
RII-pSV-Sport-1, thereby substituting the
kActRI
coding sequence for
kT
RII. For construction of wild-type
ActRI, a SmaI/HindIII fragment of SKR1 in pBluescript
was subcloned into pSV-Sport-1.
The TGF-responsive reporter
gene analyzed in cardiac myocytes,
-394/+24SkALuc(32) , contains nucleotides -394
to +24 of the chicken SkA gene as an RsaI-HindIII fragment between the SmaI and HindIII sites of the firefly luciferase reporter expression
vector pXP1(36) ; activation of the full-length promoter by
TGF
is contingent on serum response factor and the SV40
enhancer-binding protein, TEF-1, in concert(37) . p3TP-lux,
used for the mink lung epithelial cells, is a derivative of the human
PAI-1 promoter containing (5` to 3`) three copies of the tetradecanoyl
phorbol acetate-responsive element from the human collagenase promoter
(nucleotides -73 to -42), a TGF
-responsive element of
the PAI-1 promoter (nucleotides -740 to -636), and the the
adenovirus E4 promoter sequence (-38 to +38), cloned into
pGL2 (Promega)(18) . As a constitutive reporter plasmid,
CMV
-gal was used as detailed previously(32) .
Briefly, 10 µg of
receptor construct (except as noted in the text) versus the
empty expression vector, 5 µg of the appropriate luciferase
reporter plasmid, and 2.5 µg of CMV-gal were mixed with 1 ml
of DMEM, 10% NuSerum (HyClone), and 200 µg/ml DEAE-dextran. Cells
were incubated for 4 h with DNA/DEAE-dextran complexes, and then
shocked for 90 s with 10% dimethyl sulfoxide in DMEM. The cells were
cultured overnight in
-minimum essential medium containing 0.2%
fetal bovine serum. The following day, the medium was replaced, and 1
ng/ml purified porcine TGF
1 (R& Systems) or the vehicle was
added, as described(32) . Sixteen h after addition of the
growth factor, cells were harvested and assayed for luciferase and
-galactosidase activity(32) . Three to six transfections
were performed for each condition tested, using at least two
independent DNA preparations. Results were compared by analysis of
variance and the Student-Newman-Keuls multiple comparison test, using a
significance level of p < 0.05.
Figure 2:
Cell surface expression of TRII and
kT
RII. COS-1 cells transfected with T
RII,
kT
RII, or vector were affinity-labeled with
I-TGF
1 and disuccinimidyl suberate. Cell lysates
were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and autoradiography. Mv1Lu cells are included for
comparison at the right.
We transfected kT
RII into Mv1Lu mink
lung epithelial cells using p3TP-lux as the reporter gene. Mv1Lu cells
are known to be unusually sensitive to TGF
stimulation, and the
reporter gene p3TP-lux, a derivative of the PAI-1 promoter, is in turn
a construct that is highly responsive to TGF
, induced up to
20-fold ((18) and Fig. 3). Thus, this cell/reporter pair
provides an excellent responder system with which to evaluate the
potential trans-dominant effects of TGF
receptor mutations. Forced
expression of
kT
RII resulted in up to 80% inhibition of
p3TP-lux activity, compared to vector transfected cells (p = 0.0009). The cotransfected constitutive control,
CMV-lacZ, by contrast, was unaffected by the receptor mutation. Thus,
the cytoplasmic deletion mutant of T
RII functions as a dominant
inhibitor of TGF
-induced gene expression in Mv1Lu cells, without
need for concurrent mutation of T
RI.
Potentially, one reason for the residual induction seen in the Mv1Lu
cell type, but not in ventricular myocytes, might be that an incomplete
fraction of cells that have incorporated the reporter gene have also
taken up the kT
RII construct. To circumvent the risk of
excessive DNA content per transfected culture, we therefore transfected
Mv1Lu cells with decreasing ratios of the p3TP-lux reporter relative to
kT
RII. However, even using a 1:10 ratio of reporter to
receptor, residual TGF
-dependent activity remained, compared to
control, vehicle-treated cells receiving
kT
RII (p = 0.0003). Thus, residual induction in Mv1Lu cells cannot
be accounted for by failure to deliver
kT
RII to cells
receiving the luciferase reporter.
As inhibition by the
dominant-negative receptor could be reversed by titratable addition of
wild-type receptor, in both Mv1Lu cells (Fig. 3) and cardiac
myocytes(32) , we tested the alternative possibility that
differences in residual receptor function might be explained by the
respective levels of endogenous TGF receptor. The molecular
profile of TGF
receptors in mink lung epithelial cells and
neonatal rat cardiac myocytes was compared by receptor
affinity-labeling and immunoprecipitation (Fig. 4). Both cell
types expressed all three receptor components, betaglycan, type II, and
type I receptor, in concordance with prior studies of the cardiac
myocyte(6) . Whereas the levels of betaglycan were comparable
in both cell backgrounds, Mv1Lu cells contained up to 3-fold higher
content of T
RII and T
RI (Fig. 4A). The
identity of T
RII and ALK-5, respectively, as the type II and type
I receptors in cardiac myocyte cultures was corroborated by
coimmunoprecipitation (Fig. 4B). Thus, by the criteria
detailed here, both cell types have an equivalent set of
TGF
-binding proteins, but the higher content of signaling receptor
in Mv1Lu cells may account, at least in part, for the observed
differences in residual TGF
-induced transcription.
Transfection of
each point mutation of TRII into cardiac myocytes (Fig. 5A) and Mv1Lu cells (Fig. 5B)
resulted in strong inhibition of TGF
-dependent transcription, to a
similar or greater extent than the cytoplasmic deletion mutant,
kT
RII (in cardiac myocytes, p < 0.01 for each
construct, versus vector-transfected cells; in Mv1Lu cells, p < 0.05). Basal transcription of the skeletal
-actin
promoter was affected neither by full-length T
RII nor by any of
the point mutations, consistent with the prior finding that endogenous
TGF
is expressed by neonatal ventricular muscle cells
preponderantly in the latent, inactive form(6) . By contrast,
in Mv1Lu cells, basal transcription of the 3TP-luciferase gene was
augmented by wild-type receptor and inhibited by signal-defective
receptors, suggesting, in this cell background, that basal activity of
the TGF
-responsive promoter is mediated at least in part by
autocrine TGF
. In substantiation of this inference, 3TP-luciferase
activity in Mv1Lu cells was inhibited 60% (p = 0.0149)
by cotransfection with 10 µg of plasmid encoding the
NH
-terminal remnant of TGF
1 precursor protein,
1
latency-associated peptide. (
)Thus, basal activity of this
TGF
-inducible reporter gene was repressed by a high-affinity
antagonist of mature TGF
(43) .
To ensure the proper
translation and comparable expression of the mutant receptors, COS-1
cells were transfected with representative point mutations in parallel
with wild-type TRII and were subjected to cross-linking with
I-TGF
1 (Fig. 6). In each case, a 95-kDa
protein band was affinity-labeled, which was specifically displaced by
excess unlabeled ligand. Thus, the missense mutations were expressed,
transported across the surface membrane, and competent to bind
TGF
1, to a similar extent as the wild-type receptor (Fig. 6). Importantly, each missense mutation of T
RII also
was competent for presentation of ligand to T
RI. As the extent of
I-TGF
1 binding and the efficacy for competition by
unlabeled ligand each were comparable to cells transfected with the
full-length receptor, no evidence was found to suggest that the
dominant-negative deletion and substitution mutants might merely bind
TGF
more avidly than wild-type receptor. Thus, all mutations that
specifically disrupt the signaling activity of T
RII resulted in
the formation of a type II receptor that is dominant-negative for gene
induction.
To compare the
dominant-negative activities of kT
RII and
kT
RI in
more detail, we transfected each truncated receptor into Mv1Lu cells at
multiple concentrations from 1 to 16 µg/culture, using the
identical SV40-driven expression vector, and using p3TP-lux as the
reporter. Both kinase truncations resulted in inhibition of
TGF
-dependent transcription (Fig. 8); however,
kT
RI was significantly less effective than
kT
RII (p = 0.0001); a similar distinction was observed in
cardiac muscle cells (p = 0.0258). In Mv1Lu cells,
maximal inhibition at 16 µg of each construct was 88% for
kT
RII, versus 66% for
kT
RI; half-maximal
inhibition was achieved with 3 µg of
kT
RII, versus 8 µg of
kT
RI. We further tested whether
cotransfection of both kinase-truncated receptors might result in
synergistic or less-than-additive inhibition. At each plasmid
concentration examined, cotransfection of
kT
RI and
kT
RII plasmids together inhibited p3TP-lux as efficiently as
the same total plasmid concentration of
kT
RII, but was
significantly greater than inhibition produced by
kT
RI alone (p = 0.0001).
In summary, kinase-defective deletions
of both the type I and type II TGF receptors, as well as point
mutations in essential residues of the T
RII kinase domain, are
dominant-acting inhibitors of signal transduction by the TGF
receptor complex, for events that culminate in TGF
-dependent gene
expression. Taken together, the results reported here indicate that
loss of function for either type I or type II TGF
receptor is
sufficient for dominant interference with the cascade for
transcriptional activation (Fig. 9). Indeed, under the
conditions we have examined, all functionally inactive receptors serve
as dominant-negative inhibitors of this pathway.
Figure 9:
Three classes of inactive TGF
receptor are dominant inhibitors of TGF
-dependent transcription.
The prevailing genetic and biochemical evidence supports the model,
shown at the left, for TGF
signal transduction via a
heteromeric complex enabling directional phosphorylation of T
RI by
T
RII(31) . Dominant inhibitors for gene regulation by
TGF
include, from left to right, the
kinase-deficient truncation of T
RII, all inactivating point
mutations of T
RII analyzed thus far, and the kinase-deficient
truncation of T
RI.
Divergent
conclusions have been inferred, previously, using stably transfected
Mv1Lu mink lung epithelial cells. A kinase-defective truncation of the
human type II receptor was competent to disrupt growth inhibition by
TGF but failed to suppress three representative TGF
-inducible
genes including PAI-1(34) , suggesting that inactivation of
T
RII might impair only pathways important for growth control,
whereas gene regulation might be mediated exclusively by T
RI, a
model that is incompatible with our results and those of Wieser et
al.(42) , for a kinase-deficient truncation of T
RII.
As our equivalent results in Mv1Lu cells and cardiac myocytes make
clear, neither the use of different genes as end points nor differences
in the recipient cell background account for this disparity. Although
the potential for compensatory responses or fortuitous effects in
stably transfected cell lines cannot be excluded, a more plausible
explanation may be titratable differences in the levels of
dominant-negative receptor protein achieved in transiently versus stably transfected cells.
We have identified three classes of
inactive TGF receptor that block the signals for control of gene
expression by TGF
. In Mv1Lu cells, as in ventricular myocytes, a
truncation of the type II TGF
receptor interferes with
ligand-dependent activation of a TGF
-responsive gene. Beyond this
finding, seen recently by others(42) , we have engineered 10
point mutations of the T
RII kinase that are defective for
signaling activity in receptor-deficient cells; in primary cultures of
cardiac myocytes, as well as in mink lung epithelial cells, all 10
receptors are dominant inhibitors of TGF
-dependent transcription.
Finally, we have shown that a kinase-defective truncation of T
RI
likewise can impair TGF
control of gene expression in two
unrelated lineages, with no greater inhibition of these end points by
the mutation of type I receptor than by the homologous mutation of
T
RII. Thus, we find no evidence to support a
``two-pathway'' model. Although kinase-inactive variants of
T
RII are unable to rescue the mutant phenotype in
T
RII-deficient cells, the kinase activity of T
RII is not
required for ligand recognition by the type I receptor (this study and (18) ). Thus, neither a block to ligand-binding by T
RI nor
a block to appropriate expression of T
RI is tenable as a mechanism
to account for the dominant-negative phenotype produced by the
T
RII mutations.
The finding that a kinase-defective truncation
of TRII was at least as effective as truncated T
RI as a
dominant inhibitor of TGF
-dependent transcription is compatible
with the recent model for activation of the TGF
receptor complex
by directional phosphorylation of T
RI by T
RII(31) .
First, ligand binding by T
RI is contingent on presentation of
TGF
by T
RII; thus, the truncated type I receptor is expected
to function as a dominant-negative receptor only upon incorporation
into a heteromeric complex. By contrast, the truncated type II
receptor, and the inactive point mutations of T
RII, might act in
either of two ways: by sequestering T
RI, in the presence of
ligand, into a functionally inactive complex, or, alternatively, by
competing with wild-type receptor for TGF
itself. As the majority
of type II receptor may exist as homo-oligomers in both the presence or
absence of ligand(16) , a third possibility is sequestration of
wild-type T
RII. Finally, the relative activity of T
RI and
T
RII mutations is expected to depend, in part, on relative levels
of the respective endogenous receptors; the abundance of T
RI
inferred from affinity labeling data is an estimate that reflects only
the fraction of T
RI to which ligand has been presented by
T
RII. Many residues of the T
RII kinase domain that were
selected for mutagenesis (Lys-277, Arg-378/Asp-379, Lys-381, Asp-397,
Arg-528) are known to be essential for activity of cyclic AMP-dependent
protein kinase, the best characterized serine/threonine
kinase(38) ; one exception is Asp-446, which was reportedly
dispensable, notwithstanding its invariant presence throughout the
protein kinase superfamily.
Thus, all mutations which failed to
restore gene induction to receptor-deficient cells were inhibitors of
TGF-dependent transcription in wild-type mink lung epithelial
cells and in cardiac myocytes. Measurements of receptor protein kinase
activity will be needed to distinguish between inactivation of the
kinase itself and interference with a potential effector domain.
However, the meager efficiency for transfection into ventricular
myocytes (typically, 1-5%) precludes a direct comparison of
exogenous versus endogenous receptor in the two cell
backgrounds we have used, and prevents our determining the impact of
these receptor mutations on endogenous cardiac gene products or on
cardiac growth control. Providing a potential means to overcome this
impediment, we have demonstrated that virtually uniform gene transfer
to cardiac myocytes can be achieved with recombinant
adenoviruses(46) .
A more complex challenge to this
model of the heteromeric receptor complex as obligatory for all actions
of TGF is suggested by the several non-transformed cell types,
such as murine hematopoietic progenitor cells and human neutrophils,
which express negligible levels of type II receptors, yet display type
I receptor by cross-linking and respond to TGF
, measured by growth
arrest and chemotaxis,
respectively(51, 52, 53) . One potential
explanation for this paradox is the existence of low levels of type II
receptors, below the threshold for detection by conventional
means(49) . Alternatively, although isoforms of the type I and
type II receptor cDNAs for TGF
have not been isolated, comparable
to the diversity of activin receptors(28, 54) ,
immunoprecipitation of the TGF
receptor complexes in various cell
types, using antiserum against T
RI, indicates that multiple type I
receptors may exist, which interact differentially with the one known
form of T
RII(26) . Thus, it is conceivable that
alternative type I and type II receptors, which differ in their
biochemical characteristics and dimerization potential from those
presently identified, may account for some of the discrepancies in the
reported data and their interpretation. Despite the potential for
heterologous type I receptors to bind TGF
, at least under some
conditions of forced expression, no interference was produced by the
kinase-defective truncation of SKR1 (ALK-2).
As one strategy to gain insight into the biological
functions of endogenous growth factors, dominant-negative receptors for
activin have been introduced into Xenopus embryos and
explants, and substantiate a requirement for this growth factor in
establishing the body plan and inducing
mesoderm(58, 59) . Targeted expression of TGF in
its active form, in transgenic mice, supports the role of TGF
in
development of the skin (64) and mammary
gland(65, 66) , producing marked hypoplasia in both
cell backgrounds. Given the multiple functions for TGF
1 proposed
in mammalian development, including effects on proliferation, cell fate
and gene expression, adhesion, migration, extracellular matrix
production, and angiogenesis, it is particularly noteworthy that
animals homozygous for a null mutation of TGF
1 that survive until
birth possess no apparent morphological
defects(8, 9) . (The premature, but incomplete,
lethality of the mutation should not be overlooked.) Functional
redundancy (in this case, the capacity of TGF
2, TGF
3, or more
distant growth factors to substitute for TGF
1) is a potential and
inescapable impediment to ready interpretation of
``knock-out'' mutations, illustrated by the redundancy among
MyoD-like transcription factors(67, 68, 69) .
Alternatively, a more direct explanation for the lack of a more obvious
phenotype is offered by the fact that mice homozygous for the null
mutation have been found to receive significant amounts of maternal
TGF
1 across the placenta, which might rescue the developmental
consequences of the presumptive knockout(10) . Targeted
expression of dominant-negative TGF
receptors provides a potential
means to overcome the redundancy of TGF
isoforms, to obviate the
confounding effects of maternal protein, and to confine a
loss-of-function mutation to an individual organ or single cell
type-such as the cardiac myocyte. Dominant-negative receptors, in
vivo, could complement information to be gained from other genetic
models(70) .