(Received for publication, March 30, 1995; and in revised form, July 13, 1995)
From the
Transforming growth factor- (TGF-
) inhibits the
proliferation of epithelial cells by altering the expression or
function of various components of the cell cycle machinery. Expression
of one of these components, cyclin A, is inhibited by TGF-
treatment. We have identified a 760-base pair fragment of the human
cyclin A gene promoter that is sufficient to confer TGF-
responsiveness. Using this promoter fragment, we have developed a
cyclin A-based luciferase reporter assay that quantitates the growth
inhibitory effect of TGF-
in transient transfection assays. This
assay was used to determine which domains of the type I (RI) and type
II (RII) receptors were required for the antiproliferative effect of
TGF-
. In parallel, the functionality of chimeric receptors,
between RI and RII (RI-RII or RII-RI), was tested for TGF-
effect
on gene expression using a reporter assay based on the plasminogen
activator inhibitor type 1 (PAI-1) promoter. We found that
TGF-
-induced inhibition of cyclin A expression was absent in RI or
RII-deficient Mv1Lu cells and that this response was restored by
expression of wild-type type I or type II receptors in these cells.
Furthermore, expression of a single chimeric receptor, either RI-RII or
RII-RI, did not confer cyclin A regulation by TGF-
. However,
expression of two reciprocal chimeras (RI-RII and RII-RI) resulted in
growth inhibition, similarly to wild-type receptors. In addition,
chimeric receptors as well as mutant receptors with a deleted
cytoplasmic domain and kinase-negative receptors inhibited TGF-
responsiveness in the cyclin A reporter assay in a dominant negative
fashion. Finally, in both receptor types, the juxtamembrane domain
preceding the kinase domain was essential for receptor function but the
cytoplasmic tail was dispensable. Our results suggest that a functional
TGF-
receptor complex is required for TGF-
-dependent
down-regulation of cyclin A gene expression and illustrate the
identical receptor requirements for TGF-
-induced growth inhibition
and gene expression.
Transforming growth factor- (TGF-
) (
)is a
secreted protein that induces a range of cell responses which affect
growth and differentiation (for recent reviews see (1) and (2) ). TGF-
treatment of epithelial cells results in cell
cycle arrest in late G
coincident with an inhibition of
synthesis and/or activity of some cyclins and cyclin-dependent kinases (3, 4, 5, 6) . In mink lung
epithelial cells, TGF-
decreases the activities of two G
cyclin-dependent kinases, cdk2 and
cdk4(4, 6, 7) . TGF-
down-regulates the
synthesis of cdk4 and constitutive expression of cdk4 results in
resistance to TGF-
-induced growth arrest, suggesting that cdk4 is
a major downstream target in TGF-
-induced growth
inhibition(4) . The inhibition of cdk2 activity by TGF-
may involve the cdk inhibitor p27, which prevents complex formation of
cyclin E/cdk2 as well as cyclin D/cdk
4(8, 9, 10) . Another cdk inhibitor p15, that
is TGF-
-inducible, interferes with the cyclin D/cdk 4 complex in
keratinocytes(11) .
While down-regulation of cdk4 and
activation of the cdk inhibitors correlates with TGF--induced cell
cycle arrest, other components of the cell cycle machinery are also
inhibited by TGF-
. Indeed, late G
arrest by TGF-
is associated with decreased synthesis of cdc2 (12) and cyclin
A(13, 14, 15, 16, 17) .
Cyclin A plays a critical role in cell cycle progression by complexing
with and regulating the activities of cdc2 and cdk2. Cyclin A/cdc2 and
cyclin A/cdk2 complexes also interact with other proteins involved in
the regulation of the G
to S transition, including pRB, the
pRB-like proteins p107 and p130, and the transcription factor
E2F(18, 19, 20, 21, 22) .
Inhibition of pRB hyperphosphorylation by cyclin A/cdk2 may be involved
in control of G
progression by TGF-
(23) .
In addition to its antiproliferative effect, TGF- induces the
expression of many genes, including those for extracellular matrix
proteins. In fact, the induction of plasminogen activator inhibitor
type 1 (PAI-1) expression is often used as a biochemical marker for
TGF-
responsiveness. Induction of PAI-1 expression can be easily
measured using a reporter plasmid in which luciferase expression is
driven from the PAI-1 promoter in a TGF-
-dependent
manner(24, 25) . This assay is often used in
combination with transient transfection experiments to measure cellular
responsiveness to TGF-
and the corresponding receptor
requirements. In contrast, no similarly convenient reporter assay has
been developed to measure the growth inhibitory response of TGF-
,
even though considerable evidence suggests that TGF-
's
effects on growth inhibition and gene expression result from divergent
signaling cascades (for review, see (26) ).
The early
signaling events that lead to growth inhibition or extracellular matrix
production by TGF- are largely unknown. TGF-
interacts with a
set of cell surface receptors (for review, see Refs. 26, 27), including
the type I and type II receptors which are essential for signal
transduction. Both receptor types belong to an expanding family of
transmembrane serine/threonine
kinases(26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) .
Several structural features distinguish type I from type II receptors.
The extracellular domain of the type I receptor is shorter than that of
the type II receptor. The type I receptor also has a highly conserved
GS motif (SGSGSGLP) in the juxtamembrane region preceding the kinase
domain, which is absent in the type II receptor (for review, see (26) ). The type II receptor can bind TGF-
by itself, but
the type I receptors bind TGF-
only when coexpressed with the type
II receptor, presumably as a result of a physical interaction of these
receptors. To date, only one type II receptor for TGF-
has been
identified(32) , while several type I receptors, i.e. R4/ALK5(34) , Tsk7L (33) , TSR1(31) , have
been shown to bind TGF-
when coexpressed with the type II
TGF-
receptor. Despite the structural similarity, certain
functional differences among the different type I receptors have been
found. For instance, only R4/ALK-5 transduces TGF-
signals in
Mv1Lu epithelial cells(34, 39) , while Tsk7L mediates
TGF-
signaling in another epithelial cell line(40) .
The type II receptors(41, 42) , and presumably also
the type I receptors, constitutively homodimerize independent of ligand
binding. In addition, these two receptor types interact with each other (25, 43, 44) and form a heteromeric (most
likely tetrameric) complex which is stabilized by ligand
binding(45) . The cytoplasmic domain of the type II receptor is
constitutively phosphorylated, independent of ligand binding, due to
both autophosphorylation and the activity of cytoplasmic
kinases(46, 47) . In the heteromeric complex, the type
II receptor kinase phosphorylates the type I receptor (46, 47) and the two types of cytoplasmic domains
associate with each other(48) , further stabilizing the
heteromeric interaction. This heteromeric complex is likely to be the
signaling receptor unit which mediates the biological responses of
TGF-.
Epithelial Mv1Lu cells lacking either type I or type II
receptor are completely unresponsive to TGF- to induce growth
inhibition and gene expression(49, 50) . But the
TGF-
responsiveness can be restored to these mutant cell lines by
transfection with the missing receptor type (25, 39) .
These findings support the notion that the two receptors are needed for
both types of responses to TGF-
. However, separate signaling
pathways for the two types of responses have been observed in several
studies(51, 52, 53) . For example, cells in
which the type II receptor has been functionally inhibited by a
truncated type II receptor are resistant to the antiproliferative
effect of TGF-
, yet still induce the expression of PAI-1 and other
gene products(51) . Furthermore, a variety of cell lines, some
of which contain defective type II receptors (e.g.(53) ), are resistant to TGF-
-induced
antiproliferation, but remain able to induce various genes including
PAI-1 in response to TGF-
(see ``Discussion'').
To
gain insight into the role of the type I and II receptors in
TGF--mediated growth inhibition, we have developed a convenient
assay to score the antiproliferative effect of TGF-
using an
expression plasmid in which a TGF-
-responsive cyclin A promoter
segment drives a luciferase reporter gene. We used this reporter system
in transient transfection experiments to examine the function of the
TGF-
receptors in the growth inhibitory response of Mv1Lu cells.
Using chimeric and mutant receptors, we have dissected the role of the
two receptors in the growth inhibitory response of TGF-
and
compared the cyclin A reporter assay with the PAI-1 reporter assay with
respect to TGF-
responsiveness.
To amplify the RII extracellular/transmembrane (ET) domain: R2E5`, GATAGAATTCACC ATG GGT CGA GGA CTG CT; R2E3`, TCATGGATCC GAC ACG GTA GCA GTA GAA.
To amplify the RII cytoplasmic (C) domain: R2C5`, CTTAGGATCCATG CAC CGA CAG CAG AAG CTG AG; R2C3`, GATGAGGTCGACCTT GGT AGT GTT CAG AGA GC; R2K5`, TATTGGATCCATG CCC ATC GAG CTG GAC AC (for juxtamembrane deletion); R2K3`, ACTTGAGTCGAC GTC CAT ATG CTC CAG CTC AC (for tail deletion).
To amplify the RI ET domain:
R4E5`, TAATCGATGAATTC ATG GAG GCA GCA TCG GC; R4E3`,
AAAGATCTGGATCC GTT GTG GCA GAT ATA GAC.
To amplify the RI C domain: R4C5`, GATGAATTCATG CGC ACT GTC ATT CAC CAC CG;
R4C3`, CAAACTCGAGGTCGAC CAT TTT GAT GCC TTC CTG; R4K5`, GCA GGATCC ATG GTG CTA CAA GAA AGC ATC (for juxtamembrane deletion); R4K3`,
GAG GTCGAC CAA TGT CTT CTT AAT TCG CAA (for tail deletion).
To
amplify the RI ET domain: R7E5`, AGCC
GAATTCACATG GTC GAT GGA GTA AT; R7E3`, GTCTGGATCC CTT CCT GAG
AGC AAC TCC.
To amplify the RI C
domain: R7C5`, CCACGGATCCATG TTT AAG AGA CGC AAT CAA GA; R7C3`,
GGCAGTCGAC ACA GTC AGT CTT CAA TTT GTC.
Amplified receptor cDNA
sequences were cloned into the cytomegalovirus promoter-driven
mammalian expression vectors pRK5F or pRK5M. These expression vectors
are derived from pRK5 (56) but contain the sequences for a myc (57) or FLAG epitope tag (58) between the SalI
and HindIII sites, respectively (data not shown). Three
truncated receptors lacking the entire extracellular/transmembrane
domain (ET) were generated. For example, RII(ET) contains residues
1-191 corresponding to the ET domain of RII, while
RI(ET) and RI
(ET) include the first
148 residues of RI
or RI
. Sequences
containing ET domains were cloned between the EcoRI and BamHI sites, in frame with the downstream epitope tag, either
myc for RI
(ET) and RI
(ET) or FLAG
tag for RII(ET).
In the hybrid receptors, the cytoplasmic (C) domain
of one receptor, as a BamHI-SalI fragment (in case of
RI, the BglII site substituted for a BamHI site), was placed between the ET domain of a different
receptor and the epitope tag. Considering the large number of plasmids,
a detailed description of how they were made is not provided in this
report; however, further details of the cloning strategies can be
obtained upon request. A total of four chimeric receptors between RI
and RII were created from the fusion of ET domain of one type to C
domain of another. At the junction of these chimeric receptors, three
extra amino acid residues (Gly Ser Met) were created corresponding to
the restriction sites (BamHI/BglII and the introduced
start codon ATG preceding cytoplasmic domain). The nomenclature of the
chimeric receptors reflects the origin of the two fused domains. For
example, RI
(ET)-RII(C) contains ET domain of RI
and C domain of RII receptor. All receptor clones were sequenced
to ensure that there were no PCR-induced mutations.
For in vitro kinase assays,
immunoprecipitated receptors with specific deletions in the cytoplasmic
domains of RI and RII were split into two halves. While
half was directly subjected to SDS-PAGE, the other half was used in a
kinase autophosphorylation assay. The kinase assay was carried out at
room temperature for 30 min in 1
kinase buffer (10 mM HEPES-KOH, pH 7.5, 5 mM MgCl
, and 5 mM CaCl
) containing 5 µCi of
[
-
P]ATP (5000 µCi/mmol, Amersham) and
then stopped by adding equal volume of 2
SDS sample buffer (80
mM Tris, pH 6.8, 3.2% SDS, 16% glycerol, 200 mM dithiothreitol, 0.02% bromphenol blue).
R1B-L17 cells, a highly
transfectable line of R1B cells were provided by Dr. J.
Massagué. Transfection of L17 cells was carried
out using the DEAE-dextran method as described (46) at 40%
confluence in 6-well plates. For each transfection, 1 µg of
receptor expression plasmid or vector control and 3 µg of reporter
constructs (1.5 µg of pRKGAL + 1.5 µg of luciferase
expression plasmid) were used.
As a first step in developing this
assay, we identified the 5` promoter fragment of the cyclin A gene that
mediates down-regulation of cyclin A gene expression in response to
TGF-. The upstream region of the human cyclin A gene corresponding
to nucleotides -516 to +245 contains many potential
regulatory elements (54) and was isolated by PCR-based
amplification from human genomic DNA. We linked this promoter fragment
to the luciferase coding sequence to generate plasmid pCAL2. The
luciferase expression driven by this 760-bp fragment of the cyclin A
promoter was compared with the luciferase activity from the
7.5-kilobase pair promoter region (nucleotides -7300 to
+245) in plasmid CD1(54) . The cyclin A reporter plasmid
and a
-galactosidase expression plasmid pRK
Gal were
transiently transfected into TGF-
-responsive Mv1Lu cells, and the
luciferase activity was measured 48 h after transfection. Inclusion of
pRK
Gal allowed calibration of the luciferase activity against the
-galactosidase activity, providing normalization of transfection
efficiency. Both cyclin A plasmids pCAL2 and pCD1 were found to express
luciferase, and the luciferase activity was even somewhat higher when
using plasmid pCAL2 (see 0 h point in Fig. 1A), indicating that this 760-bp cyclin A promoter
fragment is functional in Mv1Lu cells.
Figure 1:
A, cyclin A-luciferase
reporter assay. Cyclin A-luciferase plasmid pCAL2 or pCD1, together
with a -galactosidase expression plasmid pRK
Gal, were
transfected into Mv1Lu cells. Four h after transfection, TGF-
was
added at 400 pM to the media for the defined times. Luciferase
and
-galactosidase activity were determined as described under
``Materials and Methods.'' Data are from two experiments with
each point determined in triplicates. The vertical bars show
the standard deviations. pCAL2 and pCD1 contain the human cyclin A
promoter sequence from nucleotide -516 to +245 and from
-7300 to +245, respectively. B, regulation of
cyclin A-luciferase activity by TGF-
in wild-type and mutant Mv1Lu
cells. Untransfected cells or DR26 cells transfected with RII or R1B
cells transfected with RI
were treated (+)
or untreated(-) with TGF-
. Transfections, TGF-
treatment (48 h), and reporter assays were carried out as in panel
A. RII, type II TGF-
receptor; RI
, type I TGF-
receptor; Mv1Lu, wild-type Mv1Lu cells; DR26, RII-deficient
cells; R1B, RI
-deficient
cells.
To evaluate the TGF-
responsiveness of the cyclin A promoter, both luciferase plasmids were
transfected into Mv1Lu cells, and the normalized luciferase activity
was calculated at defined time points after initiation of TGF-
treatment. In Mv1Lu cells transfected with pCAL2, the luciferase
activity was down-regulated by TGF-
(Fig. 1A) in a
manner similar to that in pCD1-transfected cells. This decreased
expression from the cyclin A promoter parallels the inhibition of
cyclin A expression by TGF-
in Mv1Lu (15, 16, 17) and other epithelial cell
lines(13) . The TGF-
-induced inhibition of luciferase
expression was most evident when TGF-
treatment was initiated
within 6 h after release from contact inhibition (data not shown), and
the cyclin A promoter response was maximal when the cells were exposed
to TGF-
for 48 h (Fig. 1A). These results indicate
that the 760-bp cyclin A promoter fragment in pCAL2 contains the
TGF-
-responsive element(s) and that this cyclin A-luciferase assay
can be used as transcriptional reporter system to measure the
antiproliferative effect of TGF-
in transient transfection assays.
Figure 2:
Schematic presentation of mutant TGF-
receptors. The construction and nomenclature of the receptor mutants is
described under ``Materials and Methods'' and
``Results.'' RI
, type I
TGF-
receptor R4; RI
, type I
receptor Tsk 7L; RII, type II TGF-
receptor. ET,
extracellular/transmembrane domain; C, cytoplasmic domain;
JM, juxtamembrane domain deletion;
T,
cytoplasmic tail deletion;
JMT, deletion of juxtamembrane
domain and cytoplasmic tail.
Figure 3:
A, expression of mutant receptors in COS-1
cells. COS-1 cells were transfected with pRK5 (lane 1) or
receptor expression plasmids (lanes 2-11). The receptors
were immunoprecipitated from S-labeled cell lysates using
antibodies specific for the FLAG (F) or myc (M)
epitope as indicated. The immunoprecipitated proteins were analyzed by
SDS-PAGE and autoradiography. The positions of the receptors and
molecular weight markers are shown on the side. Lane 2, RII; lane 3, RI
; lane 4,
RI
; lane 5,
RII(ET)-RI
(C); lane 6,
RII(ET)-RI
(C); lane 7,
RI
(ET)-RII(C); lane 8,
RI
(ET)-RII(C); lane 9, RII(ET); lane
10, RI
(ET); lane 11,
RI
(ET). B, ligand binding of mutant
receptors expressed in COS-1 cells. Binding of
I-TGF-
to COS-1 cells transfected with plasmids
expressing wild-type or mutant receptors was examined. Cross-linked
complexes were subjected to SDS-PAGE and autoradiography. Each lane
represents an equal amount of protein lysate as determined by BCA assay
(Bio-Rad). Migration of the receptors are indicated using the same
abreviations for receptors as in Fig. 3A. Lane
1, pRK5; lane 2, RII; lane 3,
RI
; lane 4, RI
and
RII; lane 5, RI
; lane 6,
RI
and RII; lane 7,
RII(ET)-RI
(C); lane 8,
RII(ET)-RI
(C); lane 9,
RI
(ET)-RII(C); lane 10,
RI
(ET)-RII(C) and RII; lane 11,
RI
(ET)-RII(C); lane 12,
RI
(ET)-RII(C) and RII; lane 13, RII(ET); lane 14, RI
(ET); lane 15,
RI
(ET) and RII; lane 16,
RI
(ET); lane 17,
RI
(ET) and RII.
The ability of the mutant receptors to bind
TGF- at the cell surface was determined by binding and
cross-linking of
I-TGF-
. As shown in Fig. 3B, both RII and RII(ET)-RI(C) chimeras with their
type II extracellular domain bound
I-TGF-
(lanes
2, 7, and 8). In contrast, RI and RI(ET)-RII(C)
chimeras which have RI extracellular domains did not bind TGF-
when expressed alone (lanes 3, 5, 9, and 11). However, these receptors bound ligand when cotransfected
with RII (lanes 4, 6, 10, and 12).
Similarly, RI
(ET) and RI
(ET) bound ligand
only in the presence of RII (compare lanes 14 and 16 to 15 and 17), whereas RII(ET) bound TGF-
by itself (lane 13). Taken together, these results indicate
that the mutant receptors were readily expressed at the cell surface
and displayed normal ligand binding.
The ability of the mutant
TGF- receptor to signal was then examined in the cyclin
A-luciferase assay using receptor-deficient Mv1Lu mutant cells.
Although wild-type RII restored TGF-
-dependent down-regulation of
cyclin A transcription in RII-deficient DR26 cells, none of the
chimeric receptors (RI
-RII, RI
-RII,
RII-RI
or RII-RI
), when expressed alone,
induced transcriptional regulation of cyclin A-luciferase in response
to TGF-
(Fig. 4, left panel). Similarly, the
chimeric receptors did not restore TGF-
responsiveness in the
RI-deficient R1B cells which contain endogenous RII, even though
RI
was able to restore the TGF-
-induced inhibition of
cyclin A-luciferase activity to the cells (Fig. 4, right
panel). While a single chimeric receptor was unable to confer
TGF-
responsiveness in the mutant cell lines, coexpression of the
two chimeric receptors RII(ET)-RI
(C) and
RI
(ET)-RII(C) resulted in TGF-
-dependent inhibition
of luciferase expression in these cells (Fig. 4). Similarly,
coexpression of both chimeric receptors led to TGF-
-dependent
increases of transcription from the PAI-1 promoter in a
PAI-1-luciferase assay (data not shown; Table 1). While the
coexpression of both chimeras in mutant cells restored the
TGF-
-dependent responses, significant levels of cyclin A
inhibition and PAI-1 up-regulation were already observed in the absence
of TGF-
in these cells. This constitutive signaling in the absence
of TGF-
resulted in the lower relative level of inhibition of
cyclin A expression in the presence of TGF-
, when compared with
that in the absence of TGF-
(Fig. 4). In fact,
TGF-
-independent signaling was also apparent when the two types of
cytoplasmic domains (RII and RI
) were expressed regardless
of what extracellular domains they were fused to (data not shown).
These results suggest that the high level expression of both
cytoplasmic domains is responsible for ligand-independent signaling and
that the two receptor types have an intrinsic heteromeric affinity for
each other which is consistent with previous findings(48) . In
contrast to the chimeric receptors derived from RI
,
cotransfection of RII(ET)-RI
(C) with
RI
(ET)-RII(C) did not lead to TGF-
responsiveness in
the cyclin A-luciferase assay, consistent with the inactivity of the
Tsk7L type I receptor in Mv1Lu cells(31, 39) .
Figure 4:
Signaling activity of chimeric receptors
in mutant Mv1Lu cells using TGF--dependent cyclin A-luciferase
reporter assay. Receptor expression plasmids were transfected in DR26
or R1B cells together with the cyclin A-luciferase plasmid pCAL2.
Cyclin A-luciferase data are shown as the percentage decrease in
luciferase activity relative to cells that did not receive TGF-
.
In the cases when the cyclin A activity was higher after TGF-
treatment, a negative value for the percentage inhibition was observed.
The vertical bars show the standard deviations. Receptors are
abbreviated as in Fig. 2except that hRII represents wild-type
human RII.
Figure 5:
Dominant negative inhibition of
TGF--dependent cyclin A down-regulation by mutant receptors in
wild-type Mv1Lu cells. Plasmids used for transfection of Mv1Lu cells
are as in Fig. 2except that plasmids RI
KR
and RIIKR represent the kinase-negative mutants of RI
and RII, respectively. Data are presented as in Fig. 4.
Using the TGF--responsive
wild-type Mv1Lu cells and the cyclin A-luciferase assay, we also
evaluated the effect of expression of truncated receptors lacking the
cytoplasmic domain (Fig. 5, right panel). Consistent
with the results using a truncated human type II
receptor(51, 60) , expression of the mouse RII lacking
its cytoplasmic domain, RII(ET), also blocked TGF-
-dependent
inhibition of cyclin A transcription in a dominant negative manner.
Overexpression of the truncated RI
(ET) type I receptor
lacking its cytoplasmic domain, but not RI
(ET), in Mv1Lu
cells also abolished TGF-
responsiveness (Fig. 5, right
panel). This effect was also seen in the PAI-1 assay (Table 1). However, the inhibitory effect of RI
(ET)
was less effective than RII(ET) in the PAI-1 assay (Table 1, data
not shown).
Figure 6:
Biological effects of deletion of the
cytoplasmic segments of TGF- receptors on TGF-
-dependent
down-regulation of cyclin A-luciferase expression. Cytoplasmic deletion
mutants of both RI
and RII were as shown in Fig. 2and in legend to Fig. 5. Data are presented as in Fig. 4.
When expression plasmids for these
mutant receptors were transfected into COS-1 cells and the kinase
activity of the immunoprecipitated receptors was tested in
vitro, we observed a correlation between the receptor kinase
activity and their biological functions (Fig. 7). While
RIT and RII
T maintained their kinase and
signaling activities similarly to wild-type receptors,
RI
JM and RII
JM, like the kinase-negative mutants
RI
KR and RIIKR, were also kinase inactive and unable to
autophosphorylate. Thus, mutant receptors which were inactive in the
biological assays were also inactive in the kinase assay.
Figure 7:
Expression and in vitro kinase
activity of mutant receptors with cytoplasmic deletions. COS-1 cells
were transfected with the plasmids indicated and the receptors were
immunoprecipitated from S-lysates as described in Fig. 3A. Half of the immunoprecipitated proteins were
analyzed by SDS-PAGE (left panels), whereas the other half was
subjected to in vitro kinase assay using
[gamma]
P]ATP prior to gel analysis (right panels). The positions of the receptors and molecular
weight markers are shown.
Based on the TGF- responsiveness of the 760-bp
cyclin A promoter element, we have developed a functional assay to
monitor cyclin A regulation in response to TGF-
. This assay can be
used in transient transfection assays to allow a functional evaluation
of TGF-
receptors and other signaling proteins involved in the
antiproliferative effect of TGF-
. In spite of the many
TGF-
-induced responses, transcriptional activation of the PAI-1
gene is frequently used as the only indicator for TGF-
signaling
and receptor function, primarily because of the availability of a
highly sensitive and convenient reporter assay that measures luciferase
expression from a TGF-
-responsive promoter element(25) .
Before this study, no facile reporter assay has been available to
measure the antiproliferative effect of TGF-
, even though
considerable evidence suggests that the growth inhibition and the
induction of gene expression by TGF-
result from divergent
signaling cascades. The availability of a cyclin A luciferase reporter
system allowed us to characterize the role of specific domains of
TGF-
receptors during TGF-
-mediated growth inhibition using
transient transfection assays.
Our results demonstrate that
inhibition of cyclin A transcription by TGF- requires both RII and
RI
receptors. The cyclin A transcription is inhibited by
TGF-
in Mv1Lu cells which contain endogenous RI
and
RII receptors but not in the two mutant cell lines lacking either
RI
or RII. Introduction of wild-type RI
into
RI-deficient cells or RII into RII-deficient cells restores
TGF-
-dependent cyclin A transcription. In contrast, various mutant
receptors including the chimeric receptors RII(ET)-RI
(C),
RI
(ET)-RII(C), RII(ET)-RI
(C), and
RI
(ET)-RII(C) as well as the kinase-negative point mutants
RI
KR and RIIKR, and receptors lacking the juxtamembrane
domain RI
JM and RII
JM, are unable to rescue the
ability of mutant cells to respond to TGF-
. However, coexpression
of RII(ET)-RI
(C) and RI
(ET)-RII(C), which
allows heteromerization of both extracellular and intracellular
domains(48) , restores TGF-
-dependent down-regulation of
cyclin A expression in either mutant cell line. In wild-type Mv1Lu
cells, individual expression of these mutant receptors inhibits
TGF-
-induced cyclin A down-regulation in a dominant negative
fashion. These results suggest that cyclin A expression and, thus, the
growth inhibitory effect of TGF-
need the cooperative interaction
between type I and type II TGF-
receptors.
In our study, we tested in
parallel the TGF--dependent cyclin A response and PAI-1
transcriptional response and did not observe an uncoupling of these
responses (see Table 1), which is consistent with the proposed
requirement of both cytoplasmic domains for full TGF-
responsiveness. More specifically, homomerization of cytoplasmic
domains, resulting from expression of the RII(ET)-RI
(C)
chimera in DR26 cells or the RI
(ET)-RII(C) chimera in R1B
cells, is not sufficient for TGF-
-induced regulation of cyclin A
and PAI-1 expression, suggesting that a functional interaction of the
two types of cytoplasmic domains is a prerequisite for TGF-
responsiveness. In addition, despite the heteromeric combination of
both cytoplasmic domains, homomeric combination of the
extracellular/transmembrane domains of type I or type II receptors also
fails to restore TGF-
responsiveness. In the case of the
RI
(ET)-RII(C) chimera in DR26 cells which express
endogenous RI
, this lack of signaling might be due to the
inability of the RI
extracellular domains to bind ligand
in the absence of RII. However, expression of the
RII(ET)-RI
(C) chimera in R1B cells which have endogenous
RII yet lack functional RI
also does not activate
TGF-
responsiveness even though these receptors bind TGF-
efficiently. This suggests an active function of the RI
extracellular domain in TGF-
signaling.
Coexpression of
the RII(ET)-RI(C) and RI
(ET)-RII(C) chimeras
in DR26 or R1B cells results in TGF-
responsiveness in both
reporter assays, suggesting that the coexistence of both extracellular
and cytoplasmic domains in trans provides a functional TGF-
receptor unit. TGF-
is likely to induce a conformational change in
the heteromeric complex of these chimeras in a manner similar to the
complex of wild-type RI
and RII receptors. The resulting
activation promotes the interaction of the receptors with downstream
effectors. Our data are in agreement with the recent findings of
Okadame et al.(66) , who showed the requirement of
both cytoplasmic domains for TGF-
-induced PAI-1 transcription. We
extended their findings by using the cyclin A reporter assay and by
examining the effects of chimeric receptors in wild-type Mv1Lu cells.
In wild-type Mv1Lu cells, expression of the
RI(ET)-RII(C) or RII(ET)-RI
(C) chimera
inhibits TGF-
-induced regulation of cyclin A and PAI-1 expression
in a dominant negative manner. This inhibition is likely due to the
fact that overexpression of the RI
(ET)-RII(C) or
RII(ET)-RI
(C) chimera sequesters endogenous RII and/or
RI
away from functional RI
-RII receptor
complexes. The resulting homomeric complexes of chimeric receptors and
the heteromeric complexes between endogenous receptors and the chimeras
are then unable to transduce the TGF-
signals. This dominant
negative interference resembles the inhibitory effect of truncated
receptors lacking their cytoplasmic domain and the kinase-negative
mutant receptors. The inhibition of TGF-
responsiveness by RII(ET)
is in accordance with previous
results(51, 60, 67) . A dominant negative
inhibition, albeit to a lesser extent, was also apparent with
overexpression of RI
(ET) in Mv1Lu cells, but not with
RI
(ET) even though the truncated form of Tsk7L inhibited
TGF-
-induced mesenchymal transdifferentiation in NMuMG
cells(40) . Thus, overexpression of chimeric, truncated, or
kinase-defective receptors derived from RII or RI
results
in dominant negative interference with TGF-
receptor signaling,
most likely by interfering with the assembly of functional homo- and
heteromeric complexes. After the submission of this manuscript, Vivien et al.(68) and Brand and Schneider (69) reported dominant negative inhibition of TGF-
signaling by chimeric and kinase-defective receptors, using assays for
TGF-
-induced gene expression. Their conclusions and our findings
based on cyclin A and PAI-1 assays are in agreement; moreover, our
cyclin A assay results further extend their observations to
TGF-
-induced growth inhibition.
Finally, we characterized the
function of the RI and RII receptor cytoplasmic domains
containing defined deletions. Deletion of the C-terminal tail of either
receptor did not affect the kinase and signaling activity of RI
and RII in both cyclin A and PAI-1 reporter assays. A similar
result has been observed in the case of RII using the PAI-1 reporter
assay(60) . We also demonstrated that deletion of the
juxtamembrane domain inactivates the signaling function of both
receptor types. This correlates with a loss of kinase activity as
measured by autophosphorylation. In the case of the type I receptor,
the juxtamembrane domain contains a conserved SGSGSGLP sequence. This
sequence is a major site of phosphorylation by the type II receptor,
and its deletion has been shown to inactivate TGF-
signaling(46) . Thus, our results indicate the requirement of
the juxtamembrane domains for the function and kinase activity of both
receptors. These domains may also represent binding sites for
cytoplasmic effector proteins.