(Received for publication, June 1, 1995)
From the
Transforming growth factor-1 (TGF-
1) induces
angiogenesis in vivo and capillary morphogenesis in
vitro. Two receptor serine/threonine kinases (types I and II) have
been identified as signal transducing TGF-
receptors. We explored
the possibility of inhibiting TGF-
- mediated events in glomerular
capillary endothelial cells using a TGF-
type II receptor
(T
R-II) transdominant negative mutant.
A mutant TGF- type
II receptor (T
R-II
), lacking the cytoplasmic
serine/threonine kinase domain, was produced by polymerase chain
reaction using rat T
R-II cDNA as template. Since T
R-II and
TGF-
type I receptor (T
R-I) heterodimerize for signal
transduction, the mutant receptor competes for binding to wild-type
T
R-I, hence acting in a dominant negative fashion. Glomerular
capillary endothelial cells were stably transfected with
T
R-II
, and four independent clones were expanded. That
the T
R-II
mRNA was expressed was shown by reverse
transcriptase-polymerase chain reaction, RNase protection assay, and
Northern analysis. Presence of cell surface T
R-II
protein was shown by affinity cross-linking with
I-TGF-
1. In wild-type endothelial cells, TGF-
1
(2 ng/ml) significantly inhibited [
H]thymidine
incorporation to 63 ± 10% of control (n = 4). In
transfected endothelial cells carrying T
R-II
,
TGF-
1 stimulated [
H]thymidine incorporation
to 131 ± 9% of control (n = 4, p <
0.005). Also, in wild-type endothelial cells, endogenous and exogenous
TGF-
1 induced apoptosis and associated capillary formation. Both
apoptosis and capillary formation were uniformly and entirely absent in
transfected endothelial cells carrying T
R-II
.
This
represents the first demonstration that capillary morphogenesis in
vitro is associated with apoptosis, and that interference with
TR-II signaling inhibits this process in glomerular capillary
endothelial cells.
Angiogenesis, the process of new blood vessel formation, is an integral part of development, wound repair, and tumor growth. The formation of capillary networks requires a complex series of cellular events, in which endothelial cells locally degrade their basement membrane, migrate into the connective tissue stroma, proliferate at the migrating tip, elongate and organize into capillary loops(1) . In response to angiogenic stimuli, endothelial cells in culture develop networks of capillary-like tubes.
Transforming growth factor-1
(TGF-
1) (
)is a 25-kDa homodimeric polypeptide that
belongs to a family of homologous multifunctional cytokines. TGF-
1
regulates diverse cellular functions including proliferation and
differentiation. TGF-
1 is strongly expressed during embryogenesis (2, 3) and in sites undergoing intense development and
morphogenesis(4, 5) . Moreover, TGF-
1 induces
angiogenesis in vivo(6, 7) and capillary
morphogenesis in vitro(8) . The mechanism by which
TGF-
1 induces angiogenesis is not yet well defined.
In the
early stages of angiogenesis, proteases are required for extracellular
matrix (ECM) proteolysis to facilitate endothelial cell
migration(9) . TGF-1 induces endothelial cell secretion of
plasminogen activator (PA) which activates plasmin, a protease that
degrades ECM proteins(10, 11) . Increased production
of PA has been associated with the invasive properties of cultured
endothelial cells in response to angiogenic
stimuli(10, 11) . In addition, plasmin activates
latent TGF-
1(12) , in an autocrine fashion. Furthermore,
TGF-
1 is a potent chemoattractant for macrophages and fibroblasts (13, 14) , which are postulated to release angiogenic
peptides in vivo, such as basic fibroblast growth factor
(bFGF), platelet-derived growth factor, or tumor necrosis
factor-
(15) .
Two transmembrane serine/threonine
kinases, types I and II, have been identified as signal transducing
TGF- receptors. TGF-
type II receptor (T
R-II), a
constitutively active kinase, directly binds TGF-
1, and this
ligand binding results in the recruitment and phosphorylation of
TGF-
type I receptor (T
R-I) to produce a heteromeric
signaling complex(16) . T
R-I alone is unable to bind
TGF-
1, and T
R-II is unable to signal without
T
R-I(17) .
We explored the possibility of inhibiting
TGF-1-mediated events in renal glomerular capillary endothelial
cells using a T
R-II transdominant negative mutant. A mutant
T
R-II construct (T
R-II
), lacking the cytoplasmic
serine/threonine kinase domain, but with full transmembrane spanning
and extracellular domains, was produced by polymerase chain reaction
(PCR) using rat T
R-II cDNA (18) as template. Since
T
R-II and T
R-I heterodimerize for signal transduction, the
mutant receptor competes for binding to wild-type T
R-I, hence
acting in a dominant negative
fashion(19, 20, 21) . When the transdominant
negative mutant construct was stably expressed in glomerular capillary
endothelial cells, capillary morphogenesis and associated apoptosis
were entirely blocked in these cells.
To induce
capillary tube formation, cells grown to confluence were placed in RPMI
medium containing 0.5% FBS, in the presence or absence of exogenous 2
ng/ml TGF-1 (Collaborative Biomedical Products). For transmission
electron microscopy, the cells were fixed in 3% glutaraldehyde in
phosphate-buffered saline, postfixed in 1% osmium tetroxide, dehydrated
in a graded series of ethanol, and embedded in epon directly on the
culture dish, in order to preserve morphology of the capillary tubes.
Thin sections were cut, stained with uranyl acetate and lead citrate,
and subjected to electron microscopy (Paragon Biotech Inc., Baltimore,
MD). In experiments with neutralizing antibody to TGF-
1, cells
grown to confluence were placed in RPMI medium containing 0.5% FBS in
the presence or absence of 10 ng/ml turkey anti-human TGF-
1 IgG
(Collaborative Biomedical Products).
Figure 1:
Mutant
TR-II construct. Schematic diagram of wild-type T
R-II and
mutant construct (T
R-II
), lacking the cytoplasmic
serine/threonine kinase domain, but with full transmembrane spanning
and extracellular domains, as indicated. The rat wild-type T
R-II
cDNA contains an open reading frame of 1701 nt long, corresponding to a
protein of 567 amino acid residues. T
R-II
cDNA is 573
nt long, corresponding to 191 amino acids. The horizontal arrows indicate the positions of the PCR
primers.
Figure 2:
Expression of TR-II mRNA in wild-type
and transfected glomerular endothelial cells. A, RNase
protection assay. Total RNA from wild-type and transfected glomerular
endothelial cells, treated with 1 µM dexamethasone, was
hybridized in solution with a
P-labeled rat T
R-II
cDNA probe followed by RNase digestion. As expected, no protected
fragment was observed in the wild-type cells (lane 1). In the
transfected cells (lane 2), a 211-nt long protected fragment
was seen, demonstrating that mRNA for rat T
R-II
was
expressed. Controls represent
P-labeled rat T
R-II
cDNA probe hybridized with yeast total RNA, followed by digestion with
RNase (lane 3), or without RNase treatment (lane 4).
No protected fragment was seen with RNase treatment (lane 3).
The undigested probe was 488 nt long (lane 4). Molecular size
was determined by
P-labeled 1-kb ladder DNA (not shown). B, Northern analysis. Total RNA (30 µg/lane) from
wild-type glomerular endothelial cells incubated in the absence (lane 1) or presence (lane 2) of 1 µM dexamethasone was subjected to Northern blot hybridization with a
P-labeled rat T
R-II cDNA probe. Only a 5.5-kb mRNA
was detected, corresponding to wild-type T
R-II. Lanes 3 and 4 represent total RNA from transfected endothelial
cells incubated in the absence or presence of 1 µM dexamethasone, respectively. Both the 5.5- and a 1.8-kb mRNA are
observed, reflecting wild-type T
R-II and T
R-II
,
respectively. The 1.8-kb mRNA was strongly induced by dexamethasone (lane 4). Also, a modest induction of the 5.5-kb wild-type
T
R-II mRNA was seen with dexamethasone (lanes 2 and 4). Similar densities for the 18 S signals indicate
approximate equivalence of RNA loading.
Northern
blot analysis of total RNA isolated from wild-type and transfected
glomerular endothelial cells, probed with TR-II cDNA, showed a
5.5-kb band in all cells (Fig. 2B), corresponding to
wild-type T
R-II. A new 1.8-kb band, corresponding to
T
R-II
, was observed only in cells transfected with
T
R-II
, and not present in wild-type or mock
transfected cells, and was strongly induced by dexamethasone. Also, a
modest induction of the 5.5-kb wild-type T
R-II mRNA was seen with
dexamethasone.
Figure 3:
Affinity cross-linking of I-labeled TGF-
1 to cell surface receptors. Lanes
1 and 2 represent wild-type glomerular endothelial cells
cross-linked in the presence or absence of unlabeled TGF-
1,
respectively. Lanes 3 and 4 represent transfected
glomerular endothelial cells in the presence or absence of unlabeled
TGF-
1, respectively. Specifically labeled bands are observed at
approximately 89 kDa and approximately 70 kDa corresponding to
wild-type T
R-II and T
R-I, respectively. Bands of
approximately 48 and 36 kDa are seen only in the transfected glomerular
endothelial cells.
Figure 4:
Effect of TGF-1 on
[
H]thymidine incorporation. A, cells
were incubated in the presence or absence of TGF-
1 (2 ng/ml) for
45 h, followed by [
H]thymidine incorporation into
the cells for 3 h. Lane 1, TGF-
1 inhibited
[
H]thymidine incorporation in wild-type
glomerular endothelial cells to 63 ± 10%. Lane 2, in
transfected glomerular endothelial cells carrying
T
R-II
, TGF-
1 stimulated
[
H]thymidine incorporation to 131 ± 9% (*, p < 0.005, Student's t test, n = 4). B, transfected glomerular endothelial cells
were incubated in 0.5% FBS and in the presence (
) or absence
(
) of TGF-
1 (2 ng/ml), and [
H]thymidine
incorporation was determined at various time points. Data represent
means of triplicate determinations ± S.E. (p <
0.005, analysis of variance). A second experiment gave essentially the
same results.
Figure 5:
Effect of serum deprivation and exogenous
TGF-1 on capillary morphogenesis. Formation of capillary-like
structures were observed in wild-type glomerular endothelial cells
incubated in 0.5% FBS medium, in the absence (panel A) or
presence (panel B) of exogenous TGF-
1 (2 ng/ml). Whereas,
capillary-like structures were not observed in transfected glomerular
endothelial cells carrying T
R-II
, incubated in 0.5%
FBS medium, both in the absence (panel C) or presence (panel D) of exogenous TGF-
1 (2
ng/ml).
Figure 6: Details of capillary-like structures formed by wild-type glomerular capillary endothelial cells. A, phase-contrast view of an endothelial cell cord consisting of both solid and hollow segments. The arrows indicate lumen-like translucent space. B, transmission electron micrograph demonstrating the presence of a central lumenal space (L). Clathrin-coated pits and vesicles are observed as well as cell junctional complex (arrow).
Figure 7:
Agarose gel electrophoresis of genomic
DNA. A, in wild-type glomerular endothelial cells, serum
deprivation with (+) or without(-) exogenous TGF-1
induced apoptosis as shown by genomic DNA fragmentation. This was
observed both with (+) and without(-) 24-h incubation with 1
µM dexamethasone. B, genomic DNA fragmentation
was uniformly and entirely absent in serum deprived transfected
glomerular endothelial cells carrying T
R-II
, with
(+) or without(-) exogenous TGF-
1, both with (+)
and without(-) 24-h incubation with 1 µM dexamethasone.
Figure 8:
Inhibition of capillary morphogenesis by
neutralizing antibody to TGF-1. Panel A shows
capillary-like structures formed by wild-type glomerular endothelial
cells with serum deprivation. This formation of capillary-like
structures was inhibited by the addition of neutralizing antibody to
TGF-
1, as shown in panel B.
This study sought to explore the role of TGF- receptors
in capillary morphogenesis, using renal glomerular capillary
endothelial cells stably transfected with a T
R-II construct
designed to inhibit T
R-II dependent signals through a
transdominant negative action. TGF-
receptors types I and II are
co-expressed by most cells(24) , and heterodimerize upon ligand
binding(16, 17) . Heterodimer formation was recently
shown to induce phosphorylation of T
R-I at the GS domain, an
effect dependent on T
R-II kinase activity. Phosphorylation of
T
R-I by T
R-II is thought to be essential for the propagation
of TGF-
1 signals(16) . Mutant T
R-II lacking the
cytoplasmic signaling domain can be predicted to inhibit T
R-II
dependent signals by virtue of competition for T
R-I binding, as
long as the ability to heterodimerize is conserved. Chen et al.(19) previously showed that mutant T
R-II lacking the
cytoplasmic kinase domain overexpressed in a dominant negative fashion
selectively blocked T
R-II signaling for inhibition of cell
proliferation. Wieser et al.(20) demonstrated that
truncated T
R-II lacking the cytoplasmic domain was able to bind
TGF-
, and form a complex with T
R-I, but failed to inhibit
cell proliferation, activate extracellular matrix synthesis, or
activate transcription from a promoter containing TGF-
-responsive
elements. Moreover, mutant T
R-II with kinase domain deleted was
shown to confer resistance to TGF-
control of developmentally
regulated cardiac genes(21) . In this study, essentially the
same construct was utilized, and stably expressed in cultured renal
glomerular capillary endothelial cells. Expression of T
R-II
mRNA in transfected glomerular capillary endothelial cells was
demonstrated by reverse transcriptase-PCR, RNase protection assay, and
Northern blot analysis. Although the T
R-II
construct
was under a glucocorticoid-regulated promoter, expression was observed
even in uninduced conditions, although at lower levels than that in the
presence of dexamethasone. That such promoters ``leak''
during uninduced conditions has previously been observed by
others(25) .
To demonstrate cell-surface expression and
ligand-binding by the mutant receptor, intact cells were incubated with I-TGF-
1 followed by affinity cross-linking and
analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis.
In untransfected or mock transfected cells, only the wild-type
T
R-I and T
R-II, approximately 70 and 89 kDa, respectively,
were observed. In transfected cells, two additional bands of 48 and 36
kDa in size were also observed. Mutant receptor has a predicted
molecular mass of 23 kDa. The 48-kDa band is interpreted to represent
the mutant receptor with TGF-
dimer bound to it, the 36-kDa band
could represent the same mutant receptor with TGF-
1 monomer or
possibly a degradation product of the same receptor. These data are
interpreted to show that the mutant receptor is expressed at the cell
surface and that it can bind TGF-
1.
Cross-linking to wild-type
TR-I was less in the transfected cells carrying T
R-II
(Fig. 3), when compared to untransfected or mock
transfected cells. A plausible explanation is that the mutant receptor
competes and heterodimerizes with wild-type T
R-I and is then
quickly degraded, internalized, or secreted. Alternatively, the mutant
T
R-II could interfere with TGF-
binding to T
R-I.
Inhibition of binding to wild-type T
R-II was not observed under
these conditions. This is not unexpected since wild-type T
R-II can
bind TGF-
1 in the absence of dimerization with
T
R-I(16, 17) .
Previous in vitro studies have demonstrated that TGF-1 inhibits proliferation
of many cell types(26, 27) , including endothelial
cells(28) . In wild-type or mock transfected glomerular
endothelial cells, TGF-
1 (2 ng/ml) significantly inhibited
[
H]thymidine incorporation, whereas in the
transfected endothelial cells carrying T
R-II
,
TGF-
1 stimulated [
H]thymidine incorporation.
This stimulation of [
H]thymidine incorporation by
TGF-
1 in T
R-II
transfected endothelial cells
could reflect an alternate receptor signaling pathway such as a
T
R-I-mediated response, not dependent on phosphorylation of
T
R-I by T
R-II. Time course experiments revealed that in
transfected cells, exogenous TGF-
1 stimulated
[
H]thymidine incorporation significantly above
the basal rate in 0.5% FBS at 36 and 48 h (Fig. 4B). In
comparison, 5% FBS maximally stimulated
[
H]thymidine incorporation between 12 and 24 h
(data not shown). These findings suggest that the stimulation may be a
secondary rather than a direct mitogenic effect. Our results of
stimulation of DNA synthesis are consistent with previous observations
that when cells are released from the negative growth regulatory
control of TGF-
1, cell proliferation occurs and the potential for
tumorigenesis can emerge(27, 29) .
TGF-1
inhibits proliferation by arresting cells in the G
phase
and thus interrupting progression through the cell cycle. Laiho et
al.(30) observed that TGF-
1 prevented
phosphorylation of the retinoblastoma gene product and arrested cells
in late G
phase of the cell cycle. The underphosphorylated
retinoblastoma gene product has growth-suppressive function. When
progression through the cell cycle is prevented, cells may remain
quiescent or withdraw from the cell cycle and undergo terminal
differentiation. Indeed, Zentella et al.(31) showed
that TGF-
1 inhibited cell cycle progression of skeletal myoblasts
through the G
phase and induced terminal differentiation.
In addition to the inhibition of [H]thymidine
incorporation in wild-type glomerular endothelial cells, cell
detachment was observed. Cells that remained on the plate tended to
organize into capillary-like structures, a process previously described
by others(22, 32, 33) . A possible
explanation for the observed decreased
[
H]thymidine incorporation and cell detachment
may be cell cycle arrest and withdrawal, and entry into a suicide
program, or apoptosis. In support of this, proto-oncogene
c-myc, an important positive regulator of cell growth induced
during the G
/G
phase of cell cycle, can induce
apoptosis under conditions of growth arrest, such as presence of a
negative growth regulator, TGF-
1(34) .
Indeed,
TGF-1 has been shown to inhibit cell proliferation and induce
apoptosis in rat hepatocytes in vivo(35) and in
rabbit uterine epithelial cells in vitro(36) . We
observed in wild-type glomerular endothelial cells, serum deprivation
induced apoptosis as shown by genomic DNA fragmentation and associated
capillary formation. Exogenous TGF-
1 treatment accelerated these
events. In contrast, genomic DNA fragmentation and associated capillary
formation were not observed in transfected endothelial cells expressing
T
R-II
, either with serum deprivation or exogenous
TGF-
1 treatment. Since serum deprivation acted as if exogenous
TGF-
1 had been added, and since effects of serum deprivation were
not seen in cells carrying the transdominant negative mutant receptor,
it is plausible that endogenous TGF-
1 might mediate capillary
morphogenesis with serum deprivation. In support of this hypothesis, we
observed that neutralizing antibody to TGF-
1 abolished both cell
detachment and capillary-like tube formation in serum deprived
wild-type endothelial cells as well as DNA fragmentation.
In the
process of capillary morphogenesis, our studies show that endothelial
cells, in response to TGF-1, undergo a programmed cell death and
detach from the substratum, a phenomenon called anoikis(37) .
The term anoikis is derived from the Greek word for homelessness.
Anoikis implies that once cells lose contact with underlying matrix,
they undergo programmed cell death, thus preventing these detached
cells from establishing themselves in another location. Thus, the
phenomenon of anoikis is a mechanism for homeostasis that maintain a
certain correct cell number in the body by balancing cell production
with cell death. Tumor cells escape this regulation by blocking the
apoptotic response. Anoikis may also be important in cell positioning.
For instance, in the normal maturation of the skin, the cells that are
in contact with the basement membrane proliferate and the cells that
migrate away from it into the more superficial layers undergo apoptosis (38) .
Since integrins are primarily responsible for cell
adhesion to ECM, integrin-mediated signaling has been implicated in
controlling apoptosis. Frisch and Francis (37) demonstrated
that apoptosis was induced by disruption of the interactions between
normal epithelial cells and ECM. In endothelial cells, Meredith et
al.(39) showed that cells incubated in suspension and
denied interactions with ECM rapidly underwent apoptosis. Furthermore,
when endothelial cells were plated on an integrin monoclonal antibody, apoptosis was suppressed. Therefore,
regulation of apoptosis may be mediated by disruption of cell-matrix
interactions and altered cell-cell interactions. Given this body of
evidence, it is not unreasonable to propose that our findings of cell
detachment and apoptosis may reflect TGF-
1-mediated matrix
degradation. TGF-
1 induces endothelial cell secretion of PA, which
is an enzyme that cleaves the proenzyme plasminogen to form active
plasmin. Plasmin is a protease which degrades ECM proteins. Moreover,
plasmin activates latent TGF-
1(12) , and thus providing a
mechanism for autoamplification loop. ECM proteolysis by proteases such
as plasmin facilitates endothelial cell migration and angiogenesis in vivo(11) . However, even though apoptosis may be
mediated by PA-induced matrix degradation by TGF-
1, this may not
be the sole mechanism, in view of the fact that mitogenic growth
factors which can prevent apoptosis, such as bFGF, also induces PA.
Angiogenesis is regulated by a number of cytokines in vivo,
including bFGF(40) , vascular endothelial growth
factor(41) , and TGF-1(6, 7) . In our
studies, we were able to isolate and delineate the effects of
TGF-
1 in vitro from other angiogenic growth factors. Our
findings raise the intriguing possibility that apoptosis is a
phenomenon necessary in the process of capillary morphogenesis and that
both are dependent on TGF-
receptor signaling.