(Received for publication, October 23, 1995)
From the
Transforming growth factor (TGF-
) is biosynthesized
as a membrane-bound precursor protein, pro-TGF-
, that undergoes
sequential endoproteolytic cleavages to release a soluble form of the
factor. In the present study, we have analyzed the biosynthesis and
regulation of TGF-
production in human tumor-derived cell lines
that endogenously express pro-TGF-
and the epidermal growth factor
(EGF) receptor. These cells biosynthesized membrane-anchored forms of
the TGF-
that accumulated on the cell surface. Membrane-bound
pro-TGF-
interacted with the EGF receptor, and complexes of
receptor and pro-TGF-
contained tyrosine-phosphorylated receptor.
Activation of the EGF receptor by soluble EGF or TGF-
had a dual
effect on TGF-
production: an increase in pro-TGF-
mRNA
levels and an increase in pro-TGF-
cleavage. These effects were
largely prevented by preincubation with an anti-EGF receptor monoclonal
antibody that blocked ligand binding. Growth factor autoinduction of
cleavage could be stimulated by several second messenger pathways that
are activated by the EGF receptor, including protein kinase C and
intracellular calcium, and by other alternative mechanisms.
EGF-stimulated cleavage of pro-TGF-
could be partially blocked by
inhibition of these second messenger pathways. These results suggest
that juxtacrine stimulation takes place in human tumor cells that
coexpress both the EGF receptor and membrane-anchored TGF-
and
that TGF-
is able to induce its own endoproteolytic cleavage by
activating the EGF receptor.
Transforming growth factor (TGF-
) (
)is a
50-amino acid single polypeptide, initially isolated from the culture
medium of several oncogenically transformed cell lines(1) ,
that is structurally and functionally related to the epidermal growth
factor (EGF). TGF-
binds to the 170-kDa EGF receptor, a
transmembrane glycoprotein with an extracellular ligand-binding domain
and an intracellular domain that contains a tyrosine-specific protein
kinase(2) . Upon binding of TGF-
to the EGF receptor, the
tyrosine kinase is activated, resulting in a cascade of biochemical and
physiological responses that are involved in the mitogenic signal
transduction pathway of cells(2) . TGF-
has gained
attention because of its predominant expression in tumor-derived cell
lines and in human tumors(3, 4) , suggesting that
TGF-
contributes to neoplastic growth through autocrine and
paracrine mechanisms(5) . In fact, coexpression of high levels
of TGF-
and the EGF receptor leads to a transformed cellular
phenotype (3, 6) , and increased expression of the
precursor for TGF-
in transgenic mice causes hyperplasia of
several tissues and even neoplastic
transformation(7, 8, 9) . Furthermore, the
expression of EGF receptors is elevated in many epithelial
tumor-derived cell lines(3) , and many types of epithelial
malignancies display increased EGF receptors on their cell surface
membranes(5) . This overexpression correlates with a poor
clinical outcome in a number of malignancies (10) .
TGF- is derived from a larger 20-22-kDa transmembrane
precursor (11, 12) , pro-TGF-
, which undergoes
several posttranslational modifications that include N- and O-linked glycosylation (13, 14, 15) and palmitoylation(13) .
Several molecular forms of soluble TGF-
have been reported, and
this heterogeneity appears to be due to both the type and degree of
ectoglycosylation and the preference for different sites of proteolytic
cleavage of the precursor (14, 16, 17) . In
transfected fibroblasts, proteolytic maturation of TGF-
occurs in
two steps(13, 15, 18) . At the plasma
membrane or in a cellular compartment very close to it, the first
cleavage of pro-TGF-
occurs between Ala
and
Val
(by an activity referred to as pro-TGF-
-ase-I).
This removes the NH
-terminal glycosylated extension leaving
a cell-associated 17-kDa pro-TGF-
form that contains the mature
sequence of TGF-
within the precursor. This cleavage step occurs
rapidly (t
= 15 min)(18) . Release
of soluble forms of TGF-
occurs only after a second enzymatic
activity (referred to as pro-TGF-
-ase-II) cleaves the
Ala
-Val
peptide bond that links
TGF-
to the rest of the precursor molecule. This cleavage results
in the generation of a 6-kDa soluble fragment, which accumulates in the
culture medium and corresponds to mature TGF-
, and a
cell-associated 15-kDa residual terminal fragment often referred to as
a tail.
In transformed cells such as retrovirally transformed
fibroblasts (14, 16) and hepatoma cells(17) ,
indirect evidence indicates that the basal activity of both enzymatic
activities can be relatively high. In these cells, primary activity of
pro-TGF--ase-II can lead to production of an additional soluble
20-kDa form of TGF-
, designated meso-TGF-
, which retains the
glycosylated NH
extension(14) .
Endoproteolytic
cleavage by pro-TGF--ase-II is highly regulated and depends on the
activity of several second messenger systems such as protein kinase C
(PKC), free cytosolic calcium
([Ca
]
), and other
still undefined pathways that can be stimulated by serum
factors(18, 19) . The pro-TGF-
-ase-II cleaving
activity is quite distinct from other protein maturation or degradation
processes(20) . Biochemical evidence suggests that the enzyme
belongs to the serine protease family (21) and requires ATP and
membrane association for activity(22) , and topologically, its
regulated activity depends on the presence of the enzyme and substrate
at the plasma membrane. This endoproteolytic system is likely to be
involved in the cleavage of other transmembrane proteins that undergo
regulated release of their ectodomains(23) .
Since knowledge
of the molecular properties and factors regulating release of
pro-TGF- and other membrane-anchored growth factors is of
significant biological and potentially therapeutic interest, we have
analyzed the expression, biosynthesis, and cleavage of pro-TGF-
in
several different tumor cell lines that endogenously express both the
EGF receptor and TGF-
. We show that membrane-bound pro-TGF-
accumulates at the plasma membrane and is able to interact with the EGF
receptor. Moreover, TGF-
or EGF, by acting through the EGF
receptor, is able to increase TGF-
production by a dual mechanism
that involves mRNA increase and cleavage of the membrane-bound
precursor into a soluble factor. Some of these effects are prevented by
a monoclonal antibody (mAb) that binds to the extracellular domain of
the EGF receptor and blocks ligand binding(24, 25) .
This antibody induces tumor xenograft regression in nude mice, which
bear neoplasias caused by injection of human tumor cells that
overexpress both the EGF receptor and its ligand
TGF-
(26, 27) .
After metabolic labeling and culture, cells were rinsed with
ice-cold phosphate-buffered saline (PBS) and lysed with
immunoprecipitation buffer (Hepes, containing 5 mM EDTA, 1
mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, 25
µg/ml aprotinin, 1 mM sodium orthovanadate, and 1% Triton
X-100). Lysates were incubated for 10 min on ice, and cell debris was
pelleted at 10,000 g for 10 min at 4 °C.
Supernatants were transferred to fresh tubes and incubated for 2 h with
rabbit anti-pro-TGF-
antibodies raised against a mixture of two
synthetic peptides corresponding to residues 138-151 and
145-159 in the cytoplasmatic domain of the rat pro-TGF-
sequence (28) at a 1:100 dilution. Immunocomplexes were
harvested with protein A-Sepharose and washed 4 times with
immunoprecipitation buffer. The beads were heated in 30 µl of
electrophoresis sample buffer, and proteins were analyzed in 13%
SDS-PAGE. Gels were fixed and fluorographed using Enlightning (DuPont
NEN).
Figure 1:
Molecular forms of
TGF- in tumor cell lines expressing EGF receptors. A,
CHO-TGF-
and A431 cells were metabolically labeled with
[
S]cysteine for 30 min and chased for the
depicted time points. pro-TGF-
was immunoprecipitated from cell
lysates with rabbit anti-pro-TGF-
antibodies (directed at a
pro-TGF-
COOH-terminal sequence). Immunocomplexes were processed
by SDS-PAGE and analyzed by autoradiography. The molecular weights of
the different growth factor species are shown at the left. B, a panel of malignant cell lines known to express the EGF
receptor was analyzed for pro-TGF-
expression 4 h after metabolic
labeling as above. Cell lines include: A431 squamous carcinoma; DiFi
colon carcinoma; SKRC-29 renal cell carcinoma; ME-140 and C4I cervix
carcinoma; DU-145 prostate carcinoma; MCF-10A non-malignant breast
cells; MDA-468 breast carcinoma. C, immunoprecipitations were
performed in the presence of an excess (1 mM) of competing
peptide against which the anti-pro-TGF-
antibody was raised. The
competing peptide prevented immunoprecipitation of pro-TGF-
by the
antibody.
Figure 2:
ProTGF- binds to and activates the
EGF receptor in A431 cells. Confluent cultures of A431 cells were
incubated in the absence or presence of EGF (10 nM) for 15 min
and cell lysates immunoprecipitated with anti-pro-TGF-
antibodies (A) or with anti-EGF receptor mAb 528 (B). Western
blots were probed with anti-phosphotyrosine antibody (Anti-p
tyr). IP, immunoprecipitate; EGF-R, EGF
receptor; Ab, antibody.
First, the effects of EGF
receptor stimulation on pro-TGF- mRNA accumulation were analyzed.
Addition of saturating concentrations of TGF-
(30 nM)
increased the production of a 4-kilobase pair mRNA that hybridized with
a specific human pro-TGF-
probe in A431 cells (Fig. 3).
Having established that EGF receptor stimulation resulted in increased
pro-TGF-
mRNA, we next analyzed the effects of EGF receptor
inhibition on pro-TGF-
levels. Addition of mAb 225 (10-100
nM) decreased the basal pro-TGF-
mRNA level in A431 cells (Fig. 3). Thus, the prevention of ligand-induced activation of
the receptor by a receptor-blocking antibody resulted in
down-regulation of TGF-
mRNA expression.
Figure 3:
Pro-TGF- mRNA expression is induced
by soluble TGF-
and reduced by an EGF receptor blocking antibody.
Northern analysis of pro-TGF-
expression in total RNA isolated
from A431 cells after treatment with soluble TGF-
or anti-EGF
receptor antibody (mAb) 225 for 4 h is
shown.
Studies with
metabolically radiolabeled A431 cells showed that addition of exogenous
TGF- induced cleavage of membrane pro-TGF-
(Fig. 4).
As expected from a precursor-product relationship, either a decrease of
the 17-kDa pro-TGF-
form or an increase in the 15-kDa tail form is
indicative of pro-TGF-
cleavage, and the kinetics can be followed
by analyzing the labeled 17-kDa/15-kDa ratio. Treatment with EGF or
TGF-
for as short as 15 min induced the appearance of the 15-kDa
cytosolic tail of pro-TGF-
with a concomitant decrease in the
intensity of the 17-kDa precursor form (Fig. 4A). This
cleavage did not increase by further prolonging incubation times with
either growth factor for up to 1 h. The persistence of a significant
proportion of the 17-kDa form indicates that cleavage was not complete.
Incubation with TGF-
also induced cleavage of membrane
pro-TGF-
in other tumor-derived cell lines (Fig. 4B).
Figure 4:
Soluble TGF- induces pro-TGF-
cleavage in a panel of EGF receptor-expressing cell lines. A,
A431 cells were metabolically labeled with
[
S]cysteine for 30 min and chased for the
depicted time points either with no additions or treatment with
TGF-
(10 nM) or anti-EGF receptor mAb 225 (100
nM). Pro-TGF-
was immunoprecipitated from cell lysates
with rabbit anti-pro-TGF-
antibodies directed at a pro-TGF-
COOH-terminal sequence. Immunocomplexes were processed by SDS-PAGE and
analyzed by autoradiography. B, a panel of malignant cell
lines known to express the EGF receptor was analyzed as above. See Fig. 1for a description of cell lines. Cells were metabolically
labeled with [
S]cysteine for 30 min and chased
for 45 min.
Figure 5:
Effects of A23187 and PMA on pro-TGF-
cleavage. A, 17 kDa (uncleaved) to 15 kDa (cleaved)
pro-TGF-
ratio in A431 cells. Cells were labeled with
[
S]cysteine for 30 min and chased for 45 min in
complete medium containing EGF (10 nM), PMA (1
µM), or A23187 (1 µM). Where indicated, EGTA
(10 mM) was added to the cultures 5 min before the addition of
these agents. In lanes 7 and 8, cells had been
depleted of protein kinase C by a 24-h preincubation with 1 µM PMA (PKC, -). B, control A431 cells (PKC, +)
and A431 cells that had been depleted of protein kinase C by 24-h
preincubation with 1 µM PMA (PKC, -) were labeled as
above and chased for 30 min in the presence of EGF ± EGTA as
described under A.
The participation of PKC
in pro-TGF- release from A431 cells was next investigated.
Treatment for 45 min with the tumor-promoting phorbol ester PMA, which
is known to directly activate PKC isozymes (40) , provoked 17
to 15 kDa conversion (Fig. 5A). This was prevented by
prior prolonged incubation of A431 cell cultures with PMA for 24 h, a
treatment that causes down-regulation of PKC activity (not shown).
These PKC and Ca
pathways are independent of each
other, since, on the one hand, down-regulation of PKC did not prevent
Ca
ionophore-induced cleavage (Fig. 5A) and, on the other hand, treatment with EGTA
did not prevent PMA-induced cleavage of pro-TGF-
(not shown). When
the activation of cleavage by the two second messenger systems was
compared, raising intracellular Ca
was more efficient
than increasing PKC activity (Fig. 5A, first 4
bars), and either pharmacological treatment was found to be more
efficient than EGF/TGF-
in provoking processing of the 17-kDa
form.
Cleavage of membrane-anchored pro-TGF- by activation of
the EGF receptor with EGF or TGF-
was only partially inhibited by
pharmacological inhibition of these second messenger systems (Fig. 5A, last 4 bars, and Fig. 5B, lanes 3 and 4). Although the
combined pharmacological inhibition of both second messenger systems
markedly reduced growth factor-induced cleavage (Fig. 5B, lane 5), there was still a residual
cleavage activity that neither the Ca
nor the PKC
systems could account for (Fig. 5B, compare the 17 kDa
(uncleaved) to 15 kDa (cleaved) pro-TGF-
ratio in lanes 1 and 5).
Treatment with the anti-EGF receptor blocking
mAb did not affect the general machinery responsible for pro-TGF-
biosynthesis or cleavage since (i) incubation with the mAb prior to and
during the chase did not affect the normal pattern of pro-TGF-
biosynthesis (Fig. 4A) and (ii) cleavage induced by
pharmacological activation of second messenger systems with PMA or
A23187 was insensitive to mAb 225 (Fig. 6).
Figure 6:
EGF receptor blocking antibody mAb 225
prevents TGF- mediated pro-TGF-
cleavage but does not prevent
A23187- and PMA-mediated pro-TGF-
cleavage. A431 cells were
metabolically labeled with [
S]cysteine for 30
min and chased for 45 min. During the last 30 min of the chase
TGF-
(10 nM), A23187 (1 µM), or PMA (1
µM) was added as indicated. Paired cultures were incubated
with saturating amounts of mAb 225 (100 nM), as indicated
during the 45 min of the chase period.
In experiments with cells expressing genetically engineered
mutant forms of pro-TGF- resistant to cleavage by
pro-TGF-
-ase-II, the membrane-bound 17-kDa form has been shown to
be biologically active by a proposed juxtacrine
mechanism(30, 31, 32) . Although the
mechanism of activation of the EGF receptor by soluble ligand has been
well characterized(41, 42) , little is known about the
expression and activity of pro-TGF-
in human cancer cells with an
active EGF receptor/TGF-
autocrine pathway. For this reason, we
investigated the molecular forms of pro-TGF-
in a series of
tumor-derived cell lines with putative active EGF receptor autocrine
loops and high levels of receptor expression.
In transfected
fibroblasts, generation of soluble TGF- from the 17-kDa
membrane-anchored form depends upon the activity of the transmembrane
endoprotease pro-TGF-
-ase-II, which cleaves the
Ala
-Val
peptide bond, thus eliminating
the role of membrane-bound pro-TGF-
as a juxtacrine
molecule(18) . In retrovirally transformed embryo fibroblasts (14, 16) and hepatocellular carcinoma
cells(17) , the culture medium accumulates mature 6-kDa
TGF-
as well as heterogeneous
20-kDa soluble meso-TGF-
.
Although pulse-chase analyses of pro-TGF-
in these cell lines have
not been reported, the high amount of large meso-TGF-
reflects a
high pro-TGF-
-ase-II activity, which releases some of the
pro-TGF-
molecules from the cell membrane before cleavage by
pro-TGF-
-ase-I can occur. In the tumor-derived cells that we have
analyzed, the biosynthesis of pro-TGF-
initially followed a
pattern analogous to that described for transfected cellular
models(13, 15, 18) . However, these cell
lines accumulated the 17-kDa membrane-anchored precursor form,
suggesting that the activity of pro-TGF-
-ase-II was low. In
addition, the rapid disappearance of the NH
-terminal
glycosylated extension to produce the 17-kDa molecular form indicates a
considerable pro-TGF-
-ase-I activity.
The data presented here
suggest that membrane-anchored pro-TGF- represents a significant
proportion of biosynthesized pro-TGF-
in the tumor cells that we
have studied and, in this conformation, is associated with
tyrosine-phosphorylated EGF receptor. It is possible, therefore, that
membrane-anchored forms could carry out juxtacrine stimulation that
would continually enhance growth of receptor-containing cells, and
cleavage would terminate this function and facilitate the clearance of
the factor. This could be an efficient form of receptor activation,
since down-regulation of ligand and receptor, and their subsequent
catabolism, would be precluded. The response to the addition of
saturating concentrations of exogenous EGF (Fig. 4A)
demonstrates that most EGF receptors remain inactivated when these
tumor cells are only exposed to endogenous sources of ligand.
Furthermore, soluble forms of TGF-
were reported to be more active
than membrane-bound TGF-
(30) . Nevertheless, in these
rapidly growing A431 cell cultures it is the membrane-bound form that
predominates, and interestingly, exogenous soluble ligand in saturating
amounts actually inhibits
proliferation(24, 43, 44) .
Cleavage of
membrane pro-TGF- is highly regulated(37) . Mechanisms
that can trigger the release of soluble TGF-
include rises in the
intracellular free Ca
concentration(19) ,
activation of PKC(18) , and other still undefined pathways
switched on by serum factors(19) . Although these mechanisms
are different, the cleavage event activated by all of them is probably
the same, since identical sets of protease inhibitors block cleavage
activated by the different pathways (21) and genetic mutants of
pro-TGF-
in CHO fibroblasts are resistant to cleavage activated by
several alternative mechanisms(23) . The
Ca
-dependent and PKC mechanisms of cleavage appear to
be quite universal, since we have also found them operative in human
tumor cell lines. In A431 cells, pharmacological increases in cytosolic
Ca
or PKC activity increased the conversion of 17-kDa
pro-TGF-
to the 15-kDa terminal fragment tail. Since a certain
degree of cross-talk between these two second messenger systems exists,
it was possible that their effect was mediated by a shared pathway.
This has been ruled out by using treatments that neutralize one pathway
but not the other. Thus, elevated Ca
activates
pro-TGF-
cleavage in cells that have been desensitized to PMA
action. On the other hand, PMA is able to induce cleavage in the
presence of the Ca
chelator EGTA in the culture
medium, a treatment that completely prevents calcium ionophore-induced
cleavage(19) . An interesting finding that comes out of our
data is the relative effectiveness of these second messenger systems in
inducing pro-TGF-
cleavage. In A431 cells, Ca
is
more efficient in provoking cleavage than PMA, while the opposite is
the rule for cells of fibroblastic origin(18, 19) .
This suggests potential mechanisms for specificity, since in some
tissues a cell could be highly sensitive to the stimulation of one
pathway and induce release of soluble TGF-
, while other cell types
could be largely refractory to the activation of this pathway.
What
are the mechanisms by which EGF receptor activation triggers
pro-TGF- cleavage? EGF receptor activation is known to induce the
hydrolysis of membrane polyphosphoinositides, inducing increases in
[Ca
]
and PKC
activity(39, 45) . Pharmacological manipulations of
these second messenger systems support the conclusion that both may
participate to a certain extent. Yet, the residual activity of
pro-TGF-
cleavage after neutralization of the
[Ca
]
and PKC pathways suggests
that additional unidentified mechanisms are triggered by EGF receptor
activation.
We find that TGF- production is autoregulated by a
dual mechanism in human tumor cell lines. On the one hand, activation
of the EGF receptor increased the level of pro-TGF-
mRNA, as has
been reported in other cells(33, 34, 35) ,
and, on the other hand, receptor activation rapidly induced cleavage of
the membrane-anchored factor. The latter is of special interest for at
least two reasons. (i) It demonstrates for the first time that release
of membrane-anchored pro-TGF-
can be triggered by a specific first
messenger, in addition to the well reported effect of pharmacological
manipulation of second messenger pathways; and (ii) the cleavage can be
regulated by the growth factor itself. These observations point to
achievement of a strong up-regulatory effect, using several different
mechanisms to facilitate or perpetuate autocrine stimulation of growth.
The existence of such an autocrine loop is further supported by data
obtained in treatments aimed at blocking the EGF receptor activity. We
observed that anti-EGF receptor mAb 225 can decrease resting
pro-TGF-
mRNA levels in A431 cells and prevents the cleavage of
membrane-bound pro-TGF-
induced by EGF/TGF-
.
The anchoring
of TGF- to the plasma membrane may have not only important
biological consequences upon autocrine/juxtacrine pathways in malignant
cells but therapeutic implications as well. For example, our laboratory
has found that the Fab` monovalent fragment of anti-EGF receptor mAb
225 is effective in blocking receptor activation by exogenous
TGF-
/EGF but is far less effective than the bivalent complete mAb
in blocking activation by endogenous (autocrine/juxtacrine)
TGF-
(46) . We postulate that the close proximity of the
``autojuxtacrine'' TGF-
ligand to receptors on the
surface of the same cell may give the ligand preferential access to
receptors, which is only overcome by the capacity of the bivalent, but
not monovalent, antibody to down-regulate and remove the receptors.