(Received for publication, June 25, 1994; and in revised form, December 22, 1994)
From the
The molecular basis by which transforming growth factor
(TGF)-1 protects certain tumor cells from tumor necrosis factor
(TNF) cytotoxicity was investigated. When pretreated with TGF-
1,
-
2, and -
3, murine L929S fibroblasts developed resistance to
TNF cytotoxicity. Time course experiments revealed that TGF-
1
initially induced both cellular protein-tyrosine phosphorylation and
simultaneous secretion of a novel extracellular matrix TNF-resistance
triggering (TRT) protein(s), which closely preceded the acquisition of
TNF-resistance. TGF-
2 and -
3 also increased tyrosine
phosphorylation. However, both molecules failed to stimulate TRT
secretion. The increased levels of phosphorylation, particularly to 9
specific protein tyrosine kinase inhibitor-sensitive cellular proteins,
appeared to alter the TNF killing pathway. TGF-
1-induced TRT
secretion required participation of unknown serum factors. TRT adhered
strongly to polystyrene plates and resisted treatment with heat (60
°C, 30 min), collagenase,
-macroglobulin, heparin,
antibodies against TGF-
s, and limited trypsin digestion. Notably,
TRT promoted TNF-resistance via activation of tyrosine and
serine/threonine kinase functions in L929S. Thus, the molecular pathway
involves TGF-
1-mediated initiation of a rapid tyrosine
phosphorylation of cellular protein substrates (which alters TNF
cytotoxic pathway), and a simultaneous secretion of TRT, which in turn
signals the cells to maintain the levels of phosphorylation, thereby
sustaining the TNF-resistance.
Numerous in vitro studies have demonstrated that
transforming growth factor- (TGF-
) (
)counteracts
the biological effect of tumor necrosis factor-
(TNF-
). For
example, TGF-
protects several types of cancer cells from the
cytotoxic effect of TNF-
and TNF-
(lymphotoxin)(1, 2, 3, 4) .
TGF-
suppresses TNF-
-stimulated proliferation of normal
diploid fibroblasts, WI-38 (4) . Furthermore, TGF-
inhibits the development of lymphokine-activated killer cells and
cytotoxic T cells, an action which is reversed by
TNF-
(5, 6) . However, in other reports, TGF-
and TNF-
were found to act synergistically to induce monocytic
differentiation of human leukemic cell lines(7, 8) .
The molecular mechanism by which TGF- protects some cancer
cells from TNF cytotoxicity is unknown. One possibility is that
TGF-
produces metabolic changes in cancer cells, such that part of
the TNF cytotoxic pathways is either blocked or eliminated(9) .
One report demonstrated that TGF-
-mediated growth arrest in the
G
phase of the cell cycle correlates with increased
cellular resistance to TNF in a murine fibrosarcoma cell line,
L929(2) . Furthermore, it was determined that this acquired
TNF-resistance of L929 cells is associated with TGF-
-induced de novo protein synthesis (4) . However, the
TGF-
-induced protein products which are responsible for blocking
TNF function have not been identified and their mode of action is
unknown.
To understand how the type I TGF- (TGF-
1) induces
TNF-resistance in the murine L929 fibrosarcoma cell line (L929S), we
have determined in this study that TGF-
1 induction of resistance
to TNF involves both an early protein-tyrosine phosphorylation event
and a simultaneous secretion of a novel extracellular matrix
TNF-resistance triggering (TRT) protein(s). The raised tyrosine
phosphorylation appeared to alter the TNF killing pathway, and TRT
further activated cellular protein kinases, thus maintaining the levels
of phosphorylation and prolonging the status of TNF-resistance in
TGF-
1-treated L929S cells.
The modulation of TGF-1
induction of TNF-resistance by protein kinase inhibitors was examined.
These inhibitors included genistein
(4`,5,7-trihydroxyisoflavone)(11, 12) , lavendustin
A(13) , 2-hydroxy-5-(2,5-dihydroxybenzyl)aminobenzoic acid
(BABA)(13) , tyrphostin(14) , H7, H8, and staurosporine (15, 16, 17) (Calbiochem Corp. and Life
Technologies, Inc./BRL, Gaithersburg, MD, respectively). L929S
monolayers (2
10
/ml) in 96-well microtiter plates
were treated with TGF-
1 (2 ng/ml) in the presence or absence of
protein kinase inhibitors (1-30 µM) for 1-6 h
at 37 °C prior to exposing to TNF-
/Act D for 24 h. Where
indicated, L929S cells were pretreated with TGF-
1 for 1 h,
followed by coincubation with protein kinase inhibitors for 1-6
h. Similar experiments were performed by treating L929S cells with
TGF-
1 (2 ng/ml) and/or brefeldin A (18, 19, 20) (0.5-10 µM;
Epicentre Technologies, Madison, WI) for 6 h prior to challenge with
TNF-
/Act D. Alternatively, the cells were pretreated with
TGF-
1 for 4 h followed by a 2-h exposure to brefeldin A. In
control experiments, the TNF-resistant L929R cells were treated
similarly.
In other experiments, L929S
monolayers were pretreated with or without TGF-1 (0.25-2.0
ng/ml) for 10-16 h and the cells were removed from each
individual well by treating with 100 µl of trypsin/EDTA or 0.05%
Nonidet P-40 for 10 min at room temperature. After thoroughly washing
the wells 6 times with PBS, untreated control L929S cells were then
seeded onto these wells for 12-16 h, followed by examining their
susceptibility to the cytotoxic effect of TNF-
/Act D. Similar
experiments were performed using TGF-
2 and -
3.
Figure 1:
TGF-s induction of
TNF-resistance. Murine L929S fibroblasts in 96-well microtiter plates
were pretreated with TGF-
1 (A), -
2 (B), or
-
3 (C) (0.25-2 ng/ml) for various indicated times
(10 min to 14 h). These cells were washed with RPMI 1640 and exposed to
TNF-
(5 units/ml)/Act D (1 µg/ml) for 16-24 h.
Experiments were performed in quadruplicates. Similar results were
obtained using other concentrations of TNF-
(1-20 units/ml)
in the presence of Act D (data not shown). Percentages of
TGF-
-induced TNF-resistance were calculated as described under
``Experimental Procedures.''
Figure 2:
Effect of TGF-1 on protein-tyrosine
phosphorylation. L929S or L929R cells were treated with TGF-
1 (2
ng/ml) for 0.5-8 h in the presence of
[
S]methionine, followed by immunoprecipitation
using monoclonal antiphosphotyrosine antibodies. A, shown in
the top panel are two representative SDS-PAGE, showing the
changes in the protein phosphorylation during treatment of L929S and
L929R cells with TGF-
1 for 0.5-2 h. -, control cells;
+, TGF-
1-treated cells. In the bottom panel, the
graph shows the determined changes in phosphorylation from an average
of two experiments. The result from each time point was obtained by
subtracting the total density (OD at 700 nM) of phosphorylated
proteins in each control from that of the corresponding
TGF-
1-treated cells, divided by the density of the control. B, L929S cells were metabolically labeled with
[
S]methionine and treated with TGF-
1 (2
ng/ml) for 2 h in the presence or absence of lavendustin A or
tyrphostin (10 µM) followed by processing
immunoprecipitation using antiphosphotyrosine antibodies. The arrows indicate 9 protein species which were sensitive to the
inhibition of phosphorylation by lavendustin A and
tyrphostin.
Figure 3:
Effect of protein kinase inhibitors on
reduction of TGF-1-induced TNF-resistance. A, L929S cells
were pretreated with TGF-
1 (2 ng/ml) in the presence of 10
µM of protein kinase inhibitors, including lavendustin A (laven A), BABA, tyrphostin, genistein, and staurosporine for
6 h at 37 °C, prior to performing TNF-
/Act D cytotoxic assays. B, L929S cells were pretreated with TGF-
1 for 1 h,
followed by coincubation with protein kinase inhibitors for 6 h and
exposing to TNF-
/Act D. Experiments were performed in
quadruplicates.
In contrast, staurosporine (10
µM), a potent inhibitor of protein kinase C, failed to
block the acquired TNF-resistance (Fig. 3A). Similarly,
no significant effect in blocking TGF-1 function was observed when
staurosporine was tested at lower concentrations (1 nM to 1
µM) (data not shown). At concentrations greater than 10
µM, staurosporine was cytotoxic to both L929S and L929R
cells.
Under similar experimental conditions, TGF-2 or
-
3-mediated TNF-resistance was blocked (approximately 50%) by
tyrphostin and lavendustin A (10 µM), but not by
staurosporine. The intrinsic TNF-resistance of L929R cells was not
altered by TGF-
1 and/or tyrosine kinase inhibitors (data not
shown).
Figure 4:
Effect of brefeldin A on TGF-1
induction of TNF-resistance. A, L929S cells were pretreated
with TGF-
1 (2 ng/ml) in the presence or absence of brefeldin A for
6 h, followed by challenge with TNF-
/Act D for 24 h. B,
L929S were pretreated with TGF-
1 for 4 h, followed by coincubation
with brefeldin A for 2 h, prior to conducting the TNF cytotoxic tests.
Experiments were done in quadruplicates.
TRT adhered to polystyrene-based
plastic surface and triggered cellular resistance to TNF. For example,
96-well microtiter plates were precoated with secreted proteins from
the culture supernatants of control or TGF-1-treated L929S for 3 h
at 37 °C, followed by washing the wells thoroughly with PBS and
seeding untreated control L929S cells for 14-20 h. These control
cells became TNF-resistant when seeded onto the wells precoated with
proteins secreted from TGF-
1-treated cells growing under rolling conditions, but not with proteins secreted by
untreated control cells (Fig. 5). Similarly, by growing L929S
cells in roller culture using both serum-free RPMI 1640 and HyQ-CCM2
media, the TGF-
1-treated and control cells failed to secrete TRT (Fig. 5).
Figure 5:
TRT protein(s) secreted in the
supernatants of TGF-1-treated L929S cells growing under rolling conditions. L929S cells were treated with or without
TGF-
1 (1 ng/ml) and grown in roller culture for 16 h using (A) serum-free RPMI 1640 medium, (B) serum-free
HyQ-CCM
medium, or (C) RPMI 1640 plus 10% fetal
bovine serum. Ninety-six-well microtiter plates were precoated with
100-µl aliquots of the serially-diluted culture supernatants for 3
h at 37 °C. The plates were washed 6 times with PBS prior to
seeding untreated control L929S cells overnight for testing their
susceptibility to TNF-
/Act D (in 8 duplicates). Under serum-free
conditions, TRT was not produced by TGF-
1-treated L929S. Control, without TGF-
1; Exp,
TGF-
1-treated.
In additional controls, adherent L929S
monolayers, whether or not pretreated with TGF-1, produced no TRT
proteins against TNF to the culture supernatants (data not shown). Both
serially diluted serum proteins and native or degraded fibronectin
(1-10 µg/well) when coated onto microtiter plates failed to
protect L929S from TNF killing (data not shown).
More than 95% of
TGF-1-treated L929S monolayers once detached from microtiter
plates readily became TNF-sensitive. Cell removal from microtiter
plates was achieved by using trypsin/EDTA, trypsin, or chilling the
cells at 4 °C for 30 min and repeat pipetting. However, by seeding
untreated control L929S onto the wells where the TGF-
1-treated
cells had once occupied, these control cells became TNF-resistant (Fig. 6). In this procedure, TGF-
1-treated or control L929S
monolayers were removed from each individual well by treating with
either 0.05% Nonidet P-40 or trypsin/EDTA, followed by thoroughly
washing the wells with PBS six times to remove cellular debris and
seeding control L929S overnight (14-20 h). The time required for
the control L929S cells to develop significant levels of TNF-resistance
(>10%) was at least 8 h post-seeding. When 0.01% SDS was included in
Nonidet P-40 to remove TGF-
1-treated cells from the culture
plates, the protective effects were decreased, indicating that the
adhered TRT protein(s) was partially removed from the plates (Fig. 6). High salt conditions (at 5 M NaCl) failed to
remove TRT from the microtiter plates. These results, again, provide
direct evidence that TRT adhered to the plastic surface. A time course
experiment showed that TRT was rapidly produced after treating L929S
cells with TGF-
1 for approximately 20-30 min (Fig. 7A). In contrast, both TGF-
2 and -
3
failed to induce TRT secretion (Fig. 7, B and C). A prolonged treatment of L929S cells with TGF-
2 and
-
3 for 4-16 h also failed to observe the TRT activity (less
than 10%).
Figure 6:
TGF-1 induced secretion of TRT
protein(s) to the extracellular matrix. L929S monolayers were
pretreated with or without TGF-
1 for 16 h, followed by lysing the
cells with (A) 0.05% Nonidet P-40, (B) 0.05% Nonidet
P-40 and 0.01% SDS, or (C) removing the cells with
trypsin/EDTA. The microtiter plates were then washed thoroughly with
PBS prior to seeding control L929S cells. The L929S cells acquired
TNF-resistance when plated onto the wells pre-resided with
TGF-
1-treated cells but not with the untreated controls. Data were
obtained from quadruplicate experiments.
Figure 7:
Time course induction of TRT by
TGF-1, but not by TGF-
2 and -
3. L929S cells in 96-well
microtiter plates were pretreated with TGF-
1 (A), -
2 (B), or -
3 (C) for 10 min to 2 h, followed by
removing the cells using trypsin/EDTA, washing the plates thoroughly
with PBS, and seeding untreated control L929S cells overnight for TNF
cytotoxicity assays (in 8 duplicates).
All the indicated protein tyrosine kinase inhibitors
failed to block the induction of TRT expression by TGF-1. When
L929S monolayers were coincubated with both TGF-
1 (2 ng/ml) and
protein tyrosine kinase inhibitors (10-30 µM) for
1-6 h, no significant decreases in TRT activity caused by the
protein tyrosine kinase inhibitors were found (p > 0.05;
Student's t test).
When L929R cells were treated with
or without TGF-1 in roller culture, no TRT activity was found in
the culture supernatants (data not shown). Similarly, by seeding
control L929S cells onto the wells where TGF-
1-treated L929R cells
had once resided, the L929S cells did not acquire TNF-resistance.
TRT was shown to be relatively stable. As summarized in the Table 1, TRT resisted treatments with heat, mild trypsinization,
collagenase, heparin, and -macroglobulin. However,
using a prolonged treatment for 7 h with trypsin or with 0.5 N alkaline or acid, the TRT activity was totally destroyed.
Next,
I examined whether residual TGF-1 or cellularly secreted
TGF-
s adhered to plastic wells that caused L929S resistance to
TNF. L929S cells in roller culture were treated with or
without TGF-
1 for 24 h, followed by harvesting the culture
supernatants and precoating onto microtiter plates for 3 h. The adhered
TRT and other proteins were then treated with neutralizing antibodies
against TGF-
1. These antibodies failed to abolish the TRT activity (Fig. 8A). Similarly, the TRT activity was not blocked
by another antibody, anti-TGF-
s (
1,
2, and
3) IgG.
TRT proteins in the extracellular matrix were prepared by removing
control or TGF-
1-treated L929 monolayers with trypsin/EDTA (Fig. 8B). These antibodies also failed to inhibit TRT
activity obtained from roller culture (data not shown).
Figure 8:
Antibodies against TGF-s could not
inhibit the TRT activity. A, culture supernatants from control
or TGF-
1-treated L929S (roller culture) were precoated
onto microtiter plates for 3 h followed by treating the adherent
proteins with antibodies against TGF-
1 (2 µg/ml; Collaborative
Research) for 3 h to neutralize the adhere TRT. After washing the
microtiter plates 4 times with PBS, control L929S cells were seeded and
cultured overnight prior to challenge with TNF-
/Act D (experiments
in quadruplicates). B, L929S monolayers were pretreated with
or without TGF-
1 for 16 h followed by removing cells with
trypsin/EDTA. TRT activity in the extracellular matrix was not
neutralized by the anti-TGF-
s antibodies (1 µg/ml; Genzyme;
experiments in quadruplicates). Similar results were obtained by
testing these antibodies in blocking TRT activity in the supernatants
from roller culture as described in A.
Figure 9:
Blocking of TRT-mediated TNF-resistance by
protein kinase inhibitors. A, culture supernatants from
TGF-1-treated or control cells (roller culture) were
precoated onto a 96-well microtiter plate for 3 h, followed by
thoroughly washing the plate and seeding control L929S cells. Following
adherence for 1 h, the cells were treated with lavendustin A, BABA, and
H7 (10 µM) for 3 h, washed and continuously cultured
overnight prior to exposing to TNF-
/Act D. B, L929S
monolayers were pretreated with or without TGF-
1 (2 ng/ml) and
simultaneously labeled with [
S]methionine for 3
h, in the presence or absence of lavendustin A (10 µM),
followed by performing immunoprecipitation (using antiphosphotyrosine
antibodies), SDS-PAGE, and autoradiography. Percentages of changes in
tyrosine phosphorylation were calculated by comparing to the untreated
control.
In this study we have examined the mechanisms by which
TGF-1 induces TNF-resistance in L929S cells. Our results
demonstrate that both an early TGF-
1-mediated activation of
tyrosine kinases and a simultaneous secretion of extracellular matrix
TRT protein(s) contribute to the development of TNF-resistance. Both
events occur before the development of TNF-resistance. The increased
phosphorylation levels appear to alter the TNF killing pathway. TRT is
a putative adhesion molecule and shown to provide an additional
stimulus allowing the cells to sustain the increased levels of tyrosine
phosphorylation and the status of TNF-resistance. Protein kinase C was
not involved in TGF-
1-induction of TNF-resistance, as evidenced by
the failure of staurosporine in restricting the induced resistance in
L929S cells.
In addition to TGF-1, it is determined that both
TGF-
2 and -
3 stimulate TNF-resistance in L929S cells. Time
course studies revealed that TGF-
2 and -
3 could rapidly
induce TNF-resistance faster than that of TGF-
1. However,
TGF-
1 exerts a greater extent of protection against TNF-
than
TGF-
2 and -
3 (60-70 versus 40-50%). Most
interestingly, L929S cells secreted little or no TRT by stimulating
with either TGF-
2 or -
3. These observations suggest that the
reduced efficiency for TGF-
2 and -
3 to protect L929S cells
against TNF-mediated cytotoxicity is probably due to their inability to
stimulate TRT secretion. Accordingly, the degree of TNF-resistance is
contributed, in part, by both the increased tyrosine phosphorylation
and the stimulatory effect of TRT.
Unlike TGF-1, TGF-
2 and
-
3 fail to induce TRT secretion, suggesting an undefined
difference in the signal transduction pathways among these molecules.
TGF-
signals through a heteromeric complex between the type I and
type II receptors(24) , which are known to possess intrinsic
serine/threonine kinase functions(25, 26) . Although
both the type I and type II receptors have a higher binding affinity
for TGF-
1 and -
3 than for TGF-
2(27) , such
differences in the binding probably could not account for the failure
of TGF-
2 and -
3 to induce TRT secretion. The type I receptors
have been shown to specify growth inhibitory and transcriptional
responses to TGF-
and activin in Mv1Lu lung epithelial
cells(28) . Notably, TGF-
1 activates two different signal
transduction pathways in epithelial cells(29) , and mediates
different signals for growth inhibition and extracellular matrix
protein in prostatic carcinoma cells(30) . These observations
suggest that the generation of diverse signal pathways may be related
with the kinetic formation of heteromeric receptor complex (24) .
The activation of cellular protein-tyrosine kinases
in L929S cells probably occurs downstream from the activation of
TGF- receptor kinases. This hypothesis is supported by the
observation that tyrosine kinase inhibitors restricted
TGF-
-mediated induction of TNF-resistance in L929S cells. However,
which specific protein-tyrosine kinase activates immediately subsequent
to the receptor kinases remains to be established. Both lavendustin A
and tyrphostin were shown to suppress phosphorylation of 9 identical
protein species and block the induced TNF-resistance, indicating that
phosphorylation of these proteins is critical in altering the TNF
killing pathway. These observations appear to correspond to the finding
that both the intrinsic kinase function and the extent of tyrosine
phosphorylation of epidermal growth factor receptor correlates with an
alteration of TNF cytotoxic signal transduction and control of TNF
responsiveness in ME-180 squamous carcinoma cells(31) .
However, the above postulation may not necessarily be true for other
types of cells. For instance, TGF-1 could not alter the intrinsic
TNF-resistance in L929R cells, and, most interestingly, there was a
reduced tyrosine kinase activity in TNF-resistant L929R cells upon
treatment with TGF-
s. Perhaps, TGF-
s up-regulated tyrosine
phosphatase activity in L929R. Another possibility is that there is a
functional defect in the tyrosine kinase activation cascade in L929R
cells, and this defect may be directly or indirectly associated with
the intrinsic TNF-resistance in this cell mutant.
Both the
TGF-1-mediated tyrosine phosphorylation and the secretion of TRT
are independent events, simply because protein tyrosine kinase
inhibitors failed to block the secretion of TRT. Time course studies
revealed that the occurrence of both events preceded the acquisition of
TNF-resistance. The expression of TRT requires a unknown serum
factor(s) along with TGF-
1 in the stimulation of L929S cells. In
contrast, under serum-free conditions L929S monolayers still acquire
TNF-resistance upon treatment with TGF-
1. Thus, in addition to
TRT, TGF-
1 must have induced novel intracellular proteins which
also interrupt the TNF killing pathway under either serum-free or the
present conditions. Indeed, by expression cloning I have isolated a
novel cDNA, designated TR1, whose expression is rapidly induced by
stimulating L929S cells with TGF-
1 for less than 1 h, and
transfection of L929S cells with TR1 cDNA renders the cells
TNF-resistant. (
)
Unlike the rapid increase in tyrosine
phosphorylation mediated by TGF-1, TRT slowly increased the levels
of tyrosine phosphorylation (40% increases in 3 h) and its induction of
TNF-resistance in L929S cells requires at least 8 h. Thus, the function
of TRT is mainly to provide an additional signal to sustain the high
levels of tyrosine phosphorylation and the extent of TNF-resistance.
Since the TRT-mediated tyrosine phosphorylation is a relatively
long-term process, it indicates that tyrosine phosphorylation is most
likely a secondary or a later event following TRT-mediated signal
transduction. The ability of H-7 to block the TRT-mediated
TNF-resistance, suggests that TRT may signal through receptors with
serine/threonine kinase activity(32) . Overall, the underlying
pathway is that TGF-
1 initiates both a rapid tyrosine
phosphorylation in cellular protein substrates (which is essential to
alter TNF cytotoxic pathway) and a simultaneous production of
extracellular matrix TRT, which in turn signals the cells to continue
to raise the levels of tyrosine phosphorylation necessary for
sustaining the TNF-resistance.
Although the characteristics of TRT
requires further investigation, TRT is a protein and relatively stable
as its resistance to treatments with heat, collagenase, limited
trypsinization, heparin, and -macroglobulin have
shown. Preliminary characterization of TRT by preparative isoelectric
focusing revealed that TRT complexes with high-molecular weight
extracellular matrix proteins. Precipitation by ammonium sulfate and
functional analysis showed that TRT appears to be a 46-kDa protein,
which possesses a unique N-terminal amino acid sequence, not related to
the latent form of TGF-
s, as determined by gas-phase
microsequencing.
TGF-1-mediated protection from TNF
cytotoxicity in L929 cells is not related to changes in the receptor
numbers or binding affinity(1, 4) . Our experimental
results from cellular ELISA have further verified these observations.
Similarly, in myeloid cell lines, it is determined that there is no
correlation between TNF receptor number or receptor affinity and the
cellular sensitivity to TNF(33) . Furthermore, no apparent
novel extracellular matrix proteins induced by TGF-
1 were found to
bind TNF-
as determined by functional testings and ELISA-based
binding studies. These results have been further confirmed by
separating and enriching extracellular protein species using
preparative isoelectric focusing, followed by testing their TNF
blocking activities and TNF-binding interactions using Western blotting
analyses (data not shown).
In summary, it is demonstrated in this
study that TGF-1-induced TNF-resistance in L929S cells is
associated with a rapid activation of protein tyrosine kinases as well
as secretion of an extracellular TRT protein(s). The increased tyrosine
phosphorylation levels in specific protein substrates appear to
contribute to the blockade of the TNF killing pathway. TRT, in turn,
signals for a continued high level of tyrosine phosphorylation, thus
prolonging the status of TNF-resistance. Further structural and
molecular characterizations of TRT protein(s) will increase our
understanding of TNF-resistance in tumor cells.