©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transforming Growth Factor-1 Induction of Novel Extracellular Matrix Proteins That Trigger Resistance to Tumor Necrosis Factor Cytotoxicity in Murine L929 Fibroblasts (*)

(Received for publication, June 25, 1994; and in revised form, December 22, 1994)

Nan-Shan Chang

From the Guthrie Research Institute, Laboratory of Molecular Immunology, Guthrie Medical Center, Sayre, Pennsylvania 18840

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The molecular basis by which transforming growth factor (TGF)-beta1 protects certain tumor cells from tumor necrosis factor (TNF) cytotoxicity was investigated. When pretreated with TGF-beta1, -beta2, and -beta3, murine L929S fibroblasts developed resistance to TNF cytotoxicity. Time course experiments revealed that TGF-beta1 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-beta2 and -beta3 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-beta1-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, alpha(2)-macroglobulin, heparin, antibodies against TGF-betas, 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-beta1-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.


INTRODUCTION

Numerous in vitro studies have demonstrated that transforming growth factor-beta (TGF-beta) (^1)counteracts the biological effect of tumor necrosis factor-alpha (TNF-alpha). For example, TGF-beta protects several types of cancer cells from the cytotoxic effect of TNF-alpha and TNF-beta (lymphotoxin)(1, 2, 3, 4) . TGF-beta suppresses TNF-alpha-stimulated proliferation of normal diploid fibroblasts, WI-38 (4) . Furthermore, TGF-beta inhibits the development of lymphokine-activated killer cells and cytotoxic T cells, an action which is reversed by TNF-alpha(5, 6) . However, in other reports, TGF-beta and TNF-alpha were found to act synergistically to induce monocytic differentiation of human leukemic cell lines(7, 8) .

The molecular mechanism by which TGF-beta protects some cancer cells from TNF cytotoxicity is unknown. One possibility is that TGF-beta 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-beta-mediated growth arrest in the G(1) 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-beta-induced de novo protein synthesis (4) . However, the TGF-beta-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-beta (TGF-beta1) induces TNF-resistance in the murine L929 fibrosarcoma cell line (L929S), we have determined in this study that TGF-beta1 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-beta1-treated L929S cells.


EXPERIMENTAL PROCEDURES

Cell Lines and Medium

Both murine TNF-sensitive L929S and TNF-resistant L929R cells were kindly provided by Dr. D. Beezhold of the Guthrie Research Institute. L929R cells resist the cytotoxic effect of TNF-alpha and TNF-beta (10-500 units/ml) even in the presence of actinomycin D (Act D; 1 µg/ml). These cell lines were cultured in RPMI 1640 medium (Mediatech, Washington, D. C.), supplemented with 10% fetal bovine serum (JR Scientific, Woodland, CA), in a 5% CO(2)/atmosphere, 37 °C incubator. Where indicated, a serum-free medium, HyQ-CCM2 (HyClone Laboratories, Logan, UT), was used to culture fibroblasts in some experiments.

TNF Cytotoxicity Assays

TNF cytotoxicity assays were performed using L929S fibroblasts as killing targets, as described previously(10) . Briefly, 100-µl aliquots of L929S cells (2 times 10^5/ml) were dispensed onto 96-well microtiter plates and cultured 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C. The cell monolayers were then pretreated with human TGF-beta1 (0.25-2 ng/ml; Collaborative Research Inc., Bedford, MA) for 10 min to 14 h (or prolonging up to 48 h) at 37 °C prior to washing with RPMI 1640 and challenge with recombinant TNF-alpha (2.5-10 units/ml; Genzyme Corp., Boston, MA) in the presence of Act D (1 µg/ml; Sigma). Similarly, L929S monolayers were treated with recombinant human TGF-beta2 and -beta3 (0.25-2 ng/ml; Oncogene Science, Uniondale, NY). Following 24 h in culture, measurement of TNF-mediated cellular cytotoxicity using crystal violet stain (OD at 590 nm) was performed as described(10) . The percentages of cytotoxicity were calculated as follows: % cytotoxicity = [(OD from control cells - OD from TNF-treated cells)/OD from control cells] times 100. Furthermore, the percentages of TNF-resistance induced by TGF-beta were also calculated as: % induced TNF-resistance = [1 - (% TNF killing of TGF-beta treated cells/% TNF killing of control cells)] times 100.

The modulation of TGF-beta1 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 times 10^5/ml) in 96-well microtiter plates were treated with TGF-beta1 (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-alpha/Act D for 24 h. Where indicated, L929S cells were pretreated with TGF-beta1 for 1 h, followed by coincubation with protein kinase inhibitors for 1-6 h. Similar experiments were performed by treating L929S cells with TGF-beta1 (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-alpha/Act D. Alternatively, the cells were pretreated with TGF-beta1 for 4 h followed by a 2-h exposure to brefeldin A. In control experiments, the TNF-resistant L929R cells were treated similarly.

Immunoprecipitation

To examine TGF-beta-mediated tyrosine phosphorylation of cellular protein substrates, L929S and L929R cells (5 times 10^5/ml) were treated with or without TGF-beta1, -beta2, or -beta3 (2 ng/ml) for 0.5-8 h at 37 °C in the presence of 500 µCi of [S]methionine (ICN, Costa Mesa, CA). After incubation, the cells were lysed with 1 ml of a 10 times lysis buffer (10 mM Tris-HCl, pH 7.4, containing 5 mM iodoacetamide (Sigma), 5 mM benzamidine (Sigma), 10 mM EDTA (Sigma), 0.1% SDS (Bio-Rad), 0.5% Nonidet P-40 (Calbiochem), 1 µM okadaic acid (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma)). The cell lysates were adjusted to 10 ml by adding 9 ml of PBS and precleared with 50 µl of protein A-agarose beads (Pierce) for 1 h at 4 °C. Subsequently, tyrosine-phosphorylated proteins were captured using both monoclonal antibodies against phosphotyrosine (2 µg; Life Technologies, Inc./BRL) and protein A-agarose beads (10 µl) during a 4-h period of end-over-end rotating at 4 °C. The protein A-bead-bound phosphorylated proteins were then processed for reducing SDS-PAGE, autoradiography, and densitometry as described previously (21) .

Role of TGF-beta1-induced Extracellular Matrix TRT Protein(s) in Blocking TNF Cytotoxic Function

To determine whether the TGF-beta1-induced extracellular TRT proteins that blocked TNF cytotoxic function, culture supernatants from TGF-beta1-treated and control adherent L929S monolayers were harvested after 16 h in culture. Additionally, culture supernatants were harvested from nonadherent L929S cells (3 times 10^5/ml) growing under rolling conditions in the presence or absence of TGF-beta1 (1 ng/ml) for 16 h. Ninety six-well microtiter plates were precoated with 100-µl aliquots of the serially diluted culture supernatants for 3 h at 37 °C, followed by washing each well 6 times with PBS. In controls, microtiter plates were coated with serially diluted serum, fibronectin, or its RDG-containing fragment of 120 kDa (22) (1-10 µg/well; kind gifts of Dr. D. Beezhold, Guthrie Research Institute). Untreated control L929S cells (2.5 times 10^5/ml) were then seeded onto the wells for 12-16 h prior to determining their susceptibility to the cytotoxic action of TNF-alpha/Act D. Where indicated, the culture supernatants were treated with heat (60 °C, 30 min), alkaline (0.5 M NaOH, 7 h), or acid (0.5 M HCl, 7 h) prior to coating, or treated with trypsin (250 µg/ml), alpha(2)-macroglobulin (5 µg/well or 10 µg/ml; Sigma), heparin (300 µg/ml; Sigma), neutralizing anti-TGF-beta1 IgG (1 µg/well or 2 µg/ml; Collaborative Research), anti-TGF-betas (beta1, beta2, and beta3) IgG (0.5 µg/well or 1 µg/ml; Genzyme), or collagenase (50 units/ml; Sigma) for various durations during coating onto microtiter plates. These treatments were intended to inhibit TRT and to block any residual TGF-betas from exogenous or cellular sources. alpha(2)-Macroglobulin is also known to complex with the latent form of TGF-beta1(23) .

In other experiments, L929S monolayers were pretreated with or without TGF-beta1 (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-alpha/Act D. Similar experiments were performed using TGF-beta2 and -beta3.

TNF Binding

These experiments were to examine whether TGF-beta1 induced the expression of extracellular proteins that bound TNF-alpha. Binding of TNF-alpha to 0.5% formaldehyde-fixed L929S cells pretreated with or without TGF-beta1, or to the plastic adherent extracellular matrix proteins from TGF-beta1-treated or control cells was performed by standard ELISA procedures (10) using the Pierce ELISA kit (Pierce). The plastic adherent matrix proteins were prepared by removing L929S monolayers from each individual well with trypsin/EDTA or Nonidet P-40 as described above.


RESULTS

Induction of TNF-resistance in L929S Cells by TGF-betas

TGF-beta1-treated L929S cells acquired TNF-resistance in a time- and dose-related manner (Fig. 1A). For instance, L929S developed significant levels of TNF-resistance (>10%) after treatment with TGF-beta1 (2 ng/ml) for 40 min and gained approximately 50% resistance in 3 h. L929S cells stably maintained the acquired TNF-resistance upon prolonging the treatment up to 48 h. In contrast, both TGF-beta2 and -beta3 rapidly increased TNF-resistance in L929S cells reaching approximately 40% of resistance in 40 min (Fig. 1, B and C). The intrinsic TNF-resistance of L929R cells was not altered by treatment with TGF-beta1. L929S cells also developed increasing resistance to TNF-beta when pretreated with TGF-beta1 for 1-20 h at 37 °C (data not shown). In agreement with other reports(1, 4) , TGF-beta1 did not significantly enhance proliferation of L929S cells (approximately 10% increase) during 24-48 h treatment. Similar results were observed for TGF-beta2 and -beta3 regarding the cell proliferation.


Figure 1: TGF-betas induction of TNF-resistance. Murine L929S fibroblasts in 96-well microtiter plates were pretreated with TGF-beta1 (A), -beta2 (B), or -beta3 (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-alpha (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-alpha (1-20 units/ml) in the presence of Act D (data not shown). Percentages of TGF-beta-induced TNF-resistance were calculated as described under ``Experimental Procedures.''



Protein-Tyrosine Phosphorylation

Immunoprecipitation studies revealed that TGF-beta1 increased the levels of cellular tyrosine-phosphorylated proteins in L929S cells during the initial 0.5-1 h treatment, followed by decreases with time (Fig. 2A). In contrast, protein-tyrosine phosphorylation in L929R cells was reduced after treating the cells with TGF-beta1 for 0.5-2 h at 37 °C (Fig. 2A). Similarly, by treating L929S cells with TGF-beta2 or -beta3 for 30 min, an increase in the tyrosine phosphorylation by approximately 30-50% was also observed. Both TGF-beta2 and -beta3 decreased the phosphorylation in L929R cells by approximately 15-25% in 0.5-1 h. As determined by SDS-PAGE and autoradiography, both lavendustin A and tyrphostin (10 µM) blocked the TGF-beta1-increased phosphorylation by 60 and 56%, respectively (Fig. 2B). In addition, both inhibitors concurrently restricted phosphorylation of 9 specific protein species, which were p36, p38, p44, p63, p105, p119, p138, p174, and p260 (Fig. 2B).


Figure 2: Effect of TGF-beta1 on protein-tyrosine phosphorylation. L929S or L929R cells were treated with TGF-beta1 (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-beta1 for 0.5-2 h. -, control cells; +, TGF-beta1-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-beta1-treated cells, divided by the density of the control. B, L929S cells were metabolically labeled with [S]methionine and treated with TGF-beta1 (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.



Effect of Protein Tyrosine Kinase Inhibitors on TGF-beta1-induced TNF-resistance

Pretreatment of L929S with both TGF-beta1 (2 ng/ml) and protein tyrosine kinase inhibitors, such as tyrphostin, lavendustin A, BABA, and genistein at 10 µM for 6 h, resulted in reduced TNF-resistance in these cells (Fig. 3A). Similar results were obtained by increasing the concentrations of protein tyrosine kinase inhibitors up to a non-cytotoxic concentration of 30 µM, or by treating L929S cells with both TGF-beta1 and protein tyrosine kinase inhibitors for 1-5 h (data not shown). However, when L929S cells were pretreated with TGF-beta1 for 1 h followed by coincubation with protein tyrosine kinase inhibitors for 6 h, the acquired TNF-resistance was not abolished (Fig. 3B).


Figure 3: Effect of protein kinase inhibitors on reduction of TGF-beta1-induced TNF-resistance. A, L929S cells were pretreated with TGF-beta1 (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-alpha/Act D cytotoxic assays. B, L929S cells were pretreated with TGF-beta1 for 1 h, followed by coincubation with protein kinase inhibitors for 6 h and exposing to TNF-alpha/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-beta1 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-beta2 or -beta3-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-beta1 and/or tyrosine kinase inhibitors (data not shown).

TGF-beta1-induced Secretion of TRT Proteins to the Extracellular Matrix

In the following studies, evidence is presented that TGF-beta1 induced secretion of TRT protein(s) to the extracellular matrix. As an inhibitor of protein secretion(18, 19, 20) , brefeldin A inhibited the acquisition of TNF-resistance. By treating L929S cells with both TGF-beta1 and brefeldin A for 6 h prior to challenge with TNF-alpha/Act D resulted in inhibition of TGF-beta1-mediated protection of TNF cytotoxicity in a dose-dependent manner (Fig. 4). However, when L929S cells were treated with TGF-beta1 for 4 h before exposure to brefeldin A for 2 h, the cells developed TNF-resistance (Fig. 4).


Figure 4: Effect of brefeldin A on TGF-beta1 induction of TNF-resistance. A, L929S cells were pretreated with TGF-beta1 (2 ng/ml) in the presence or absence of brefeldin A for 6 h, followed by challenge with TNF-alpha/Act D for 24 h. B, L929S were pretreated with TGF-beta1 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-beta1-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-beta1-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-beta1-treated and control cells failed to secrete TRT (Fig. 5).


Figure 5: TRT protein(s) secreted in the supernatants of TGF-beta1-treated L929S cells growing under rolling conditions. L929S cells were treated with or without TGF-beta1 (1 ng/ml) and grown in roller culture for 16 h using (A) serum-free RPMI 1640 medium, (B) serum-free HyQ-CCM(2) 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-alpha/Act D (in 8 duplicates). Under serum-free conditions, TRT was not produced by TGF-beta1-treated L929S. Control, without TGF-beta1; Exp, TGF-beta1-treated.



In additional controls, adherent L929S monolayers, whether or not pretreated with TGF-beta1, 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-beta1-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-beta1-treated cells had once occupied, these control cells became TNF-resistant (Fig. 6). In this procedure, TGF-beta1-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-beta1-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-beta1 for approximately 20-30 min (Fig. 7A). In contrast, both TGF-beta2 and -beta3 failed to induce TRT secretion (Fig. 7, B and C). A prolonged treatment of L929S cells with TGF-beta2 and -beta3 for 4-16 h also failed to observe the TRT activity (less than 10%).


Figure 6: TGF-beta1 induced secretion of TRT protein(s) to the extracellular matrix. L929S monolayers were pretreated with or without TGF-beta1 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-beta1-treated cells but not with the untreated controls. Data were obtained from quadruplicate experiments.




Figure 7: Time course induction of TRT by TGF-beta1, but not by TGF-beta2 and -beta3. L929S cells in 96-well microtiter plates were pretreated with TGF-beta1 (A), -beta2 (B), or -beta3 (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-beta1. When L929S monolayers were coincubated with both TGF-beta1 (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-beta1 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-beta1-treated L929R cells had once resided, the L929S cells did not acquire TNF-resistance.

Characteristics of TRT Protein(s)

To determine whether blocking of TNF function by TRT was due to binding interactions, I have examined the binding of TNF-alpha to extracellular matrix proteins by ELISA. Compared to controls, there were no significant increases (p > 0.05; Student's t test) in the binding of TNF-alpha to the plastic adherent extracellular matrix proteins from TGF-beta1-treated L929S cells, or to the formaldehyde-fixed L929S cells pretreated with TGF-beta1.

TRT was shown to be relatively stable. As summarized in the Table 1, TRT resisted treatments with heat, mild trypsinization, collagenase, heparin, and alpha(2)-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-beta1 or cellularly secreted TGF-betas adhered to plastic wells that caused L929S resistance to TNF. L929S cells in roller culture were treated with or without TGF-beta1 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-beta1. These antibodies failed to abolish the TRT activity (Fig. 8A). Similarly, the TRT activity was not blocked by another antibody, anti-TGF-betas (beta1, beta2, and beta3) IgG. TRT proteins in the extracellular matrix were prepared by removing control or TGF-beta1-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-betas could not inhibit the TRT activity. A, culture supernatants from control or TGF-beta1-treated L929S (roller culture) were precoated onto microtiter plates for 3 h followed by treating the adherent proteins with antibodies against TGF-beta1 (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-alpha/Act D (experiments in quadruplicates). B, L929S monolayers were pretreated with or without TGF-beta1 for 16 h followed by removing cells with trypsin/EDTA. TRT activity in the extracellular matrix was not neutralized by the anti-TGF-betas 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.



TRT Promoted TNF-resistance by Increasing Protein Kinase Activation

Evidence was obtained that TRT triggered L929S resistant to TNF by increasing protein phosphorylation in these cells. For example, control L929S cells become TNF-resistant when seeded onto the TRT-containing wells of microtiter plates (Fig. 9A). Inhibitors of protein kinases, such as lavendustin A, BABA, and H7, blocked this induction of TNF-resistance (Fig. 9A). In controls (without TGF-beta1 treatment), no TRT activity was found and the kinase inhibitors alone also failed to induce the production of TRT (Fig. 9A). Moreover, by metabolic labeling and immunoprecipitation, L929S cells, when seeded onto TRT-containing Petri dishes for 3 h, had a 44.7% increase in protein-tyrosine phosphorylation which was significantly inhibited by lavendustin A (Fig. 9B). The increase in phosphorylation could go up to 100% after 16 h culturing of L929S cells on the TRT-containing matrix.


Figure 9: Blocking of TRT-mediated TNF-resistance by protein kinase inhibitors. A, culture supernatants from TGF-beta1-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-alpha/Act D. B, L929S monolayers were pretreated with or without TGF-beta1 (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.




DISCUSSION

In this study we have examined the mechanisms by which TGF-beta1 induces TNF-resistance in L929S cells. Our results demonstrate that both an early TGF-beta1-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-beta1-induction of TNF-resistance, as evidenced by the failure of staurosporine in restricting the induced resistance in L929S cells.

In addition to TGF-beta1, it is determined that both TGF-beta2 and -beta3 stimulate TNF-resistance in L929S cells. Time course studies revealed that TGF-beta2 and -beta3 could rapidly induce TNF-resistance faster than that of TGF-beta1. However, TGF-beta1 exerts a greater extent of protection against TNF-alpha than TGF-beta2 and -beta3 (60-70 versus 40-50%). Most interestingly, L929S cells secreted little or no TRT by stimulating with either TGF-beta2 or -beta3. These observations suggest that the reduced efficiency for TGF-beta2 and -beta3 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-beta1, TGF-beta2 and -beta3 fail to induce TRT secretion, suggesting an undefined difference in the signal transduction pathways among these molecules. TGF-beta 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-beta1 and -beta3 than for TGF-beta2(27) , such differences in the binding probably could not account for the failure of TGF-beta2 and -beta3 to induce TRT secretion. The type I receptors have been shown to specify growth inhibitory and transcriptional responses to TGF-beta and activin in Mv1Lu lung epithelial cells(28) . Notably, TGF-beta1 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-beta receptor kinases. This hypothesis is supported by the observation that tyrosine kinase inhibitors restricted TGF-beta-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-beta1 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-betas. Perhaps, TGF-betas 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-beta1-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-beta1 in the stimulation of L929S cells. In contrast, under serum-free conditions L929S monolayers still acquire TNF-resistance upon treatment with TGF-beta1. Thus, in addition to TRT, TGF-beta1 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-beta1 for less than 1 h, and transfection of L929S cells with TR1 cDNA renders the cells TNF-resistant. (^2)

Unlike the rapid increase in tyrosine phosphorylation mediated by TGF-beta1, 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-beta1 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 alpha(2)-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-betas, as determined by gas-phase microsequencing.^2

TGF-beta1-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-beta1 were found to bind TNF-alpha 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-beta1-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.


FOOTNOTES

*
This work was supported in part by the Guthrie Foundation for Medical Research, the Wendy Will Case Cancer Fund, and National Institutes of Health grant 1R01CA-61879. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: TGF-betas, transforming growth factor-beta types 1, 2, and 3; TNF, tumor necrosis factor; TNF-beta, tumor necrosis factor-beta (lymphotoxin); BABA, 2-hydroxy-5-(2,5-dihydroxybenzyl)aminobenzoic acid; L929S, a TNF-sensitive murine L929 fibrosarcoma cell line; L929R, a subline of L929S resistant to TNF cytotoxicity; ActD, actinomycin D; TRT, TNF-resistance triggering protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.

(^2)
N-S. Chang, manuscript in preparation.


ACKNOWLEDGEMENTS

I thank Dr. D. Beezhold for providing the L929 cell lines, Drs. R. S. Aronstam and D. A. Kostyal for critical review and discussions, and M. Ou, E. Chu, T. Dinh, and J. Mattison for technical assistance.


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