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Immunology |
Radiation Biology Branch [Y. V., W. D., D. A. W.], and Laboratory of Cell Regulation and Carcinogenesis [L. C., S-J. K., B. R.], National Cancer Institute, Bethesda, Maryland 20892; Life Sciences Division, Lawrence Berkeley Laboratory, Berkeley, California 94720 [H. C., M. H. B-H.]; Laboratory of Neurotoxicology, National Institute of Mental Health, Bethesda, Maryland 20892 [J. T. S.]; and Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 [G. W. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-ß1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-ß1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-ß1. A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-ß1 in a time- and dose-dependent fashion at 24–48 h after treatment. Exposure of latent TGF-ß1 to solution sources of NO neither led to recombinant latent TGF-ß1 activation nor modified recombinant TGF-ß1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-ß1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-ß1 and raise the possibility that NO production may augment TGF-ß1 activity by modifying a naturally occurring neutralizing peptide. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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plus LPS3
or other agents produce superoxide via the activity of NADPH:ubiquinone oxidoreductase (NADPH oxidase) (1)
, whereas NO is produced by NOS2 (3)
. NO released from activated macrophages (5
, 6)
or from chemical donors (7, 8, 9, 10, 11)
can suppress the proliferation of tumor cells. This effect is due, at least in part, to inhibition of mitochondrial respiration (5)
, DNA synthesis (12
, 13)
, and iron metabolism (14)
. NO also triggers apoptosis in tumor cells (15)
, stimulates their differentiation (7, 8, 9, 10)
, and may block metastasis by reducing the adhesion of tumor cells to blood vessels (4)
. Additionally, coculture of macrophages and tumor cells has been shown to lead to increased production of NO by the macrophages and concomitant cytotoxicity toward the tumor cells (16
, 17)
. It has been hypothesized that the production of superoxide and/or NO by host macrophages may inhibit tumor progression and metastasis (4 , 18 , 19) . If this were true, agents that suppress the expression or activity of NOS2 or NADPH oxidase might be reasonable targets of intervention in cancer therapy. The TGF-ßs constitute a family of three highly homologous isoforms that together exert pleiotropic effects on different cell types (20 , 21) , including the suppression of proliferation and function of many cells of the immune system (22 , 23) . The most prominent isoform, TGF-ß1, suppresses the production of both superoxide (24) and NO (25) by macrophages. The role of TGF-ß1 in tumor biology is complex, but it has been implicated in the progression of certain tumors (20 , 21) .
We have focused our attention on the interaction between TGF-ß1 and NOS2. TGF-ß1 is a potent suppressor of the expression of NOS2 by multiple mechanisms in numerous cell types, including macrophages (25) . This suppression has relevance in vivo in that TGF-ß1 null mice exhibit spontaneously elevated expression of NOS2 mRNA and protein as well as augmented systemic levels of metabolites of NO (26) . In contrast, systemic and macrophage NO production stimulated by i.p. administration of LPS is suppressed in transgenic mice overexpressing TGF-ß1 (27) .
For TGF-ß1 to exert these effects on the expression and/or activity of NOS2, it must first be activated, because TGF-ß1 is secreted in a latent form (28) . Several studies have demonstrated that activated macrophages in culture convert latent TGF-ß1 to an active status after exposure to cytokines similar to those that induce NO production (29, 30, 31, 32, 33) . One possible mechanism of activation is through reactive oxygen production by macrophages because recombinant latent TGF-ß1 is efficiently activated by exposure to solution sources of reactive oxygen (34) . Additional sources of oxidants like hemin and nitroxyl anion are also effective in eliciting activation.4 With appropriate temporal regulation, one could envisage the possibility of negative feedback control if activated macrophage production of reactive oxygen species and NO are both regulated by TGF-ß1 and in turn mediate the activation of latent TGF-ß1. Because the biological spectrum of action of nitrosative stress congeners is often different from that induced by chemical oxidants (35) , we investigated whether elevated concentrations of NO could affect the production or activity of TGF-ß1 in A549 lung adenocarcinoma cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Coculture of A549 and ANA-1 Cells.
The human lung adenocarcinoma cell line A549 was obtained from the American Culture Collection (Rockville, MD) and was cultured as recommended in DMEM (high glucose; Life Technology, Gaithersburg, MD) supplemented with 10% FCS and 10,000 units/ml penicillin and 10,000 mg/ml streptomycin, except where indicated. The mouse macrophage cell line ANA-1 was established and cultured as described previously (36)
. A549 cells were cocultured with activated ANA-1 macrophages as follows. ANA-1 macrophages (106/ml; 10 ml total) were cultured in loosely capped, 50-ml conical bottomed polypropylene centrifuge tubes for 18–24 h with medium only (supplemented as above), 100 units/ml IFN-, 10 µg/ml LPS, or IFN- plus LPS. These treatments were shown previously to induce high-level expression of NOS2 and tumoricidal activity in ANA-1 cells (37)
. After treatment, supernatant was collected for nitrite assay, and cells were washed twice with HBSS and resuspended to 2 x 106 cells/ml in DMEM containing 0.5% FCS, antibiotics (as above), and either 0.5 mM L-NMA or D-NMA. The macrophages (106/well) were then added to individual wells of a 24-well plate in a total volume of 1 ml/well in the absence or presence of 5 x 105 A549 cells.
Treatments of A549 Cells with Chemical NO Donors.
The human lung adenocarcinoma cell line A549 was obtained from the American Type Culture Collection and was cultured as recommended in DMEM (high glucose; Life Technologies, Inc.) supplemented with 10% FCS and 10,000 units/ml penicillin and 10,000 mg/ml streptomycin, except where indicated. The chemical NO donor DEANO was prepared in NaOH. The negative controls for DEANO were either PBS or expired (released) DEANO. Expired DEANO was prepared by diluting the stock of DEANO to 1 mM in culture medium containing 10 mM HEPES for 4 h. The reagents were added to cells at concentrations of 0.1 or 1 mM for up to 48 h in normal culture conditions.
Nitrite Assay.
An aliquot of the conditioned medium was removed for assay of NO-2 content by the Griess reaction (38, 39, 40)
. Briefly, 100 µl of Griess reagent (38, 39, 40)
were added to 100 µl of each supernatant in triplicate. The plates were read using either a Vmax ELISA plate reader (Molecular Devices, Menlo Park, CA) or a MR5000 ELISA plate reader (Dynatech, Chantilly, VA) at 550 nm against a standard curve of NaNO2.
Determination of Secreted TGF-ß1 Concentrations.
TGF-ß1 concentration in conditioned media in experiments in which A549 cells were treated with DEANO was determined using the PAI-L mink lung epithelial cells transfected with a PAI-luciferase construct (41)
, generously provided by D. Rifkin (New York University Medical Center, New York, NY). The cells were seeded in a 96-well plate in 0.5% serum containing media and incubated for 3 h. Samples of conditioned media collected from various cell lines were added to the cells in three serial dilution concentrations, using 0.2% fetal bovine serum/DMEM as the diluent. After a 14–19-h incubation with the sample conditioned media, the cells were washed and lysed, and luciferin substrate (Promega, Madison, WI) was injected to generate luminescence, which was recorded as relative light units produced from the reaction (EG&G Berthold Microlumat LB 96P, Oak Ridge, TN). Recombinant active TGF-ß1 standard curves were performed with each experiment. Latent TGF-ß1 present in the sample-conditioned medium was determined after heat activation at 80°C for 5 min (28)
.
In Vitro Treatments of Latent or Active TGF-ß1 and Determination of Bioactivity.
The bioactivity of TGF-ß1 after in vitro treatments with NO (see below) was assayed by its antiproliferative effect on CCL-64 mink lung epithelial cells as described previously (42)
. Carrier-free latent TGF-ß1 (10 mg/ml in 1 mM HEPES) or active TGF-ß1 was treated with 0.01–1 mM DEANO 30–60 min h at 37°C. The TGF-ß1 was diluted in a dose curve from 0.002 to 2 ng/ml in 1 mM HEPES and added to CCL-64 cells for 20–22 h. The cells were pulsed with [3H]thymidine for 2 h and then processed as described (42)
.
Immunocytochemistry.
Determination of latent versus active TGF-ß1 on cells was carried out by a modification of a method described previously (33
, 43)
. A549 cells (1 x 105) were seeded in 24-well plates in 1 ml of DMEM + 10% FCS and then treated with 0.01–1 mM DEANO. The cultures were processed after a 48-h incubation as follows. The cells were washed three times with PBS, fixed in 4% paraformaldehyde, washed with PBS, and permeabilized with 0.2% Triton X-100 in PBS. Anti-LAP antibody was used for the immunodetection of latent TGF-ß1 on cell cultures. The antibody was produced in goat immunized to recombinant human LAP (43)
and used at 50 µg/ml. LC (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
, a polyclonal antibody derived from rabbit antiserum raised to the NH2-terminal 30 amino acids of the mature TGF-ß1 (44)
, was used at 10 µg/ml for the detection of active TGF-ß1 (43)
. Affinity-purified chicken anti-TGF-ß1 antibody was provided by Dr. Monica Tsang (R&D Systems, Minneapolis, MN) and was used at 15 µg/ml (33
, 45)
. Rabbit anti-goat IgG labeled with fluorescein (Southern Biotechnology Associates, Inc., Birmingham, AL) was used at a concentration of 4 µg/ml for the detection of anti-LAP. Texas Red-labeled goat-anti-chicken IgG secondary antibody (34 µg/ml) and anti-rabbit (40 µg/ml) were used for the detection of active TGF-ß1 (Sigma). As above, the nuclei of the cells were counterstained with 4',6-diamidino-2-phenylindole.
Nonspecific sites were blocked by treating the cells for 1 h with blocking buffer consisting of the supernatant of 0.5% casein stirred for 1 h in PBS. After blocking, 50 µl of primary antibody diluted in blocking buffer were added to the cells and covered with Parafilm (American National Can, Greenwich, CT) coverslips. The cells were incubated overnight (18–20 h) at 4°C with primary antibody. Both primary antibodies were incubated at the same time at the appropriate dilutions. Secondary antibodies were incubated sequentially in a dark chamber for 1 h at room temperature. The cells were then washed with PBS and counterstained with 4',6-diamidino-2-phenylindole nuclear dye during the last wash. Finally, cells were mounted with Vectashield (Vector Laboratories, Palo Alto, CA) mounting medium, coverslipped, and viewed under the microscope.
Image Acquisition and Processing.
Triple fluorescence was viewed using a x40 (0.75 numerical aperture) objective on a Zeiss Axiovert microscope equipped with epifluorescence and multiband pass filters and a differential wavelength filter wheel using a scientific grade, 12-bit charge coupled device (KAF-1400, 1317 x 1035 6.8-µm square pixels) camera (Xillix, Vancouver, British Columbia, Canada). This camera has a linear response suitable for quantitative imaging. The images were captured using Scilimage software (TNO Institute of Applied Physics, Delft, the Netherlands) so that intensities for a given experiment fell within the 12-bit linear range. Relative intensity was maintained when constructing figures by scaling the data set to a common 8-bit scale. Internal standardization was achieved by comparing only images stained with the same antibodies in the same immunostaining procedure and captured with identical parameters and at identical times.
Detection of Nitrosothiols.
LAP was treated with a saturated solution of NO in PBS for 30 min. The presence of protein nitrosothiols was then assayed as described previously (46)
.
Statistical Analysis.
All values are presented as mean ± SE, unless indicated otherwise. Statistical comparisons among multiple groups were carried out using one-way ANOVA, followed by the Bonferroni post-hoc test. Statistical significance was determined at the 95% confidence levels (P < 0.05).
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RESULTS |
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By immunocytochemistry, both A549 tumor cells (Fig. 2A)
and ANA-1 cells cultured alone without stimulation with IFN- + LPS (Fig. 1B)
expressed predominantly latent TGF-ß1, visualized as green fluorescence. Upon stimulation with IFN- and LPS, ANA-1 cells expressed predominantly active TGF-ß1, visualized as either red or orange fluorescence (Fig. 1C)
. When A549 cells were cultured with unstimulated macrophages, no conversion to the active form was observed in either cell type (data not shown). However, dramatically higher expression of active TGF-ß1 was observed when ANA-1 cells were activated prior to coculture (Fig. 1D)
. As a control, omission of the primary antibody resulted in little spontaneous fluorescence (Fig. 1A)
.
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. Very little NO-2 was detected in the cultures of unstimulated ANA-1 cells or in A549 cells treated with inflammatory stimuli. Treatment of ANA-1 with IFN- + LPS for 48 h resulted in the production of high levels of NO. Coculture of ANA-1 and A549 cells resulted in reduced NO-2 levels by 50% in one experiment and 35% in another (Fig. 1F)
. These results suggested that some suppressive factor(s) was being produced in these cocultures.
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, treatment of cells with DEANO resulted in a dose-dependent induction and activation of latent TGF-ß1 detected immunocytochemically 48 h after exposure. Time course experiments demonstrated only latent TGF-ß1 30 min after exposure to 1 mM DEANO, whereas expression of active TGF-ß1 was observed by 24 h after exposure to this concentration of the NO donor (data not shown).
The immunocytochemical data presented in Fig. 2
showing a reciprocal shift in immunodetectable latent versus active TGF-ß1 and the cell-associated nature of the active TGF-ß1 are consistent with an effect of NO largely on activation and to a lesser degree on synthesis of TGF-ß1. Treatment of A549 cells with doses of DEANO ranging from 0.01 to 1 mM also did not result in any appreciable change in detection of either active (Fig. 3A)
or latent TGF-ß1 (Fig. 3B)
in the conditioned medium as compared with controls at either 24 or 48 h. Thus, NO derived from a chemical donor did not affect secretion of TGF-ß1 into the culture medium of A549 cells, which suggests that exposure to NO leads to activation of latent TGF-ß1 bound at or near the cell surface. It should be noted that cells engineered to overexpress either active or latent TGF-ß1 and which secrete their respective proteins into the culture medium at high levels also exhibit a high degree of cell-associated immunostaining (33)
.
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suggested a long-term, perhaps indirect, effect of NO on activation of latent TGF-ß1. Nonetheless, one possible mechanism by which TGF-ß1 could be activated might be a direct one, as has been reported for reactive oxygen species (34)
, hemin, and nitroxyl anion.4
Latent TGF-ß1 did not demonstrate significant biological activity at doses ranging from 0.002 to 2 ng/ml, although suppression of up to 20% of proliferation of CCL-64 cells was observed at the highest doses (Fig. 4A)
. Treatment of latent TGF-ß1 with 0.01 or 1 mM DEANO did not result in activation of latent TGF-ß1 as assessed by decreased proliferation of CCL-64 cells (Fig. 4A)
. We next examined whether NO would alter the activity of recombinant active TGF-ß1. As seen in Fig. 4B
, DEANO at 0.01 or 1 mM had no effect on the bioactivity of active TGF-ß1. The ability of NO to suppress the proliferation of numerous cell types (3)
raises the potential artifactual situation in which DEANO may have suppressed the activity of TGF-ß1 in this last experiment, but that this effect was masked by an antiproliferative effect of NO. However, treatment of CCL-64 cells with DEANO alone at either 0.01 or 1 mM did not affect the proliferation of CCL-64 cells (Fig. 4B)
.
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, a 3-fold molar excess of LAP reduced the bioactivity of 2 ng/ml of TGF-ß1 by half after a 10-min incubation (compare 
and 
columns). Simultaneous incubation of active TGF-ß1, LAP, and 1 mM DEANO did not alter the capacity of LAP to neutralize active TGF-ß1 (Fig. 5
, compare and 
columns). However, preincubation of LAP with 1 mM DEANO for 30 min, followed by the addition of active TGF-ß1, resulted in reduced neutralizing capacity of LAP (Fig. 5
, compare and 
columns). These findings suggest that latent TGF-ß1 forms more rapidly than the rate of reaction of NO with LAP and are in agreement with the inability of NO to activate preformed latent TGF-ß1. However, these findings suggest that in the open configuration, i.e., not associated with TGF-ß1, certain sites on LAP are susceptible to nitrosylation.
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and suggested a biochemical reason for the ability of NO to interfere with the neutralizing capacity of LAP (Fig. 5)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-1 as well as bacterial products such as LPS (28
, 29
, 31, 32, 33)
. These same agents can induce the production of both reactive oxygen and nitrogen species (1
, 3)
. Previous reports indicate that active TGF-ß1 suppresses the production of both reactive oxygen species (24)
and NO (25)
, forming half of a negative feedback cycle. Oxidative stress, caused by reactive oxygen species (34)
, nitroxyl anion, or heme (Y. Vodovotz et al., manuscript in preparation), or nitrosative stress, as suggested by our experiments, leads to activation of latent TGF-ß1. We hypothesize that this constitutes the other half of a putative cycle that may occur in solid tumors infiltrated by host macrophages (Fig. 7)
.
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The activation of latent TGF-ß1 by tumor cells may suppress the production of NO by neighboring cells and thus contribute to tumor progression or metastasis (3 , 4) . A recent study showed that tumor cells that could activate TGF-ß1 suppressed cytokine-inducible NO production by endothelial cells, whereas those that did not activate latent TGF-ß1 did not suppress NO production (57) . Other studies have demonstrated that activation of latent TGF-ß1 by tumor cells suppresses the NO-dependent cytotoxic capacity of macrophages (57 , 60 , 61) . We found that levels of NO-2 in cocultures of A549 cells and activated macrophages were lower than in cultures of macrophages alone, which raises the possibility that some suppressive factor was produced in the cocultures; the nature of such factor(s) has not yet been determined.
Other data also support our findings. Two groups have demonstrated that NO or chemical NO donors could bring about inhibition of NOS2 mRNA and protein in macrophages (62 , 63) , whereas others observed that endogenous activation of latent TGF-ß1 suppresses NO production in activated macrophages (64) . Furthermore, the expression of NOS2 and the capacity of endothelial cells to produce NO are suppressed after treatment with spermine-NONOate or in coculture with astrocytes activated to produce NO with cytokines and LPS (65) . Taken together, these data suggest that a feedback loop may indeed exist to inhibit the action of tumor-associated macrophages. Interestingly, A549 cells treated with DEANO are not deficient in their clonogenic potential.5 This finding raises the possibility that any activation of latent TGF-ß1 concomitant with this treatment does not affect the growth of the tumor cells in vitro but instead may serve to deactivate infiltrating macrophages.
Others have described both transcriptional and posttranscriptional modulation of expression of various cytokines by NO. These cytokines include positive regulators of the expression NOS2, such as tumor necrosis factor-
(66)
, IL-1 (67, 68, 69)
, and IL-12 (70)
, as well as cytokines that suppress the expression of NOS2, such as IL-8 (71, 72, 73)
. A recent report has demonstrated the effect of NO on the promoter of the IL-8 gene by transient transfection (73)
, whereas another report described a posttranslational modification of IL-1 (69)
. In our studies, we observed the possible inactivation of LAP through nitrosylation. The induction of cytokines that enhance the expression of NOS2 (66, 67, 68, 69, 70)
occurred at much lower concentrations of NO (1–100 µM) than those required to increase the production of cytokines which suppress the expression of NOS2 (Refs. 71, 72, 73
) and this manuscript). It is therefore possible that NO has a biphasic effect on the expression of NOS2 through the modulation of distinct subsets of cytokines (Fig. 7)
. A biphasic modulation of the expression of NOS2 in macrophages treated with DEANO has indeed been reported (63)
.
The nitrosylation of LAP suppresses its known biological activity, that of neutralizing active TGF-ß1. Moreover, the recent demonstrations that nitrosylated serum albumin (74) , tissue-type plasminogen activator (75) , and hemoglobin (76) were endowed with novel biological activity raise the possibility that the modification of LAP by NO may also endow this protein with heretofore unknown biological activity. Several studies on the function of p21ras have suggested that this protein is subject to a redox regulation through modification by NO, as demonstrated by ESI (77 , 78) . Although we were able to analyze recombinant latent and active TGF-ß1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and active TGF-ß1 by ESI, only ESI is suitable for demonstrating nitrosylation. We did not observe, using mass spectrometry, any evidence of modification of active TGF-ß1 by NO. Unfortunately, attempts to analyze latent TGF-ß1 and LAP by ESI on our instrumentation proved unsuccessful.6 However, the absence of modification of active TGF-ß1 by NO, coupled with the previously mentioned inactivation of the ability of LAP to neutralize active TGF-ß1 after treatment with NO, strongly suggests a modification of LAP by NO.
The activation of latent TGF-ß1 by oxidative stress congeners may also be relevant in vivo and might occur through direct oxidative activation of TGF-ß1 LAP, an extremely efficient mechanism as compared with that mediated by plasmin (34) .4 Such a mechanism may be implicated in role of TGF-ß1 in chronic inflammatory tissue responses in addition to cancer (34) and may suggest that TGF-ß1 may be involved in the cellular response to both oxidative and nitrosative stress.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Cardiovascular Research Foundation and Medlantic Research Institute, Washington, DC 20010. 
2 To whom requests for reprints should be addressed, at Cardiovascular Research Foundation, 110 Irving St., NW, Suite 4B-1, Washington, DC 20010. Phone: (202) 877-3867; Fax: (202) 877-3339; E-mail: yxv1{at}mhg.edu
3 The abbreviations used are: LPS, bacterial lipopolysaccharide; DEANO, diethylamine-NONOate; ESI, electrospray ionization; IL, interleukin; LAP, latency-associated peptide; NOS2, inducible isoform of NO synthase; TGF, transforming growth factor.
4 Y. Vodovotz et al., manuscript in preparation.
5 W. DeGraff, D. A. Wink, and J. Mitchell, unpublished observations.
6 J. T. Simpson, Y. Vodovotz, and D. A. Wink, unpublished observations.
Received 5/28/98. Accepted 3/ 4/99.
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, latent TGF-ß1 treated with heat; •, latent TGF-ß1 treated with 0.01 mM DEANO; 
) express NOS2 and NADPH:ubiquinone oxidoreductase (NADPH oxidase). NOS2 produces NO, which in turn can react to form reactive nitrogen species (NOx). These NOx then bring about a series of reactions in the target tumor cell, which result in the activation of latent TGF-ß1. NADPH oxidase produces superoxide (ONO-2), which in turn can react to form reactive oxygen species (ROS). These ROS can activate latent TGF-ß1 directly. Active TGF-ß1 can then feed back on the macrophage to reduce the expression and/or activity of NOS2 and NADPH oxidase. Lines indicate enhancement, with solid lines indicating known reactions and dotted arrows indicating unknown reactions. Lightning bolts, suppression.