©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of Reactive Oxygen Species in Cytokine and Growth Factor Induction of c-fos Expression in Chondrocytes(*)

Yvonne Y. C. Lo , Tony F. Cruz (§)

From the (1) Connective Tissue Research Group, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5 and the Department of Cellular and Molecular Pathology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cytokine tumor necrosis factor (TNF) and the growth factor basic fibroblast growth factor (bFGF) are known to induce early response genes such as c-fos and c-jun in various cell types. Activation of AP-1, a heterodimeric complex of Fos and Jun proteins, is required for matrix metalloproteinase production and cell proliferation. However, the signaling pathways by which these two factors influence the expression and activities of AP-1 remain currently poorly characterized. Several studies have shown that cytokines induce reactive oxygen species (ROS) production, but growth factor induction of ROS has not been reported. In the present study we demonstrate that both TNF and bFGF induce ROS production, and that this is a common signaling event involved in the stimulation of c-fos gene expression in chondrocytes. To our knowledge, this is the first report directly demonstrating ROS production upon stimulation with a growth factor. TNF and bFGF induction of ROS production is mediated through flavonoid-containing enzymes such as NADPH oxidase. Moreover, the ROS nitric oxide is not responsible for the induction of c-fos expression by TNF and bFGF. In addition, the inhibitory effects of antioxidants on c-fos expression may account for their protective roles against proliferative and inflammatory diseases such as cancer, cardiovascular diseases, and arthritis.


INTRODUCTION

Tumor necrosis factor (TNF)() is a polypeptide hormone originally identified as a mediator of hemorrhagic necrosis of tumors in bacillus Calmette-Guerin-infected mice (1) . It is synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators, or cytokines such as interleukin-1 (2) . TNF has been shown to elicit a wide variety of biological effects. It may be mitogenic, cytostatic, or cytotoxic, depending on the cell type and the growth state of the cells (1, 3, 4, 5, 6) . The molecular basis for these interesting effects on cell growth and differentiation is unknown as the intracellular mediators of TNF action are poorly characterized. Some of the known signal transduction pathways of TNF action include coupling to G proteins (7, 8) , activation of phospholipase A(4, 9, 10, 11) , and calcium mobilization (12, 13) .

The multitude of responses elicited by TNF, however, is not unique to the cytokine family of regulatory peptides. Many of the cytokine responses such as mitogenesis and differentiation are also shared by the large family of growth factors (14) . One of these, basic fibroblast growth factor (bFGF), utilizes a receptor with tyrosine kinase activity, a distinctive feature of the growth factor family (15, 16) . bFGF has potent growth and angiogenic activities (17) and is produced by various mesodermal and neuroectodermal tissues, including cartilage, brain, and retina (18, 19) . Unlike other growth factors, bFGF does not possess a signal peptide and thus does not appear to be secreted (20) . Although signaling by bFGF may involve the autophosphorylating tyrosine kinase domain of the receptor which can interact with phospholipase C- 1 (21) , a G protein-coupled phospholipase A activity has also been reported (22) . In fact, a mutant bFGF receptor that retains the tyrosine kinase activity shows diminished ability to stimulate plasminogen activator production, indicating that the receptor kinase activity is not sufficient for the full biological activity of bFGF (23) . Both TNF and bFGF are known to induce the expression of early response gene c-fos in various cell types (24, 25) . However, the downstream signaling events through which TNF and bFGF elicit the induction of c-fos expression remain to be elucidated. The protooncoproteins Fos and Jun also referred to as the activator protein-1 (AP-1) complex form homodimers (e.g. Jun-Jun) or heterodimers (e.g. Fos-Jun) and bind to the AP-1-responsive element in the regulatory domains of several genes including collagenase, stromelysin, metallothionein (26, 27) , and other cellular and viral genes involved in proliferation (28) .

Emerging evidence has pointed to the involvement of reactive oxygen species (ROS) as signaling intermediates for cytokines (29, 30) . The term ROS encompasses many species including singlet oxygen, superoxide, hydrogen peroxide (HO), nitric oxide (NO), and hydroxyl radical which are small, diffusible, and ubiquitous molecules, being produced by virtually every type of cell using diverse enzyme systems (31) . Furthermore, it has been demonstrated in several cell types, such as HeLa cells (32) and muscle osteoblasts (33) , that HO can stimulate c-fos and c-jun gene expression and enhance AP-1 binding activity. Production of ROS upon growth factor activation, on the other hand, has not been reported. In the present study, we examined the involvement of ROS in TNF- and bFGF-mediated c-fos induction in a primary culture system of articular chondrocytes.


MATERIALS AND METHODS

Reagents

Recombinant human TNF and bFGF were from Sigma. HO was from Fisher Scientific. N-Acetylcysteine (NAC), ascorbic acid (Asc), and L-N-monomethylarginine (L-NMMA) were also from Sigma. S-Nitroso-N-acetylpenicillamine (SNAP) was purchased from Biomol Research Laboratories. Diphenyleneiodonium (DPI) was from Toronto Research Chemicals. Isotope was from DuPont NEN. Sulfanilamide and naphthylethylenediamine used in assaying nitrite content were from Sigma.

Cell Culture

Primary cultures of bovine articular chondrocytes were isolated from bovine articular cartilage as described in Ref. 34. The cells were plated at 2 10 cells/ml in 12 ml of Ham's F-12 media containing 3% antibiotics and 5% fetal bovine serum. The cells were allowed to recover for 24 h at 37 °C in a humidified atmosphere supplemented with 5% CO.

Northern Blot Analysis

Total RNA was isolated by the acidified guanidine isothiocyanate method (35) and subjected to electrophoresis on a denaturing gel. Denatured RNA samples (12 µg) were analyzed by gel electrophoresis in a denaturing 1% agarose gel, transferred to a nylon membrane (Bio-Rad), cross-linked with an ultraviolet cross-linker (Stratagene UV Stratalinker 1800), and hybridized with P-labeled rat c-fos cDNA. The blots were subsequently stripped and reprobed with P-labeled rat tubulin cDNA.

Fluorescence-activated Cell Sorting (FACS) Analysis

Chondrocytes were treated with TNF (30 ng/ml) or bFGF (10 ng/ml) for 4 h in the presence of dihydrorhodamine 123 (DHR) (2 µM) (Molecular Probes) with or without DPI (2 µM). Both DHR and DPI were dissolved in dimethyl sulfoxide. During the cellular production of ROS, the intracellular DHR was irreversibly converted to the green fluorescent compound rhodamine 123 (R123) (500-540 nm). R123 was membrane-impermeable and accumulated in the cells. Chondrocytes were fixed for 20 min in 1.5% paraformaldehyde (Sigma) and the cellular R123 fluorescence intensity of 5000 chondrocytes was measured for each sample by flow cytometry using a fluorescein isothiocyanate argon laser with the excitation source at 488 nm (Coulter Epics C flow cytometer) (36).

Measurement of NO Production

NO production was measured as the amount of nitrite, the stable end product of NO, released into the culture supernatant. Nitrite concentration was determined in cell-free culture supernatants using the spectrophotometric method based on the Griess reaction (37) . Briefly, samples were reacted in equal volume with 1% sulfanilamide, 0.1% naphthylethylenediamine (Sigma), and 5% phosphoric acid at room temperature for 10 min. The nitrite concentration was determined by absorbance at 540 nm in comparison with standard solutions of sodium nitrite prepared in the same medium.

RESULTS AND DISCUSSION

In the current study, we examined the effects of TNF and bFGF on ROS production and its relationship with c-fos expression in chondrocytes. Three criteria were utilized to assess the relevance of ROS as putative second messengers in the induction of c-fos by the two factors: (i) addition of ROS should mimic the inducers' biological effects, (ii) decreasing ROS production or inactivating ROS should inhibit the induction of c-fos expression by TNF and bFGF, and finally (iii) TNF and bFGF should stimulate ROS production. Hydrogen peroxide, a membrane-permeable reagent physiologically produced in large amounts by granulocytes and macrophages during inflammatory processes (31, 38, 39) , has been widely used to assess the effects of ROS. HO is capable of inducing c-fos(32, 33) and DNA synthesis (40) and activating the transcription factor NFB in various systems (41) . We therefore first examined the effect of HO on c-fos mRNA expression in primary cultures of bovine articular chondrocytes. Cells were treated with 100 µM hydrogen peroxide for various time points, and c-fos mRNA levels were determined by Northern blot analysis. As shown in Fig. 1A, we found that HO increased c-fos mRNA levels in chondrocytes optimally at 30 min and levels decreased to base-line levels by 2 h. These results are consistent with reactive oxygen species involvement in c-fos induction. Similar transient increases in c-fos mRNA levels were observed with TNF and bFGF (data not shown).


Figure 1: Effects of HO and antioxidants on c-fos expression. A, effect of HO on c-fos mRNA levels. Chondrocyte cultures were stimulated with HO (100 µM) at different time points as indicated. B, the antioxidants NAC and Asc inhibit TNF- and bFGF-induced c-fos mRNA levels. Chondrocyte cultures were preincubated with NAC (30 mM) or Asc (100 µM) for 2 h before the addition of bFGF (10 ng/ml) or TNF (30 ng/ml) for 30 min. Both human recombinant TNF and bFGF were dissolved in phosphate-buffered saline with 0.1% bovine serum albumin. NAC and Asc were first dissolved in Ham's F-12 medium containing 5% (v/v) fetal bovine serum, then neutralized with sodium hydroxide. Total RNA from bovine articular chondrocytes was isolated, and the c-fos mRNA levels were determined by Northern blot analysis as described under ``Materials and Methods.'' The blots were subsequently stripped of DNA and reprobed with P-labeled rat tubulin cDNA.



We next determined the effects of two antioxidants, NAC and Asc, on the induction of c-fos expression. NAC and Asc are effective free radical scavengers; the former is known to increase intracellular glutathione levels, which in turn control the concentration of ROS within cells via glutathione peroxidase (31) . On the other hand, Asc itself is highly reactive toward radicals and proven to be a versatile scavenger (42) . Fig. 1B demonstrates that TNF (30 ng/ml) and bFGF (10 ng/ml) increased c-fos mRNA levels in chondrocytes. However, the addition of antioxidants NAC (30 mM) and Asc (100 µM) 2 h prior to stimulation with TNF and bFGF attenuated the induction of c-fos mRNA levels. NAC was more effective than Asc in reducing the induction of c-fos mRNA expression. Both antioxidants did not affect the mRNA levels of the housekeeping gene tubulin. Taken together, these results suggest that decreasing ROS levels by antioxidants suppresses TNF and bFGF induction of c-fos expression in chondrocytes.

Cytokines such as TNF and interleukin 1 have been shown to stimulate HO production by fibroblasts and chondrocytes (29, 30); however, the effects of growth factors on ROS production in cells have not been reported. To directly determine whether TNF and bFGF induce ROS production, chondrocyte cultures loaded with DHR (2 µM) were stimulated with TNF or bFGF and then examined by FACS analysis. Although c-fos expression was induced at 30 min, ROS production induced by either TNF and bFGF or the ROS HO itself could not be detected by FACS analysis at this time point (data not shown), probably due to experimental limitations of the assay. However, both TNF and bFGF stimulated ROS production in chondrocytes after a 4-h incubation as shown by a shift in the logarithmic fluorescence intensity to the right (Fig. 2, d and f) as compared to constitutive ROS production (Fig. 2c). These data provide direct evidence of ROS production by chondrocytes after stimulation with both TNF and bFGF. One class of enzymes that are known to give rise to various types of ROS is the flavonoid-containing enzymes. Therefore, we examined the effects of DPI, a potent inhibitor of flavonoid-containing enzymes such as NADPH oxidase and nitric oxide synthase (NOS) (43) , on TNF and bFGF induction of ROS production. Pretreatment of chondrocytes with DPI (2 µM) completely abolished the induction of ROS production as demonstrated by a backward shift of green fluorescence to that of basal levels (Fig. 2, e and g). The inhibition by DPI of both TNF- and bFGF-induced ROS production indicates a role for flavonoid-containing enzymes in the induction process. Although further research is necessary to elucidate the mechanisms by which these factors stimulate ROS production, it is possible that increases in arachidonic acid or its metabolites in response to cytokines and growth factors (22, 44, 45) may serve as intermediates in the activation of enzymes such as NADPH oxidase (46, 47) .


Figure 2: DPI inhibits TNF- and bFGF-induced ROS production in chondrocytes. With time, DHR by itself caused a shift in fluorescence to the right as shown in panels a-c. A dottedline was drawn through the mean fluorescence intensity of the control (panel c) with DHR alone for 4 h. After incubation with TNF or bFGF for 4 h in the presence of DHR, there was an additional shift in logarithmic fluorescence intensity as indicated in panels d and f. In panels e and g, DPI abolished the fluorescent shift stimulated by TNF and bFGF, respectively.



Next, the effects of DPI on TNF- and bFGF-induced c-fos mRNA levels were examined by Northern blot analysis. Pretreatment of chondrocytes with DPI significantly decreased TNF and bFGF induction of c-fos mRNA levels (Fig. 3). Under identical conditions, tubulin mRNA levels were not altered by DPI. These data indicate that flavonoid-containing enzymes are involved in the regulation of c-fos expression mediated by TNF and bFGF in chondrocytes. The concomitant inhibitory effects of DPI on ROS release and on induction of c-fos support the involvement of ROS as a shared mechanism by which TNF and bFGF induce c-fos expression.


Figure 3: Diphenyleneiodonium also inhibits the induction of c-fos expression by TNF and bFGF. Chondrocyte cultures were pretreated with DPI (2 µM) for 30 min before the addition of TNF (30 ng/ml) or bFGF (10 ng/ml) for 30 min. Measurements of c-fos and tubulin mRNA levels were as described under Fig. 1.



TNF has been shown to stimulate nitric oxide (NO) production in a variety of cell types (48, 49) . The free radical NO has received increasing recognition as an important physiologic messenger molecule with regulatory roles in the nervous, immune, and cardiovascular systems (50) . Thus, we investigated the capacity of bovine chondrocytes to produce NO and the potential influences of NO, if any, on c-fos expression (Fig. 4). TNF was found to increase NO production by 11-fold when compared with that of the control (Fig. 4A). As expected, the NOS inhibitor L-NMMA blocked the stimulation of NO production by 75% (Fig. 4A). In contrast, bFGF did not induce NO production in chondrocytes; thus, NO is not involved in bFGF induction of c-fos expression. Since TNF increased NO production, we examined the effect of L-NMMA on TNF-induced c-fos mRNA levels (Fig. 4B). The data demonstrated that L-NMMA did not alter constitutive or TNF- or bFGF-induced c-fos mRNA expression. In summary, NO does not appear to be involved in the induction of c-fos expression by cytokines or growth factors. Furthermore, the inability of increased levels of extracellular NO using SNAP to induce c-fos expression provides further evidence that NO production is not involved in the regulation of c-fos mRNA expression in chondrocytes. However, we could not exclude the possibility that NO released as the result of TNF stimulation may participate in other signaling processes important in regulating the expression of other genes. Moreover, abnormal levels of NO production in response to heightened cytokine secretion may also result in cytotoxicity and tissue damage, a process implicated in some pathological situations (50, 51) .


Figure 4: Role of NO in regulation of c-fos expression. A, effects of TNF and bFGF on nitric oxide production. Chondrocytes were first treated with 250 µML-NMMA 2 h prior to the addition of either TNF (30 ng/ml) or bFGF (10 ng/ml). After 72 h of incubation, nitrite content was determined as described under ``Materials and Methods.'' Values shown are means (± S.E.) of three independent experiments, each done in triplicate. B, L-NMMA has no effect on TNF- and bFGF-induced c-fos mRNA levels. Chondrocyte cultures were first incubated with L-NMMA (250 µM) for 2 h before adding TNF (30 ng/ml) or bFGF (10 ng/ml). After 30 min, total RNA was isolated and c-fos mRNA levels were determined as described in Fig. 1. C, the organic NO donor, SNAP, cannot induce c-fos mRNA levels. Chondrocytes were stimulated with 100 µM SNAP for the time periods as indicated. HO (lane 2) was used as a positive control in this experiment.



Although cytokine and growth factor stimulation of c-fos transcription is thought to be mediated by mitogen-activated protein (MAP) kinase phosphorylation of the transcription factor ELK-1/TCF (52-54), a second messenger common to both factors in this early gene response has not been identified. In our study, we demonstrated that cytokine and growth factor stimulation of ROS production by flavonoid-containing enzymes is a common signaling mechanism involved in the induction of c-fos expression in chondrocytes. More important, this is the first report directly demonstrating ROS production upon stimulation with a growth factor. Recently, it was demonstrated that ROS produced by HO or ionizing irradiation are capable of stimulating MAP kinases (55, 56) . Therefore, it is conceivable that ROS induction of c-fos mRNA levels occurs through activation of MAP kinases, which phosphorylate and thus activate transcription factors such as ELK-1/TCF, which in turn regulate c-fos promoter activity (52-54). Although ROS production may be a component of normal signal transduction in many cell types, it is likely that abnormal production of ROS stimulated by elevated levels of cytokines and growth factors may inappropriately activate early response genes such as c-fos and c-jun leading to the overexpression of metalloproteinases and uncontrolled cell proliferation. The inhibition of TNF- and bFGF-induced c-fos mRNA levels by antioxidants also led us to hypothesize that antioxidants may prove useful to impede disease progress by down-regulating AP-1 and their responsive genes such as metalloproteinases. Hence, antioxidant inhibition of c-fos expression may serve to explain, at least in part, the ability of antioxidant mixtures to improve health conditions in patients with diabetes, arthritis, hypertension, and other age-related diseases (57) .


FOOTNOTES

*
This work was supported by the Medical Research Council of Canada. 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.

§
To whom correspondence should be addressed: Rm. 992B, 600 University Ave., Toronto, Ontario M5G 1X5, Canada. Tel.: 416-586-8537; Fax: 416-586-8628.

The abbreviations used are: TNF, tumor necrosis factor ; bFGF, basic fibroblast growth factor; AP-1, activator protein-1; ROS, reactive oxygen species; NAC, N-acetylcysteine; Asc, ascorbic acid; L-NMMA, L-N-monomethylarginine; SNAP, S-nitroso-N-acetylpenicillamine; DPI, diphenyleneiodonium; FACS, fluorescence-activated cell sorting; DHR, dihydrorhodamine 123; R123, rhodamine 123; NOS, nitric oxide synthase; MAP, mitogen-activated protein.


ACKNOWLEDGEMENTS

We thank A. Bansil for technical assistance with the FACS analysis, and M. Breitman for the gift of c-fos and -tubulin cDNAs.


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