![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan
(Received September 29, 2005; Accepted January 24, 2006)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
) and has been extensively used for identifying the physiological roles of the extracellular signal-regulated kinase (ERK) 1/2. PD98059 binds to the inactive form of MAPK kinase (MEK), the kinase immediately upstream of ERK1/2, preventing its activation by Raf-1 and other upstream activators (Alessi et al., 1995). The inhibitor neither competes with ATP nor inhibits the phosphorylation of MEK, and thus is likely to have a distinct binding site on MEK. In a comparison of multiple kinase inhibitors, PD98059 appeared to be the most specific kinase inhibitor tested (Davies et al., 2000), although the inhibitor has been shown to inhibit activation of the ERK5 pathway through direct effects on MEK5 (Karihaloo et al., 2001).
We previously used PD98059 as a MEK1 inhibitor in studies of the signaling pathways responsible for the stimulation of glutathione (GSH) synthesis in response to insulin (Kim et al., 2004). In that study we found that PD98059, at a concentration commonly used for complete inhibition of ERK1/2 activation, enhanced the insulin-mediated increase in GSH levels in primary cultured rat hepatocytes. However, whether the PD98059-mediated inhibition of MEK/ERK was responsible for the elevation of GSH levels was unknown.
PD98059 is a derivative of flavone, which belongs to the chemical class of flavonoids, naturally occurring compounds found in fruits and vegetables that may exert common biological functions. Flavonoids have attracted attention as potential chemopreventive and chemotherapeutic agents in inflammatory diseases, cardiovascular diseases and cancers (Harborne and Williams, 2000; Middleton et al., 2000; Havsteen, 2002). The possible mechanisms of action of flavonoids include an increase in antioxidant capacity by directly scavenging oxidizing species and indirectly modulating drug-metabolizing enzyme activities (Middleton et al., 2000; Frei and Higdon, 2003). In fact, flavone, 2'-amino-3'-methoxyflavone (PD98059), naphthoflavone, and 3'-methoxy-4'-nitroflavone induce a G1 arrest in the cell cycle, increase Ah receptor-mediated gene expression, and activate CCAAT/enhancer-binding protein ß resulting in 
-class glutathione S-transferase (GST) induction (Reiners et al., 1999; Kang et al., 2003). However, little is known concerning the effect of flavonoids on GSH homeostasis.
GSH serves several vital intracellular functions, including detoxifying electrophiles, scavenging free radicals, maintaining the essential thiol status of proteins, and providing a reservoir for cysteine (Lu, 1998). Thus, maintenance of GSH levels is pivotal for cellular defense against oxidative injury and for cellular integrity. The maintenance of cellular GSH is a dynamic process involving its synthesis, utilization, and export to blood and bile (Lu, 1998; Ookhtens and Kaplowitz, 1998). 
-Glutamylcysteine ligase (GCL), which consists of a regulatory subunit and a catalytic subunit (GCLC), is a major determinant of GSH synthesis capacity, as it is the rate-limiting step in GSH synthesis. It has been suggested that regulation of GCLC expression is critical for GSH homeostasis (Cai et al., 1997).
The objective of this study was to determine whether MEK inhibition is responsible for the PD98059-mediated elevation of GSH levels. The MEK inhibitor U0126, which does not have a flavone structure, failed to affect GSH levels. Flavone, on the other hand, elevated GSH levels, but was without effect on ERK phosphorylation. Together, these results suggest that the PD98059-mediated elevation of GSH levels occurs via mechanism(s) unrelated to MEK inhibition. Neither PD98059 nor flavone had any significant effect on GCL activity or GCLC protein levels. In contrast, PD98059 and flavone resulted in significant elevation of cellular cysteine levels, suggesting a role of the increased cysteine levels in the PD98059- and flavone-mediated increase in GSH levels.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Primary Rat Hepatocyte Culture. Hepatocytes were isolated from the livers of male Sprague-Dawley rats (200–300 g) using collagenase perfusion as described previously (Woodcroft and Novak, 1997). Hepatocytes were plated onto dishes covalently coated with Vitrogen, and modified Chee's medium was fortified as described (Woodcroft and Novak, 1997) and supplemented with 0.1 µM dexamethasone and 1 µM insulin. Cells were plated at a density of 3 x 106 cells/60-mm dish. Four hours after plating, cells were washed with insulin-free medium several times and cultured in insulin-free medium thereafter. Cells were treated with PD98059 (0–100 µM), U0126 (0–50 µM), or flavone (0–50 µM) for up to 24 h. These chemicals inhibitors were dissolved in DMSO and the final DMSO concentration in the medium was 1 µl/ml (0.1%). This concentration of DMSO did not affect the GCLC protein level, GCL activity, or GSH level relative to untreated hepatocytes. In some instances, PD98059, U0126, or flavone was added 1.5 h before addition of insulin (10 nM) for up to 24 h. None of the chemicals resulted in increased cell toxicity, compared with untreated cells, at the concentrations used in this study. The Wayne State University Animal Investigation Committee approved all experimental procedures involving animals.
Determination of GSH and Cysteine Levels. The concentration of GSH was measured as described previously (Kim et al., 2003b). Reaction mixtures (final volume of 1 ml) contained 5 mM EDTA, 0.6 mM 5,5'-dithiobis(2-nitrobenzoic acid), 0.2 mM NADPH, 0.2 ml of supernatant, and 3 units if GSH reductase in 0.1 M potassium phosphate buffer (pH 7.5). Reaction mixtures were incubated for 4 min followed by the addition of GSH reductase to the mixtures. The rate of formation of 2-nitro-5-thiobenzoic acid was measured immediately following the addition of GSH reductase at 412 nm. None of the protein kinase inhibitors interfered with the determination of GSH. Cysteine concentration was determined using the HPLC method of Carducci et al. (1999). HPLC equipment was from Shimadze: pump, model LC-10AT; system controller, model SCL-10A; injector, Rheodyne equipped with a 20-µl loop; RF-10A fluorescence detector. Following the reduction of cysteine by 2-mercaptoethanol, cysteine was then derivatized with O-phthalaldehyde/iodoacetate and quantified using a HPLC equipped with a fluorescence detector and a 3.5-µm Kromasil C18 column (4.6 x 100 mm; Eka, Bohus, Sweden).
GCL Activity Determination. The activity of GCL was measured as described previously (Kim et al., 2003b). Reaction mixtures contained 0.1 M Tris-HCl buffer (pH 8.0), 150 mM KCl, 5 mM ATP, 2 mM phosphoenolpyruvate, 10 mM L-glutamate, 10 mM L--aminobutyrate, 20 mM MgCl2, 2 mM EDTA, 0.2 mM NADH, 17 µg of pyruvate kinase, 17 µg of lactate dehydrogenase, and 0.1 mg of protein of enzyme solution in a final volume of 1 ml. Absorbance at 340 nm was monitored for 5 min. The enzyme activity was calculated using a molar extinction coefficient of 6.3 x 102 mol–1 mm–1.
Immunoblot Analysis. Whole cell lysates were prepared as described previously (Kim et al., 2003d). For immunoblot analysis of GCLC, lysates (20 µg) were resolved by 10% SDS-PAGE, transferred to nitrocellulose (Bio-Rad, Hercules, CA), blocked in 5% milk powder in PBS-T (0.05% Tween 20 in PBS) and incubated with anti-rat GCLC (1:10,000 in 5% milk powder in PBS-T) overnight at room temperature. To determine the phosphorylation state of ERK, cell lysates were prepared by scraping cells directly into 500 µl of SDS-PAGE sample buffer (Kim et al., 2003c). Lysates (10 µl) were separated by 10% SDS-PAGE, transferred to nitrocellulose, blocked in 5% milk powder in TBS-T (0.05% Tween 20 in Tris-HCl-buffered saline) and probed with phospho-specific antibodies (1:250 in 5% bovine serum albumin in TBS-T) overnight at 4°C. Blots were stripped and reprobed with phosphorylation state-independent antibodies to ERK. Proteins were detected by enhanced chemiluminescence on Kodak X-OMAT film (Sigma Chemical) and quantified by densitometry with a Molecular Dynamics scanning laser densitometer and ImageQuant analysis program (Amersham Biosciences, Piscataway, NJ).
Statistical Analysis. Significant differences between groups were determined by analysis of variance followed by the Newman-Keuls multiple comparison test (p < 0.05). Statistical analysis was performed on three to five cell lysates from a single hepatocyte preparation. Reproducibility of results was confirmed in two to four separate hepatocyte preparations.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
43% increase in GSH levels relative to controls lacking insulin (Fig. 1, B and C), which is consistent with our previous findings (Kim et al., 2004). PD98059 pretreatment substantially augmented the insulin-mediated increase in GSH levels in a concentration-dependent manner (Fig. 1B). GSH levels in hepatocytes cultured in the absence of insulin were increased to 106, 130, 159, or 152%, relative to untreated cells, in the presence of 1, 10, 50, or 100 µM PD98059, respectively, (Fig. 1B, B1), while cells treated with PD98059 and cultured in the presence of insulin also exhibited a progressive increase in GSH levels, except at the highest concentration used in this study (Fig. 1B, B2).
Treatment of cells with U0126 had a different effect on GSH levels. In contrast to PD98059, U0126, up to 25 µM (sufficient to inhibit the insulin-mediated ERK1/2 phosphorylation), failed to increase GSH levels in hepatocytes cultured in either the presence or absence of insulin (Fig. 1C, C1 and C2). GSH levels were slightly but significantly elevated (124%, relative to untreated cells) only by the highest concentration of U0126 (50 µM) used in this study (Fig. 1C). These results suggest that PD98059, but not U0126, increases GSH levels in primary cultured rat hepatocytes, independent of the presence of insulin and independent of their inhibition of ERK1/2 phosphorylation.
We next investigated whether the flavone structure of PD98059 (Fig. 2A) might be responsible for the elevated GSH levels in response to PD98059 treatment. In the absence of insulin, GSH levels were increased to 106, 122, 167, or 200%, relative to untreated cells, in the presence of 0.1, 1, 10, or 50 µM flavone, respectively (Fig. 2B). Thus, flavone appears to be more potent than PD98059 (Fig. 1B) at increasing GSH levels in cultured hepatocytes. The presence of PD98059 (100 µM) or flavone (100 µM) in the GSH samples did not change the rate of increasing absorbance caused by reduction of 5,5'-dithiobis-2-nitrobenzoic acid (data not shown), indicating that neither PD98059 nor flavone interfered in the determination of GSH.
|
To determine whether flavone has MEK inhibitory activity, the phosphorylation of ERK1/2 was examined in hepatocytes cultured in the absence of insulin for 1.5 h following addition of 50 µM flavone, 50 µM PD98059, or 25 µM U0126 (Fig. 2C). Basal ERK1/2 phosphorylation was decreased by 50 µM PD98059 and more markedly by 25 µM U0126 but was unaffected by flavone at concentrations up to 50 µM (Fig. 2C). Insulin-mediated phosphorylation of ERK1/2 was marginally decreased (maximally 15%) by 50 µM flavone (data not shown). Thus, it is apparent that the flavone-mediated elevation of GSH levels is not associated with MEK1 inhibition. These results do not rule out the possibility that the MEK/ERK cascade modulates GSH levels in hepatocytes; however, these data do demonstrate that PD98059 produces dramatic changes in GSH homeostasis in hepatocytes through a mechanism(s) unrelated to MEK inhibition.
Effects of PD98059 and Flavone on GCLC Expression, GCL Activity, GSH Efflux, and Cellular Cysteine Levels. To investigate possible mechanisms for the GSH elevation by PD98059 and flavone, the effects of PD98059 and flavone on GCLC protein level and GCL activity were examined (Fig. 3). Treatment of hepatocytes with 10 nM insulin increased both GCLC protein level and GCL activity to 200 and 160%, respectively, relative to those in untreated hepatocytes, which is consistent with our previous findings (Kim et al., 2004). However, neither flavone, up to 50 µM, nor PD98059, up to 100 µM, altered GCLC protein levels or GCL activity in hepatocytes (Fig. 3). These data suggest that the mechanism(s) for PD98059- or flavone-induced elevation of GSH levels differs from that of insulin.
|
). The possible involvement of GSH efflux in the PD98059- or flavone-induced effect on GSH homeostasis was examined by determining the GSH concentration in hepatocyte culture medium (Fig. 4A). GSH levels in the culture medium were also increased in the presence of PD98059 or flavone in a concentration-dependent manner. These results indicate that PD98059 and flavone do not prevent GSH efflux from hepatocytes and suggest that the elevated cellular GSH levels induced by PD98059 or flavone may be associated with increased GSH synthesis.
|
), GSH levels were determined at 1, 4, 12, and 24 h following treatment with 50 µM PD98059 or 50 µM flavone (Fig. 4B). Neither PD98059 nor flavone caused a decrease in GSH levels, and the PD98059- or flavone-mediated increases in GSH levels were observed as early as 4 h after initiation of treatment. These results suggest that the mechanism(s) of PD98059- or flavone-induced elevation of GSH synthesis differs from that of oxidative stress. Because the supply of cysteine, a limiting substrate for GSH biosynthesis, is directly related to the synthesis of GSH, cysteine levels in hepatocytes were determined at 1, 4, 12, and 24 h following treatment with 50 µM PD98059 and 50 µM flavone (Fig. 4C). PD98059 and flavone increased cellular cysteine levels as early as 1 to 4 h after the initiation of treatment. These data suggest that one possible mechanism for the PD98059- and flavone-mediated elevation of GSH synthesis in hepatocytes is the increased cellular cysteine levels, which occurred in response to PD98059 and flavone treatment.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Middleton et al., 2000; Havsteen, 2002). The possible cancer chemopreventive effects of flavonoids, some of which have proven effective in clinical trials (Ferry et al., 1996), may result from the modulation of antioxidant enzyme activity as well as inhibition of enzymes that generate reactive metabolites (Middleton et al., 2000; Frei and Higdon, 2003). A number of studies have demonstrated that hepatic GST and UDP-glucuronyltransferase enzyme activities are induced in rats fed dietary flavone (Brouard et al., 1988; Nijhoff et al., 1995). Siess et al. (1989) showed that flavone was the most potent inducer of drug-metabolizing enzymes among the tested flavonoids. In the present study we demonstrate that flavone and PD98059 regulate GSH homeostasis in primary cultured rat hepatocytes. To our knowledge, this is the first report of increased GSH synthesis induced by PD98059. PD98059 treatment resulted in increased GSH levels in hepatocytes cultured in both the presence and absence of insulin. However, U0126, at concentrations sufficient to completely inhibit ERK1/2 phosphorylation in response to insulin, failed to affect GSH levels. Moreover, flavone did not inhibit basal ERK1/2 phosphorylation, but GSH levels were more markedly increased by flavone than PD98059, indicating that elevation of GSH levels by flavone is not due to MEK1 inhibition. Taken together, these data suggest that PD98059 produces dramatic changes in GSH homeostasis in hepatocytes through a mechanism(s) unrelated to MEK inhibition.
PD98059 and flavone caused the elevation of GSH levels in medium as well as in hepatocytes, suggesting that the cellular GSH elevation could not be accounted for by the inhibition of GSH efflux into medium. The elevation of GSH levels in response to both PD98059 and flavone was observed as early as 4 h, without an initial depletion of GSH content. In addition, these chemicals did not affect GCLC protein level or GCL activity, suggesting that the mechanism(s) for PD98059- or flavone-induced GSH synthesis is different from that of chemicals causing oxidative stress. Cellular cysteine levels were elevated by both flavone and PD98059 as early as 1 to 4 h. Hepatic synthesis of GSH is limited by the availability of cysteine as well as by the activity of GCL (Lu, 1998). We have reported that betaine treatment and protein-calorie malnutrition result in a marked decrease in hepatic GSH levels through alteration of cysteine availability, although GCL activity was increased (Kim et al., 2003b,e). Our previous study also suggested that the reduction of cysteine catabolism to taurine could salvage the cysteine supply needed for the synthesis of GSH in mice supplemented betaine for 2 weeks (Kim and Kim, 2005). The Chee's medium used in this study contains 62.6 mg/l L-cystine·2HCl (0.2 mM) and 60 mg/l L-methionine (0.4 mM) but does not contain any cysteine. It has been reported that GSH levels in hepatocytes are increased by the addition of cysteine or methionine in a concentration-dependent manner up to 2 mM (Wang et al., 1997). These results, in conjunction with the results of the present study, suggest that the elevation of cellular cysteine levels induced by PD98059 and flavone may be responsible for the increased GSH synthesis in primary cultures of rat hepatocytes. These results also warrant further studies to elucidate the effect(s) of PD98059 and flavone on the cellular transport and metabolism of cysteine.
GSH, the predominant cellular low molecular weight thiol (normal cellular levels of 0.5–10 mM), scavenges free radicals and other reactive oxygen/nitrogen species (e.g., hydroxyl radical, lipid peroxyl radical, peroxynitrite, and H2O2) directly and indirectly through enzymatic reactions mediated by GSH peroxidase and GSTs. GSH also reacts with various electrophiles to form mercapturates, a reaction initiated by GSTs. Thus, maintenance of GSH levels is critical for cellular defense against oxidative injury and for cellular integrity. Moreover, it is important to note that shifting the GSH redox state toward the oxidizing state regulates several signaling pathways including MAPKs, phosphatidylinositol 3-kinase/Akt, protein tyrosine kinases, nuclear factor 
B, apoptosis signal-regulated kinase 1 and receptor tyrosine kinases (Kang et al., 2000; Chiarugi and Cirri, 2003; Torres and Forman, 2003; Haddad, 2004). These results raise the possibility that flavone and PD98059 shift the cellular redox state by elevating GSH synthesis, which may be associated with regulation of cell proliferation and apoptosis.
It has been reported that oxidative stress resulting in decreased GSH availability may produce the transcriptional induction of antioxidant enzymes such as GST, heme oxygenase-1, quinone reductase, and GCLC, which is associated with an increase in transcription factor binding to the antioxidant response element (ARE) (Masuya et al., 1998; Yu et al., 1999; Kang et al., 2000; Zipper and Mulcahy, 2000; Nguyen et al., 2003). Protein phosphorylation is of major importance in the response to oxidative stress that stimulates ARE-mediated transcription (Nguyen et al., 2003). A number of studies using PD98059 for ERK inhibition suggested that transcription factor binding to the ARE is a downstream consequence of ERK activation and that elevation of antioxidant enzyme expression induced by oxidative stress is inhibited by pretreatment with PD98059 through inhibition of ERK activation (Masuya et al., 1998; Zipper and Mulcahy, 2000; Owuor and Kong, 2002). Considering that GSH plays a critical role in regulation of the cellular redox state and detoxification of reactive oxygen/nitrogen species and electrophiles, the suppressive effect of PD98059 on antioxidant enzyme induction mediated by oxidative stress may be due to PD98059-induced elevation of GSH levels. In fact, GSH and N-acetylcysteine, a GSH precursor, inhibit ARE-related gene expression in response to oxidative stress (Day et al., 2002; Buckley et al., 2003; Kim et al., 2003a).
In summary, the results of the present study indicate that PD98059 and flavone produce dramatic changes in GSH homeostasis in hepatocytes through a mechanism(s) unrelated to MEK inhibition. Furthermore, the present study suggests that the increase in cysteine induced by PD98059 and flavone may be responsible, at least partially, for the elevation of GSH synthesis. These results, in conjunction with those of previous studies (Brouard et al., 1988; Siess et al., 1989; Nijhoff et al., 1995), suggest that flavone-like flavonoids result in elevation of GSH synthesis as well as modulation of drug-metabolizing enzyme expression, which may be associated with flavonoid-induced chemopreventive and chemotherapeutic effects. These results also raise the possibility that the suppressive effect of PD98059 on ARE-mediated gene expression in response to oxidative stress may be due to inhibition of oxidative stress through PD98059-induced elevation of GSH levels.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: PD98059, 2'-amino-3'-methoxyflavone; MAPK, mitogen activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase; GSH, glutathione; GST, glutathione S-transferase; GCL, -glutamylcysteine ligase; GCLC, GCL catalytic subunit; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacryamide gel electrophoresis; PBS, phosphate-buffered saline; ARE, antioxidant response element.
1 Current affiliation: College of Pharmacy and Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon, South Korea. 
Address correspondence to: Dr. Raymond F. Novak, Institute of Environmental Health Sciences, Wayne State University, 2727 Second Ave., Room 4000, Detroit, MI 48201. E-mail: r.novak{at}wayne.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-glutamylcysteine synthetase heavy and light subunit gene expression. Biochem J 326: 167–172.-glutamylcysteine synthetase in pulmonary artery endothelial cells. Am J Physiol 282: L1349–L1357.-glutamylcysteine ligase catalytic subunit in primary cultured rat hepatocytes. J Pharmacol Exp Ther 311: 99–108.-angelicalactone and flavone. Carcinogenesis 16: 607–612.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
