1 Department of Medicine and 2 Free Radical and Radiation Biology Program, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
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ABSTRACT |
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Extracellular signal-regulated kinases (ERKs) are key regulatory proteins that mediate cell survival, proliferation, and differentiation. Reactive oxygen species (ROS) may play a role in activation of the ERK pathway. Because mitochondria are a major source of ROS, we investigated whether mitochondria-derived ROS play a role in ERK activation. Diazoxide, a potent mitochondrial ATP-sensitive K+ (KATP) channel opener, is known to depolarize the mitochondrial membrane potential and cause a reversible oxidation of respiratory chain flavoproteins, thus increasing mitochondrial ROS production. Using THP-1 cells as a model, we postulated that opening mitochondrial KATP channels would increase production of ROS and, thereby, regulate the activity of the ERK kinase. We found that opening mitochondrial KATP channels by diazoxide induced production of ROS as determined by an increased rate of dihydroethidium and dichlorofluorescein fluorescence. This increased production of ROS was associated with increased phosphorylation of ERK kinase in a time-dependent fashion. The MEK inhibitors PD-98059 and U-0126 blocked ERK activation mediated by diazoxide. N-acetylcysteine, but not diphenyleneiodonium, attenuated ERK activation mediated by diazoxide. Adenovirus-mediated overexpression of manganese superoxide dismutase, which is expressed in mitochondria, decreased the rate of dihydroethidium oxidation as well as ERK activation. We conclude that mitochondrial KATP channel openers trigger ERK activation via mitochondria-derived ROS.
reactive oxygen species; cell metabolism; electron transport chain
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INTRODUCTION |
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A VARIETY OF STUDIES suggest that low levels of reactive oxygen species (ROS) can exert a physiological role in cell signaling and cell proliferation (32). In particular, ROS are important signaling molecules for activation of mitogen-activated protein kinases (MAPKs) (4, 30, 51). MAPKs are a family of serine/threonine protein kinases that regulate a wide array of cellular processes. Three major classes of MAPKs have been identified: extracellular signal-regulated kinases (ERK), Jun NH2-terminal kinases (JNK), and p38 MAPKs. JNK and p38 MAPKs participate in the cellular response to environmental stress and are cumulatively known as stress-activated protein kinases (24, 28, 47). In contrast, ERK activation is associated with cell survival and cell proliferation in response to growth factors such as platelet-derived growth factor and epidermal growth factor (47). The classic pathway of ERK activation is characterized by the sequential phosphorylation of upstream kinases, namely, Raf-1 and mitogen-activated kinase kinase (MEK) (4, 24, 28, 30, 47). Recently, it has been shown that the ERK pathway can be activated in response to ROS such as H2O2 (29, 30).
ROS are generated by several pathways during cell metabolism.
Mitochondria generate cellular energy in the form of ATP by the process
of oxidative phosphorylation through an elaborate electron transport
chain (ETC), in which O2 accepts electrons and is reduced
to water (22, 56). The released energy from the flow of
electrons from NADH and FADH2 through the ETC to
O2 is used to pump protons across the mitochondrial inner
membrane and create a transmembrane electrical potential ().
Through this process, a proton motive force is generated as a result of a pH gradient and
. ATP is synthesized when protons flow back to
the mitochondrial matrix. It has been reported that, in normal conditions, 1-2% of consumed O2 undergoes incomplete
reduction and generates superoxide anion (O
- and H2O2-mediated
signal transduction (15).
The amount of mitochondria-derived ROS can be modified by inhibition of
the ETC, change of pH (pH), and reduction of
. Recently, an
ionic channel highly selective for K+ has been identified
in the inner membrane of rat liver mitochondria (33). This
channel is blocked by ATP and sulfonylurea derivatives (27,
52). It has been postulated that mitochondrial ATP-sensitive K+ (KATP) channels play a role in mitochondrial
and
pH and regulation of mitochondrial volume
(52). Several pharmacological agents stimulate ion flux
though the K+ channels in mitochondrial membranes. Among
these drugs, diazoxide opens mitochondrial KATP channels
2,000 times more effectively than the surface KATP channels
in cardiac myocytes and rat liver (23). A number of
studies have shown that mitochondrial KATP channel openers
(KCO, diazoxide and pinacidil) can mimic the effect of ischemic
preconditioning (IPC), thus protecting cardiac tissue against prolonged
ischemic episodes (3). Their protective effect was
abolished by coadministration of antioxidants (13).
In this study, we tested the hypothesis that opening mitochondrial KATP channels leads to ERK activation via the generation of mitochondria-derived ROS. We demonstrated in THP-1 cells, a monocytic cell line, that opening mitochondrial KATP channels leads to ROS production and activation of ERK. Furthermore, we observed that ERK activation is blocked by antioxidants, as well as adenovirus-mediated overexpression of Mn-SOD, which is expressed in mitochondria. In contrast, diphenyleneiodonium (DPI), which effectively inhibits cell membrane-associated NADPH oxidase, had no effect on diazoxide-mediated ERK activation. These studies demonstrate that agents that open mitochondrial KATP channels activate ERK kinase in part via mitochondria-derived ROS.
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MATERIALS AND METHODS |
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Cell culture. The THP-1 cell line was obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 medium supplemented with gentamicin and 10% fetal calf serum (FCS; Life Technologies). The tissue culture medium was changed every 3 days.
Chemicals. Diazoxide, pinacidil, and N-acetylcysteine (NAC) were purchased from Sigma Chemical (St. Louis, MO). Diazoxide and pinacidil were dissolved in DMSO before each experiment. NAC was dissolved in water corrected to pH 7.3 with equimolar bicarbonate solution. Dihydroethidium (DHE) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR). PD-98059, U-0126, and DPI were obtained from Calbiochem (La Jolla, CA). Mn-SOD antibody was obtained from Upstate Biotechnology (Lake Placid, NY).
Infection with recombinant adenovirus constructs.
Recombinant adenovirus vectors expressing enhanced green fluorescent
protein (AdEGFP) or Mn-SOD (AdMnSOD) (61) were used for
these studies. All adenovirus vectors were purchased from the Vector
Core Facility at the University of Iowa. Recombinant adenovirus stocks
were stored in 10 mM Tris with 20% glycerol at 80°C. The particle
titers of adenovirus stocks were typically 1013 DNA
particles/ml. DNA particle-to-infectious unit ratio was 100. Multiplicity of infection (MOI) is given in infectious units. Adenovirus infection was performed for 2 h at 37°C in RPMI 1640 medium without FCS. After infections, an equal volume of RPMI 1640 medium containing 20% fetal bovine serum was added to reach a serum
concentration of 10%, and the infections were continued for 36-48
h. We studied various MOI to determine the most effective concentration
of virus particles for infection of THP-1 cells. Adenovirus infection
at an MOI of 50 in THP-1 cells resulted in >95% infection (determined
by fluorescence of cells infected with AdEGFP vector) without any
effect on cell viability at 48 h as measured by trypan blue
staining (data not shown).
Measurement of intracellular ROS.
Intracellular oxidant production was monitored with two different
fluorescence probes: DHE and DCFH-DA. Intracellular
O
DCFH-DA fluorescence reflects intracellular ROS.
Oxidation by ROS, in particular H2O2 and ·OH,
yields the fluorescence product DCF. DCFH-DA was dissolved in DMSO and
diluted in PBS to a working concentration of 5 µM. After 30 min of
incubation of THP-1 cells in 5 µM DCFH-DA at 37°C, the cells were
washed twice with PBS and exposed to 100 µM pinacidil or 100 µM
diazoxide for different times. Fluorescence levels were monitored by
flow cytometry analysis. DCF fluorescence was measured by analyzing 10,000 events by using a FACS flow cytometer (Becton-Dickinson), as
described previously (35).
Western analysis.
Western analysis for the presence of specific proteins or for
phosphorylated forms of proteins was performed on whole cell sonicates
and lysates from THP-1 cells (37-39). Protein
(30-100 µg) was mixed 1:1 with 2× sample buffer (20% glycerol,
4% SDS, 10% -mercaptoethanol, 0.05% bromphenol blue, and 1.25 M
Tris · HCl, pH 6.8; all from Sigma Chemical), loaded onto a
10% SDS-polyacrylamide gel, and run at 40 V for 2 h. Cell
proteins were transferred to nitrocellulose (ECL, Amersham, Arlington
Heights, IL) overnight at 30 V. Equal loading of proteins on the blots
was evaluated using nonphosphorylated antibody or Ponceau S, a staining
solution designed for detecting proteins on nitrocellulose membranes
(Sigma Chemical). Images of the Ponceau S stain are included to
demonstrate equal loading of the samples. The nitrocellulose was then
blocked with 5% milk in TTBS (Tris-buffered saline with 0.1% Tween
20) for 1 h, washed, and then incubated with the primary antibody [antiphosphorylated ERK (Sigma Chemical) and Mn-SOD antibody (Upstate Biotechnology)] overnight at 4°C. The blots were washed four times with TTBS and incubated for 1 h with horseradish
peroxidase-conjugated anti-IgG antibody (Jackson Laboratories, West
Grove, PA). Immunoreactive bands were developed using a
chemiluminescent substrate (ECL Plus or ECL, Amersham). An
autoradiograph was obtained, with exposure times of 10 s to 2 min.
Statistical analysis. Data were analyzed with GraphPad Prism software (GraphPad) using Student's paired t-test. P < 0.05 was considered to be significant.
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RESULTS |
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Mitochondrial KCO activate ERK.
To evaluate the effect of opening mitochondrial KATP
channels, we performed the following experiments. THP-1 cells were
grown in 10% FCS and then cultured in 1% FCS 16 h before each
experiment to decrease the baseline ERK activity. To demonstrate that
opening mitochondrial KATP channels activates ERK, cells
were stimulated with 100 µM diazoxide or 100 µM pinacidil for
various periods of time. Garlid et al. (23) showed that,
at this concentration, diazoxide preferentially opens the mitochondrial
KATP channels and not the cell surface K+
channels. After protein extraction, 30 µg of cell lysates were subjected to SDS-PAGE and standard Western blotting. ERK activity was
assessed using the diphosphorylated ERK antibody. We found that ERK
activity increased as early as 5 min and was maximally active at 30 min
(Fig. 1). Western blot analysis of total
ERK demonstrated equal loading of the proteins on the blots and no increase in total ERK. There was no significant difference between diazoxide and pinacidil in the time response or fold increase of ERK
activation. Pretreatment of cells for 30 min with 5 µM PD-98509 or
U-0126, MEK inhibitors (2), before treatment with diazoxide, abolished the KCO-mediated ERK activation (Fig.
2). These data demonstrate that agents
that open mitochondrial KATP channels can activate ERK and
that the MEK inhibitors abolish this activation.
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ERK phosphorylation is oxidant dependent.
It has been demonstrated that blocking of the diazoxide effect by
antioxidants blocked the ischemic preconditioning effect of KCO
in cardiac myocytes (21, 44). To determine whether the
phosphorylation of ERK is oxidant dependent, we preincubated the THP-1
cells with 30 mM NAC for 90 min before exposure to 100 µM
diazoxide or 100 µM pinacidil. NAC is a glutathione precursor and a potent scavenger of ROS (46). We found that NAC
attenuated the activation of ERK mediated by pinacidil and diazoxide in
a time-dependent fashion (Fig. 3). By
contrast, preincubation of THP-1 cells with the NAD(P)H oxidase
inhibitor DPI (20 µM) 90 min before diazoxide exposure failed to
block the ERK activation (Fig. 4). These
results suggest that the activation of ERK mediated by mitochondrial
KCO is oxidant dependent and that the ROS were not derived from
membrane-associated NADPH oxidase or mitochondrial complex I.
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Exposure to mitochondrial KCO generates intracellular ROS.
It has been postulated that opening of mitochondrial KATP
channels causes reversible oxidation of flavoproteins and possible inhibition of respiratory chain complex II (26, 45). This inhibition may cause univalent electron transfer to O2 and
thus generate O
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Adenovirus-mediated overexpression of Mn-SOD decreases
mitochondria-derived ROS.
To further localize the subcellular location of KCO-mediated ROS
production, we evaluated the effect of overexpression of Mn-SOD. We
infected THP-1 cells with adenovirus expressing Mn-SOD. Mn-SOD is
located in the mitochondrial matrix and removes
O
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Overexpression of Mn-SOD blocks KCO-mediated ERK activation.
Because Mn-SOD is able to scavenge superoxide generated after exposure
of THP-1 cells to KCO, we determined whether the activation of ERK was
triggered by mitochondria-derived ROS. We infected THP-1 cells with
AdMnSOD or AdEGFP at an MOI of 50 (61). At 36 h after
infection, the cells were treated with 100 µM pinacidil for 30 min.
Total cell protein was isolated, and Western blotting was performed to
detect activated ERK (Fig. 9). We
observed that Mn-SOD-overexpressing cells significantly decreased ERK
activity in response to pinacidil. These results suggest that
mitochondria-derived ROS activate the ERK pathway in response to KCO.
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DISCUSSION |
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Our studies demonstrate that KCO can activate the ERK MAPK in THP-1 cells. In this system, activation of ERK is rapid (by 5 min) and reaches a peak at 30 min. This activation is inhibited in cells overexpressing Mn-SOD or cells pretreated with the oxygen radical scavenger NAC. DPI, which inhibits membrane-associated NADPH oxidase, had no effect on KCO-mediated ERK activation. Parallel to these findings, we were able to measure an increase of ROS production in THP-1 cells on treatment with KCO with two different fluorescence probes. An increase in superoxide production, as measured by DHE oxidation, was not detected in cells that overexpressed Mn-SOD. Overexpression of Mn-SOD suppressed KCO-mediated ERK activation. Taken together, our results suggest that KCO activate ERK via mitochondria-derived ROS.
The activation of ERK is a critical signal for cell survival, proliferation, and differentiation. Receptors for peptide growth factors, such as platelet-derived growth factor, epidermal growth factor, and insulin, are protein tyrosine kinases that undergo phosphorylation in response to ligand binding. This triggers sequential phosphorylation and activation of the Ras-Raf-MEK-ERK pathway (8, 11, 24, 47). Recent studies support the notion that the ERK pathway can be redox regulated (29, 30). The activation of a number of cell surface receptors elicits rapid production of ROS (4, 5). Furthermore, extracellular administration of H2O2 can activate the MAPK pathway (30). Recently, Bogoyevitch and co-workers (7) showed that intact mitochondrial transport function is essential for signaling by H2O2 in cardiac myocytes. Our studies show that ERK activation in response to KCO also depends on mitochondria-derived ROS.
In designing this study, we took advantage of observations using the IPC model and the knowledge that compounds that open mitochondrial KATP channels are capable of mimicking the effect of IPC. It is known that ROS can function as second messengers during IPC (18, 40, 54) and that KCO, which are specific for mitochondrial KATP channels, are able to simulate the preconditioning effect by generating ROS (3, 44). Most previous studies have been performed in intact hearts and have shown that the protective effect of ischemic preconditioning, as well as KCO, is abolished by administration of free radical scavengers, implying a role for ROS as signaling molecules. The mechanisms by which the mitochondrial KCO might alter ROS generation are not well understood. It has been suggested that K+ influx into the mitochondrial matrix would dissipate the potential across the inner membrane and uncouple the ETC (27, 45, 52). In addition, diazoxide exhibits a direct effect on mitochondrial energy metabolism by inhibition of respiratory chain complex II in liver mitochondria (26). It also enhances respiration and augments electron flux through the Q cycle (31). One interesting aspect of the effect of diazoxide is the reversible depolarization of mitochondrial membrane potential. This might enhance superoxide production without increasing cell death compared with other mitochondrial ETC inhibitors that are very toxic to cells.
In these studies, we showed that the opening of KATP channels is associated with an early increase in the rate of superoxide production. These findings parallel recent studies showing an increase in mitotracker fluorescence in cardiac myocytes on treatment with diazoxide (13). To test whether the KCO-mediated superoxide generation is mitochondrial in origin, we infected the THP-1 cells with AdMnSOD. Mn-SOD-overexpressing cells showed decreased superoxide production on treatment with KCO. The effect of overexpression of Mn-SOD confirms the mitochondrial origin of KCO-mediated superoxide, since mitochondria-localized Mn-SOD is only known to be active in the mitochondrial matrix and dismutates the fraction of superoxide that arises in this compartment (1, 17, 58).
We also assessed the effect of DPI on ERK activation. DPI is an NAD(P)H oxidase inhibitor (36) that also inhibits superoxide formation from the flavin moiety of nitric oxide synthase (59). It had no effect on KCO-mediated ERK activation. The lack of an effect of DPI on ERK activation on treatment with KCO suggests that NAD(P)H oxidase, mitochondrial complex I, and nitric oxide synthase are unlikely sources of ROS that mediate ERK activation. We are presently determining which mitochondrial ETC complex is important for the increased production of superoxide mediated by KCO.
Although excessive amounts of ROS can lead to tissue injury and inflammation, low levels of ROS may be useful for cell signaling. Quantitative differences in amounts of ROS may have different impacts on the redox environment of the cell and may initiate different signaling pathways (46). This can be demonstrated by comparing the effects of ischemia-reperfusion injury and ischemic preconditioning. In the first instance, ROS triggers signaling pathways that result in cell injury and cell death. In the latter instance, signaling pathways are initiated that protect the cells from subsequent ROS-mediated injury. The extent of ischemia-reperfusion injury can also be diminished by free radical scavengers and overexpression of Mn-SOD (16). The beneficial effect of IPC also is abolished with administration of free radical scavengers (42, 44, 53). Recently, Ozcan and co-workers (43) showed that KCO protect the isolated cardiac mitochondria by attenuation of oxidant stress at reoxygenation (similar to the effect of SOD in the ischemia-reperfusion model). These findings suggest that effects of KCO on isolated mitochondria may be different from those in the whole cell. Our findings are supported by findings of other investigators that KCO triggers generation of oxygen radicals (13, 21, 44). We hypothesize that low levels of ROS generated during KCO exposure trigger protective cell mechanisms, whereas higher levels of ROS result in tissue injury (ischemia-reperfusion).
An important finding of this study is that KCO, which mimic IPC, trigger ERK activation. ERK activation has been shown to enhance cell survival. Furthermore, free radical scavengers inhibited this effect of KCO. These observations might help elucidate signaling pathways that are activated during IPC. Protein kinase C (PKC) is held as the central mediator of IPC (49, 50). Recent studies suggest that mitochondria-derived superoxide may play a critical role as an upstream activator of PKC (41). In addition, it has been postulated that PKC exerts its protective effect by activating mitochondrial KATP channels (49). PKC is known to be an activator of the ERK pathway. Thus it is possible that the effect of mitochondria-derived ROS is mediated via activation of one or more PKC isoforms. We are presently investigating these possibilities.
Mitochondria-derived ROS may also affect activation of the ERK pathway at sites other than PKC. The extent of phosphorylation (activation) of the kinases in the ERK pathway reflects the balance between kinases and relevant phosphatases. Activation of kinases and/or inhibition of phosphatases may result in MAPK activation. Recent studies indicate that phosphatases are regulated by a redox mechanism. Most phosphatases contain in their active site cysteine residues and/or metal centers (Fe+2-Zn+2), which are targets of oxidation by various ROS, including superoxide and H2O2 (12, 19, 57). The inhibition of phosphatases is potentially important, since phosphatases exhibit a much higher specific activity than the kinases (20).
In summary, our studies show that KCO trigger ERK activation via the production of mitochondria-derived ROS. Although our study was not designed to differentiate between the signaling cascade by superoxide and that by H2O2, our data indicate that mitochondria-derived superoxide is necessary to activate ERK, since Mn-SOD overexpression abolished the effect of KCO. Our primary interest in these studies was to determine whether mitochondria-derived oxidants activate the ERK pathway in monocytic cells. However, these studies have broader implications. They also provide one mechanism (increased expression of the ERK MAPK) to explain how KCO protect cells from subsequent ROS-mediated cell injury.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Samavati, Div. of Pulmonary and Critical Care Medicine, Rm. 100, EMRB, University of Iowa Hospital and Clinic, Iowa City, IA 52242 (E-mail: lobelia-samavati{at}uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 13, 2002;10.1152/ajpcell.00514.2001
Received 25 October 2001; accepted in final form 6 March 2002.
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