Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576
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ABSTRACT |
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In the current study, we investigated links between O2-regulated H2O2 formation and the hypoxic induction of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in O2-sensitive PC-12 cells. During exposure of PC-12 cells to 5% O2, H2O2 concentration decreased by 40% as measured with 2',7'-dichlorofluorescein (DCF). Treatment with H2O2 reduced TH mRNA during normoxia and prevented the induction of TH mRNA during hypoxia. Treatment with catalase or N-(2-mercaptopropionyl)-glycine, a reducing antioxidant agent that decreases H2O2 concentration, also induced TH mRNA. Deferoxamine (DF), an iron chelator, failed to affect H2O2 formation but induced TH mRNA in normoxia and hypoxia. CoCl2 led to a decrease in H2O2 at 20 h of treatment but induced TH mRNA during normoxia and hypoxia before it affected H2O2. In conclusion, TH gene expression correlates inversely with H2O2 formation. DF and Co2+ seem to affect TH gene expression in the mechanism downstream from the H2O2 formation rather than by interfering with the H2O2-generating activity of the O2 sensor.
oxygen-dependent regulation; catecholamines; dopamine; carotid body; chemoreceptors; pheochromocytoma
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INTRODUCTION |
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THE FUNCTION OF TYROSINE HYDROXYLASE (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by decrease in oxygen tension (PO2). Induction of TH mRNA during hypoxia has been demonstrated in the O2-sensitive type I cells of the carotid body (10) and in the pheochromocytoma-derived clonal cell line PC-12 (13), which is used as an experimental model system for the type I cells (9, 10). The increase in TH mRNA leads to an increase in TH protein and its enzymatic activity (18, 25, 29) and results in enhanced synthesis (19) and release of dopamine (20). Dopamine, in the feedback mechanism, regulates the sensitivity of O2-sensitive cells to hypoxia (41). This regulation is specific for the O2-sensitive cells and does not occur in other catecholaminergic cells (10).
The molecular mechanisms that regulate expression of TH gene in the O2-dependent manner are studied intensively. In PC-12 cells, the hypoxic induction of TH mRNA occurs in a dual mechanism that results from an augmented rate of TH gene transcription and from increased stability of TH mRNA (13). This regulation involves binding of the hypoxia-sensitive proteins to the TH gene promoter (39) and to the stability element in the 3'-untranslated region of TH mRNA (11, 12, 14). In contrast, little is known about the intracellular signal transduction pathways that link decrease in the environmental PO2 to the expression of the TH gene.
Regulation of gene expression by O2 requires an O2 sensor that detects PO2 in the environment and activates specific intracellular signal transduction pathways. Because many actions of molecular O2 are mediated by heme-containing proteins, a heme protein is believed to be the O2 sensor. Initially, Goldberg and collaborators (22, 23) proposed that during hypoxia the heme-containing O2 sensor changes its conformation from oxy to deoxy and in this way initiates intracellular signal transduction pathways that lead to induction of the hypoxia-inducible gene erythropoietin (EPO). In the most recent hypothesis, Acker and his collaborators (1-3, 8, 26, 27) proposed that the O2 sensor is a membrane-bound cytochrome b coupled to an NAD(P)H oxidase that generates H2O2 in an O2-dependent manner. During hypoxia, the H2O2-generating activity of oxidase is inhibited. The decrease in H2O2 formation results in a shift of the cellular proteins' redox state toward the reduced forms and thereby affects regulatory proteins that control different cell functions, including gene expression. The evidence for the potential role of NAD(P)H oxidase in O2 sensing was presented in erythropoietin-synthesizing hepatoblastoma (Hep G2) cells (26, 27), in the O2-sensitive cells of carotid body (3, 36), and in the pulmonary neuroendocrine cells (44).
The experimental methods for changing the activity of the heme-containing O2 sensor to study its effects on gene expression are limited. It is well established that two compounds, an iron chelator (deferoxamine, DF) and cobaltous ion (Co2+), induce expression of the hypoxia-sensitive genes similarly to hypoxia. The molecular mechanism by which DF and Co2+ induce gene expression is unclear. Several different mechanisms were proposed. In this respect, Co2+ is believed to replace iron in the O2-sensing heme protein, locking it into the deoxy conformation (22, 30), to activate an H2O2-degrading enzyme, glutathione peroxidase (GPO) (15), or to interact with cytochrome b-558 within the NADPH oxidase (26). It was proposed that DF inhibits heme synthesis by iron chelation (22). More recently, however, DF has been shown to interfere with iron-mediated degradation of H2O2 in the Fenton reaction without influence on cytochrome b activity (15, 16). Most of the studies on the molecular nature of the O2 sensor attempt to couple its activity with the O2-induced regulation of EPO gene expression (15, 17, 22, 30).
In the present study, we investigate the hypothesis that H2O2 levels change during hypoxia and that this change regulates expression of the TH gene in the O2-sensitive neuroendocrine PC-12 cells. Indeed, we show that during hypoxia the H2O2 level decreases and that steady-state levels of TH mRNA correlate inversely with H2O2 formation. We have also attempted to determine whether DF and Co2+ affect formation of H2O2 and TH gene expression.
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MATERIALS AND METHODS |
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Tissue cultures and treatment of cells with drugs. Rat PC-12 cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium-Ham's F-12 medium with 10% fetal calf serum, as described previously (11-14). For all experiments, 50% confluent cells were used. Immediately before each experiment, the regular medium was replaced with serum- and iron-free RPMI 1640 medium. We verified that the change did not affect the viability of cells for at least 24 h. Exposures to normoxia (room air + 5% CO2) or hypoxia (5% O2, 5% CO2, and 90% N2) were performed as described previously (11-14). N-(2-mercaptopropionyl)-glycine (NMPG) and N-acetylcysteine (NAC) solutions were prepared fresh before each experiment by dissolving the drugs in water and adjusting the pH to 7.5. Drug concentrations and exposure times are reported in RESULTS. The doses of each drug were determined experimentally to give maximal responses without affecting cell viability, as evaluated with the trypan blue exclusion test.
Hydrogen peroxide measurements. H2O2 was measured with the 2',7'-dichlorofluorescin diacetate (DCFDA) method (Molecular Probes, Eugene, OR) (5, 6). DCFDA penetrates easily into the cells; there it is cleaved to 2',7'-dichlorofluorescin (DCF) and reacts with H2O2 to form fluorescent 2',7'-dichlorofluoroscein, which is detected with spectrofluorometry. This assay is chemically specific for hydroperoxides. Because H2O2 is the major peroxide in the cells, it is generally accepted that DCF is proportional to H2O2 concentration (5, 6).
Cells were plated at a density of 2.5 × 106 cells/ well (50% confluence) and subjected to either normoxic or hypoxic conditions or were treated appropriately with the drugs in serum-free RPMI 1640 medium for the specified periods. After each treatment, cells were washed in buffer [(in mM) 150 NaCl, 5 KCl, 1 MgSO4, 1 NaH2PO4, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1.8 CaCl2, and 10 glucose, pH 7.4] and were loaded with 10 µM DCFDA in the same loading buffer for 5 min at 37°C. The DCFDA-buffer was then removed, and the cells were washed with loading buffer and analyzed by spectrofluorometer. Measurements were taken at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Emission from cell samples that were not loaded with DCFDA was the same as for those with the loading buffer.Northern blot analysis.
TH mRNA stability was measured using actinomycin D as described before
(13). For analysis of total RNA, cells from each treatment were
collected in TRI reagent (Molecular Research Center, Cincinnati, OH) at
the indicated times. Total cellular RNA was extracted, and equal
amounts of RNA were processed for gel electrophoresis on 1% agarose
gel containing 0.41 M formaldehyde. The RNA was transferred onto a
synthetic membrane with a downward capillary blotting system in
10× saline sodium citrate for at least 1 h. The
filters were hybridized with 5 × 106 counts/min of TH or actin cDNA
probes labeled with
[-32P]dCTP, using
nick translation. They were washed and quantified with either X-ray
film autoradiography or phosphoimager (Molecular Dynamics, Sunnyvale,
CA). Radioactivities were normalized to the optical density of lower
ribosomal bands, as determined in the ethidium bromide-stained gels.
The differences in the amount of RNA loaded were usually <10%.
Hybridization with the actin probe is used to demonstrate specificity
of the effects for TH mRNA.
Data presentation. Measurements of H2O2 formation and TH mRNA are presented as percentage changes from the control. Each experimental time point has its own control, which is accepted as 100%. Data are presented as means ± SE, and n is number of independent experiments performed in duplicate or triplicate. Statistical analyses were performed with Student's t-test, and P < 0.05 was accepted as significant.
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RESULTS |
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Gene expression for TH correlated with H2O2 formation in PC-12 cells. Analysis of H2O2 levels in PC-12 cells using DCF fluorescence showed significant and readily detectable levels of H2O2 during normoxia. Fig. 1 shows that the constitutive formation of H2O2 declined markedly when the PC-12 cells were exposed to 5% O2. This response started as early as 1 h after the beginning of hypoxia, reached a nadir of 60% after 6 h, and remained in a decreased state for up to 20 h (the longest time studied).
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Reducing antioxidants that decrease
H2O2 induce TH
mRNA similarly to hypoxia.
We tested various antioxidative agents for their ability to reduce
H2O2
levels in PC-12 cells and to induce TH
gene expression similarly to hypoxia. Table
1 summarizes the results of these experiments. In particular, we tested two reducing drugs,
NMPG--mercaptoethanol derivative (38) and NAC-cysteine derivative
(4, 40), that increase the number of reduced thiol groups. NMPG proved
the most potent in decreasing intracellular
H2O2
and increasing TH mRNA levels. Figure 5
shows the effects of NMPG on
H2O2
(Fig. 5A) and on TH mRNA (Fig.
5B) in PC-12 cells. NMPG decreased
the
H2O2
concentration (Fig. 5A) and induced
the expression of TH mRNA (Fig. 5B).
These changes are similar in time course and magnitude to the effects of hypoxia. The effect of NMPG on TH mRNA was completely abolished when
ATZ was applied simultaneously to inhibit catalase and to raise
intracellular
H2O2
(Fig. 5B). NAC, another reducing
antioxidant, also reduced
H2O2
and induced TH mRNA, but it did so less effectively than NMPG (Table
1).
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Effects of DF and Co2+ on H2O2 and TH mRNA. The iron chelator DF failed to decrease H2O2 in PC-12 cells over 1-20 h of treatment (Table 1). It also failed to affect the hypoxia-mediated decrease in H2O2. Figure 6 shows the effects of DF treatment on TH mRNA. Treatment with DF from 3 to 24 h caused induction of TH mRNA (Fig. 6A), with the largest increase at 24 h. Simultaneous treatment with hypoxia and with DF resulted in an augmented response (Fig. 6B). Treatment of PC-12 cells with 1.5 mM FeCl2 resulted in a decrease in the constitutive expression of TH mRNA (Fig. 6C, lanes 5 and 6) and an attenuation of the DF effect on TH gene expression (Fig. 6C, lanes 7 and 8).
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DISCUSSION |
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The present study shows that hypoxia decreases the concentrations of H2O2 in PC-12 cells and that O2-dependent regulation of TH mRNA correlates inversely with the levels of H2O2. Increased levels of intracellular H2O2 correlate with decreased expression of TH mRNA, whereas decreased formation of H2O2 accompanies elevated expression of TH mRNA. This finding supports the hypothesis that a decrease in H2O2 during hypoxia mediates induction of TH mRNA. The results also show that during normoxia H2O2 has an additional suppressing effect on TH gene expression. This is the first evidence that TH gene expression is regulated by reactive oxygen intermediates (ROIs). EPO is the other gene whose O2-dependent expression has been shown to be regulated by decreases in H2O2 and potentially by next-step ROIs (15-17).
In the present study we have not attempted to determine specifically whether treatments affecting H2O2 levels regulate TH gene expression at the transcriptional or posttranscriptional level. It is most likely, however, that H2O2 regulates TH gene expression at the transcriptional level, because our initial experiments showed that the stability of TH mRNA did not change with alterations in H2O2. This conclusion also may be supported by the finding that ATZ treatment abolished hypoxic induction of TH mRNA during brief exposures to hypoxia but was less effective during longer exposures, when a twofold induction was still visible (see Fig. 3C). Because the long-term increase in TH mRNA results primarily from increased TH mRNA stability, this component seems not to be affected by peroxide. On the other hand, ATZ treatment abolished even late induction of TH mRNA by NMPG, which does not affect mRNA stability (compare Figs. 3 and 5).
The potential significance of our finding in regulation of respiratory and cardiovascular function is supported by the recent report showing that long-term exposure to oxidative stress (ozone) impairs catecholamine metabolism and TH activity, specifically in noradrenergic neurons of caudal nucleus tractus solitarii (A2) and locus coeruleus (A6) (7). In addition, decrease in TH gene expression can be linked to oxidative stress in the substantia nigra neurons during development of Parkinson's disease (35). Thus PC-12 cells may represent a valuable model system for studying the effects of ROIs on catecholaminergic phenotype. The broad implication of these data is that H2O2 and perhaps other ROIs have modulatory effects on the expression of various proteins; thus they serve as modulators of cell phenotype and function in response to changes in environmental PO2.
Finally, the results provide insights into the molecular nature of interactions between the putative O2 sensor and O2-dependent regulation of gene expression. The original assumption, following Acker's hypothesis (1-3, 8, 26, 27), implied that H2O2 is generated in an O2-dependent manner by NAD(P)H oxidase. In our experiments, however, an inhibitor of NAD(P)H oxidase, DPI, failed to affect either H2O2 or TH mRNA levels during either normoxia or hypoxia. Thus either DPI is ineffective in PC-12 cells or the decrease in H2O2 measured during hypoxia results from the inhibited activity of a different oxidase system. This last possibility is supported by a recent finding that the hypoxic induction of genes for vascular endothelial growth factor (VEGF) and the glycolytic enzyme aldolase does not involve cytochrome b-558-coupled NAD(P)H oxidase and is maintained in the B cell lines derived from chronic granulomatous patients who lack functional oxidase (42). Furthermore, there is also conflicting evidence as to whether the O2-dependent induction of erythropoietin in the Hep 3B cell line is also DPI sensitive (21, 24). The inhibition of other oxidases, such as xanthine oxidase, cyclooxygenase, or mitochondrial ubisemiquinone, did not affect H2O2 concentrations in PC-12 cells. Thus none of these oxidase systems is a likely candidate for the hypoxia-dependent regulation of H2O2.
Another possible explanation for the decrease in H2O2 during hypoxia is increased scavenging of H2O2 by either catalase or GPO or by reactions with small-molecule antioxidants. Neither the activities of GPO nor those of catalase have been measured specifically in the PC-12 cells or carotid body type I cells during hypoxia; however, the exposure of rats to a 10% O2 environment substantially augmented the activity of catalase and selenium-containing GPO in the rats' lungs (43). H2O2 also may be scavenged nonenzymatically by small-molecule sulfhydryl-containing compounds and antioxidants (28). Increased direct scavenging of H2O2 by sulfhydryl groups is the major mechanism by which NMPG and other reducing agents reduce H2O2 concentration in PC-12 cells (4, 38, 40).
The most widely used approach to studying the O2 sensor includes use of DF and Co2+, which are believed to mimic effects of hypoxia on gene expression by interfering with the activity of the O2 sensor heme molecules. Thus we expected that both DF and Co2+ would increase TH gene expression and simultaneously would decrease formation of H2O2. Yet, although both DF and Co2+ induced expression of TH gene similarly to hypoxia, these effects must be independent of H2O2 formation, because DF failed to decrease H2O2 formation and Co2+ decreased its formation after the onset of TH mRNA induction. In the case of DF, our results differ from those in Hep G2 cells, in which DF decreased H2O2 concentration by 25% (16). The time course of Co2+-mediated effects on TH gene expression agrees with previously published results regarding EPO and VEGF gene expression (23). Co2+-induced decrease in H2O2 in PC-12 cells is similar to that measured in Hep G2 cells after 18 h (26) or 24 h (16) of treatment. The effects of DF or Co2+ augmented the effects of hypoxia on TH mRNA, an indication that DF, Co2+, and hypoxia may act through separate but complementary pathways. A similar augmenting effect of DF on the hypoxia-induced synthesis of EPO was observed in Hep G2 cells (16). We hypothesize here that the effects of DF and Co2+ on TH gene expression may be mediated by altering formation of the next-step ROI-hydroxyl radical (OH ·) rather than by interfering with the synthesis of heme-containing O2 sensor (22).
The following interpretation of our results supports this hypothesis. Hydroxyl radicals are generated from H2O2 in the Fenton reaction catalyzed by intracellular iron (28). Thus treatment of PC-12 cells with iron or H2O2 increases formation of the hydroxyl radicals and in this way leads to a decrease in TH gene expression. On the other hand, decrease in H2O2 formation (hypoxia or NMPG) or chelation of iron (DF) attenuates formation of hydroxyl radicals and causes induction of TH mRNA expression. A similar interpretation of the molecular mechanism of DF effects was proposed in the case of O2-induced expression of the EPO gene (15, 16). The effects of Co2+ also may be interpreted in the context of OH · formation. Co2+, like iron, is a transition metal that catalyzes the in vitro formation of OH · from H2O2 through the Fenton reaction. At physiological pH levels, however, Co2+ is unable to catalyze the classic Fenton reaction in either chemical or microsomal systems; it catalyzes the production of superoxide rather than OH · (34). Thus, under physiological conditions, Co2+ may compete with iron in the Fenton reaction; at sufficiently high concentrations of Co2+, the superoxide ion is generated from H2O2 rather than from OH ·. Thus iron-dependent OH · formation decreases and leads to the induction of TH gene expression. In support of this hypothesis, iron (with enhanced OH · formation) abolishes Co2+-dependent induction of TH mRNA. On the other hand, the decrease in H2O2 after long-term treatment with Co2+ may be due to Co2+-induced activity of GPO similar to the effect reported in the Hep G2 cells (15).
The hypothesis implying the role of ROIs in gene expression regulation requires intracellular subcompartmentalization of ROI formation and ROI reactions with cellular proteins and glutathione. Although little is known about compartmentalization of ROIs, it is clear that the glutathione-related redox state is compartmentalized within cells (32, 33). For example, the ratio of oxidized to reduced glutathione is higher in the endoplasmic reticulum, where it serves in formation of protein disulfide bridges during protein synthesis, than in cytosol (32).
In summary, our results demonstrate clearly that TH gene expression correlates inversely with H2O2 formation in PC-12 cells. DF and Co2+ seem to affect TH gene expression in the mechanism downstream from the H2O2 formation rather than by interfering with the H2O2-generating activity of the O2 sensor.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Paul for the use of the spectrofluorometer and Glenn Doerman for preparation of the figures.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-51078 and HL-58687, American Heart Association Grant-in-Aid 94017440, and the NARSAD Young Investigator Award. S. L. Kroll was supported by NHLBI Training Grant HL-07571.
Address reprint requests to M. F. Czyzyk-Krzeska.
Received 4 June 1997; accepted in final form 20 September 1997.
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