Mechanism of Doxorubicin-Induced Inhibition of Sarcoplasmic Reticulum Ca2+-ATPase Gene Transcription
Abstract—Doxorubicin (DOX)–induced cardiomyopathy has been found to be associated with impaired Ca2+ handling in the sarcoplasmic reticulum (SR), leading to reduced cardiac function. We have recently demonstrated that expression of mRNA encoding sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2), a major Ca2+ transport protein in SR, is markedly decreased in DOX-treated hearts. To extend this observation, we have dissected the molecular mechanisms by which DOX downregulates SERCA2 gene transcription. Using cultured rat neonatal cardiac myocytes, we found that the antioxidant N-acetylcysteine blocked the DOX-induced decrease in SERCA2 mRNA levels, as well as the DOX-induced increase in H2O2 concentration; thus, H2O2 is an intracellular mediator of DOX activity. Using a luciferase reporter assay, we found that the sequence from −284 to −72 bp in the 5′ flanking region of the SERCA2 gene has a DOX-responsive element. Although several transcription factors have putative binding motifs in this region of the SERCA2 gene, only the expression of Egr-1 mRNA and the binding of Egr-1 protein to the 5′ regulatory sequence of SERCA2 gene increased markedly after DOX administration. We also found that overexpression of Egr-1 was associated with a significant reduction in SERCA2 gene transcription. In addition, Egr-1 antisense oligonucleotides blocked the DOX-induced reduction in SERCA2 mRNA, suggesting that Egr-1 is a transcriptional inhibitor of the SERCA2 gene in DOX-induced cardiomyopathy. We observed activation of 3 mitogen-activated protein kinases (MAPKs), p44/42 MAPK, p38 MAPK, and stress-activated MAPK/Jun N-terminal kinase, by DOX, but only a specific inhibitor of the p44/42 MAPK kinase suppressed the effects of DOX on Egr-1 and SERCA2 mRNA expression. These findings indicate that reactive oxygen intermediates, the transcription factor Egr-1, and p44/42 MAPK are critical elements in the transcriptional regulation of the SERCA2 gene in response to DOX.
Doxorubicin (DOX) is widely used to treat patients with neoplastic diseases. Its clinical utility is limited, however, by its cardiotoxicity, inducing a condition often called “DOX cardiomyopathy.” Recently, we have reported that a decrease in mRNA expression for sarcoplasmic reticulum (SR) Ca2+ transport proteins is responsible for the impaired Ca2+ handling, and thus, for the reduced cardiac function seen in DOX cardiomyopathy.1
SR Ca2+-ATPase is a key protein in the SR that regulates intracellular Ca2+ concentrations. Muscle relaxation is triggered by ATP-dependent Ca2+ transport into the SR by the SR Ca2+-ATPase. In cardiac myocytes, the SR Ca2+-ATPase protein is encoded by the sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) gene.2 3 The expression of the SERCA2 gene is regulated under a variety of pathophysiological conditions. In our previous study, we demonstrated that the levels of SERCA2 mRNA and protein, as well as the ability of SERCA2 protein to take up Ca2+ were significantly diminished in DOX-treated hearts.1 We also showed that Ca2+ transport activity significantly correlates with SERCA2 mRNA levels,1 suggesting that genetic regulation of the SERCA2 gene is critically important for the impaired Ca2+ transport activity observed in DOX cardiomyopathy.
To clarify the molecular mechanisms by which DOX controls transcription of the SERCA2 gene, we have dissected the signal transduction pathway mediating the effect of DOX on SERCA2 gene regulation. We have also ascertained the identity of the transcription factor responsible for the DOX-induced decrease in SERCA2 mRNA, and we have determined the regulatory sequence of the SERCA2 gene targeted by the DOX-activated transcription factor.
Materials and Methods
Primary cultures of neonatal rat cardiac ventricular myocytes were prepared as described,4 with minor modifications. Total cellular RNA was isolated by the guanidine thiocyanate procedure.5 For DNA transfection, neonatal rat cardiocytes were grown to 60% to 80% confluence. Two micrograms of each SERCA2 luciferase test plasmid and 20 ng of pRL-CMV control plasmid (Promega), an internal standard for the variation of transfection efficiency, were preincubated with 35 μg of Tfx-50 cationic liposomes (Promega) and transfected into 3 dishes of cells. To determine the role of transcription factors, 1 μg of expression plasmid encoding each transcription factor was cotransfected into the cells. The firefly luciferase activity of the SERCA2 test plasmid and the Renilla luciferase activity of pRL-CMV in each culture were sequentially measured 24 hours after transfection.
The cDNA fragments used as hybridization probes and for protein expression were the following: rabbit SERCA2 cDNA,2 rat skeletal α-actin cDNA,6 rat Sp1 cDNA,7 8 mouse Egr-1 cDNA,9 human p300 cDNA,10 Drosophila AP2 cDNA,11 and human GAPDH cDNA.12 Deletion constructs within the 5′ upstream region of the SERCA2 gene (nucleotides −1810 to +350 relative to the transcription initiation site)3 13 and the −407 to +80–bp sequence of the mouse Egr-1 gene were used to test the transcriptional activity of each gene.
Electrophoretic mobility shift assays were performed by incubating 5′-end–labeled SERCA2 fragment from nucleotides −206 to −91 with nuclear extracts (3 μg)14 for 30 minutes at 4°C. Competition experiments were performed with 10-fold and 100-fold excess of unlabeled double-strand Egr-1 consensus oligonucleotide (5′-GGATCCAG-CGGGGGCGAGCGGGGGCGA-3′) and Egr-1 mutant oligonucleotide (5′-GGATCCAGCTAGGGCGAGCTAGGGCGA-3′) (Santa Cruz Biotechnology). For the supershift experiment, nuclear extracts were preincubated with an antiserum against Egr-1 (sc-110x, Santa Cruz Biotechnology) for 30 minutes at 4°C before the binding reaction.
Phosphothioate-modified oligonucleotides were generated to target the translation initiation site of the Egr-1 gene. The sequences utilized were as follows: sense, 5′-ATGGCAGCGGCC-AAGGCCGA-3′; mismatch, 5′-TCGACCGTGGCTGCTGCCCT-3′; scramble, 5′-GCTCGTCCGAGTCGTCCTGC-3′; and antisense, 5′-TCGGCCTTGGCCGCTGCCAT-3′. Oligonucleotides, at concentrations of 0.1 to 1 μmol/L, were preincubated with 140 μg Tfx50 cationic liposomes and were incubated with cultured myocytes for 1 hour. Twenty-four hours later, the cells were harvested, and total RNA was isolated.
Intracellular H2O2 generated by DOX was measured in situ using a fluorogenic probe, 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (H2DCFDA-AM) (Molecular Probes). The cells were incubated for 30 minutes with 1 μmol/L DOX, with or without 10 mmol/L N-acetylcysteine, or 0.1 to 10 μmol/L H2O2.
For immunohistological detection of kinase activation, the cells were incubated with rabbit anti-phosphospecific p42/42 mitogen-activated protein kinase (MAPK) (Thr202/Tyr204), anti-phosphospecific p38 MAPK (Thr180/Tyr182), or anti-phosphospecific SAPK/JNK (Thr183/Tyr185) antibody (New England Biolabs). To distinguish cardiocytes from fibroblasts, cultures were simultaneously incubated with mouse monoclonal antibody to α-sarcomeric actinin (Sigma).
Data are expressed as mean±SD. Overall differences within groups were determined by 1-way ANOVA. Individual experimental groups were compared by the Bonferroni test.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
DOX Decreases SERCA2 mRNA Levels by Inhibiting Gene Transcription
We found that administration of DOX to cultured rat cardiocytes reduced the steady-state level of SERCA2 mRNA in a time- and dose-dependent manner (Figure 1⇓). To determine whether this decrease is due to impaired gene transcription, transcriptional activity from the 5′ regulatory region of the SERCA2 gene (ie, nucleotides −1810 to +350 relative to the transcription initiation site) was determined. We found that 0.25 μmol/L DOX decreased transcription by 64% (Figure 2a⇓).
Reactive Oxygen Intermediates Mediate DOX Activity
To determine the signaling pathway that mediates DOX inhibition of SERCA2 gene transcription, we utilized several specific inhibitors of signal transduction. Both calphostin C, a protein kinase C inhibitor, and staurosporin, an inhibitor of protein kinases A and C, markedly depressed the basal transcription of the SERCA2 gene. We found that wortmannin, a PI3 kinase inhibitor; genistein, a tyrosine kinase inhibitor; tautomycin, a protein phosphatase 1 inhibitor; and okadaic acid, a protein phosphatase 2 inhibitor, were unable to reverse the effect of DOX on SERCA2 gene transcription (Figure 2⇑, black columns). In the presence of the antioxidant N-acetylcysteine (10 mmol/L), however, DOX did not diminish transcription and mRNA expression of the SERCA2 gene, suggesting that reactive oxygen intermediates may mediate the inhibitory effect of DOX on SERCA2 gene expression (Figure 2b⇑ and 2c⇑).
When we assayed H2O2 concentration in cardiocytes, we found that, in the absence of DOX, the H2O2 concentration was 0.7 μmol/L. DOX significantly increased the intracellular H2O2 concentration, up to 2.0 μmol/L, in a dose-dependent manner (Figure 3a⇓). Addition of N-acetylcysteine, however, completely blocked the DOX-induced increase in H2O2 concentration, even at the highest DOX concentrations.
To determine whether H2O2 can inhibit transcription of the SERCA2 gene, cultured cardiac myocytes were incubated with exogenously administered H2O2 and SERCA2 gene transcription, and message levels were measured. We observed that both SERCA2 gene transcription and steady-state mRNA levels were suppressed by H2O2 in a dose-dependent manner (Figure 3b⇑ and 3c⇑).
DOX-Susceptible Elements Are Present in the −284- to −72-bp Region of the SERCA2 Gene
To elucidate the sequence in the 5′ upstream regulatory region of the SERCA2 gene that reacts with DOX, we transfected cardiac myocytes with a vector containing the firefly luciferase gene under the regulatory control of sequences representing deletions in the 5′ regulatory region of the SERCA2 gene. We then measured luciferase activity in cells cultured in the presence or absence of 1 μmol/L DOX. The effect of DOX, which was evident in plasmid containing the −284 to +350 sequence of the SERCA2 gene, was not present in plasmid containing the −72 to +350 sequence, suggesting that the −284 to −72 sequence of the SERCA2 gene contains a DOX-susceptible element (Figure 4a⇓).
The Transcription Factor Egr-1 May Regulate DOX-Induced Inhibition of SERCA2 Gene Transcription
Several putative consensus sequences to which transcription factors can bind are present in the −284- to −72-bp sequence of the SERCA2 gene (Figure 4b⇑). To identify the transcription factor that mediates the inhibitory action of DOX on SERCA2 gene transcription, we assayed the expression levels of mRNA encoding the transcription factors Egr-1, Sp1, and AP2, as well as that encoding p300, a coactivator of transcription factors in cultured cardiocytes, 0, 1, 3, and 24 hours after DOX administration. Although message abundance of Sp1, p300, and AP2 did not change significantly, Egr-1 mRNA increased as early as 1 hour after administration of DOX (Figure 5a⇓). Additionally, when we utilized a plasmid-containing luciferase assay under the transcriptional control of the −407- to +80-bp sequence of the Egr-1 gene, we found that DOX strongly activated transcription of this gene (Figure 5b⇓).
To determine whether Egr-1 can suppress transcription of the SERCA2 gene, we cotransfected the Egr-1 expression vector together with the SERCA2 gene reporter construct (ie, the −1810- to +350-bp region of SERCA2 ligated to the luciferase gene) into rat primary cardiocytes. We found that overexpression of Egr-1 significantly reduced the transcriptional activity of the SERCA2 gene, whereas overexpression of Sp1 or p300 markedly increased SERCA2 transcription (Figure 5c⇑), suggesting that Egr-1 may downregulate SERCA2 gene transcription in response to DOX.
We also assayed the effect of reactive oxygen intermediates on Egr-1 expression. We found that 2 μmol/L H2O2 increased the level of Egr-1 mRNA, as well as the transcriptional activity of the Egr-1 gene. We also found that preloading the cardiocytes with N-acetylcysteine partially abolished the DOX-induced increases in Egr-1 mRNA and transcription (Figure 6⇓).
To determine whether Egr-1 recognizes and binds to the 5′-regulatory region of SERCA2 gene, electrophoretic mobility shift assay was performed on the 116-bp fragment of SERCA2 gene, nucleotides −206 to −91, with nuclear proteins prepared from cardiac myocytes cultured in the presence or absence of 1 μmol/L DOX. A significantly increased formation of DNA/protein complexes was observed in assays using nuclear extracts from DOX-treated cardiac myocytes (Figure 7⇓, lanes 4 and 5). Bound proteins were identified as Egr-1 by the competition with unlabeled Egr-1 consensus and mutant oligonucleotides and by the supershift assay using an antibody against Egr-1 (Figure 7⇓, lanes 6 to 9).
Finally, to test whether Egr-1 has a physiological role in the DOX-induced downregulation of SERCA2 gene transcription in living cardiac myocytes, we transfected the cells with antisense oligonucleotides and measured expression levels of SERCA2 mRNA. We found that Egr-1 antisense oligonucleotides blocked decrease in SERCA2 mRNA induced by DOX in a dose-dependent manner (Figure 8a⇓). The specificity of this inhibition was shown by the inability of sense oligonucleotides, randomly scrambled antisense oligonucleotides, having the same nucleotide ratio as the antisense nucleotides, and mismatch oligonucleotides, having a sequence 20% different from the antisense oligonucleotides, to inhibit the DOX-induced decrease of SERCA2 mRNA expression (Figure 8b⇓).
MAPKs Play a Key Role in DOX-Induced Downregulation of SERCA2 Gene Transcription
Recently, it was reported that exposure of neutrophils to oxidants increases the activity of MEK, an upstream activator of MAPK (mitogen-activated protein kinase/extracellular signal–regulated kinase [ERK]).15 MAPKs have also been shown to activate the Egr-1 gene in renal cells.16 We therefore used MAPK-specific antibodies to determine whether these protein kinases are involved in the signaling pathway utilized by DOX to regulate SERCA2 gene activity. In nonstimulated cells, the binding of antibodies directed against p44/42 MAPK (ERK1/ERK2), p38 MAPK, and stress-activated MAPK/Jun N-terminal kinase (SAPK/JNK) were weak and scattered around the cytoplasm. Incubation of the cardiocytes with DOX, however, led to increased binding of antibodies directed against these kinases. In addition, the signals were translocated to the nuclei, suggesting that these kinases are activated by DOX (Figure 9a⇓). To determine which of the MAPKs is functionally relevant to the regulation of SERCA2 gene, we tested the effect of a specific inhibitor of each kinase on the expression of Egr-1 and SERCA2 mRNA. We found that SB203580, a specific inhibitor of p38 MAPK, did not affect DOX-induced downregulation of SERCA2 mRNA. In contrast, PD98059, a specific inhibitor of MEK1 (a kinase upstream of p44/42 MAPK), blocked the DOX-induced activation of Egr-1 mRNA, as well as the DOX-induced downregulation of SERCA2 mRNA expression (Figure 9b⇓), thus suggesting that p44/42 MAPK may play a key role in the signaling pathway utilized by DOX in regulating the SERCA2 mRNA expression.
Several hypotheses have been proposed to explain the cardiotoxicity of DOX, including the intercalation of DOX between DNA base pairs, free radical formation, a mitochondrial effect, and the inhibition of myocardial Ca2+ transport.17 Our finding, that the antioxidant N-acetylcysteine prevented the DOX-induced decrease in SERCA2 mRNA expression, suggests that reactive oxygen intermediates mediate the inhibitory action of DOX on SERCA2 gene expression. DOX has been reported to produce reactive oxygen intermediates, including hydroxyl18 and superoxide19 radicals, as well as hydrogen peroxide.19 In addition, catalase, an enzyme that hydrolyzes hydrogen peroxide, was shown to be more effective in maintaining cardiac function than either mannitol, a hydroxyl radical scavenger, or a superoxide dismutase,19 suggesting to us that hydrogen peroxide has a role in modulating the transcriptional activity of the SERCA2 gene. In testing this hypothesis, we found that DOX induced a 3-fold increase in H2O2 concentration and that exogenously administered H2O2 significantly decreased the transcription of the SERCA2 gene and thus the expression level of its mRNA. These findings suggest that the H2O2 generated by DOX in cardiac myocytes inhibits transcription of the SERCA2 gene. It should be noted, however, that N-acetylcysteine was only partially effective in counteracting the DOX-induced decrease in SERCA2 mRNA. In addition, whereas DOX increased the concentration of H2O2 significantly, it was not high enough to account for the reduction of SERCA2 mRNA levels to that observed after DOX administration. These results therefore suggest that H2O2 is not the sole intracellular messenger mediating the effects of DOX on SERCA2 gene expression.
To attempt to identify DOX-responsive elements in the 5′ flanking region of the SERCA2 gene, we constructed deletions in this region and measured the transcriptional activity of each in the presence or absence of DOX. Transcription from both the −284- to +350-bp and −1810- to +350-bp sequences was diminished 10-fold by DOX, suggesting that the SERCA2 gene has 2 DOX-susceptible regions. Our findings, however, identified the −284- to −72-bp region as the primary DOX-susceptible region of the SERCA2 gene. This region is highly GC rich and contains the consensus sequences that bind the transcription factors Sp1 (5′-GGGGCGGGGC-3′), Egr-1 (5′-GCGGGGGCG-3′), and AP2 (5′-GCCCGCGG-3′). We found that overexpression of Sp1 increased transcription of the SERCA2 gene 5-fold in cardiac myocytes. Interestingly, Baker et al13 have demonstrated that Sp1 sites in this region are essential for the muscle-specific expression of the SERCA2 gene in the Sol8 mouse skeletal muscle cell line. Because it has been reported that DOX inhibits the myogenesis of C2 myoblasts, a cell line established from mouse skeletal muscle, by preventing muscle-specific gene expression without significantly altering nonmuscle gene transcripts,20 we predicted that DOX suppresses the function of Sp1 and thus decreases transcription of the SERCA2 gene. To test this hypothesis, we assayed Sp1 mRNA expression after DOX administration, but we observed no alteration in Sp1 mRNA level.
We also found that p300, which was recently cloned and characterized as a transcriptional adapter protein,10 and which has been reported to associate with MyoD, CREB, c-Jun, c-Fos, or Sp1 and to function as a coactivator of these transcription factors,19 21 22 23 24 did not function in place of Sp1. Specifically, the level of expression of p300 mRNA, which was low in untreated cells, was not altered significantly in DOX-treated myocytes, suggesting that p300 does not play a role in regulating DOX-modified transcription of the SERCA2 gene.
In contrast, we found that the expression of Egr-1 mRNA increased significantly as early as 1 hour after DOX administration and that overexpression of Egr-1 strongly inhibited SERCA2 gene transcription in cardiac myocytes. These findings strongly suggested that Egr-1 serves as an inhibitor of SERCA2 gene transcription. Further characterization of Egr-1 revealed that N-acetylcysteine partially abolished DOX-induced increases in Egr-1 transcription and that H2O2, which decreased SERCA2 mRNA levels, increased the levels of Egr-1 mRNA. These findings further indicate that Egr-1 is a negative regulator of the SERCA2 gene. In fact, Egr-1 recognizes and binds to the 5′-regulatory region of SERCA2 gene. Finally, our findings, that Egr-1 antisense oligonucleotides prevented the DOX-induced decrease in SERCA2 mRNA, indicate that Egr-1 is a key transcription factor in the DOX signaling cascade that induces inhibition of SERCA2 gene transcription.
We also found that transcription of the Egr-1 gene was markedly activated by DOX. The 5′ flanking sequence of the Egr-1 gene has 5 domains of serum-responsive elements (SREs), which are the sequence responsible for activation of the c-fos gene after serum stimulation.9 25 The c-fos SRE, which comprises a core sequence and a nearby Ets motif, binds serum response factor over the core sequence, as well as the ternary complex factor, Elk-1, over the Ets motif.26 27 Because MAPKs phosphorylate the Elk-1, as part of the mechanism by which c-fos transcription is activated through the SRE,28 we sought to determine whether DOX activates MAPKs and whether these activated MAPKs upregulate the transcription of the Egr-1 gene. We found that DOX induces strong phosphorylation of all 3 MAPKs, p44/42 MAPK (also known as ERK), p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). We also observed translocation of these protein kinases from cytoplasm to nuclei in DOX-treated cardiac myocytes. Experiments to determine which MAPK is responsible for the DOX-induced upregulation of Egr-1 mRNA expression showed that PD98059, a specific inhibitor of p44/42 MAPK kinase, prevented the DOX-induced increase of Egr-1 mRNA and the decrease of SERCA2 mRNA, suggesting that this kinase is activated by DOX, subsequently activating Egr-1 mRNA expression and leading to a decrease of SERCA2 mRNA expression. Because Egr-1 antisense oligonucleotides were only partially effective in preventing a decrease in SERCA2 mRNA expression, these findings suggest that the signaling pathway via Egr-1 is important in mediating the effects of DOX on SERCA2 gene regulation, but that it is not the only such pathway. Other transcription factors, which bind to the distal part of DOX-responsive sequences in the SERCA2 gene, may also be involved in regulating expression of the gene.
Our findings suggest a molecular mechanism by which DOX inhibits the transcription of the SERCA2 gene (Figure 10⇓). In this model, DOX inhibits the transcription of SERCA2 gene, thereby decreasing its expression, and H2O2 is an intracellular mediator of the DOX signaling pathway. The model further predicts that DOX activates 3 MAPKs, p44/42 MAPK (ERK), p38 MAPK, and SAPK/JNK, the first of which is a key transcriptional inhibitor of the SERCA2 gene. This model also shows that Egr-1, which is activated by p44/42 MAPK, is the transcription factor responsible for the DOX-induced downregulation of SERCA2 gene expression and that Egr-1 binding motifs in the −284- to −72-bp 5′ flanking sequence of the SERCA2 gene are the targets of DOX. This model may have important implications for the prevention of the abnormal calcium handling and cardiac dysfunction observed in DOX cardiomyopathy.
This study was supported in part by a Grant-in-Aid (09670692) for Scientific Research from the Ministry of Education, Science and Culture of Japan and by grants from the Study Group of Molecular Cardiology of the Japan Heart Foundation and from the Ichiro Kanehara Foundation. We greatly thank Dr Muthu Periasamy, University of Cincinnati for providing the SERCA2 gene.
- Received August 17, 1999.
- Accepted October 13, 1999.
- © 2000 American Heart Association, Inc.
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