Mechanism of doxorubicin-induced inhibition of sarcoplasmic reticulum Ca(2+)-ATPase gene transcription.
Doxorubicin (DOX)-induced cardiomyopathy has been found to be associated with impaired Ca(2+) handling in the sarcoplasmic reticulum (SR), leading to reduced cardiac function. We have recently demonstrated that expression of mRNA encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 (SERCA2), a major Ca(2+) 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 H(2)O(2) concentration; thus, H(2)O(2) 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.
Arai
M
M
Second Department of Internal Medicine, Gunma University School of Medicine, Gunma, Japan. araim@akagi.sb.gunma-u.ac.jp
Yoguchi
A
A
Takizawa
T
T
Yokoyama
T
T
Kanda
T
T
Kurabayashi
M
M
Nagai
R
R
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D051381:Rats; D017382:Reactive Oxygen Species / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects* / Q000502:physiology
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D004317:Doxorubicin; D020928:Mitogen-Activated Protein Kinases; D000252:Calcium-Transporting ATPases