Mechanism of Doxorubicin-Induced Inhibition of Sarcoplasmic Reticulum Ca 2+ -ATPase Gene Transcription

Abstract

Abstract —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.