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Aims Cardiotoxicity by doxorubicin predicts worse prognosis of patients. Accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. SIRT1, an NAD+-dependent histone/protein deacetylase, protects cells by deacetylating target proteins. We investigated whether SIRT1 counteracts doxorubicin-induced cardiotoxicity by mediating Ser139 phosphorylation of histone H2AX, a critical signal of the DNA damage response. Methods and results Doxorubicin (5 mg/kg per week, x4) was administered to mice with intact SIRT1 (Sirt1f/f) and mice that lack SIRT1 activity in cardiomyocytes (Sirt1f/f; MHCcre/+). Reductions in left ventricular fractional shortening and ejection fraction by doxorubicin treatment were more severe in Sirt1f/f; MHCcre/+ than in Sirt1f/f. Myocardial expression level of type-B natriuretic peptide was 2.5-fold higher in Sirt1f/f; MHCcre/+ than in Sirt1f/f after doxorubicin treatment. Sirt1f/f; MHCcre/+ showed larger fibrotic areas and higher nitrotyrosine levels in the heart after doxorubicin treatment. Although doxorubicin-induced DNA damage evaluated by TUNEL staining was enhanced in Sirt1f/f; MHCcre/+, the myocardium from Sirt1f/f; MHCcre/+ showed blunted Ser139 phosphorylation of H2AX by doxorubicin treatment. In H9c2 cardiomyocytes, SIRT1 knockdown attenuated Ser139 phosphorylation of H2AX, increased DNA damage, and enhanced caspase-3 activation under doxorubicin treatment. Immunostaining revealed that acetylation level of H2AX at Lys5 was higher in hearts from Sirt1f/f; MHCcre/+. In H9c2 cells, acetyl-Lys5-H2AX level was increased by SIRT1 knockdown and reduced by SIRT1 overexpression. Ser139 phosphorylation in response to doxorubicin treatment was blunted in a mutant H2AX with substitution of Lys5 to Gln (K5Q) that mimics acetylated lysine compared with that in wild-type H2AX. Expression of K5Q-H2AX as well as S139A-H2AX, which cannot be phosphorylated at Ser139, augmented doxorubicin-induced caspase-3 activation. Treatment of mice with resveratrol, a SIRT1 activator, attenuated doxorubicin-induced cardiac dysfunction, which was associated with a reduction in acetyl-Lys5-H2AX level and a preserved phospho-Ser139-H2AX level. Conclusion These findings suggest that SIRT1 counteracts doxorubicin-induced cardiotoxicity by mediating H2AX phosphorylation through its deacetylation in cardiomyocytes. Translational perspective Doxorubicin-induced cardiotoxicity limits the further use of doxorubicin for cancer treatment and determines prognosis of patients. This work shows for the first time the protective effect of SIRT1, an NAD+-dependent deacetylase, on doxorubicin-induced cardiotoxicity using a genetically modified mouse model. We identified histone H2AX as a target of SIRT1 for proper DNA damage response. Therefore, DNA repair by SIRT1 could be a potential therapeutic target to attenuate doxorubicin cardiotoxicity. SIRT1 activity may also help to predict a risk of developing cardiotoxicity in patients treated with doxorubicin.

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STATa, a Dictyostelium homologue of metazoan signal transducer and activator of transcription, is important for the organizer function in the tip region of the migrating Dictyostelium slug. We previously showed that ecmF gene expression depends on STATa in prestalk A (pstA) cells, where STATa is activated. Deletion and sitedirected mutagenesis analysis of the ecmF/lacZ fusion gene in wild-type and STATa null strains identified an imperfect inverted repeat sequence, ACAAATANTATTTGT, as a STATa-responsive element. An upstream sequence element was required for efficient expression in the rear region of pstA zone; an element downstream of the inverted repeat was necessary for sufficient prestalk expression during culmination. Band shift analyses using purified STATa protein detected no sequence-specific binding to those ecmF elements. The only verified upregulated target gene of STATa is cudA gene; CudA directly activates expL7 gene expression in prestalk cells. However, ecmF gene expression was almost unaffected in a cudA null mutant. Several previously reported putative STATa target genes were also expressed in cudA null mutant but were downregulated in STATa null mutant. Moreover, mybC, which encodes another transcription factor, belonged to this category, and ecmF expression was downregulated in a mybC null mutant. These findings demonstrate the existence of a genetic hierarchy for pstA-specific genes, which can be classified into two distinct STATa downstream pathways, CudA dependent and independent. The ecmF expression is indirectly upregulated by STATa in a CudAindependent activation manner but dependent on MybC, whose expression is positively regulated by STATa.

Dictyostelium harbors multiple expansin-like genes with generally unknown functions. Thus, we analyzed the expansin-like 3 (expL3) gene and found that its expression was reduced in a null mutant for a STATa gene encoding a transcription factor. The expression of expL3 was developmentally regulated and its transcript was spliced only in the multicellular stages. The expL3 promoter was activated in the anterior prestalk region of the parental strain and downregulated in the STATa null slug, although the expL3 promoter was still expressed in the prestalk region. The expL3 overexpressing strain exhibited delayed development and occasionally formed an aberrant structure, i.e., a fruiting body-like structure with a short stalk. The ExpL3-myc protein bound cellulose.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-0964-0) contains supplementary material, which is available to authorized users.