Abstract
Type I interferons (IFNs) induce expression of multiple genes that control innate immune responses to invoke both antiviral and antineoplastic activities. Transcription of these interferon-stimulated genes (ISGs) occurs upon activation of the canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. Phosphorylation and acetylation are both events crucial to tightly regulate expression of ISGs. Here, using mouse embryonic fibroblasts and an array of biochemical methods including immunoblotting and kinase assays, we show that sirtuin 2 (SIRT2), a member of the NAD-dependent protein deacetylase family, is involved in type I IFN signaling. We found that SIRT2 deacetylates cyclin-dependent kinase 9 (CDK9) in a type I IFN-dependent manner and that the CDK9 deacetylation is essential for STAT1 phosphorylation at Ser-727. We also found that SIRT2 is subsequently required for the transcription of ISGs and for IFN-driven antiproliferative responses in both normal and malignant cells. These findings establish the existence of a previously unreported signaling pathway whose function is essential for the control of JAK-STAT signaling and the regulation of IFN responses. Our findings suggest that targeting sirtuin activities may offer an avenue in the development of therapies for managing immune-related diseases and cancer.
Highlights
Type I interferons (IFNs) induce expression of multiple genes that control innate immune responses to invoke both antiviral and antineoplastic activities
We found that sirtuin 2 (SIRT2) deacetylates cyclin-dependent kinase 9 (CDK9) in a type I IFN– dependent manner and that the CDK9 deacetylation is essential for STAT1 phosphorylation at Ser-727
SIRT2 is predominantly localized in the cytoplasm, 7 The abbreviations used are: IFN, interferon; JAK, Janus kinase; STAT, signal transducer and activator of transcription; interferon-stimulated genes (ISGs), interferon-stimulated gene; histone deacetylases (HDACs), histone deacetylase; CDK9, cyclin– dependent kinase 9; mouse embryonic fibroblasts (MEFs), mouse embryonic fibroblast; shRNA, short hairpin RNA; FBS, fetal bovine serum; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase
Summary
At the outset we examined whether IFN treatment of Sirt2Ϫ/Ϫ mouse embryonic fibroblasts (MEFs) leads to phosphorylation of STAT1. We examined the role of SIRT2 in the generation of type I IFN– dependent anti-leukemic activity in cells expressing the JAK2(V617F) mutation, which is a critical pathogenic mutation in myeloproliferative neoplasms [31] For this purpose, we performed studies involving siRNA-mediated knockdown of SIRT2 in human erythroleukemia HEL cells and in SET-2 cells derived from an essential thrombocythemia patient at the megakaryoblastic. IFN␣ treatment suppressed the growth of primitive malignant hematopoietic precursors from HEL cells transfected with control siRNA, but this inhibition was suppressed by SIRT2 knockdown (Fig. 5C and Fig. S2B), indicating a requirement for SIRT2 in the generation of the inhibitory effects of type I IFNs on JAK2(V617F)-transformed cells. These data indicate that SIRT2 activity is required for induction of type I IFN-driven antiproliferative effects in both normal and malignant cells
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