SIRT1 is a conserved NAD-dependent deacetylase that regulates life span in accord with nutritional provision. In mammalian cells, SIRT1 also down-regulates stress-induced p53 and FoxO pathways for apoptosis, thus favoring survival under stress. The functioning of SIRT1 under normal, nonstressed conditions of cell growth is unknown. Here we have asked if SIRT1 has the capacity to influence cell viability in the absence of applied stress. For this purpose we used synthetic small interfering RNA to silence SIRT1 gene expression by RNA interference (RNAi). We show that the process of RNAi, by itself, does not affect cell growth and is not sufficient to activate a cellular stress response (indicated by lack of activation of endogenous p53). We also show that, in the absence of applied stress, SIRT1 silencing induces growth arrest and/or apoptosis in human epithelial cancer cells. In contrast, normal human epithelial cells and normal human diploid fibroblasts seem to be refractory to SIRT1 silencing. Combined gene knockout with RNAi cosilencing experiments indicate that SIRT1 and Bcl-2 may suppress separable apoptotic pathways in the same cell lineage and that the SIRT1-regulated pathway is independent of p53, Bax, and caspase-2. Alternatively, SIRT1 may suppress apoptosis downstream from these apoptotic factors. In either case, we show that FoxO4 (but not FoxO3) is required as proapoptotic mediator. We further identify caspase-3 and caspase-7 as downstream executioners of SIRT1/FoxO4-regulated apoptosis. Our work identifies SIRT1 as a novel target for selective killing of cancer versus noncancer epithelial cells. (Cancer Res 2005; 65(22): 10457-63)

Mammalian SIRT1 is an NAD-dependent deacetylase with critical roles in the maintenance of homeostasis and cell survival. Elevated levels of SIRT1 protein are evident in cancer in which SIRT1 can function as a cancer-specific survival factor. Here we demonstrate that elevated SIRT1 protein in human cells is not attributable to increased SIRT1 mRNA levels but, instead, reflects SIRT1 protein stability. RNAi-mediated depletion of JNK2 reduced the half-life of SIRT1 protein from >9 h to <2 h and this correlated with lack of SIRT1 protein phosphorylation at serine 27. In contrast, depletion of JNK1 had no effect upon SIRT1 protein stability and SIRT1 phosphorylation at serine 47 showed no correlation with SIRT1 protein stability. Thus we show that JNK2 is linked, directly or indirectly, with SIRT1 protein stability and that this function is coupled with SIRT1 phosphorylation at serine 27. Our observations identify a route for therapeutic modulation of SIRT1 protein levels in SIRT1-linked diseases including cancer, neurodegeneration and diabetes.

BackgroundThe NAD-dependent deacetylase SIRT1 is a nutrient-sensitive coordinator of stress-tolerance, multiple homeostatic processes and healthspan, while p53 is a stress-responsive transcription factor and our paramount tumour suppressor. Thus, SIRT1-mediated inhibition of p53 has been identified as a key node in the common biology of cancer, metabolism, development and ageing. However, precisely how SIRT1 integrates such diverse processes remains to be elucidated.Methodology/Principal FindingsHere we report that SIRT1 is alternatively spliced in mammals, generating a novel SIRT1 isoform: SIRT1-ΔExon8. We show that SIRT1-ΔExon8 is expressed widely throughout normal human and mouse tissues, suggesting evolutionary conservation and critical function. Further studies demonstrate that the SIRT1-ΔExon8 isoform retains minimal deacetylase activity and exhibits distinct stress sensitivity, RNA/protein stability, and protein-protein interactions compared to classical SIRT1-Full-Length (SIRT1-FL). We also identify an auto-regulatory loop whereby SIRT1-ΔExon8 can regulate p53, while in reciprocal p53 can influence SIRT1 splice variation.Conclusions/SignificanceWe characterize the first alternative isoform of SIRT1 and demonstrate its evolutionary conservation in mammalian tissues. The results also reveal a new level of inter-dependency between p53 and SIRT1, two master regulators of multiple phenomena. Thus, previously-attributed SIRT1 functions may in fact be distributed between SIRT1 isoforms, with important implications for SIRT1 functional studies and the current search for SIRT1-activating therapeutics to combat age-related decline.

SIRT1 is an NAD-dependent deacetylase and epigenetic regulator essential for normal mammalian development and homeostasis. Here we describe a human SIRT1 splice variant, designated SIRT1-⌬2/9, in which the deacetylase coding sequence is lost due to splicing between exons 2 and 9. This work aimed to determine if SIRT1-⌬2/9 is a novel functional product of the SIRT1 gene. Endogenous SIRT1-⌬2/9 protein was identified in human cell lysate by immunoblotting and splice variant-specific RNA interference (RNAi). SIRT1-⌬2/9 mRNA is bound by CUGBP2, which downregulates its translation. Using pulldown assays, we demonstrate that SIRT1-⌬2/9 binds p53 protein. SIRT1-⌬2/9 maintains basal p53 protein levels and supports p53 function in response to DNA damage, as evidenced by RNAi-mediated depletion of SIRT1-⌬2/9 prior to damage. In turn, basal p53 downregulates SIRT1-⌬2/9 RNA levels, while stress-activated p53 eliminates SIRT1-⌬2/9. Loss of wild-type (wt) p53 has been correlated with overexpression of SIRT1-⌬2/9 in a range of human cancers. Exogenous SIRT1-⌬2/9 protein associates with specific promoters in chromatin and can regulate cancer-related gene expression, as evidenced by chromatin immunoprecipitation analysis and RNAi/genomic array data. SIRT1 is of major therapeutic importance, and potential therapeutic drugs are screened against SIRT1 deacetylase activity. Our discovery of SIRT1-⌬2/9 identifies a new, deacetylase-independent therapeutic target for SIRT1-related diseases, including cancer. Mammalian SIRT1 belongs to the sirtuin family of proteins that was first identified and characterized in yeast and subsequently found to be highly conserved through evolution (2,23,43,47,51). The Saccharomyces cerevisiae homologue of SIRT1 is Sir2, which stabilizes yeast chromosomes and impacts yeast aging. In mammals, SIRT1 is an epigenetic regulator of normal development, gametogenesis, homeostasis, and aging-related processes (3,22,30,34,41,54). Mammalian genes that fall within the scope of SIRT1 regulation include key genes linked, for example, with hormonal control of metabolism and insulin signaling (e.g., PGC-1␣), the ability of cells to respond to stress (e.g., p53, Foxo, and p300), and the processing of amyloid precursor protein in neuronal cells of the brain (ADAM10) (6,15,17,20,31,37,42,52,53). These genes in turn link SIRT1 with disease processes, including diabetes, cancer, and neurodegeneration (4,17,54).Given the multifunctional roles of SIRT1 in health and disease, it is not surprising that SIRT1 is now recognized as an important therapeutic target across a range of age-related diseases, and this is a strong driving force for understanding the pathways subject to SIRT1 activity. For example, with a mouse model of Alzheimer's disease, Guarente's group recently demonstrated that SIRT1 suppresses the production of ␤-amyloid protein and the formation of amyloid plaques in the brain. This is achieved via SIRT1-dependent transcriptional activation of ␣-secretase ADAM10, which is involved in the cellular cleavage of amyloi...