Nancy Olashaw

SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress

SIRT1 is the closest mammalian homologue of yeast SIR2, an important ageing regulator that prolongs lifespan in response to caloric restriction. Despite its importance, the mechanisms that regulate SIRT1 activity are unclear. Our study identifies a novel post-translational modification of SIRT1, namely sumoylation at Lys 734. In vitro sumoylation of SIRT1 increased its deacetylase activity. Conversely, mutation of SIRT1 at Lys 734 or desumoylation by SENP1, a nuclear desumoylase, reduced its deacetylase activity. Stress-inducing agents promoted the association of SIRT1 with SENP1 and cells depleted of SENP1 (but not of SENP1 and SIRT1) were more resistant to stress-induced apoptosis than control cells. We suggest that stress-inducing agents counteract the anti-apoptotic activity of SIRT1 by recruiting SENP1 to SIRT1, which results in the desumoylation and inactivation of SIRT1 and the consequent acetylation and activation of apoptotic proteins.

PARTICIPATION OF REACTIVE OXYGEN SPECIES IN THE LYSOPHOSPHATIDIC ACID-STIMULATED MITOGEN-ACTIVATED PROTEIN KINASE KINASE ACTIVATION PATHWAY

Recent evidence suggests that reactive oxygen species (ROS) may function as second messengers in intracellular signal transduction pathways. We explored the possibility that ROS were involved in lysophosphatidic acid (LPA)-induced mitogen-activated protein (MAP) kinase signaling pathway in HeLa cells. Antioxidant N-acetylcysteine inhibited the LPA-stimulated MAP kinase kinase activity. Direct exposure of HeLa cells to hydrogen peroxide resulted in a concentration- and time-dependent activation of MAP kinase kinase. Inhibition of catalase with aminotriazole enhanced the effect of LPA on induction of MAP kinase kinase. Further, LPA stimulated ROS production in HeLa cells. These findings suggest that ROS participate in the LPA-elicited MAP kinase signaling pathway.

Deacetylation of cortactin by SIRT1 promotes cell migration.

Cortactin binds F-actin and promotes cell migration. We showed earlier that cortactin is acetylated. Here, we identify SIRT1 (a class III histone deacetylase) as a cortactin deacetylase and p300 as a cortactin acetylase. We show that SIRT1 deacetylates cortactin in vivo and in vitro and that the SIRT1 inhibitor EX-527 increases amounts of acetylated cortactin in ovarian cancer cells. We also show that p300 acetylates cortactin in vivo and that cells lacking or depleted of p300 express less-acetylated cortactin than do control cells. Deletion analysis mapped the SIRT1-binding domain of cortactin to its repeat region, which also binds F-actin. Mouse embryo fibroblasts (MEFs) lacking sir2α (the mouse homolog of SIRT1) migrated more slowly than did wild-type cells. The expression of SIRT1 in sir2α-null cells restored migratory capacity, as did expression of a deacetylation-mimetic mutant of cortactin. SIRT1 and cortactin were more abundant in breast tumor tissue than in their normal counterparts, whereas SIRT1 expression inversely correlates with the ratio of acetylation cortactin versus total cortactin. These data suggest that deacetylation of cortactin is associated with high levels of SIRT1 and tumorigenesis. Finally, breast and ovarian cancer cell lines expressing an acetylation mimetic mutant of cortactin are less motile than that of control cells, whereas cells expressing the deacetylation mimetic mutant of cortactin migrate faster than that of control cells in Transwell migration assays. In summary, our results suggest that cortactin is a novel substrate for SIRT1 and p300 and, for the first time, a possible role for SIRT1 in cell motility through deacetylation of cortactin.

Cell adhesion-mediated drug resistance (CAM-DR) is associated with activation of NF-κB (RelB/p50) in myeloma cells

The microenvironment has been shown to influence tumor cell phenotype with respect to growth, metastasis, and response to chemotherapy. We have utilized oligonucleotide microarray analysis to identify signal transduction pathways and gene products altered by the interaction of myeloma tumor cells with the extracellular matrix component fibronectin that may contribute to the antiapoptotic phenotype conferred by the microenvironment. Genes with altered expression associated with fibronectin cell adhesion, either induced or repressed, were numerically ranked by fold change. FN adhesion repressed the expression of 469 gene products, while 53 genes with known coding sequences were induced by twofold or more. Of these 53 genes with two fold, or greater increase in expression, 11 have been reported to be regulated by the nuclear factor-kappa B (NF-κB) family of transcription factors. EMSA analysis demonstrated NF-κB binding activity significantly increased in cells adhered to fibronectin compared to cells in suspension. This DNA binding activity consisted primarily of RelB-p50 heterodimers, which was distinct from the NF-κB activation of TNFα. These data demonstrate the selectivity of signal transduction from the microenvironment that may contribute to tumor cell resistance to programmed cell death.

SIRT1 deacetylates the DNA methyltransferase 1 (DNMT1) protein and alters its activities.

ABSTRACT DNA methylation and histone acetylation/deacetylation are distinct biochemical processes that control gene expression. While DNA methylation is a common epigenetic signal that inhibits gene transcription, histone deacetylation similarly represses transcription but can be both an epigenetic and nonepigenetic phenomenon. Here we report that the histone deacetylase SIRT1 regulates the activities of DNMT1, a key enzyme responsible for DNA methylation. In mass spectrometry analysis, 12 new acetylated lysine sites were identified in DNMT1. SIRT1 physically associates with DNMT1 and can deacetylate acetylated DNMT1 in vitro and in vivo. Interestingly, deacetylation of different lysines on DNMT1 has different effects on the functions of DNMT1. For example, deacetylation of Lys1349 and Lys1415 in the catalytic domain of DNMT1 enhances DNMT1s methyltransferase activity, while deacetylation of lysine residues in the GK linker decreases DNMT1s methyltransferase-independent transcriptional repression function. Furthermore, deacetylation of all identified acetylated lysine sites in DNMT1 abrogates its binding to SIRT1 and impairs its capability to regulate cell cycle G2/M transition. Finally, inhibition of SIRT1 strengthens the silencing effects of DNMT1 on the expression of tumor suppressor genes ER-α and CDH1 in MDA-MB-231 breast cancer cells. Together, these results suggest that SIRT1-mediated deacetylation of DNMT1 is crucial for DNMT1s multiple effects in gene silencing.

Activation of the Growth-Differentiation Factor 11 Gene by the Histone Deacetylase (HDAC) Inhibitor Trichostatin A and Repression by HDAC3

ABSTRACT Histone deacetylase (HDAC) inhibitors inhibit the proliferation of transformed cells in vitro, restrain tumor growth in animals, and are currently being actively exploited as potential anticancer agents. To identify gene targets of the HDAC inhibitor trichostatin A (TSA), we compared the gene expression profiles of BALB/c-3T3 cells treated with or without TSA. Our results show that TSA up-regulates the expression of the gene encoding growth-differentiation factor 11 (Gdf11), a transforming growth factor β family member that inhibits cell proliferation. Detailed analyses indicated that TSA activates the gdf11 promoter through a conserved CCAAT box element. A comprehensive survey of human HDACs revealed that HDAC3 is necessary and sufficient for the repression of gdf11 promoter activity. Chromatin immunoprecipitation assays showed that treatment of cells with TSA or silencing of HDAC3 expression by small interfering RNA causes the hyperacetylation of Lys-9 in histone H3 on the gdf11 promoter. Together, our results provide a new model in which HDAC inhibitors reverse abnormal cell growth by inactivation of HDAC3, which in turn leads to the derepression of gdf11 expression.

ERK couples chronic survival of NK cells to constitutively activated Ras in lymphoproliferative disease of granular lymphocytes (LDGL)

Chronic NK lymphoproliferative disease of large granular lymphocytes (LDGL) is characterized by the expansion of activated CD3−, CD16+ or CD56+ lymphocytes. The mechanism of survival of NK cells from LDGL patients is unknown but may be related to antigenic stimulation. There is currently no standard effective therapy for LDGL, and the disease is characteristically resistant to standard forms of chemotherapy. We found evidence of constitutive activation of extracellular-regulated kinase (ERK) in NK cells from 13/13 patients with NK-LDGL (one patient with aggressive and 12 patients with chronic disease). Ablation of ERK activity by inhibitors or a dominant-negative form of MEK, the upstream activator of ERK, reduced the survival of patient NK cells. Ras was also constitutively active in patient NK cells, and exposure of cells to the Ras inhibitor FTI2153 or to dominant-negative-Ras resulted not only in ERK inhibition but also in enhanced apoptosis in both the presence and absence of anti-Fas. Therefore, we conclude that a constitutively active Ras/MEK/ERK pathway contributes to the accumulation of NK cells in patients with NK-LDGL. These findings suggest that strategies to inhibit this signaling pathway may be useful for the treatment of the NK type of LDGL.

Inhibition of JNK by cellular stress- and tumor necrosis factor alpha-induced AKT2 through activation of the NF kappa B pathway in human epithelial Cells

Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor α (TNFα) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNFα-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNFα-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates IκB kinase α. The phosphorylation of IκB kinase α and activation of NFκB mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNFα-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFα stimulation with an accompanying decreased JNK and p38 activity. These results indicate that activated AKT2 protects epithelial cells from stress- and TNFα-induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NFκB pathway.

Tumor Necrosis Factor-α-induced Proliferation of Human Mo7e Leukemic Cells Occurs via Activation of Nuclear Factor κB Transcription Factor

Tumor necrosis factor-α (TNF-α) stimulates proliferation of Mo7e, CMK, HU-3, and M-MOK human leukemic cell lines. We report here the signal transduction pathway involved in TNF-α-induced Mo7e cell proliferation. Mo7e cells spontaneously die in the absence of growth factors, but treating the cells with interleukin (IL)-3, IL-6, thrombopoietin, granulocyte/macrophage colony-stimulating factor, or TNF-α promotes their survival and proliferation. Although most of these factors activate MAP kinase and Jun NH2-terminal kinase/signal transducer and activators of transcription signaling pathways, TNF-α fails to activate either pathway. When Mo7e cells were treated with TNF-α, nuclear factor κB (NF-κB) was activated transiently. The activated NF-κB consisted of heterodimers of p65 and p50 subunits. The degradation of IκBα coincided with activation of NF-κB in TNF-α-treated cells. To investigate the role of activated NF-κB in TNF-α-induced Mo7e proliferation, a cell-permeable peptide (SN50) carrying the nuclear localization sequence of p50 NF-κB was used to block nuclear translocation of activated NF-κB. Pretreating Mo7e cells with SN50 blocked TNF-α-induced nuclear translocation of NF-κB and inhibited TNF-α-induced Mo7e cell survival and proliferation. A mutant SN50 peptide did not affect TNF-α-induced Mo7e cell growth. SN50 had no effects on IL-3- or granulocyte/macrophage colony-stimulating factor-induced Mo7e cell proliferation. The results indicate that activation of NF-κB is involved in TNF-α-induced Mo7e cell survival and proliferation.

Inhibition of JNK by Cellular Stress- and Tumor Necrosis Factor α-induced AKT2 through Activation of the NFκB Pathway in Human Epithelial Cells

Abstract Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor α (TNFα) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNFα-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNFα-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates IκB kinase α. The phosphorylation of IκB kinase α and activation of NFκB mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNFα-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFα stimulation with an accompanying decreased JNK and p38 activity. These results indicate that activated AKT2 protects epithelial cells from stress- and TNFα-induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NFκB pathway.