
We investigated whether SIRT1 is associated with reactive oxygen species (ROS) accumulation during CK2 downregulation-mediated senescence. SIRT1 overexpression suppressed ROS accumulation, reduced transcription of FoxO3a target genes, and nuclear export and acetylation of FoxO3a, which were induced by CK2 downregulation in HCT116 and MCF-7 cells. Conversely, overexpression of a dominant-negative mutant SIRT1 (H363Y) counteracted decreased ROS levels, increased transcriptional activity of FoxO3a, and increased nuclear import and decreased acetylation of FoxO3a, which were induced by CK2 upregulation. CK2 downregulation destabilized SIRT1 protein via an ubiquitin-proteasome pathway in human cells, whereas CK2 overexpression reduced ubiquitination of SIRT1. Finally, the SIRT1 activator resveratrol attenuated the accumulation of ROS and lipofuscin as well as lifespan shortening, and reduced expression of the DAF-16 target gene
Reactive oxygen species (ROS) induce DNA damage, which results in the stabilization of p53 and subsequent overexpression of p21Cip1/WAF1, which then proceeds to inhibit cell cycle progression. Cellular senescence is the state of irreversible cell cycle arrest at the G1 phase (1–4). We previously reported that downregulation of protein kinase CK2 (CK2) activity induces premature senescence of various human cells through the ROS–p53–p21Cip1/WAF1 pathway (5, 6). In addition, we demonstrated that the PI3K–AKT–mTOR pathway is involved in CK2 inhibition-mediated ROS generation (7) and that CK2 downregulation stimulated ROS accumulation via AKT-mediated phosphorylation of FoxO3a, which resulted in reduced transcriptional activity of FoxO3a (8). CK2 downregulation resulted in reduced longevity and onset of age-related biomarkers associated with both ROS generation and the AGE-1/PI3K-AKT-1/AKT-DAF-16/FoxO pathway in nematodes (9).
Forkhead box O (FoxO) class proteins are transcription factors, which modulate expression of various antioxidant genes. Humans express four FoxO class members (FoxO1a, FoxO3a, FoxO4, and FoxO6); of these, FoxO3a is primarily involved in resistance to oxidative stress (10). The transcriptional activity of FoxO3a can be regulated by post-translational modifications such as phosphorylation and acetylation. For example, AKT phosphorylates Ser 253 on FoxO3a, causing nuclear export of FoxO3a (11). The complex of cyclic AMP response element binding protein (CBP) and its associated protein p300 (CBP/p300) acetylates FOXO3a, resulting in the exclusion of FOXO3a from the nucleus (12). It has been demonstrated that the deacetylation of FOXO3a by SIRT1, a paralog of silent information regulator 2 (Sir2), represses the activity of FOXO3a (13). However, SIRT1 has additionally been reported to stimulate the transcription of FoxO3a target genes via deacetylation of FOXO3a (14). Therefore, the detailed mechanism for SIRT1-mediated FoxO3a regulation remains unclear. We investigated the physiological significance of a SIRT1-FoxO3a axis in ROS accumulation during cellular senescence and nematode aging induced by CK2 downregulation. CK2 downregulation mediated inhibition of the SIRT1-FoxO3a axis, which resulted in ROS accumulation.
To analyze whether SIRT1 is involved in CK2 inhibition-mediated ROS production, HCT116 and MCF-7 cells were transfected with SIRT1 cDNA in the presence of CK2α siRNA (
To analyze the impact of SIRT1 on the transcription of FoxO3a target genes during CK2 inhibition-mediated senescence, we introduced CK2α siRNA and/or pECE-Flag-SIRT1 into HCT116 and MCF-7 cells. CK2α knockdown decreased mRNA levels of major antioxidant genes, including Cu/ZnSOD, MnSOD, catalase, thioredoxin-2, and peroxiredoxin-5, but ectopic expression of SIRT1 attenuated this result (Fig. 1C,
We examined whether SIRT1 regulates nuclear localization of FoxO3a during CK2 downregulation-mediated senescence. Immunocytochemical analysis clearly showed that, consistent with a previous study (8), CK2α knockdown stimulated cytoplasmic sequestration of FoxO3a in cells. Ectopic expression of SIRT1 abolished the CK2 downregulation-mediated nuclear export of FoxO3a (Fig. 2A). Further densitometric analysis of immunofluorescence images confirmed this result. In contrast, the nuclear import of FoxO3a was stimulated in ectopic CK2α expressing cells, but concomitant overexpression of SIRT1 mutant (H363Y) abrogated FoxO3a nuclear localization in ectopic CK2α expressing cells (Fig. 2B). Collectively, these data demonstrate that CK2 downregulation results in cytoplasmic retention of FoxO3a by inhibiting SIRT1 in cells.
It has been reported that the acetylation status of Lys residues on FoxO3a can be modulated by the histone acetyl transferase CBP/p300 and the deacetylase SIRT1 (12–14). To determine the effect of SIRT1 on FoxO3a acetylation during CK2 downregulation-mediated senescence, we immunoprecipitated total proteins containing acetylated Lys from cell extracts using anti-acetylated Lys antibodies and then probed the immunoprecipitates with anti-FoxO3a antibodies. Cell extracts immunoprecipitated with IgG were used as a control. Higher levels of acetylated FoxO3a protein were observed in cells downregulating CK2α compared with control cells. Ectopic expression of SIRT1, however, abrogated the induction in acetylated FoxO3a levels caused by CK2 downregulation (Fig. 2C). In contrast, CK2α overexpression (Fig. 2D) or treatment with 15 mM nicotinamide (
It has been previously reported that SIRT1 deacetylase activity can be stimulated by CK2 phosphorylation (17, 18). In this study, however, we observed that CK2 regulates the protein levels of SIRT1 (Fig. 3A). To analyze whether CK2 regulates expression of SIRT1 at the transcription level, we extracted total RNA from cells transfected with CK2α siRNA or pcDNA3.1-HA-CK2α and then performed RT-PCR using specific primers for SIRT1. Knockdown or overexpression of CK2α did not change the mRNA levels of SIRT1 compared with control cells, suggesting that CK2 regulates SIRT1 at a post-transcription level (
Because
We have previously reported that CK2 is downregulated in senescent human lung fibroblast IMR-90 cells, aged rat tissues, and aging nematodes (5–9, 23). CK2 downregulation induces premature senescence in IMR-90, HCT116, and MCF-7 cells (23, 24), and
The previous studies have shown that CK2 promotes SIRT1 deacetylase activity through its phosphorylation (17, 18) and that phosphorylation of SIRT1 increases its substrate-binding affinity (17). The present study importantly indicates that CK2 downregulation stimulates degradation of SIRT1 protein through promoting SIRT1 ubiquitination in cells and, conversely, CK2 upregulation inhibits SIRT1 ubiquitination and increases the levels of SIRT1 protein. Ubiquitinated SIRT1 protein was increased by treatment with the proteasome inhibitor MG132, indicating that the proteasome pathway was responsible for degradation of SIRT1 protein induced by CK2 downregulation (Fig. 3). Collectively, the results found in this study and our previous work show that CK2 can modulate the activity and protein levels of SIRT1 through phosphorylation and ubiquitination. It has been reported that JNK1 and MEK1 signaling modulates SIRT1 stability through SIRT1 ubiquitination (25, 26). Ubiquitination is required for SIRT1 function during the DNA damage response (27). However, little is known about the molecular mechanism of SIRT1 ubiquitination. How does CK2 activity modulate the ubiquitination of SIRT1? Because it has been determined that CK2 phosphorylates E3 ubiquitin ligases such as MDM2 and SCF(cyclin F) complex (28, 29), we speculate that CK2 regulates SIRT1 ubiquitination via CK2-mediated phosphorylation of SIRT1 and/or E3 ubiquitin ligases. Although further studies are required to identify more regulators for SIRT1 ubiquitination, the discovery of CK2 regulation of SIRT1 ubiquitination provides a new avenue of research in the fields of SIRT1 regulation and senescence.
HCT116 human colon cancer and MCF-7 human breast cancer cells were cultured in Dulbecco’s modified Eagle medium containing 10% (v/v) fetal bovine serum under a humidified atmosphere of 5% (v/v) CO2 at 37°C. siRNAs, pcDNA-HA-CK2α, pECE-Flag-SIRT1, and pECE-Flag-SIRT1 (H363Y) were transfected into cells using Lipofectamine (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer’s instruction. siRNA for CK2α was 5′-UCAAGAUGACUACCAGCUGdTdT-3′. siRNA for the negative control was 5′-GCUCAGAUCAAUACGGAG AdTdT-3′. At 48 h after transfection, the cells were harvested.
Intracellular ROS levels were measured as described previously (6).
Antibodies to CK2α, SIRT1, ubiquitin, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against FoxO3a and acetylated Lys were obtained from Cell Signaling Technology (Beverly, MA, USA). Western blotting was performed as described previously (6).
Cells were seeded on four-well micro-chamber slides (Thermo Fisher Scientific, NY, USA) and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at 25°C and permeabilized in 0.25% Triton X-100 before blocking with 2% bovine serum albumin in PBS. Primary antibodies (anti-FoxO3a; 1:50) were added at 25°C for 1 h. The secondary antibodies were rhodamine-conjugated, goat anti-rabbit IgG (1:200, Invitrogen). Next, 4′,6-diamidino-2- phenylindole (DAPI; Invitrogen) was used to counterstain nuclei and fluorescence signals were detected using a Carl Zeiss Axioplan 2 microscope (Carl Zeiss, Jena, Germany). Fluorescence images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/).
Total RNA was extracted from HCT116 and MCF-7 cells. RNA was reverse transcribed using gene-specific primers and reverse transcriptase (Takara Bio Inc., Kyoto, Japan), and resulting cDNAs were PCR-amplified. The PCR primer sequences used for Cu/ZnSOD, MnSOD, catalase, thioredoxin-2, peroxiredoxin-5, and β-actin have been reported previously (8). The PCR primer sequences used for SIRT1 were: forward (5′-GCAGATTAG TAGGCGGCTTG-3′) and reverse (5′-TCTGGCATGTCCCACTA TCA-3′). PCR products were resolved on a 2% agarose gel. Quantification of the reverse transcribed-PCR bands was performed using densitometry. Levels of β-actin RNA were used to normalize the amount of RNA in each sample.
Cell lysates were pre-cleared with normal mouse or rabbit IgG and protein A sepharose beads (Amersham Pharmacia Biotech, Korea) for 1 h at 4°C. The supernatant was then incubated with anti-acetylated Lys antibodies (Cell Signaling Technology, Danvers, MA, USA) and protein A sepharose beads with mixing for 12 h at 4°C. Then, the beads were collected by centrifugation and washed three times with PBS.
Lifespan assays were performed as described previously (30). Synchronized L4 larvae were placed on HT115-seeded NGM plates containing FUdR. Surviving nematodes were counted daily and were moved to fresh HT115-seeded NGM plates. Death was scored as the absence of a response to slight touch using a thin platinum wire. Three independent experiments were performed.
ROS levels in synchronized (day 1 of adulthood) nematodes were measured as described previously (9). Intestinal lipofuscin accumulation in nematodes was determined by autofluorescence (9) using a fluorescence microscope (ZEISS AxioCam MRc, Jena, Germany) with excitation and emission wavelengths of 350 nm and 470 nm, respectively. The relative fluorescence intensity was quantified using ImageJ software to determine lipofuscin levels.
Synchronized (L4 larva) nematodes expressing
Data were analyzed by one-way analysis of variance with the SPSS package program (IBM, Armonk, NY). The results were considered significant if the P value was < 0.05. Duncan’s multiple-range test was performed if the differences between the groups were identified as α = 0.05. For all bar graphs, bars that do not share a common letter (a, b, c) are significantly different among the groups at P < 0.05.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning [grant number NRF-2015R1A2A2A01004593].
The authors have no conflicting interests.
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