XBP1 (X-box-binding protein 1) is a key modulator of the UPR (unfolded protein response), which is involved in a wide range of pathological and physiological processes. The mRNA encoding the active spliced form of XBP1 (XBP1s) is generated from the unspliced form by IRE1 (inositol-requiring enzyme 1) during the UPR. However, the post-translational modulation of XBP1s remains largely unknown. In the present study, we demonstrate that XBP1s is a target of acetylation and deacetylation mediated by p300 and SIRT1 (sirtuin 1) respectively. p300 increases the acetylation and protein stability of XBP1s, and enhances its transcriptional activity, whereas SIRT1 deacetylates XBP1s and inhibits its transcriptional activity. Deficiency of SIRT1 enhances XBP1s-mediated luciferase reporter activity in HEK (human embryonic kidney)-293 cells and the up-regulation of XBP1s target gene expression under ER (endoplasmic reticulum) stress in MEFs (mouse embryonic fibroblasts). Consistent with XBP1s favouring cell survival under ER stress, Sirt1−/− MEFs display a greater resistance to ER-stress-induced apoptotic cell death compared with Sirt1+/+ MEFs. Taken together, these results suggest that acetylation/deacetylation constitutes an important post-translational mechanism in controlling protein levels, as well as the transcriptional activity, of XBP1s. The present study provides a novel insight into the molecular mechanisms by which SIRT1 regulates UPR signalling.
- cell death
- sirtuin 1 (SIRT1)
- unfolded protein response (UPR)
- X-box-binding protein 1 (XBP1)
XBP1 (X-box-binding protein-1), a bZIP (basic-region leucine zipper) transcription factor of the CREB (cAMP-response-element-binding protein)/ATF (activating transcription factor) protein family, is a key mediator of the UPR (unfolded protein response). When cells experience ER (endoplasmic reticulum) stress due to disturbance of ER homoeostasis, the UPR is activated to re-establish ER homoeostasis or to induce apoptosis of overly stressed cells [1,2]. The UPR consists of three parallel signalling branches initiated by PERK [PKR (double-stranded-RNA-dependent protein kinase)-like ER kinase], IRE1 (inositol-requiring enzyme 1) and ATF6. IRE1, the best-conserved proximal sensor of ER stress, executes unconventional splicing of XBP1 mRNA to generate XBP1s (the spliced form of XBP1) mRNA. The XBP1s mRNA encodes an active transcription factor that activates the expression of a wide variety of genes required for biogenesis of the protein secretory pathway, protein folding and secretion, as well as clearance of misfolded proteins from the ER [3,4]. Functionally, IRE1/XBP1s signalling supports cell survival under ER stress .
XBP1 is implicated in a wide range of human physiological and pathological processes. The critical function of XBP1 was first shown by a gene-knockout mouse model in which Xbp1-deficient embryos died in utero due to fatal anaemia caused by liver hypoplasia and apoptosis . XBP1s induces interleukin-6 production  and expands secretory apparatus in B-cells during their differentiation into plasma cells . Moreover, XBP1s drives cancer pathogenesis and is required for survival of MM (multiple myeloma) cells . XBP1s also plays an essential role in regulating adipogenesis and hepatic lipogenesis [10,11]. Although the pathophysiological significance of XBP1s has been well-established, the molecular mechanisms responsible for regulating XBP1s protein expression and its transcriptional activity remain to be elucidated.
The SIR2 (silent information regulator 2) family is a group of histone deacetylases that are highly conserved among species from prokaryotes to eukaryotes . There are three distinct classes of histone deacetylases including the class I, class II and the NAD+-dependent class III histone deacetylase families . SIRT1 (sirtuin 1), the closest mammalian homologue to SIR2 in Caenorhabditis elegans, belongs to the class III histone deacetylases and is the best-characterized SIR2 family member. It has been shown that SIRT1 also deacetylates non-histone proteins, including transcription factors, to modulate stress-responsive signalling pathways . For instance SIRT1 deacetylates FOXO3, a member of the forkhead box O family of transcription factors, which grants cells a greater resistance to oxidative stress . In addition, SIRT1 represses p53, via deacetylating it, to facilitate cellular recovery from genotoxic stress . In C. elegans, SIR2 was shown to regulate UPR genes through an unidentified mechanism . However, there is no evidence as yet to indicate whether SIRT1 is involved in regulating the mammalian UPR, especially its proximal modulators such as XBP1s.
Acetylation/deacetylation of transcription factors is emerging as an important post-translational regulatory mechanism to modulate their protein expression and transcriptional activity . Like XBP1s, ATF2 and ATF4 also belong to the CREB/ATF family [18,19]. Both ATF2 and ATF4 have been shown to be acetylated by the histone acetyltransferase p300 [20,21]. SREBP (sterol-regulatory-element-binding protein), a transcription factor critical for lipid and sterol homoeostasis in eukaryotes, has been shown to be a target of SIRT1-mediated deacetylation [22,23]. Since XBP1s, ATF4 and SREBP1 are all ER-stress-inducible molecules , and both XBP1s and SREBP1 share similar functions in regulating cellular lipid synthesis , we hypothesized that XBP1s may also be acetylated/deacetylated. In the present study, we demonstrate that XBP1s can be acetylated and deacetylated by p300 and SIRT1 respectively. These post-translational modifications regulate the protein level and transcriptional activity of XBP1s. The present study provides a novel mechanistic link between SIRT1 and UPR signalling.
Reagents and antibodies
DMEM (Dulbecco's modified Eagle's medium), FBS (fetal bovine serum), L-glutamine, MEM (minimal essential medium) plus NEAA (non-essential amino acid mix), 200× penicillin/streptomycin antibiotics, Lipofectamine™ reagent, Rhodamine-Red™-X-labelled anti-(mouse Ig) antibody (catalogue number R6393) and Alexa Fluor® 488-labelled anti-(rabbit Ig) antibody (catalogue number A11034) were purchased from Invitrogen. TSA (trichostatin A) was purchased from Cayman Chemical. EX-527 was purchased from Tocris Bioscience. Thapsigargin, tunicamycin, DTT (dithiothreitol), cycloheximide, the protease inhibitor cocktail, Trypan Blue (catalogue number T8154), the anti-β-actin antibody (catalogue number A5316), the anti-FLAG M2 antibody (catalogue number F1804), anti-FLAG M2 affinity gel (catalogue number A2220) and the FLAG peptide (catalogue number F3290) were from Sigma–Aldrich. The anti-acetylated-lysine antibody (catalogue number 9441) and the anti-caspase-3 antibody (catalogue number 9662) were from Cell Signaling Technology. The anti-SIRT1 antibody (catalogue number sc-15404) and the anti-XBP1 antibody (catalogue number sc-7160) were from Santa Cruz Biotechnology. HRP (horseradish peroxidase)-conjugated IgG secondary antibodies were from GE Healthcare Life Sciences. Protein G–agarose beads and PVDF membranes were from Millipore.
HEK (human embryonic kidney)-293, HEK-293T [HEK-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and Cos-7 cells were grown in DMEM supplemented with 10% (v/v) FBS and 1× penicillin/streptomycin antibiotics. Sirt1+/+ and Sirt1−/− MEFs (mouse embryonic fibroblasts)  were kindly provided by Dr Frederick W. Alt (Harvard University, Boston, MA, U.S.A.) via Dr Paul Robbins (University of Pittsburgh, Pittsburgh, PA, U.S.A.) and were grown in DMEM supplemented with 10% (v/v) FBS, 1× NEAA, 2 mM L-glutamine and 1× penicillin/streptomycin antibiotics.
Plasmids and transfection
The expression construct for XBP1s was generated by inserting mouse XBP1s coding regions with or without a FLAG tag into a pQCXIP-based vector (Clontech). The HA (haemagglutinin)–p300 construct was a gift from Dr Ming Hu (University of South Florida, Tampa, FL, U.S.A.). The FLAG–SIRT1 construct was obtained from AddGene . XBP1s-mediated transcriptional activity was measured using the 5× UPRE (UPR element) luciferase reporter (a gift from Dr Randal J. Kaufman, University of Michigan, Ann Arbor, MI, U.S.A.). The SIRT1 shRNA (small-hairpin RNA) plasmid was constructed by inserting a double-stranded oligonucleotide containing 5′-GAAGTTGACCTCCTCATTGT-3′  into the BglII/XhoI sites of the pSUPER.neo+gfp vector (OligoEngine). Transfection of cells was performed using Lipofectamine™ reagent.
Detection of acetylated XBP1s and the in vitro deacetylation assay
HEK-293 cells overexpressing FLAG–XBP1s were lysed in lysis buffer [20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM EGTA, 1 mM EDTA, 50 mM NaF, 1 mM 2-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM orthovanadate, 1× protease inhibitor cocktail, 10 mM nicotinamide and 1 mM TSA]. Cell extracts were subjected to immunoprecipitation with the anti-FLAG M2 antibody. The immune complexes eluted from Protein G–agarose beads were analysed by Western blotting with an anti-acetylated-lysine antibody. The stripped membranes were re-probed with the rabbit polyclonal antibody against XBP1.
For the in vitro deacetylation assay, acetylated XBP1s was purified from HEK-293T cells transfected with FLAG–XBP1s and p300. To maximize levels of acetylation, 10 mM NAM (nicotinamide) was added to the medium for 5 h before the cell harvest. SIRT1 was purified from HEK-293T cells transfected with FLAG–SIRT1. After immunoprecipitation using the anti-FLAG M2 affinity gel, epitope products were eluted using FLAG peptide. The eluted acetylated XBP1s was then incubated in reaction buffer (25 mM Tris/HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.2 mM PMSF and 0.5 mM DTT) at 30 °C for 1 h in the presence or absence of SIRT1 and 5 mM NAD+. The acetylated and total amounts of XBP1s were then measured by Western blotting.
HEK-293 cells were seeded into 24-well plates. After transfection and the indicated treatment, cells were harvested using 1× passive lysis buffer (Promega). XBP1s transcriptional activity was measured by dual luciferase assay with normalization using a pRL-CMV control reporter vector (Promega).
Western blotting and co-immunoprecipitation
Protein samples were obtained with the lysis buffer. Nuclear proteins were extracted using the CelLytic™ NuCLEAR™ extraction kit (Sigma–Aldrich). Approx. 10 μg samples were loaded per lane for analysis by SDS/PAGE (8% gels). The separated proteins were transferred on to PVDF membranes. The following primary antibodies and dilutions were used: anti-XBP1 antibody (1:1000), anti-caspase 3 antibody (1:1000), antiSIRT1 antibody (1:1000) and anti-β-actin antibody (1:20000). Signals were detected using HRP-conjugated secondary antibodies (1:2000) and ECL (enhanced chemiluminescence) reagents (GE Healthcare Life Sciences). Band intensity measurements were performed using the TotalLab100 software (Nonlinear Dynamics). For the co-immunoprecipitation of XBP1s and SIRT1, we transfected either FLAG–XBP1s or an empty vector into HEK-293 cells. Cell lysates were immunoprecipitated using anti-FLAG M2 affinity gel and were then analysed for endogenous SIRT1 with an anti-SIRT1 antibody.
Cells grown on coverslips were fixed with 4% (w/v) paraformaldehyde in PBS for 20 min and then permeabilized with 0.1% Triton X-100 for 5 min. Cells were washed and incubated in 10% (v/v) normal goat serum solution for 20 min. Primary anti-XBP1 antibody (1:200) and anti-FLAG antibody (1:500) were used to stain XBP1s and FLAG–SIRT1 respectively for 60 min. Rinsed cells were loaded with Alexa Fluor® 488-labelled anti-(rabbit Ig) antibody (1:200) and Rhodamine Red™-X-labelled anti-(mouse Ig) antibody (1:200) for 45 min. After extensive PBS washing, samples were mounted and observed under an Olympus Fluoview 500 confocal microscope.
RNA extraction and qRT-PCR (quantitative real-time PCR)
Total RNA was isolated using the TRIzol® reagent according to the manufacturer's instructions (Invitrogen). Reverse transcription was conducted using a reverse transcription system kit (catalogue number A3500; Promega). An aliquot (8 μl) of the product cDNA was used for qRT-PCR with the iQ™ SYBR Green Supermix and the iCycler iQ PCR detection system (Bio-Rad Laboratories). 18S rRNA was applied as an internal control for data analysis. The nucleotide sequences of primers used for PCR are as follows: 18S rRNA, 5′-CGCTTCCTTACCTGGTTGAT-3′ and 5′-GAGCGACCAAAGGAACCATA-3′; Gadd34 (growth-arrest and DNA-damage-inducible protein 34), 5′-GAGATTCCTCTAAAAGCTCGG-3′ and 5′-CAGGGACCTCGACGGCAGC-3′; Grp78 (glucose-regulated protein of 78 kDa)/BiP (immunoglobulin heavy-chain-binding protein), 5′-CATGGTTCTCACTAAAATGAAAGG-3′ and 5′-GCTGGTACAGTAACAACTG-3′; Ero1a (ER oxidation 1α), 5′-TCAGTGGACCAAGCATGATGA-3′ and 5′-TCCACATACTCAGCATCGGG-3′; Sec61a (Sec61α subunit), 5′-CTATTTCCAGGGCTTCCGAGT-3′ and 5′-AGGTGTTGTACTGGCCTCGGT-3′; Edem1 (ER degradation enhancer, mannosidase-α-like 1), 5′-AAGTCTCAGGAGCTCAGAGTCATTAA-3′ and 5′-CGATCTGGCGCATGTAGATG-3′; Xbp1s, 5′-GAGTCCGCAGCAGGTG-3′ and 5′-GTGTCAGAGTCCATGGGA-3′; and total Xbp1, 5′-AAGAACACGCTTGGGAATGG-3′ and 5′-ACTCCCCTTGGCCTCCAC-3′.
Assay for cell viability
Cells were treated with tunicamycin for 24 h and subsqently stained with Trypan Blue. The number of live and dead cells was counted using a haemacytometer under the microscope.
All the experiments were repeated at least twice. Data are presented as means S.E.M. Statistical analyses between groups were done with a Student's t test using GraphPad (http://www.graphpad.com) software.
XBP1s is a target of acetylation
To determine whether XBP1s is a target for p300-mediated acetylation, we transfected FLAG–XBP1s with increasing amounts of p300 into HEK-293 cells. It was found that overexpression of p300 increased the acetylation of XBP1s and promoted XBP1s protein level in a dose-dependent manner (Figure 1A). We then determined whether p300 regulates XBP1s protein levels via modulating its protein stability. With cycloheximide blocking new protein synthesis, we observed a quick degradation of XBP1s in the absence of p300. However, co-transfection with p300 significantly raised the basal protein levels of XBP1s and strongly stabilized XBP1s protein (Figure 1B). These results demonstrate that XBP1s is subjected to p300-mediated acetylation.
SIRT1 deacetylates XBP1s
Since acetylation and deacetylation are coupled biological processes, we determined whether XBP1s is also subjected to deacetylation. Histone deacetylases consist of the class I, class II and the NAD+-dependent class III histone deacetylase families . In order to determine which class of histone deacetylases was involved in regulating XBP1s, we examined the effects on XBP1s acetylation of TSA and NAM, two inhibitors of histone deacetylases class I/II and class III respectively. We found that NAM enhanced XBP1s acetylation, whereas TSA alone did not significantly affect XBP1s acetylation. We calculated the ratio of acetylated to total XBP1s to indicate the extent of acetylation (Figure 2A). This result suggests that it is a class III histone deacetylase that plays a dominant role in regulating XBP1s deacetylation. Since SIRT1 is the closest mammalian homologue of class III histone deacetylase SIR2 in C. elegans, we tested whether SIRT1 regulates XBP1s deacetylation via three strategies.
First, we studied the effect of the SIRT1-specific inhibitor EX-527 on XBP1s acetylation status. It was found that EX-527 increased the acetylation of XBP1s and caused accumulation of XBP1s protein (Figure 2B). In addition, we evaluated the effects of SIRT1-knockdown on XBP1s acetylation. It was observed that co-transfection with an shRNA expression vector targeting SIRT1 appreciably increased XBP1s acetylation in HEK-293 cells (Figure 2C).
Secondly, we determined whether overexpression of SIRT1 could lead to deacetylation of XBP1s. XBP1s and p300 were co-transfected either with or without SIRT1 into HEK-293 cells. It was shown that overexpression of SIRT1 completely diminished the p300-mediated acetylation of XBP1s (Figure 2D).
Thirdly, we performed an in vitro deacetylation assay, using purified acetylated XBP1s and SIRT1, to determine whether XBP1s is a direct target of SIRT1. As expected, the purified SIRT1 deacetylated XBP1s protein in an NAD+-dependent manner (Figure 2E). These results demonstrate that SIRT1 is a bona fide NAD+-dependent deacetylase for XBP1s and can directly deacetylate XBP1s.
These findings prompted us to determine whether XBP1s and SIRT1 co-localize and/or physically bind each other. As determined by Western blotting, endogenous XBP1s protein induced by the ER-stress-activator thapsigargin accumulated within the nucleus of HEK-293 cells. The endogenous SIRT1 was also detected in the nuclear compartment (Figure 3A). Immunofluorescence staining demonstrated that the overexpressed XBP1s and SIRT1 were co-localized within the nucleus of all of the transfected Cos-7 cells (Figure 3B). In addition, we found that there was a physical interaction between exogenous XBP1s and endogenous SIRT1 (Figure 3C).
SIRT1 represses the transcriptional activity of XBP1s
We next sought to assess whether SIRT1 regulates the transcriptional activity of XBP1s. We used a 5× UPRE luciferase reporter to determine the transcriptional activity of XBP1s. It was found that SIRT1 co-transfection significantly attenuated the XBP1s-mediated 5× UPRE luciferase reporter activity (Figure 4A). SIRT1 also significantly compromised the stimulatory effect of p300 on XBP1s-mediated luciferase reporter activity (Figure 4B). To test further the role of endogenous SIRT1, we used an shRNA strategy to knockdown the endogenous SIRT1 in HEK-293 cells. Co-transfection of the shRNA targeting SIRT1 significantly increased the transcriptional activity of XBP1s 2.4-fold, compared with co-transfection of the control vector, whereas SIRT1 shRNA did not affect basal levels of 5× UPRE luciferase reporter activity (Figure 4C). These results demonstrate that endogenous SIRT1 specifically regulates transcriptional activity of overexpressed XBP1s rather than exerting non-specific effects on the 5× UPRE luciferase reporter activity.
Next, we determined whether SIRT1 affects XBP1 target gene transcription. For this purpose, we employed Sirt1+/+ (wild-type) and Sirt1−/− (Sirt1-knockout) MEFs in a pharmacological ER-stress model. The ER stressor, either thapsigargin or tunicamycin, induced mRNA expression of the UPR target genes, including Edem1, Ero1a, Sec61a, Bip and Gadd34 (Figure 4D). Among them, Edem1, Ero1a and Sec61a are regarded as transcription targets of XBP1s [10,28,29], whereas Bip and Gadd34 are regarded as targets of ATF6 and PERK signalling respectively . Intriguingly, ER stress triggered a greater induction of the XBP1s-dependent UPR target genes Edem1, Ero1a and Sec61a in Sirt1−/− MEFs compared with Sirt1+/+ MEFs. In contrast, the induction of the ATF6 target gene Bip and the PERK pathway target gene Gadd34 was less in Sirt1−/− MEFs than in Sirt1+/+ MEFs (Figure 4D). Furthermore, we noticed that Sirt1−/− MEFs did not display higher Xbp1 mRNA splicing, as determined by the ratio of spliced to total Xbp1 mRNA (Figure 4D), excluding the possibility that the stronger induction of XBP1s target genes in Sirt1−/− was caused by a compensatory over-activation of the IRE1/XBP1 pathway due to reduced ATF6 and/or PERK signalling. Taken together, these results demonstrate that SIRT1 deficiency specifically augments XBP1s-mediated gene expression rather than universally affecting gene expression of all three signalling branches of the UPR in the same manner. In other words, these results support the hypothesis that SIRT1 exerts a repressive regulation specific for the XBP1s signalling branch of the UPR.
SIRT1 sensitizes cells to ER-stress-induced cell death
IRE1/XBP1s signalling prevents activation of cell death pathways and determines the cell fate during sustained ER stress [5,31,32]. To evaluate the physiological significance of the enhanced XBP1s signalling in Sirt1−/− cells, we measured cell viability in Sirt1+/+ and Sirt1−/− MEFs under ER stress induced by tunicamycin treatment for 24 h. As expected, ER stress induced cell death in both cell types (Figure 5A). However, importantly, Sirt1−/− MEFs displayed approx. 50% less cell death compared with the Sirt1+/+ control cells, as shown by a quantitative cell viability assay (Figure 5B). There was also decreased processing of pro-caspase-3 to active cleaved caspase-3 in Sirt1−/− cells compared with the wild-type Sirt1+/+ cells (Figure 5C), providing further evidence for decreased cell apoptosis in these cells. In agreement with a greater resistance to ER-stress-induced cell apoptosis, the Sirt1−/− MEFs displayed higher levels of XBP1s protein (Figure 5C). These results suggest that SIRT1 sensitizes cells to ER-stress-induced cell apoptosis, possibly via repressing XBP1s signalling.
The UPR modulator XBP1 plays important roles in a wide range of human physiological and pathological processes [1,2]. Although XBP1 transcription and mRNA splicing have been extensively studied , the post-translational modifications of XBP1s remain unclear, although a recent study has revealed that XBP1s is subjected to SUMO (small ubiquitin-related modifier) modification . In the present study we have demonstrated, for the first time, that XBP1s is a target of acetylation and deacetylation mediated by p300 and SIRT1 respectively. During preparation of the manuscript, p300 has been shown to physically interact with XBP1s but not with the unspliced form of XBP1, XBP1u . The results of the present study coupled with that finding suggest a role of p300 specific for XBP1s. We observed that overexpression of p300 increased acetylation of XBP1s and elevated its protein levels (Figure 1), whereas SIRT1 deacetylated XBP1s and decreased its protein levels (Figures 2D and 2E). Moreover, the p300-induced XBP1s protein levels resulted from the stabilization of XBP1s (Figure 1B), echoing the previous reports that acetylation stabilizes transcription factors such as SREBP1a [21,23]. We demonstrated further that acetylation of XBP1s increases XBP1s-mediated 5× UPRE luciferase reporter activity (Figure 4B). Given we have observed that increasing the protein levels of XBP1s, by use of the proteasome inhibitor MG132, failed to stimulate XBP1s-mediated 5× UPRE luciferase reporter activity (results not shown), it is likely that acetylation regulates XBP1s transcriptional activity independently of its regulation of XBP1s protein levels. By the same token, overexpression of SIRT1 repressed XBP1s-mediated luciferase reporter activity (Figures 4A and 4B). The results of the present study suggest that, like SUMOylation , acetylation and/or deacetylation also constitutes an important regulatory mechanism controlling the protein levels of XBP1s and its transcription function. However, further studies are necessary to identify the acetylated lysine residue(s) in XBP1s, and determine whether the modification is unique to XBP1s as compared with XBP1u, whether acetylation competes with SUMOylation for the same lysine residue(s) in XBP1s and how the modification of the lysine residue(s) regulates the multiple functions of XBP1s. The answers to these questions are critical for elucidating the post-translational regulation of the UPR transcription factor XBP1s.
Emerging evidence has implied that an intricate relationship exists between the ER stress response and lifespan or aging [35,36]. SIR2, a homologue of mammalian SIRT1 in C. elegans, has been shown previously to regulate the lifespan of C. elegans and the effect has been associated with its capacity to regulate ER-stress-response genes . SIRT1 has also been implicated in promoting longevity in mammals . However, it remains unclear whether SIRT1 regulates mammalian UPR signalling. The results of the present study have demonstrated that SIRT1 is a deacetylase for XBP1s. Overexpression of SIRT1 inhibited the activity of XBP1s (Figures 4A and 4B) and, conversely, knockdown of SIRT1 increased its activity (Figure 4C). Furthermore, Sirt1-deficiency led to enhanced up-regulation of XBP1 target gene expression and less apoptotic cell death in MEFs upon ER-stress challenge. These novel results provide a solid biochemical and functional connection between SIRT1 and the best-conserved UPR signalling branch, i.e. IRE1/XBP1 signalling.
XBP1s has been shown to play a crucial role in pathogenesis of MM, a neoplastic disease of B-cell origin; a high level of XBP1s expression has been linked to rapid tumour expansion and a poor prognosis [37,38]. Inhibition of XBP1 splicing, by inhibiting IRE1α activity, increased apoptosis in MM cells in response to ER stress . Intriguingly, several lines of evidence, including our own unpublished results, have shown that the SIRT1 activator resveratrol also induced apoptotic cell death in MM cells [40,41]. Although the detailed molecular mechanism underlying the therapeutic effects of resveratrol remains unclear, the present study, demonstrating that SIRT1 represses XBP1s and sensitizes cells to ER-stress-induced apoptosis, suggests that resveratrol might act through activating SIRT1 and consequently inhibiting XBP1s signalling, an absolute requirement for MM tumour growth , to achieve its ‘killing’ effects. It would be interesting to determine whether the SIRT1/XBP1s signalling pathway mediates the therapeutic effect of resveratrol on MM cells and other cancers that are responsive to resveratrol, such as breast cancer and colon cancer, which are also associated with an up-regulation of XBP1 [42–44].
It has also been was shown recently that XBP1s plays a critical role for adipogenesis via directing expression of the CEBPA (CCAAT/enhancer-binding protein α) gene, an important transcription factor required for adipogenesis . Either overexpression of SIRT1 or XBP1s-knockdown attenuates adipogenesis. In contrast, RNA interference of Sirt1 enhances adipogenesis [11,45]. However, it remains unknown whether there is a functional connection between SIRT1 and XBP1s in regulating adipogenesis. The results of the present study indicate that SIRT1 regulates XBP1s post-translationally and represses its transcriptional activity, suggesting that XBP1s might serve as an important downstream effector through which SIRT1 exerts its anti-adipogenic effects. Elucidation of the mechanism by which SIRT1 co-ordinates with XBP1s in the regulation of adipogenesis might provide a framework for the development of promising therapeutic strategies that target the SIRT1/XBP1s signalling axis to treat human diseases associated with deregulated fat metabolism and/or UPR signalling, such as obesity and Type 2 diabetes.
In summary, the results of the present study have demonstrated that mammalian XBP1s is subjected to acetylation and deacetylation mediated by p300 and SIRT1 respectively. Acetylation/deacetylation constitutes an important post-translational mechanism controlling the protein stability and activity of XBP1s (Figure 5D). The biochemical and functional linkage between SIRT1 and XBP1s reveals novel insights into the molecular mechanisms by which SIRT1 regulates UPR signalling and suggests SIRT1 is a potential therapeutic target for treating diseases where deregulation of UPR/XBP1s signalling is implicated.
Feng-Ming Wang and Hong-Jiao Ouyang conceived the project. Feng-Ming Wang performed the experiments and analysed the results. Yi-Jiun Chen constructed the XBP1-related plasmids and assisted in the experiments. Hong-Jiao Ouyang supervised the study and wrote the manuscript together with Feng-Ming Wang.
This work is supported by the National Institute of Dental and Craniofacial Research [grant number DE017439 (to H.-J. O.)]; and the start-up fund provided by the Department of Medicine, School of Medicine at University of Pittsburgh (to H.-J. O.); F.-M. W. is supported in part by the National Natural Science Foundation of China [grant number 30901677].
We are grateful to Dr Randy Kaufman, Dr Paul Robbins, and Dr Ming Hu for generously providing plasmids and cells. We thank the co-workers in Dr Ouyang's laboratory for technical support and stimulating discussion. We also thank Mimi Li (University of South Florida, Tampa, FL, U.S.A.) and Donna Gaspich (University of Pittsburgh, PA, U.S.A.) for assistance in the preparation of the manuscript.
Abbreviations: ATF, activating transcription factor; BiP, immunoglobulin heavy-chain-binding protein; CREB, cAMP-response-element-binding protein; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; ER, endoplasmic reticulum; Edem1, ER degradation enhancer, mannosidase-α-like 1; Ero1a, ER oxidation α; FBS, fetal bovine serum; Gadd34, growth-arrest and DNA-damage-inducible protein 34; Grp78, glucose-regulated protein of 78 kDa; HA, haemagglutinin; HEK, human embryonic kidney; HEK-293T, HEK-293 cells expressing the large T-antigen of SV40 (simian virus 40); HRP, horseradish peroxidise; IRE1, inositol-requiring enzyme 1; MEF, mouse embryonic fibroblast; MM, multiple myeloma; NAM, nicotinamide; NEAA, non-essential amino acid mix; PERK, PKR (double-stranded-RNA-dependent protein kinase)-like ER kinase; qRT-PCR, quantitative real-time PCR; shRNA, small-hairpin RNA; SIR2, silent information regulator 2; SIRT1, sirtuin 1; SREBP, sterol-regulatory-element-binding protein; SUMO, small ubiquitin-related modifier; TSA, trichostatin A; UPR, unfolded protein response; UPRE, UPR element; XBP1, X-box-binding protein 1; XBP1s, spliced form of XBP1; XBP1u, unspliced form of XBP1
- © The Authors Journal compilation © 2011 Biochemical Society