Human cDC1s display constitutive activation of the UPR sensor IRE1.

The intracellular mechanisms safeguarding DC function are of biomedical interest in several immune-related diseases. Type 1 conventional DCs (cDC1s) are prominent targets of immunotherapy typified by constitutive activation of the unfolded protein response (UPR) sensor IRE1. Through its RNase domain, IRE1 regulates key processes in cDC1s including survival, ER architecture and function. However, most evidence linking IRE1 RNase with cDC1 biology emerges from mouse studies and it is currently unknown whether human cDC1s also activate the enzyme to preserve cellular homeostasis. In this work, we report that human cDC1s constitutively activate IRE1 RNase in steady state, which is evidenced by marked expression of IRE1, XBP1s, and target genes, and low levels of mRNA substrates of the IRE1 RNase domain. On a functional level, pharmacological inhibition of the IRE1 RNase domain curtailed IL-12 and TNF production by cDC1s upon stimulation with TLR agonists. Altogether, this work demonstrates that activation of the IRE1/XBP1s axis is a conserved feature of cDC1s across species and suggests that the UPR sensor may also play a relevant role in the biology of the human lineage.

Activation of the XBP1s/O-GlcNAcylation Pathway Improves Functional Outcome After Cardiac Arrest and Resuscitation in Young and Aged Mice.

ABSTRACT: After cardiac arrest (CA) and resuscitation, the unfolded protein response (UPR) is activated in various organs including the brain. However, the role of the UPR in CA outcome remains largely unknown. One UPR branch involves spliced X-box-binding protein-1 (XBP1s). Notably, XBP1s, a transcriptional factor, can upregulate expression of specific enzymes related to glucose metabolism, and subsequently boost O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation). The current study is focused on effects of the XBP1 UPR branch and its downstream O-GlcNAcylation on CA outcome. Using both loss-of-function and gain-of-function mouse genetic tools, we provide the first evidence that activation of the XBP1 UPR branch in the post-CA brain is neuroprotective. Specifically, neuron-specific Xbp1 knockout mice had worse CA outcome, while mice with neuron-specific expression of Xbp1s in the brain had better CA outcome. Since it has been shown that the protective role of the XBP1s signaling pathway under ischemic conditions is mediated by increasing O-GlcNAcylation, we then treated young mice with glucosamine, and found that functional deficits were mitigated on day 3 post CA. Finally, after confirming that glucosamine can boost O-GlcNAcylation in the aged brain, we subjected aged mice to 8 min CA, and then treated them with glucosamine. We found that glucosamine-treated aged mice performed significantly better in behavioral tests. Together, our data indicate that the XBP1s/O-GlcNAc pathway is a promising target for CA therapy.

Targeting the IRE1α/XBP1 Endoplasmic Reticulum Stress Response Pathway in ARID1A-Mutant Ovarian Cancers.

The SWI/SNF chromatin-remodeling complex is frequently altered in human cancers. For example, the SWI/SNF component ARID1A is mutated in more than 50% of ovarian clear cell carcinomas (OCCC), for which effective treatments are lacking. Here, we report that ARID1A transcriptionally represses the IRE1α-XBP1 axis of the endoplasmic reticulum (ER) stress response, which confers sensitivity to inhibition of the IRE1α-XBP1 pathway in ARID1A-mutant OCCC. ARID1A mutational status correlated with response to inhibition of the IRE1α-XBP1 pathway. In a conditional Arid1aflox/flox/Pik3caH1047R genetic mouse model, Xbp1 knockout significantly improved survival of mice bearing OCCCs. Furthermore, the IRE1α inhibitor B-I09 suppressed the growth of ARID1A-inactivated OCCCs in vivo in orthotopic xenograft, patient-derived xenograft, and the genetic mouse models. Finally, B-I09 synergized with inhibition of HDAC6, a known regulator of the ER stress response, in suppressing the growth of ARID1A-inactivated OCCCs. These studies define the IRE1α-XBP1 axis of the ER stress response as a targetable vulnerability for ARID1A-mutant OCCCs, revealing a promising therapeutic approach for treating ARID1A-mutant ovarian cancers. SIGNIFICANCE: These findings indicate that pharmacological inhibition of the IRE1α-XBP1 pathway alone or in combination with HDAC6 inhibition represents an urgently needed therapeutic strategy for ARID1A-mutant ovarian cancers.

The XBP1‒MARCH5‒MFN2 Axis Confers Endoplasmic Reticulum Stress Resistance by Coordinating Mitochondrial Fission and Mitophagy in Melanoma.

Melanoma cells are relatively resistant to endoplasmic reticulum (ER) stress, which contributes to tumor progression under stressful conditions and renders tolerance to ER stress‒inducing therapeutic agents. Mitochondria are tightly interconnected with ER. However, whether mitochondria play a role in regulating ER stress resistance in melanoma remains elusive. In this study, we reported that the XBP1‒MARCH5‒MFN2 axis conferred ER stress resistance by coordinating mitochondrial fission and mitophagy in melanoma. Our integrative bioinformatics first revealed that the downregulation of mitochondrial genes was highly correlated with unfolded protein response activation in melanoma. Then we proved that mitochondrial fission and mitophagy were prominently induced to contribute to ER stress resistance both in vitro and in vivo by maintaining mitochondrial function. Mechanistically, the activation of IRE1α/ATF6-XBP1 branches of unfolded protein response promoted the transcription of E3 ligase MARCH5 to facilitate the ubiquitination and degradation of MFN2, which thereby triggered mitochondrial fission and mitophagy under ER stress. Together, our findings show a regulatory axis that links mitochondrial fission and mitophagy to the resistance to ER stress. Targeting mitochondrial quality control machinery can be exploited as an approach to reinforce the efficacy of ER stress‒inducing agents against cancer.

The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon beta.

The open reading frame 8 (orf8) is an accessory protein of SARS-CoV-2. It has 121 amino acids with two genotypes, orf8L and orf8S. In this study, we overexpressed the orf8L and orf8S of SARS-CoV-2 as well as the orf8b of SARS-CoV to investigate their roles in the regulation of endoplasmic reticulum (ER) stress and the inhibition of interferon beta (IFNß) production. We found that the two genotypes of SARS-CoV-2 orf8 are capable of inducing ER stress without significant difference by triggering the activating transcription factor 6 (ATF6) and inositol-requiring enzymes 1 (IRE1) branches of the ER stress pathway. However, the third branch of ER stress pathway, i.e. the protein kinase-like ER kinase (PERK), was unaffected by the overexpression of SARS-CoV-2 orf8L or orf8S. Moreover, both orf8L and orf8S of SARS-CoV-2 are capable of down regulating the production of IFNß and interferon-stimulated genes (ISG), ISG15 and ISG56 induced by polyinosinic-polycytidylic acid (poly (I:C)). Moreover, we also found decreased nuclear translocation of Interferon regulatory factor 3 (IRF3), after overexpressing orf8L and orf8S induced by poly (I:C). Our data demonstrated that SARS-CoV-2 orf8 protein could induce ER stress by activating the ATF6 and IRE1 pathways, but not the PERK pathway, and functions as an interferon antagonist to inhibit the production of IFNß. However, these functions appeared not to be affected by the genotypes of SARS-CoV-2 orf8L and orf8S.

Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) influences adipocytes injuries through triggering XBP1 and activating mitochondria-mediated apoptosis.

Obesity is an important public-health problem worldwide. This study aimed to determine effects of porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on adipocytes injuries and explore associated mechanisms. Adipocytes were isolated from SD rats. pLVX-XBP1 (XBP1 over-expression) and pLVX-XBP1-RNAi (silencing XBP1) were structured and transfected into adipocytes. All adipocytes were divided into pLVX-NC, pLVX-XBP1, pLVX-NC+Pg-LPS and pLVX-XBP1+ Pg-LPS group. Oil-Red O staining was employed to identify isolated adipocytes. Quantitative real-time PCR (qRT-PCR) was used to examine gene transcription of IL-6, TNF-α, leptin, adiponectin. Western blotting was used to detect Bax and caspase-3 expression. Adipocytes were successfully isolated and identified with Oil-Red O staining. Both XBP1 mimic and XBP1 RNAi were effectively transfected into adipocytes with higher expressing efficacy. XBP1 over-expression significantly aggravated Pg-LPS induced inflammatory response compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS significantly enhanced leptin and inhibited adiponectin expression by up-regulating XBP1 expression (p<0.05). XBP1 silence significantly alleviated Pg-LPS induced inflammatory response and reduced leptin, enhanced adiponectin expression in Pg-LPS treated adipocytes compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS induced apoptosis of adipocytes by enhancing XBP1 expression and modulating Bcl-2/Bax pathway associated molecules. In conclusion, Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) induces adipocytes injuries through modulating XBP1 expression and initialling mitochondria-mediated apoptosis.

X-box binding protein 1 (XBP1) function in diseases.

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes endoplasmic reticulum stress (ERS), which is characteristic of cells with high levels of secretory activity and is involved in a variety of diseases. In response to ERS, cells initiate an adaptive process named the unfolding protein response (UPR) to maintain intracellular homeostasis and survival. However, long term and unresolved ERS can also induce apoptosis. As the most conserved signaling branch of UPR, the IRE1-XBP1 pathway plays an important role in both physiological and pathological states, and its activity has a profound impact on disease progression and prognosis. Here, the latest research progress of IRE1-XBP1 pathway in cancer, metabolic diseases, and other diseases was briefly introduced, and the relationship between several diseases and this pathway was analyzed. Besides, the new understanding and prospect of IRE1-XBP1 pathway regulating male reproduction were reviewed.

Targeting the IRE1-XBP1 axis to overcome endocrine resistance in breast cancer: Opportunities and challenges.

Estrogen receptor 1 (ESR1, which encodes estrogen receptor-alpha) is a key driver gene for the initiation and progression of hormone receptor-positive breast cancer. Estrogen receptor-alpha (ER) is expressed in up to 70% of cases, and patients are routinely treated with endocrine therapies. However, the development of resistance over time is common and occurs in one-third of ER-positive breast tumors, leading to disease progression and death. X-box binding protein 1 (XBP1), a key component of the unfolded protein response (UPR) and ER signaling pathway, generates a positive feedback regulatory loop that leads to increased expression of XBP1 and ER in luminal breast cancer. In this review, we highlight new insights into the mechanisms of crosstalk between XBP1 and ER signaling and its clinical implications. Next, we describe the key signaling nodes that play an important role in XBP1-mediated endocrine resistance in breast cancer. Further, we discuss XBP1 gene mutations in breast cancer and the role of these mutations in the emergence of endocrine resistance and response to treatment. Finally, we discuss the current state and future directions for targeting XBP1 in combination with standard endocrine therapy to improve clinical outcomes in endocrine-resistant breast cancer patients.

The emerging role of XBP1 in cancer.

X-box binding protein 1 (XBP1) is a unique basic-region leucine zipper (bZIP) transcription factor whose dynamic form is controlled by an alternative splicing response upon disturbance of homeostasis in the endoplasmic reticulum (ER) and activation of the unfolded protein response (UPR). XBP1 was first distinguished as a key regulator of major histocompatibility complex (MHC) class II gene expression in B cells. XBP1 communicates with the foremost conserved signalling component of the UPR and is essential for cell fate determination in response to ER stress (ERS). Here, we review recent advances in our understanding of this multifaceted translation component in cancer. In this review, we briefly discuss the role of XBP1 mediators in the UPR and the transcriptional function of XBP1. In addition, we describe how XBP1 operates as a key factor in tumour progression and metastasis. We mainly review XBP1's expression, function and prognostic value in research on solid tumours. Finally, we discuss multiple approaches, especially those involving XBP1, that overcome the immunosuppressive effect of the UPR in cancer that could potentially be useful as antitumour therapies.

Repression of viral gene expression and replication by the unfolded protein response effector XBP1u.

The unfolded protein response (UPR) is a cellular homeostatic circuit regulating protein synthesis and processing in the ER by three ER-to-nucleus signaling pathways. One pathway is triggered by the inositol-requiring enzyme 1 (IRE1), which splices the X-box binding protein 1 (Xbp1) mRNA, thereby enabling expression of XBP1s. Another UPR pathway activates the activating transcription factor 6 (ATF6). Here we show that murine cytomegalovirus (MCMV), a prototypic ß-herpesvirus, harnesses the UPR to regulate its own life cycle. MCMV activates the IRE1-XBP1 pathway early post infection to relieve repression by XBP1u, the product of the unspliced Xbp1 mRNA. XBP1u inhibits viral gene expression and replication by blocking the activation of the viral major immediate-early promoter by XBP1s and ATF6. These findings reveal a redundant function of XBP1s and ATF6 as activators of the viral life cycle, and an unexpected role of XBP1u as a potent repressor of both XBP1s and ATF6-mediated activation.

Cells survive by making many different proteins that each carry out specific tasks. To work correctly, each protein must be made and then folded into the right shape. Cells carefully monitor protein folding because unfolded proteins can compromise their viability. A protein called XBP1 is important in controlling how cells respond to unfolded proteins. Normally, cells contain a form of this protein called XBP1u, while increasing numbers of unfolded proteins trigger production of a form called XBP1s. The change from one form to the other is activated by a protein called IRE1. Viruses often manipulate stress responses like the unfolded protein response to help take control of the cell and produce more copies of the virus. Murine cytomegalovirus, which is known as MCMV for short, is a herpes-like virus that infects mice; it stops IRE1 activation and XBP1s production during the later stages of infection. However, research had shown that the unfolded protein response was triggered for a short time at an early stage of infection with MCMV, and it was unclear why this might be. Hinte et al. studied the effect of MCMV on cells grown in the laboratory. The experiments showed that a small dose of cell stress, namely activating the unfolded protein response briefly during early infection, helps to activate genes from the virus that allow it to take over the cell. Together, XBP1s and another protein called ATF6 help to switch on the viral genes. The virus also triggers IRE1 helping to reduce the levels of XBP1u, which could slow down the infection. Later, suppressing the unfolded protein response allows copies of the virus to be made faster to help spread the infection. These findings reveal new details of how viruses precisely manipulate their host cells at different stages of infection. These insights could lead to new ways to manage or prevent viral infections.

12-h clock regulation of genetic information flow by XBP1s.

Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s's ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

Endoplasmic reticulum stress, an important factor in the development of Parkinson's disease.

Similar to other types of neuronal degeneration, Parkinson's disease (PD) is characterized by the aggregation of a pathological protein, α-synuclein. The endoplasmic reticulum (ER) is the principal site of protein synthesis, quality control and degradation. Genetic mutants, environmental insults and other factors disturb ER balance and induce the accumulation of misfolded/unfolded proteins, which initiate ER stress and disturb normal cell function. ER stress perturbs Ca2+ homeostasis and initiates the activation of autophagy and inflammasomes, which have been identified as risk factors for the development of PD. However, the mechanisms by which ER stress contributes to the processed of PD pathogenesis and development remain unclear. This review summarizes current knowledge of ER stress and highlights the principal role of ER stress in PD pathogenesis which may help reveal novel sight to illustrate the pathomechanism of PD.

HLA-B27-mediated activation of TNAP phosphatase promotes pathogenic syndesmophyte formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a type of axial inflammation. Over time, some patients develop spinal ankylosis and permanent disability; however, current treatment strategies cannot arrest syndesmophyte formation completely. Here, we used mesenchymal stem cells (MSCs) from AS patients (AS MSCs) within the enthesis involved in spinal ankylosis to delineate that the HLA-B27-mediated spliced X-box-binding protein 1 (sXBP1)/retinoic acid receptor-ß (RARB)/tissue-nonspecific alkaline phosphatase (TNAP) axis accelerated the mineralization of AS MSCs, which was independent of Runt-related transcription factor 2 (Runx2). An animal model mimicking AS pathological bony appositions was established by implantation of AS MSCs into the lumbar spine of NOD-SCID mice. We found that TNAP inhibitors, including levamisole and pamidronate, inhibited AS MSC mineralization in vitro and blocked bony appositions in vivo. Furthermore, we demonstrated that the serum bone-specific TNAP (BAP) level was a potential prognostic biomarker to predict AS patients with a high risk for radiographic progression. Our study highlights the importance of the HLA-B27-mediated activation of the sXBP1/RARB/TNAP axis in AS syndesmophyte pathogenesis and provides a new strategy for the diagnosis and prevention of radiographic progression of AS.

Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model.

Endoplasmic reticulum (ER) stress is a major pathology encountered after hypoxic-ischemic (HI) injury. Accumulation of unfolded proteins triggers the unfolded protein response (UPR), resulting in the activation of pro-apoptotic cascades that lead to cell death. Here, we identified Bax inhibitor 1 (BI-1), an evolutionarily conserved protein encoded by the transmembrane BAX inhibitor motif-containing 6 (TMBIM6) gene, as a novel modulator of ER-stress-induced apoptosis after HI brain injury in a neonatal rat pup. The main objective of our study was to overexpress BI-1, via viral-mediated gene delivery of human adenoviral-TMBIM6 (Ad-TMBIM6) vector, to investigate its anti-apoptotic effects as well as to elucidate its signaling pathways in an in vivo neonatal HI rat model and in vitro oxygen-glucose deprivation (OGD) model. Ten-day-old unsexed Sprague Dawley rat pups underwent right common carotid artery ligation followed by 1.5 h of hypoxia. Rat pups injected with Ad-TMBIM6 vector, 48 h pre-HI, showed a reduction in relative infarcted area size, attenuated neuronal degeneration and improved long-term neurological outcomes. Furthermore, silencing of BI-1 or further activating the IRE1α branch of the UPR, using a CRISPR activation plasmid, was shown to reverse the protective effects of BI-1. Based on our in vivo and in vitro data, the protective effects of BI-1 are mediated via inhibition of IRE1α signaling and in part via inhibition of the second stress sensor receptor, PERK. Overall, this study showed a novel role for BI-1 and ER stress in the pathophysiology of HI and could provide a basis for BI-1 as a potential therapeutic target.

XBP1 inhibits mesangial cell apoptosis in response to oxidative stress via the PTEN/AKT pathway in diabetic nephropathy.

Diabetic nephropathy (DN) is a complication of diabetes mellitus (DM) that frequently results in renal disease, and is characterized by a variety of symptoms, including albuminuria. It has been shown that apoptosis of glomerular mesangial cells (MCs) can aggravate albuminuria and contribute to the development of diabetic glomerulosclerosis. Hence, determination of the mechanisms leading to MC apoptosis may help us gain insights into the pathogenesis of DN. As our understanding of the role of high glucose (HG) in MC apoptosis remains elusive, we explored the interplay between X-box binding protein 1 (XBP1) and MC apoptosis in this study. XBP1 was observed to be downregulated both in vivo and in vitro. Treatment of XBP1-overexpressing cells with HG resulted in a decrease of reactive oxygen species (ROS) and a suppression of cell apoptosis, concomitant with decreases in cleaved caspase-3 and Bax. Subsequent analyses demonstrated that XBP1 overexpression inhibited the expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and enhanced the activation of AKT in MCs exposed to HG. In addition, XBP1-induced injuries in MC were reversed by overexpression of PTEN, and XBP1 inhibited apoptosis, which was mediated by the activated PTEN/AKT signaling pathway. Thus, our data indicate that XBP1 can activate the PTEN/AKT signaling pathway, thereby alleviating oxidative stress caused by HG or MC apoptosis. These findings suggest that XBP1 may have potential in the development of treatment methods for DN.

The role of the XBP-1/AGR2 signaling pathway in the regulation of airway Mucin5ac hypersecretion under hypoxia.

Oversecretion of Mucin5ac (MUC5AC), which is primarily synthesized by goblet cells and is the major gel-forming mucin, is a hallmark of various pulmonary inflammatory diseases. Hypoxia is considered a common pathophysiologic feature in various pulmonary inflammatory diseases. It has been suggested that hypoxia-inducible factor 1α (HIF-1α) acts as a key factor in hypoxia-induced MUC5AC hypersecretion; however, the exact mechanisms that maintain the stability of HIF-1α and support oversecretion by airway epithelial cells under hypoxia are still unclear. With immunohistochemistry, we found overexpression of anterior gradient 2 (AGR2) in the bronchial epithelial cells of hypoxia-treated mice. With specific shRNA transduction, AGR2 was demonstrated to be a key factor in MUC5AC hypersecretion in vitro. Additionally, co-immunoprecipitation, cell immunochemistry and confocal microscopy experiments were performed to explore the interaction between HIF-1α and AGR2 during hypoxia-induced MUC5AC hypersecretion in vitro. The results indicated increased binding and intracytoplasmic colocation of HIF-1α and AGR2. Our findings suggest that AGR2 acts as a key regulator in hypoxia-induced airway MUC5AC hypersecretion by increasing the stability of HIF-1α. Additionally, the elevated expression of AGR2 induced by hypoxia in bronchial epithelial cells likely depends on an XBP-1-associated pathway.

[S100 calcium binding protein A16 promotes fat synthesis through endoplasmic reticulum stress in HepG2 cells].

The aim of this study was to investigate the role of S100 calcium binding protein A16 (S100A16) in lipid metabolism in hepatocytes and its possible biological mechanism. HepG2 cells (human hepatoma cell line) were cultured with fatty acid to establish fatty acid culture model. The control model was cultured without fatty acid. Each model was divided into three groups and transfected with S100a16 over-expression, shRNA and vector plasmids, respectively. The concentration of triglyceride (TG) in the cells was measured by kit, and the lipid droplets was observed by oil red O staining. Immunoprecipitation and mass spectrometry were used to find the interesting proteins interacting with S100A16, and the interaction was verified by immunoprecipitation. The further mechanism was studied by Western blot and qRT-PCR. The results showed that the intracellular lipid droplet and TG concentrations in the fatty acid culture model were significantly higher than those in the control model. The accumulation of intracellular fat in the S100a16 over-expression group was significantly higher than that in the vector plasmid transfection group. There was an interaction between heat shock protein A5 (HSPA5) and S100A16. Over-expression of S100A16 up-regulated protein expression levels of HSPA5, inositol-requiring enzyme 1α (IRE1α) and pIREα1, which belong to endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway. Meanwhile, over-expression of S100A16 up-regulated the mRNA expression levels of adipose synthesis-related gene Srebp1c, Acc and Fas. In the S100a16 shRNA plasmid transfection group, the above-mentioned protein and mRNA levels were lower than those of vector plasmid transfection group. These results suggest that S100A16 may promote lipid synthesis in HepG2 cells through endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway.

Role of X-Box Binding Protein-1 in Fructose-Induced De Novo Lipogenesis in HepG2 Cells.

BACKGROUND: A high consumption of fructose leads to hepatic steatosis. About 20-30% of triglycerides are synthesized via de novo lipogenesis. Some studies showed that endoplasmic reticulum stress (ERS) is involved in this process, while others showed that a lipotoxic environment directly influences ER homeostasis. Here, our aim was to investigate the causal relationship between ERS and fatty acid synthesis and the effect of X-box binding protein-1 (XBP-1), one marker of ERS, on hepatic lipid accumulation stimulated by high fructose. METHODS: HepG2 cells were incubated with different concentrations of fructose. Upstream regulators of de novo lipogenesis (i.e., carbohydrate response element-binding protein [ChREBP] and sterol regulatory element-binding protein 1c [SREBP-1c]) were measured by polymerase chain reaction and key lipogenic enzymes (acetyl-CoA carboxylase [ACC], fatty acid synthase [FAS], and stearoyl-CoA desaturase-1 [SCD-1]) by Western blotting. The same lipogenesis-associated factors were then evaluated after exposure of HepG2 cells to high fructose followed by the ERS inhibitor tauroursodeoxycholic acid (TUDCA) or the ERS inducer thapsigargin. Finally, the same lipogenesis-associated factors were evaluated in HepG2 cells after XBP-1 upregulation or downregulation through cell transfection. RESULTS: Exposure to high fructose increased triglyceride levels in a dose- and time-dependent manner and significantly increased mRNA levels of SREBP-1c and ChREBP and protein levels of FAS, ACC, and SCD-1, concomitant with XBP-1 conversion to an active spliced form. Lipogenesis-associated factors induced by high fructose were inhibited by TUDCA and induced by thapsigargin. Triglyceride level in XBP-1-deficient group decreased significantly compared with high-fructose group (4.41 ± 0.54 µmol/g vs. 6.52 ± 0.38 µmol/g, P < 0.001), as mRNA expressions of SREBP-1c (2.92 ± 0.46 vs. 5.08 ± 0.41, P < 0.01) and protein levels of FAS (0.53 ± 0.06 vs. 0.85 ± 0.05, P = 0.01), SCD-1 (0.65 ± 0.06 vs. 0.90 ± 0.04, P = 0.04), and ACC (0.38 ± 0.03 vs. 0.95 ± 0.06, P < 0.01) decreased. Conversely, levels of triglyceride (4.22 ± 0.54 µmol/g vs. 2.41 ± 0.35 µmol/g, P < 0.001), mRNA expression of SREBP-1c (2.70 ± 0.33 vs. 1.00 ± 0.00, P < 0.01), and protein expression of SCD-1 (0.93 ± 0.06 vs. 0.26 ± 0.05, P < 0.01), ACC (0.98 ± 0.09 vs. 0.43 ± 0.03, P < 0.01), and FAS (0.90 ± 0.33 vs. 0.71 ± 0.02, P = 0.04) in XBP-1s-upregulated group increased compared with the untransfected group. CONCLUSIONS: ERS is associated with de novo lipogenesis, and XBP-1 partially mediates high-fructose-induced lipid accumulation in HepG2 cells through augmentation of de novo lipogenesis.

Sulforaphane Improves Abnormal Lipid Metabolism via Both ERS-Dependent XBP1/ACC &SCD1 and ERS-Independent SREBP/FAS Pathways.

SCOPE: To investigate the effect of sulforaphane (SFN) on the abnormal lipid metabolism and underlying mechanisms. METHODS AND RESULTS: Models with abnormal lipid metabolism are established both in rats and human hepatocytes. Hepatic steatosis is detected by hematoxylin and eosin and oil red O staining. The structure of endoplasmic reticulum is visualized by transmission electron microscopy. The expressions of X-box binding protein 1 (XBP1), protein kinase-like ER kinase (PERK), sterol regulatory element binding protein-1c (SREBP1c), and lipogenic enzymes are determined by real-time PCR and western blot analysis. SFN lowers the content of triglyceride and cholesterol. SFN alleviates the swelling of ER and decreases the perimeter of ER. SFN significantly decreases the expressions of acetyl CoA carboxylase 1 (ACC1), stearoyl-CoA desaturase 1 (SCD1), and fatty acid synthase. SFN inhibits SREBP1c by blocking the PERK. Meanwhile, SFN suppresses ACC1 and SCD1 via blocking the formation of splicing-type XBP1. The key roles of XBP1 and SREBP1c in SFN-reduced lipid droplets are confirmed by a timed sequence of measurement according to time points. CONCLUSION: SFN improves abnormal lipid metabolism via both ER-stress-dependent and -independent pathways.