Salivary Endocannabinoid Profiles in Chronic Orofacial Pain and Headache Disorders: An Observational Study Using a Novel Tool for Diagnosis and Management.

The endocannabinoid system is involved in physiological and pathological processes, including pain generation, modulation, and sensation. Its role in certain types of chronic orofacial pain (OFP) has not been thoroughly examined. By exploring the profiles of specific salivary endocannabinoids (eCBs) in individuals with different types of OFP, we evaluated their use as biomarkers and the influence of clinical parameters and pain characteristics on eCB levels. The salivary levels of anandamide (AEA), 2-arachidonoyl glycerol (2-AG), and their endogenous breakdown product arachidonic acid (AA), as well as the eCB-like molecules N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), were assessed in 83 OFP patients and 43 pain-free controls using liquid chromatography/tandem mass spectrometry. Patients were grouped by diagnosis: post-traumatic neuropathy (PTN), trigeminal neuralgia (TN), temporomandibular disorder (TMD), migraine, tension-type headache (TTH), and burning mouth syndrome (BMS). Correlation analyses between a specific diagnosis, pain characteristics, and eCB levels were conducted. Significantly lower levels of 2-AG were found in the TN and TTH groups, while significantly lower PEA levels were found in the migraine group. BMS was the only group with elevated eCBs (AEA) versus the control. Significant correlations were found between levels of specific eCBs and gender, health-related quality of life (HRQoL), BMI, pain duration, and sleep awakenings. In conclusion, salivary samples exhibited signature eCBs profiles for major OFP disorders, especially migraine, TTH, TN, and BMS. This finding may pave the way for using salivary eCBs biomarkers for more accurate diagnoses and management of chronic OFP patients.

Reduced Endocannabinoid Tone in Saliva of Chronic Orofacial Pain Patients.

BACKGROUND: the endocannabinoid system (ECS) participates in many physiological and pathological processes including pain generation, modulation, and sensation. Its involvement in chronic orofacial pain (OFP) in general, and the reflection of its involvement in OFP in salivary endocannabinoid (eCBs) levels in particular, has not been examined. OBJECTIVES: to evaluate the association between salivary (eCBs) levels and chronic OFP. METHODS: salivary levels of 2 eCBs, anandamide (AEA), 2-arachidonoylglycerol (2-AG), 2 endocannabinoid-like compoundsN-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA), and their endogenous precursor and breakdown product, arachidonic acid (AA), were analyzed using liquid chromatography/tandem mass spectrometry in 83 chronic OFP patients and 43 pain-free controls. The chronic OFP patients were divided according to diagnosis into musculoskeletal, neurovascular/migraine, and neuropathic pain types. RESULTS: chronic OFP patients had lower levels of OEA (p = 0.02) and 2-AG (p = 0.01). Analyzing specific pain types revealed lower levels of AEA and OEA in the neurovascular group (p = 0.04, 0.02, respectively), and 2-AG in the neuropathic group compared to controls (p = 0.05). No significant differences were found between the musculoskeletal pain group and controls. Higher pain intensity was accompanied by lower levels of AA (p = 0.028), in neuropathic group. CONCLUSIONS: lower levels of eCBs were found in the saliva of chronic OFP patients compared to controls, specifically those with neurovascular/migraine, and neuropathic pain. The detection of changes in salivary endocannabinoids levels related to OFP adds a new dimension to our understanding of OFP mechanisms, and may have diagnostic as well as therapeutic implications for pain.

Endocannabinoid Levels in Ulcerative Colitis Patients Correlate With Clinical Parameters and Are Affected by Cannabis Consumption.

Background: Inflammatory bowel diseases (IBDs) are chronic, idiopathic, inflammatory, gastrointestinal disorders. The endocannabinoid system may have a role in the pathogenesis of IBD. We aimed to assess whether cannabis treatment influences endocannabinoids (eCBs) level and clinical symptoms of IBD patients. Methods: Blood samples and biopsies were taken from IBD patients treated by either cannabis or placebo for 8 weeks. Immunohistochemistry for N-acyl-phosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH) expression was done on colon biopsies, and sample levels of anandamide (AEA), eCB2-arachidonylglycerol (2-AG), arachidonic acid (AA), palmitoylethanolamine (PEA), and oleoylethanolamine (OEA) were measured in patient's sera before and after cannabis treatment. Caco-2 cells were cultured with extracts of cannabis with/without tetrahydrocannabinol (THC) and their proteins extracted, and Western blotting for NAPE-PLD and FAAH expression was done. Results: Thirteen patients with Crohn's disease (CD) and nine patients with ulcerative colitis (UC) were treated with cannabis. Seventeen patients with CD and 10 with UC served as placebo groups. In all CD patients, the levels of eCBs remained unaltered during the treatment period. In UC patients treated with placebo, but not in those treated with cannabis, the levels of PEA, AEA, and AA decreased significantly. The percent reduction in bowel movements was negatively correlated with changes observed in the circulating AEA and OEA, whereas improvement in quality of life was positively correlated with the levels of 2-AG. In the biopsies from UC patients, FAAH levels increased over the study period. In Caco-2 cells, both cannabis extracts increased NAPE-PLD levels but reduced FAAH expression levels. Conclusion: Our study supports the notion that cannabis use affects eCB "tone" in UC patients and may have beneficial effects on disease symptoms in UC patients.

CB1R regulates soluble leptin receptor levels via CHOP, contributing to hepatic leptin resistance.

The soluble isoform of leptin receptor (sOb-R), secreted by the liver, regulates leptin bioavailability and bioactivity. Its reduced levels in diet-induced obesity (DIO) contribute to hyperleptinemia and leptin resistance, effects that are regulated by the endocannabinoid (eCB)/CB1R system. Here we show that pharmacological activation/blockade and genetic overexpression/deletion of hepatic CB1R modulates sOb-R levels and hepatic leptin resistance. Interestingly, peripheral CB1R blockade failed to reverse DIO-induced reduction of sOb-R levels, increased fat mass and dyslipidemia, and hepatic steatosis in mice lacking C/EBP homologous protein (CHOP), whereas direct activation of CB1R in wild-type hepatocytes reduced sOb-R levels in a CHOP-dependent manner. Moreover, CHOP stimulation increased sOb-R expression and release via a direct regulation of its promoter, while CHOP deletion reduced leptin sensitivity. Our findings highlight a novel molecular aspect by which the hepatic eCB/CB1R system is involved in the development of hepatic leptin resistance and in the regulation of sOb-R levels via CHOP.

When the human body has stored enough energy from food, it releases a hormone called leptin that travels to the brain and stops feelings of hunger. This hormone moves through the bloodstream and can affect other organs, such as the liver, which also help control our body's energy levels. Most people with obesity have very high levels of leptin in their blood, but are resistant to its effects and will therefore continue to feel hungry despite having stored enough energy. One of the proteins that controls the levels of leptin is a receptor called sOb-R, which is released by the liver and binds to leptin as it travels in the blood. Individuals with high levels of this receptor often have less free leptin in their bloodstream and a lower body weight. Another protein that helps the body to regulate its energy levels is the cannabinoid-1 receptor, or CB1R for short. In people with obesity, this receptor is overactive and has been shown to contribute to leptin resistance, which is when the brain becomes less receptive to leptin. Previous work in mice showed that blocking CB1R reduced the levels of leptin and allowed mice to react to this hormone normally again, but it remained unclear whether CB1R affects how other organs, such as the liver, respond to leptin. To answer this question, Drori et al. blocked the CB1R receptor in the liver of mice eating a high-fat diet, either by using a drug or by deleting the gene that codes for this protein. This caused mice to have higher levels of sOb-R circulating in their bloodstream. Further experiments showed that this change in sOb-R was caused by the levels of a protein called CHOP increasing in the liver when CB1R was blocked. Drori et al. found that inhibiting CB1R caused these obese mice to lose weight and have healthier, less fatty livers as a result of their livers no longer being resistant to the effects of leptin. Scientists, doctors and pharmaceutical companies are trying to develop new strategies to combat obesity. The results from these experiments suggest that blocking CB1R in the liver could allow this organ to react to leptin appropriately again. Drugs blocking CB1R, including the one used in this study, will be tested in clinical trials and could provide a new approach for treating obesity.

Author Correction: Bone Anabolic Response in the Calvaria Following Mild Traumatic Brain Injury is Mediated by the Cannabinoid-1 Receptor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reversal of diet-induced hepatic steatosis by peripheral CB1 receptor blockade in mice is p53/miRNA-22/SIRT1/PPARα dependent.

OBJECTIVE: The endocannabinoid (eCB) system is increasingly recognized as being crucially important in obesity-related hepatic steatosis. By activating the hepatic cannabinoid-1 receptor (CB1R), eCBs modulate lipogenesis and fatty acid oxidation. However, the underlying molecular mechanisms are largely unknown. METHODS: We combined unbiased bioinformatics techniques, mouse genetic manipulations, multiple pharmacological, molecular, and cellular biology approaches, and genomic sequencing to systematically decipher the role of the hepatic CB1R in modulating fat utilization in the liver and explored the downstream molecular mechanisms. RESULTS: Using an unbiased normalized phylogenetic profiling analysis, we found that the CB1R evolutionarily coevolves with peroxisome proliferator-activated receptor-alpha (PPARα), a key regulator of hepatic lipid metabolism. In diet-induced obese (DIO) mice, peripheral CB1R blockade (using AM6545) induced the reversal of hepatic steatosis and improved liver injury in WT, but not in PPARα-/- mice. The antisteatotic effect mediated by AM6545 in WT DIO mice was accompanied by increased hepatic expression and activity of PPARα as well as elevated hepatic levels of the PPARα-activating eCB-like molecules oleoylethanolamide and palmitoylethanolamide. Moreover, AM6545 was unable to rescue hepatic steatosis in DIO mice lacking liver sirtuin 1 (SIRT1), an upstream regulator of PPARα. Both of these signaling molecules were modulated by the CB1R as measured in hepatocytes exposed to lipotoxic conditions or treated with CB1R agonists in the absence/presence of AM6545. Furthermore, using microRNA transcriptomic profiling, we found that the CB1R regulated the hepatic expression, acetylation, and transcriptional activity of p53, resulting in the enhanced expression of miR-22, which was found to specifically target SIRT1 and PPARα. CONCLUSIONS: We provide strong evidence for a functional role of the p53/miR-22/SIRT1/PPARα signaling pathway in potentially mediating the antisteatotic effect of peripherally restricted CB1R blockade.

Bone Anabolic Response in the Calvaria Following Mild Traumatic Brain Injury is Mediated by the Cannabinoid-1 Receptor.

Brain trauma was clinically associated with increased osteogenesis in the appendicular skeleton. We showed previously in C57BL/6J mice that mild traumatic brain injury (mTBI) transiently induced bone formation in the femur via the cannabinoid-1 (CB1) receptor. Here, we subjected ICR mice to mTBI and examined the bone response in the skull using microCT. We also measured mast cell degranulation (MCD)72 h post-injury. Finally, we measured brain and calvarial endocannabinoids levels post-mTBI. mTBI led to decreased bone porosity on the contralateral (untouched) side. This effect was apparent both in young and mature mice. Administration of rimonabant (CB1 inverse agonist) completely abrogated the effect of mTBI on calvarial porosity and significantly reduced MCD, compared with vehicle-treated controls. We also found that mTBI resulted in elevated levels of anandamide, but not 2-arachidonoylglycerol, in the contralateral calvarial bone, whereas brain levels remained unchanged. In C57BL/6J CB1 knockout mice, mTBI did not reduce porosity but in general the porosity was significantly lower than in WT controls. Our findings suggest that mTBI induces a strain-specific CB1-dependent bone anabolic response in the skull, probably mediated by anandamide, but seemingly unrelated to inflammation. The endocannabinoid system is therefore a plausible target in management of bone response following head trauma.

Prediction and Experimental Confirmation of Novel Peripheral Cannabinoid-1 Receptor Antagonists.

Small molecules targeting peripheral CB1 receptors have therapeutic potential in a variety of disorders including obesity-related, hormonal, and metabolic abnormalities, while avoiding the psychoactive effects in the central nervous system. We applied our in-house algorithm, iterative stochastic elimination, to produce a ligand-based model that distinguishes between CB1R antagonists and random molecules by physicochemical properties only. We screened ∼2 million commercially available molecules and found that about 500 of them are potential candidates to antagonize the CB1R. We applied a few criteria for peripheral activity and narrowed that set down to 30 molecules, out of which 15 could be purchased. Ten out of those 15 showed good affinity to the CB1R and two of them with nanomolar affinities (Ki of ∼400 nM). The eight molecules with top affinities were tested for activity: two compounds were pure antagonists, and five others were inverse agonists. These molecules are now being examined in vivo for their peripheral versus central distribution and subsequently will be tested for their effects on obesity in small animals.

Cannabinoid-1 receptor regulates mitochondrial dynamics and function in renal proximal tubular cells.

AIMS: To evaluate the specific role of the endocannabinoid/cannabinoid type-1 (CB1 R) system in modulating mitochondrial dynamics in the metabolically active renal proximal tubular cells (RPTCs). MATERIALS AND METHODS: We utilized mitochondrially-targeted GFP in live cells (wild-type and null for the CB1 R) and electron microscopy in kidney sections of RPTC-CB1 R-/- mice and their littermate controls. In both in vitro and in vivo conditions, we assessed the ability of CB1 R agonism or fatty acid flux to modulate mitochondrial architecture and function. RESULTS: Direct stimulation of CB1 R resulted in mitochondrial fragmentation in RPTCs. This process was mediated, at least in part, by modulating the phosphorylation levels of the canonical fission protein dynamin-related protein 1 on both S637 and S616 residues. CB1 R-induced mitochondrial fission was associated with mitochondrial dysfunction, as documented by reduced oxygen consumption and ATP production, increased reactive oxygen species and cellular lactate levels, as well as a decline in mitochondrial biogenesis. Likewise, we documented that exposure of RPTCs to a fatty acid flux induced CB1 R-dependent mitochondrial fission, lipotoxicity and cellular dysfunction. CONCLUSIONS: CB1 R plays a key role in inducing mitochondrial fragmentation in RPTCs, leading to a decline in the organelle's function and contributing to the renal tubular injury associated with lipotoxicity and other metabolic diseases.

Magel2 Modulates Bone Remodeling and Mass in Prader-Willi Syndrome by Affecting Oleoyl Serine Levels and Activity.

Among a multitude of hormonal and metabolic complications, individuals with Prader-Willi syndrome (PWS) exhibit significant bone abnormalities, including decreased BMD, osteoporosis, and subsequent increased fracture risk. Here we show in mice that loss of Magel2, a maternally imprinted gene in the PWS critical region, results in reduced bone mass, density, and strength, corresponding to that observed in humans with PWS, as well as in individuals suffering from Schaaf-Yang syndrome (SYS), a genetic disorder caused by a disruption of the MAGEL2 gene. The low bone mass phenotype in Magel2-/- mice was attributed to reduced bone formation rate, increased osteoclastogenesis and osteoclast activity, and enhanced trans-differentiation of osteoblasts to adipocytes. The absence of Magel2 in humans and mice resulted in reduction in the fatty acid amide bone homeostasis regulator, N-oleoyl serine (OS), whose levels were positively linked with BMD in humans and mice as well as osteoblast activity. Attenuating the skeletal abnormalities in Magel2-/- mice was achieved with chronic administration of a novel synthetic derivative of OS. Taken together, Magel2 plays a key role in modulating bone remodeling and mass in PWS by affecting OS levels and activity. The use of potent synthetic analogs of OS should be further tested clinically as bone therapeutics for treating bone loss. © 2018 American Society for Bone and Mineral Research.

Modulation of Renal GLUT2 by the Cannabinoid-1 Receptor: Implications for the Treatment of Diabetic Nephropathy.

Altered glucose reabsorption via the facilitative glucose transporter 2 (GLUT2) during diabetes may lead to renal proximal tubule cell (RPTC) injury, inflammation, and interstitial fibrosis. These pathologies are also triggered by activating the cannabinoid-1 receptor (CB1R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB1R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB1R blockade or genetically inactivating CB1Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB1R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB1R or inhibiting GLUT2 dynamics in RPTCs has the potential to treat and ameliorate DN. These findings may support the rationale for the clinical testing of peripherally restricted CB1R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN.

Proximal Tubular Cannabinoid-1 Receptor Regulates Obesity-Induced CKD.

Obesity-related structural and functional changes in the kidney develop early in the course of obesity and occur independently of hypertension, diabetes, and dyslipidemia. Activating the renal cannabinoid-1 receptor (CB1R) induces nephropathy, whereas CB1R blockade improves kidney function. Whether these effects are mediated via a specific cell type within the kidney remains unknown. Here, we show that specific deletion of CB1R in the renal proximal tubule cells did not protect the mice from obesity, but markedly attenuated the obesity-induced lipid accumulation in the kidney and renal dysfunction, injury, inflammation, and fibrosis. These effects associated with increased activation of liver kinase B1 and the energy sensor AMP-activated protein kinase, as well as enhanced fatty acid ß-oxidation. Collectively, these findings indicate that renal proximal tubule cell CB1R contributes to the pathogenesis of obesity-induced renal lipotoxicity and nephropathy by regulating the liver kinase B1/AMP-activated protein kinase signaling pathway.

Targeting the endocannabinoid/CB1 receptor system for treating obesity in Prader-Willi syndrome.

OBJECTIVE: Extreme obesity is a core phenotypic feature of Prader-Willi syndrome (PWS). Among numerous metabolic regulators, the endocannabinoid (eCB) system is critically involved in controlling feeding, body weight, and energy metabolism, and a globally acting cannabinoid-1 receptor (CB1R) blockade reverses obesity both in animals and humans. The first-in-class CB1R antagonist rimonabant proved effective in inducing weight loss in adults with PWS. However, it is no longer available for clinical use because of its centrally mediated, neuropsychiatric, adverse effects. METHODS: We studied eCB 'tone' in individuals with PWS and in the Magel2-null mouse model that recapitulates the major metabolic phenotypes of PWS and determined the efficacy of a peripherally restricted CB1R antagonist, JD5037 in treating obesity in these mice. RESULTS: Individuals with PWS had elevated circulating levels of 2-arachidonoylglycerol and its endogenous precursor and breakdown ligand, arachidonic acid. Increased hypothalamic eCB 'tone', manifested by increased eCBs and upregulated CB1R, was associated with increased fat mass, reduced energy expenditure, and decreased voluntary activity in Magel2-null mice. Daily chronic treatment of obese Magel2-null mice and their littermate wild-type controls with JD5037 (3 mg/kg/d for 28 days) reduced body weight, reversed hyperphagia, and improved metabolic parameters related to their obese phenotype. CONCLUSIONS: Dysregulation of the eCB/CB1R system may contribute to hyperphagia and obesity in Magel2-null mice and in individuals with PWS. Our results demonstrate that treatment with peripherally restricted CB1R antagonists may be an effective strategy for the management of severe obesity in PWS.

HUR mRNA expression in ovarian high-grade serous carcinoma effusions is associated with poor survival.

The objective of this study was to analyze the expression and clinical role of the RNA-binding molecule HuR in metastatic high-grade ovarian serous carcinoma (HGSC). HUR mRNA expression by reverse-transcription polymerase chain reaction was analyzed in 66 effusions from patients diagnosed with HGSC. Protein expression was analyzed in 262 HGSC effusions using immunohistochemistry. HUR mRNA was detected in all 66 effusions. HUR mRNA levels were unrelated to clinicopathological parameters. However, higher HUR mRNA levels were significantly related to poor overall survival in the entire cohort (P=.023), as well as in analysis limited to patients with prechemotherapy primary diagnosis specimens (P=.001) in univariate analysis. Cox multivariate analysis showed an independent prognostic role for HUR mRNA in the entire cohort (P=.033) and in patients with prechemotherapy primary diagnosis specimens (P=.002). HuR protein was detected in the nucleus and cytoplasm of tumor cells in 258 (98%) of 262 and 153 (58%) of 262 effusions, respectively. Higher HuR protein expression was associated with higher serum Cancer Antigen (CA) 125 levels at diagnosis (P=.01), but its presence at both cellular compartments was otherwise unrelated to clinicopathological parameters or survival. In conclusion, HuR is widely expressed in metastatic HGSC at both the mRNA and protein level. Higher HUR mRNA levels are associated with poor survival in metastatic HGSC, whereas protein expression has no prognostic value.

Regulated IRE1-dependent decay participates in curtailing immunoglobulin secretion from plasma cells.

Inositol-requiring enzyme 1 (IRE1) is a kinase and ribonuclease that executes the splicing of X box binding protein 1 (XBP-1) mRNA in response to the accumulation of unfolded protein in the ER, a signal cascade termed the unfolded protein response. Recently, IRE1 has been implicated in mRNA and miRNA cleavage and degradation, a pathway termed regulated IRE1-dependent decay (RIDD). Deletion of XBP-1 in the liver and pancreas strongly enhances RIDD by upregulating IRE1 protein levels and enhancing its ribo-nuclease activity. Because XBP-1 is essential for generating plasma cells with developed secretory capacity, we sought to evaluate the contribution of RIDD to this regulation. Mice were conditionally deleted for XBP-1 and/or IRE1 in their B-cell lineage. Similarly to the liver, deletion of XBP-1 induces IRE1 expression in LPS-treated B cells. In vitro, IRE1 cleaves the mRNA of secretory µ chains, which explains the reduction in secretory µ mRNA and its synthesis in XBP-1 KO plasma cells. In accordance, the IgM response is partially restored in XBP-1/IRE1 double KO mice relative to XBP-1 KO mice. Interestingly, the IgG1 response is reduced to a similar level in XBP-1 KO, IRE1 KO, and their double knockout animals. Our data demonstrate a specific contribution by RIDD in curtailing immunoglobulin synthesis and secretion.

Endoplasmic reticulum stress induces a caspase-dependent N-terminal cleavage of RBX1 protein in B cells.

Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.

SUMOylation of Blimp-1 promotes its proteasomal degradation.

B lymphocyte induced maturation protein-1 (Blimp-1) is a transcription repressor of the Krueppel-like family. Blimp-1 plays important roles in developmental processes, such as of germ cells and hair follicle stem cells. In B lymphocytes Blimp-1 orchestrates the terminal differentiation into plasma cells. We discovered that Blimp-1 undergoes SUMOylation by SUMO-1. This SUMOylation is modulated by the SUMO protease SENP1. While Blimp-1 is relatively stable in 293T cells, a fusion with SUMO1 rendered it to rapid proteasomal degradation. Increase in SENP1 activity stabilized Blimp-1, while a decrease promoted its degradation. Our data indicate that SUMOylation of Blimp-1 regulates its intracellular stability.

Direct proteasome binding and subsequent degradation of unspliced XBP-1 prevent its intracellular aggregation.

The non-canonical splicing of XBP-1 mRNA is a hallmark of the mammalian unfolded protein response (UPR). The proteasomal degradation of unspliced XBP-1 (XBP-1u) facilitates the termination of the UPR. Thus, understanding the mechanism of XBP-1u degradation may allow control over UPR duration and intensity. We show that XBP-1u interacts with purified 20S proteasomes through its unstructured C-terminus, which leads to its degradation in a manner that autonomously opens the proteasome gate. In living cells, the C-terminus of XBP-1u accumulates in aggresome structures in the presence of proteasome inhibitors. We propose that direct proteasomal degradation of XBP-1u prevents its intracellular aggregation.

De novo ceramide synthesis is required for N-linked glycosylation in plasma cells.

Plasma cells (PCs) are terminally differentiated B lymphocytes responsible for the synthesis and secretion of Igs. The differentiation of B cells into PCs involves a remarkable expansion of both lipid and protein components of the endoplasmic reticulum. Despite their importance in many signal transduction pathways, the role of ceramides, and of complex sphingolipids that are derived from ceramide, in PC differentiation has never been directly studied. To assess their putative role in PC differentiation, we blocked ceramide synthesis with fumonisin B1, a specific inhibitor of ceramide synthase. Under fumonisin B1 treatment, N-linked glycosylation was severely impaired in LPS-activated, but not in naive, B cells. We also show that ceramide synthesis is strongly induced by XBP-1 (X box-binding protein-1). In the absence of ceramide synthesis, ER expansion was dramatically diminished. Our results underscore ceramide biosynthesis as a key metabolic pathway in the process of PC differentiation and reveal a previously unknown functional link between sphingolipids and N-linked glycosylation in PCs.