Optimization of α-amido boronic acids via cryo-electron microscopy analysis: Discovery of a novel highly selective immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual inhibitor.

The immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit ß5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over ß5c may be desirable for treating autoimmune diseases. In this study, we present the optimization and discovery of α-amido boronic acids using cryo-electron microscopy (cryo-EM). The exploitation of structural differences between the proteasome subunits led to the identification of a highly selective LMP7/LMP2 dual inhibitor 19. Molecular dynamics simulation based on cryo-EM structures of the proteasome subunits complexed with 19 explained the inhibitory activity profile. In mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin, results indicate that 19 is orally bioavailable and shows promise as potential treatment for autoimmune diseases.

CERS6 required for cell migration and metastasis in lung cancer.

Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

Evaluation and Comparison of Daiokanzoto and Lubiprostone for Constipation: A Retrospective Cohort Study.

Daiokanzoto (DKT) and lubiprostone (LPS) are drugs used for constipation, but few studies have compared them. This study examined the effectiveness, adverse events, and medical economic efficiency of DKT and LPS for constipation. Patients who received DKT (DKT group) and those who received LPS (LPS group) during admission to Ogaki Municipal Hospital between November 2012 and May 2016 were enrolled. Drug efficacy was evaluated based on the median value of bowel movement frequency over 1 week before and after drug administration, and their safety was evaluated by the presence or absence of diarrhea, abdominal pain, nausea, and vomiting. To assess medical economic efficiency, drug costs for constipation per week were calculated. The median values (quartile ranges) of bowel movement frequency at 1 week after drug administration were 8.5 (6.0-12.0) in the DKT group and 5 (3.0-7.0) in the LPS group, which was significantly different (p < 0.01). Diarrhea occurred significantly less often in the DKT group (4 cases) than in the LPS group (17 cases) (p < 0.01). The median cost of drugs administered for constipation for 1 week was significantly lower in the DKT group (631 [quartile range, 513-653] yen) than in the LPS group (1431 [1135-2344] yen) (p < 0.01). DKT had a higher immediate effect on constipation and was associated with more frequent bowel movement and fewer adverse events of diarrhea than LPS, suggesting that it may be effective and safe for treating constipation, and DKT is inexpensive.

Feasibility of real-time intestinal bloodstream evaluation using probe-based confocal laser endomicroscopy in a porcine intestinal ischemia model.

BACKGROUND: Intestinal ischemia can lead to fatal complications if left unrecognized during surgery. The current techniques of intraoperative microvascular assessment remain subjective. Probe-based confocal laser endomicroscopy (pCLE) has the potential to objectively evaluate microvascular blood flow in real-time setting. The present study evaluated the technical feasibility of real-time intestinal bloodstream evaluation using pCLE in a porcine intestinal ischemia model. METHODS: Seven pigs were used. The intestinal ischemia model was prepared by sequentially dividing the mesenteric blood vessels. The intestinal bloodstream was evaluated on its serosal surface using pCLE (Cellvizio 488 probe, Ultra Mini O) at every 1-cm segment from a vessel-preservation border (i.e., the cut end of the vessel). Images of the blood vessels and flow of red blood cells (RBCs) in each visualized vessel were semi-qualitatively assessed using a 3-scale scoring system. In addition, 25 surgeons blindly assessed the 10 movies recorded at 0, 1, 2, 3, and 5 cm from a vessel-preservation border using a 4-scale scoring system to confirm the consistency of the evaluation of the pCLE system. RESULTS: Images of the blood vessels were successfully obtained from the cut end of the vessel to the segment 4 cm away. Good unidirectional flow of RBCs was observed from the cut end to the 2-cm segment, whereas the flow became bidirectional between 2 and 3 cm segments. Beyond 4 cm, no flow images were obtained. The specimen obtained from the segment beyond 4 cm showed remarkable mucosal color change, which was confirmed as a necrotic change histologically. The evaluations from the cut end of the vessel to the segment 1 cm away by surgeons were excellent or good and it was almost consistent. CONCLUSIONS: Real-time bloodstream evaluation using pCLE is feasible and potentially effective for predicting intestinal ischemia during surgery.

Imaging mass spectrometry for toxicity assessment: a useful technique to confirm drug distribution in histologically confirmed lesions.

To evaluate the usefulness of imaging mass spectrometry (IMS) technology for assessing drug toxicity, we analyzed animal tissues in an amiodarone (AMD)-induced phospholipidosis model by IMS and confirmed the relationship between the distribution of AMD, its metabolites, and representative phospholipids (phosphatidylcholine, PC) and histological changes. AMD was administered to rats for 7 days at 150 mg/kg/day. The lung, spleen, and mesenteric lymph node were histologically examined and analyzed using IMS. The detection intensities of AMD, its metabolites, and typical PCs were higher in regions infiltrated by foamy macrophages compared with normal areas. This tendency was common in all three organs analyzed in this study. For the spleen, signals for AMD, its metabolites, and typical PCs were significantly more intense in the marginal zone, where foamy macrophages and vacuolated lymphocytes are abundant, than in the other areas. These results indicate that AMD, its metabolites, and PCs accumulate together in foamy or vacuolated cells, which is consistent with the mechanism of AMD-induced phospholipidosis. They also indicate that IMS is a useful technique for evaluating the distribution of drugs and biological components in the elucidation of toxicity mechanisms.

Appropriate Follow-Up Strategies for Gastrointestinal Stromal Tumor Patients Based on the Analysis of Recurrent Interval and Patterns.

BACKGROUND/AIMS: Previous studies have proposed risk classifications for patients with gastrointestinal stromal tumor (GIST) after resection and have contributed to the prediction of its prognosis. However, optimal postoperative surveillance has not yet been established. METHODS: We retrospectively analyzed data from 115 GIST patients who experienced recurrence after complete resection. The relationships between clinicopathological characteristics and the first recurrence sites, or time to recurrence (TTR), were investigated. We also compared the characteristics between 2 subgroups based on a TTR of ≤5 or >5 years. RESULTS: The first recurrence occurred in the abdomen in 114 of 115 patients (99.1%); one case of esophageal GIST recurred in the lung. Gastric and small intestinal GISTs recurred most frequently in the liver or peritoneum, while the most common recurrences of colorectal GISTs were found to be local. Fourteen patients (12.2%) experienced recurrence after >5 years. Smaller tumors and those categorized as lower risk were significantly more frequent in the TTR >5 years group than in the TTR ≤5 years group. In the TTR >5 years group, local recurrence was the most frequent type of recurrence (42.9%). CONCLUSION: Based on abdominal examination, postoperative surveillance after complete resection for primary GISTs may be recommended for >5 years.

Intravitreal Tissue Plasminogen Activator, Ranibizumab, and Gas Injection for Submacular Hemorrhage in Polypoidal Choroidal Vasculopathy.

PURPOSE: To investigate the efficacy of intravitreal injection of recombinant tissue plasminogen activator (rt-PA), ranibizumab, and gas without vitrectomy for submacular hemorrhage. DESIGN: Prospective, interventional, consecutive case series. PARTICIPANTS: Twenty consecutive patients (20 eyes) with submacular hemorrhage secondary to exudative age-related macular degeneration (AMD) or polypoidal choroidal vasculopathy (PCV). METHODS: Ranibizumab, rt-PA (25 µg/0.05 ml), and 100% perfluoropropane (0.3 ml) were injected intravitreally, followed by 2-day prone positioning. MAIN OUTCOME MEASURES: The primary outcome measure was best-corrected visual acuity (BCVA) 6 months after treatment. Secondary outcome measures included central retinal thickness (CRT), central pigment epithelial detachment (PED) thickness, central ellipsoid zone, recurrence rate, and complications. RESULTS: Underlying disease was exudative AMD in 1 eye and PCV in 19 eyes. Submacular hemorrhage ranged in size from 2 to 31 disc diameters. Complete displacement of submacular hemorrhage was achieved in 17 eyes (85%), and partial displacement was achieved in 3 eyes (15%). Snellen BCVA improved from 20/139 before treatment to 20/65 at 6 months (P = 0.0061). Mean change in Early Treatment Diabetic Retinopathy Study score from baseline was +13 letters (P = 0.0040). Mean CRT decreased from 599 µm before treatment to 208 µm at 6 months (P < 0.0001), and central PED thickness decreased from 188 to 88 µm (P = 0.0140). Three eyes developed vitreous hemorrhage, and 1 eye developed retinal detachment; all were treated surgically, and Snellen BCVA improved at 6 months (P = 0.0012). Recurrence was observed in 10 eyes (50%) within 6 months, but visual acuity was preserved with intravitreal injection of anti-vascular endothelial growth factor (VEGF) pro re nata (PRN). The factors that affect BCVA at 6 months after treatment were pre- and posttreatment central ellipsoid zone (P = 0.0366 and P = 0.0424), pretreatment BCVA (P = 0.0015), and pre- and posttreatment central PED thickness (P = 0.0046, P = 0.0021). CONCLUSIONS: Subretinal hemorrhage treatment by intravitreal injection of rt-PA, ranibizumab, and gas is useful to achieve hemorrhage displacement and lesion improvement. To preserve visual acuity, early detection of posttreatment recurrence and intravitreal anti-VEGF injection PRN are necessary.

Hypoxia remodels the composition of the constituent ceramide species of HexCer and Hex2Cer with phytosphingosine and hydroxy fatty acids in human colon cancer LS174T cells.

Oxygen-requiring enzymes, such as Δ4-desaturase (dihydroceramide desaturase), sphingolipid Δ4-desaturase/C-4-hydroxylase, and fatty acid 2-hydroxylase are involved in ceramide synthesis. We prepared free ceramides, sphingomyelins and glycosphingolipids (GSLs) from cancer cells cultivated under conditions of normoxia and hypoxia, and analyzed these compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Human colon cancer LS174T cells were employed because these cells highly express hydroxyl fatty acids and phytosphingosine (t18:0) which are expected to be greatly influenced by changes in oxygen levels. As expected, the populations of dihydro-species of free ceramide and sphingomyelin with C16:0 non-hydroxy fatty acid were elevated, and the populations of HexCers and Hex2Cers, composed of C16:0 or C16:0 hydroxy fatty acid (C16:0h), and sphingosine (d18:1) or t18:0, were decreased under hypoxia. However, appreciable populations of HexCer and Hex2Cer species of C24:0 or C24:0h and t18:0 remained. These results suggest that the individual species of GSLs with fatty acids possessing different alkyl chain lengths, either non-hydroxy fatty acids or hydroxyl fatty acids, may be metabolized individually.

Individual profiles of free ceramide species and the constituent ceramide species of sphingomyelin and neutral glycosphingolipid and their alteration according to the sequential changes of environmental oxygen content in human colorectal cancer Caco-2 cells.

We previously performed a systematic analysis of free ceramide (Cers) species, the constituent ceramide species of sphingomyelins and neutral glycosphingolipids (NGSLs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with high-energy collision-induced dissociation. As a result, distinct species differences were found among Cers, sphingomyelins and NGSLs in the kidneys. Using this method, we investigated various sphingolipid species from human colon cancer Caco-2 cells as well as the influence of environmental oxygen on these species in detail. Unexpectedly, even in normoxia, all Cers species were composed of dihydrosphingosine (d18:0) and non-hydroxy fatty acid (NFA), and 34% of sphingomyelins were composed of dihydrosphingomyelins with NFA. In contrast, major constituent ceramide species of NGSLs were composed of the usual long-chain base of sphingosine (d18:1) and hydroxy fatty acid (HFA). When the cells were cultured under hypoxic condition for 3 days, all the Cers and nearly 80% of the sphingomyelins were dihydrosphingolipids composed of d18:0-NFAs, but a significant proportion of d18:1-HFAs still remained in the NGSLs. When the cells were transferred from conditions of hypoxia to normoxia again (reoxygenation), Cer species composed of d18:1-NFAs, which were not found in Cers under the original normoxic conditions, appeared. Such Cers were probably synthesized as precursors for the constituent ceramides of sphingomyelins and NGSLs.

Self-reported assessment of health-related quality of life in children who underwent restorative proctocolectomy with ileal J-pouch anal anastomosis for ulcerative colitis.

PURPOSE: To clarify health-related quality of life (HRQOL) by self-evaluation after restorative proctocolectomy with ileal J-pouch anal anastomosis (IPAA) in children with ulcerative colitis, a questionnaire using the Pediatric Quality of Life Inventory™ 4.0 (PedsQL) was administered. METHODS: The PedsQL was administered to 13 consecutive children (mean age 14.5 years) who underwent IPAA between 2005 and 2010 in our hospital and age-matched healthy controls. The mean duration after IPAA was 2.5 years (range 0.08-6 years) at the time of this study. Healthy children completed the same questionnaire by retrospective imaging during the past 1 month by the PedsQL evaluation policy. RESULTS: Patients' total score and each functioning score after IPAA reached the same levels as those in healthy controls. Soiling, pouchitis occurrence, and bowel movements had no significant relationship to the PedsQL total score and each functioning score. CONCLUSIONS: Interference of physical activity, emotional status, and social life caused by refractory ulcerative colitis (UC) worsens patients' HRQOL. IPAA could resolve these problems in children with UC and result in an HRQOL comparable with that in healthy children.

Quantitative transcriptomic profiling of branching in a glycosphingolipid biosynthetic pathway.

Cellular biosynthesis of macromolecules often involves highly branched enzyme pathways, thus cellular regulation of such pathways could be rather difficult. To understand the regulatory mechanism, a systematic approach could be useful. We genetically analyzed a branched biosynthetic pathway for glycosphingolipid (GSL) GM1 using correlation index-based responsible enzyme gene screening (CIRES), a novel quantitative phenotype-genotype correlation analysis. CIRES utilizes transcriptomic profiles obtained from multiple cells. Among a panel of B cell lines, expression of GM1 was negatively correlated with and suppressed by gene expression of CD77 synthase (CD77Syn), whereas no significant positive correlation was found for enzymes actually biosynthesizing GM1. Unexpectedly, a GM1-suppressive phenotype was also observed in the expression of catalytically inactive CD77Syn, ruling out catalytic consumption of lactosylceramide (LacCer) as the main cause for such negative regulation. Rather, CD77Syn seemed to limit other branching reaction(s) by targeting LacCer synthase (LacCerSyn), a proximal enzyme in the pathway, because they were closely localized in the Golgi apparatus and formed a complex. Moreover, turnover of LacCerSyn was accelerated upon CD77Syn expression to globally change the GSL species expressed. Collectively, these data suggest that transcriptomic assessment of macromolecule biosynthetic pathways can disclose a global regulatory mechanism(s) even when unexpected.

Hybrid liposomes affect cellular lipid constituents and caveolae structures.

We examined alterations of lipid constituents induced by hybrid liposomes (HLs) in cancer cells. As early as 1h after HL treatment, amounts of the raft/caveolae lipids sphingomyelin, ceramide, and ether-type PC were altered. In addition, the structures of caveolae on the cytoplasmic surface of the cell membrane were significantly changed. Our results suggest that alterations of lipid composition in caveolae mediate HL signaling for apoptosis.

Intracorporeal circular-stapled Billroth I anastomosis in single-incision laparoscopic distal gastrectomy.

PURPOSE: The intracorporeal anastomotic procedure is one of the most difficult components in single-incision laparoscopic gastrectomy. We describe a simple surgical technique for intracorporeal circular-stapled Billroth I gastroduodenostomy with transumbilical introduction of the instrument. SURGICAL TECHNIQUE: After standard laparoscopic mobilization of the distal stomach using a mini-loop retractor (diameter, 2 mm), a semi-circumference duodenotomy was made just distal to the pyloric ring. The anvil of a circular stapling device, secured with a Prolene suture with a needle, was introduced via the duodenotomy. The suture was advanced anteriorly such that an anvil shaft transfixed the anterior duodenal wall. The duodenum was staple-transected at this point, and the anvil shaft was removed from the duodenum by pulling the thread. The anvil shaft was advanced against the posterior wall of the stomach and tightly grasped by the mini-loop retractor to avoid slippage. After the cartridge-carrying instrument was introduced transumbilically with the pneumoperitoneum maintained using a surgical glove, the anvil shaft was connected with the center rod of the instrument under fine laparoscopic view. The instrument was fired to complete the circular-stapled gastroduodenostomy. RESULTS: We employed this technique in 20 patients. Neither postoperative complications, including anastomotic leakage and stricture, nor postoperative mortality were observed. CONCLUSIONS: Our modified technique for intracorporeal Billroth I reconstruction in single-incision laparoscopic distal gastrectomy is safe and feasible. This technique could be an attractive surgical option for all laparoscopic reconstructive procedures using circular stapling devices.

Double balloon enteroscopy for pediatric inflammatory bowel disease.

BACKGROUND: The aim of the present study was to evaluate the efficacy and safety of double balloon enteroscopy (DBE) in children with inflammatory bowel disease (IBD). METHODS: A total of 106 DBE procedures in 67 patients were performed at Mie University Hospital from January 2008 to June 2011. Of these, 17 procedures in 12 children and adolescents with established or suspected Crohn's disease (CD) were included in the present study. The procedure, sedation, efficacy, and safety of DBE were evaluated. RESULTS: Mean patient age was 12.9 years (range, 3-19 years). Patients ≤ 15 years old had general anesthesia. The procedures included the oral approach (n= 9), the anal approach (n= 4), and the ileostomal approach (n= 4). The mean procedure duration was 60 min. Accurate diagnosis was obtained in 7/8 cases (88%) of suspected CD. Only one case was diagnosed as indeterminate colitis, although the total small and large bowel was examined on DBE and pathology. Procedure tolerance was acceptable and recovery was uneventful in all cases. No serious complications were encountered. CONCLUSIONS: With regard to the present limited IBD pediatric case series, DBE is a safe and effective procedure.

Imaging Mass Spectrometry (IMS) for drug discovery and development survey: Results on methods, applications and regulatory compliance.

Imaging mass spectrometry (IMS) is increasingly used for drug discovery and development to understand target enagement, tissue distribution, drug toxicity, and disease mechanisms, etc. However, this is still a relatively new technique that requires further development validation before it will be an acceptable technique to support regulated development of new drugs. Thus, best practices will need to be established to build more confidence and gain wider acceptance by the scientific community, pharmaceutical industry, and regulatory authorities. The Imaging Mass Spectrometry Society (IMSS) and the Japan Association for Imaging Mass Spectrometry (JAIMS) have conducted a thorough survey to gather information on the current state of IMS and to identify key issues. The survey was sent to researchers or managers in the position who are currently using IMS techniques in support of their drug discovery and development efforts and/or who plan to use such tools as best practices are established. The survey probes questions related to details regarding technical aspects of IMS, which includes data acquisition, data analysis and quantitation, data integrity, reporting, applications, and regulatory concerns. This international survey was conducted online through the Survey Monkey (https://www.surveymonkey.com) in both English and Japanese from September 14 through September 30, 2020.

Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID.

Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated (3,5)A ions, indicating Hex 1-4 Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.

Efficacy of Seprafilm® in abdominal surgery for ventriculoperitoneal shunt malfunction: a report of two pediatric cases.

Temporary external drainage with adequate antibiotic treatment followed by ventriculoperitoneal (VP) shunt reinsertion is necessary to treat VP shunt malfunction with infection. In surgeries for VP shunt reinsertion, the key factors that determine whether the reinserted VP shunt will function sufficiently are adequate adhesiolysis and safe placement of the VP shunt within a satisfactory peritoneal location. Based on these factors, the prevention of postoperative adhesions after surgery for VP shunt malfunction is very important. We present two cases of shunt malfunction related to infection and report their successful treatment using Seprafilm®. Seprafilm may improve the safety of VP shunt reinsertion and preserve the function of the reinserted VP shunt after VP shunt malfunction with infection.

HsMAD2 mRNA expression may be a predictor of sensitivity to paclitaxel and survival in neuroblastoma.

PURPOSE: We investigated the associations between hsMAD2 mRNA expression in tumor cells and sensitivity to paclitaxel or patient prognosis in neuroblastoma. METHODS: Fifty-one formalin-fixed paraffin-embedded tumor samples were manually microdissected to collect tumor cells, and RNA was purified. Nineteen clinical samples of advanced neuroblastoma showed appropriate quality of the isolated RNA for real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses. The hsMAD2 expression levels were determined by real-time RT-PCR in 4 neuroblastoma cell lines and 19 clinical samples. The sensitivity to paclitaxel was assessed by WST-8 colorimetric assays and flow cytometry. HsMAD2 expression of the clinical samples was investigated for its association with prognosis in advanced neuroblastoma patients. RESULTS: There was a significant positive correlation between hsMAD2 mRNA expression and the sensitivity to paclitaxel in four neuroblastoma cell lines. High hsMAD2 expression may be correlated with paclitaxel-induced apoptosis. Kaplan-Meier survival curves were stratified by hsMAD2 expression using the median value as a cut-off point and analyzed for prognostic significance by the log-rank test (P = 0.0467). Furthermore, multivariate survival analysis revealed that only hsMAD2 expression had a significant impact on the overall survival rate. CONCLUSIONS: Our results may warrant clinical application of paclitaxel in neuroblastoma treatment for poor prognosis patients.

Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast cancer cell line, MCF-7, induced by the anti-cancer drug, daunorubicin.

Mg(2+)-dependent neutral SMases (NSMases) have emerged as prime candidates for stress-induced ceramide production. Among isoforms identified, previous reports have suggested the importance of NSMase2. However, its activation mechanism has not been precisely reported. Here, we analyzed the mechanism of NSMase2 gene expression by the anti-cancer drug, daunorubicin (DA). DA increased cellular ceramides (C16, C18 and C24) and NSMase activity of a human breast cancer cell line, MCF-7. DA remarkably increased the NSMase2 message and protein, whereas little change in NSMase1 and NSMase3 mRNAs and only a mild increase in acid SMase mRNA were observed. Overexpression and a knock down of NSMase2 indicated that NSMase2 played a role in DA-induced cell death. NSMase2 promoter analysis revealed that three Sp1 motifs located between -148 and -42bp upstream of the first exon were important in basic as well as in DA-induced promoter activity. Consistently, luciferase vectors containing three consensus Sp1-motifs but not its mutated form showed DA-induced transcriptional activation. DA-treated MCF-7 showed increased Sp3 protein. In SL2 cells lacking Sp family proteins, both Sp1 and Sp3 overexpression increased NSMase promoter activity. Increased binding of Sp family proteins by DA to three Sp1 motifs was shown by electrophoresis mobility shift and ChIP assays.

ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH-SY5Y cells: the role of COUP-TFI.

Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1-phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all-trans retinoic acid (ATRA)-induced differentiation of a human neuroblastoma cell line, SH-SY5Y. ATRA reduced CERK mRNA and protein levels. Over-expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA-induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5'-promoter of CERK. Truncation and mutation study suggests that ATRA-responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between -40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), RARalpha, and RXRalpha, respectively. DNA pull-down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP-TFI and siRNA transfection of these genes revealed that COUP-TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co-repressors as well as three transcription factors. These results suggest that COUP-TFI was the ATRA-responsive suppressive transcription factor of CERK gene transcription.