Characteristics of sphingomyelin metabolism in the MCF7 and BT474 radiotherapy­resistant HER2­positive breast cancer cell lines.

Breast cancer is the most common cancer globally in terms of incidence. This cancer is classified into subtypes based on histological or immunological characteristics. HER2-positive cases account for 15-25% of breast cancer cases, and one of the first events in breast carcinogenesis is HER2 upregulation. Furthermore, HER2 expression increases the detection rate of metastatic or recurrent breast cancers by 50-80%. The epidermal growth factor receptor family includes HER2, which is a transmembrane receptor protein. In our previous case report, patients who were resistant to anti-HER2 monoclonal antibody therapy, chemotherapy and radiotherapy had higher concentrations of phospholipid metabolites such as phosphatidylcholine and sphingomyelin (SM), which was associated with cancer recurrence progression. To better understand the relationship between radiotherapy resistance and SM expression, breast cancer cell lines with and without HER2 expression (MCF7 and BT474) after exposure to ionizing radiation (IR) were examined. In the cell culture supernatant, similar levels of SM in MCF7 cells were identified after 1-4 Gy exposure. However, SM levels in BT474 cells were upregulated compared with those of in the control group. Intracellular SM levels were upregulated in BT474 cells exposed to 1 and 4 Gy compared with the non-irradiated control group. Furthermore, significantly increased mRNA expression levels of sphingomyelin synthase 2 (SGMS2) in BT474 cells exposed to IR were observed compared with those in nonirradiated cells; however, the SGMS2 levels in MCF7 cells did not differ significantly among the 0, 2 and 4 Gy groups. These findings suggested that a higher dose of IR induced the secretion of SM and its associated gene expression in HER2-positive breast cancer cells.

Disruption of adipocyte HIF-1α improves atherosclerosis through the inhibition of ceramide generation.

Atherosclerosis is a chronic multifactorial cardiovascular disease. Western diets have been reported to affect atherosclerosis through regulating adipose function. In high cholesterol diet-fed ApoE -/- mice, adipocyte HIF-1α deficiency or direct inhibition of HIF-1α by the selective pharmacological HIF-1α inhibitor PX-478 alleviates high cholesterol diet-induced atherosclerosis by reducing adipose ceramide generation, which lowers cholesterol levels and reduces inflammatory responses, resulting in improved dyslipidemia and atherogenesis. Smpd3, the gene encoding neutral sphingomyelinase, is identified as a new target gene directly regulated by HIF-1α that is involved in ceramide generation. Injection of lentivirus-SMPD3 in epididymal adipose tissue reverses the decrease in ceramides in adipocytes and eliminates the improvements on atherosclerosis in the adipocyte HIF-1α-deficient mice. Therefore, HIF-1α inhibition may constitute a novel approach to slow atherosclerotic progression.

Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity.

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.

Regulation of human sphingomyelin synthase 1 translation through its 5'-untranslated region.

Bcr-abl1 oncogene causes a shift in the transcription start site of the SMS1 gene (SGMS1) encoding the sphingomyelin (SM) synthesizing enzyme, sphingomyelin synthase 1 (SMS1). This results in an mRNA with a significantly shorter 5'-UTR, called 7-SGMS1, which is translated more efficiently than another transcript (IIb-SGMS1) with a longer 5'UTR in Bcr-abl1-positive cells. Here, we determine the effects of these alternative 5'UTRs on SMS1 translation and investigate the key features underlying such regulation. First, the presence of the longer IIb 5'UTR is sufficient to greatly impair translation of a reporter gene. Deletion of the upstream open reading frame (-164 nt) or of the predicted stem-loops in the 5'UTR of IIb-SGMS1 has minimal effects on SGMS1 translation. Conversely, deletion of nucleotides -310 to -132 enhanced transcription of IIb-SGMS1 to reach that of 7-SGMS1. We thus suggest that regulatory features within nucleotides -310 and -132 modulate IIb-SGMS1 translation efficiency.

Deficiency of sphingomyelin synthase 1 but not sphingomyelin synthase 2 reduces bone formation due to impaired osteoblast differentiation.

BACKGROUND: There are two isoforms of sphingomyelin synthase (SMS): SMS1 and SMS2. SMS1 is located in the Golgi apparatus only while SMS2 is located in both the plasma membrane and the Golgi apparatus. SMS1 and SMS2 act similarly to generate sphingomyelin (SM). We have undertaken the experiments reported here on SMS and osteoblast differentiation in order to better understand the role SMS plays in skeletal development. METHODS: We analyzed the phenotype of a conditional knockout mouse, which was generated by mating a Sp7 promoter-driven Cre-expressing mouse with an SMS1-floxed SMS2-deficient mouse (Sp7-Cre;SMS1f/f;SMS2-/- mouse). RESULTS: When we compared Sp7-Cre;SMS1f/f;SMS2-/- mice with C57BL/6, SMS2-deficient mice (SMS1f/f;SMS2-/-) and SP7-Cre positive control mice (Sp7-Cre, Sp7-Cre;SMS1+/+;SMS2+/- and Sp7-Cre;SMS1+/+;SMS2-/-), we found that although cartilage formation is normal, Sp7-Cre;SMS1f/f;SMS2-/- mice showed reduced trabecular and cortical bone mass, had lower bone mineral density, and had a slower mineral apposition rate than control mice. Next, we have used a tamoxifen-inducible knockout system in vitro to show that SMS1 plays an important role in osteoblast differentiation. We cultured osteoblasts derived from ERT2-Cre;SMS1f/f SMS2-/- mice. We observed impaired differentiation of these cells in response to Smad1/5/8 and p38 that were induced by bone morphogenic protein 2 (BMP2). However, Erk1/2 phosphorylation was unaffected by inactivation of SMS1. CONCLUSIONS: These findings provide the first genetic evidence that SMS1 plays a role in bone development by regulating osteoblast development in cooperation with BMP2 signaling. Thus, SMS1 acts as an endogenous signaling component necessary for bone formation.

Sphingomyelin synthase 1 enhances BCR signaling to promote lupus-like autoimmune response.

BACKGROUND: Sphingomyelin synthase 1 (SMS1) has been reported to participate in hepatitis and atherosclerosis. However, its role in autoimmune response is not clear. This study investigates the possible involvement of SMS1 in B-cell activation and lupus-like autoimmunity. METHODS: SMS1 knockout lupus-like animal model and SLE patient samples were utilized. B-cell activation and associated signal transduction were detected by flow cytometry, confocal analysis and western blotting. The SMS1 expression in B cells was measured by real-time qPCR. FINDINGS: SMS1 deficiency suppressed B-cell activation in culture, which was restored by exogenous SM supplementation. The BCR-mediated early signal transduction including the colocalization of BCR with F-actin or pY/pBtk, and the phosphorylation of intracellular Fyn and Syk were impaired in SMS1 knockout B cells. Furthermore, SMS1 knockout mice showed reduced production and deposition of autoantibodies, accompanied by less severe kidney pathological changes after pristane induction. SMS1 deficiency also displayed lower autoantibody titers and 24 h urine protein excretion in bm12-induced lupus, which were associated with reduced B-cell activation. Adoptively transferred wide-type B cells partially recovered B-cell activation and autoantibody production in SMS1 deficient bm12-induced lupus mice. Moreover, the SMS1 mRNA level in B cells of SLE patients was increased and positively correlated with the serum anti-dsDNA level, IgG and globulin titers. INTERPRETATION: These data suggest that SMS1 is involved in lupus-like autoimmunity via regulating BCR signal transduction and B cell activation. (Word count for the abstract: 230).

Sphingomyelin synthase 1 regulates the epithelial­to­mesenchymal transition mediated by the TGF­ß/Smad pathway in MDA­MB­231 cells.

Breast cancer is the most common cancer in women and a leading cause of cancer­associated mortalities in the world. Epithelial­to­mesenchymal transition (EMT) serves an important role in the process of metastasis and invasive ability in cancer cells, and transforming growth factor ß1 (TGF­ß1) have been investigated for promoting EMT. However, in the present study, the role of the sphingomyelin synthase 1 (SMS1) in TGF­ß1­induced EMT development was investigated. Firstly, bioinformatics analysis demonstrated that the overexpression of SMS1 negatively regulated the TGFß receptor I (TßRI) level of expression. Subsequently, the expression of SMS1 was decreased, whereas, SMS2 had no significant difference when MDA­MB­231 cells were treated by TGF­ß1 for 72 h. Furthermore, the present study constructed an overexpression cells model of SMS1 and these cells were treated by TGF­ß1. These results demonstrated that overexpression of SMS1 inhibited TGF­ß1­induced EMT and the migration and invasion of MDA­MB­231 cells, increasing the expression of E­cadherin while decreasing the expression of vimentin. Furthermore, the present study further confirmed that SMS1 overexpression could decrease TßRI expression levels and blocked smad family member 2 phosphorylation. Overall, the present results suggested that SMS1 could inhibit EMT and the migration and invasion of MDA­MB­231 cells via TGF­ß/Smad signaling pathway.

Complex formation of sphingomyelin synthase 1 with glucosylceramide synthase increases sphingomyelin and decreases glucosylceramide levels.

Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile α-motif of SMS1 reduced the stability of the SMS1-GCS complex, resulting in a significant reduction in SM synthesis in vivo In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo These results suggest that formation of the SMS1-GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1-GCS complex was confirmed by CRISPR/Cas9-mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.

Circular RNAs-one of the enigmas of the brain.

Circular RNAs (circRNAs) provide a new and relatively unexplored class of noncoding RNAs that are predominantly found in mammalian cells. In this review, we present the latest data regarding the structural organization, possible mechanisms of synthesis, and functions of circRNAs. These transcripts were isolated as an RNA fraction that was resistant to RNase R treatment, which selectively destroys the linear forms of RNA molecules. circRNAs are encoded by orthologous genes in different organisms and show tissue- and organ-specific expression. Currently, the biogenesis and functional properties of circRNAs remain unclear; transcripts of this class, however, remain highly promising targets of research. Some of them have been ascribed the function of "molecular sponges" that can absorb microRNAs, RNA-binding proteins, and small nuclear RNAs. circRNAs are often formed from the RNA portions of protein-coding genes in the course of alternative splicing. Some features of the circRNAs of mammals were demonstrated using 11 circRNAs of the human sphingomyelin synthase 1 gene (SGMS1), which were discovered by us in the brain. These circRNAs consist mainly of portions of the multi-exon 5' untranslated region (5'UTR) of the SGMS1 gene and include one to five exons. The synthesis of circRNAs may be new, previously unknown, function of the multi-exon 5'UTR of genes. This feature is most clearly manifested in the brain, where the level of circRNAs is significantly higher.

Circular RNA of the human sphingomyelin synthase 1 gene: Multiple splice variants, evolutionary conservatism and expression in different tissues.

The human sphingomyelin synthase 1 gene (SGMS1) encodes an essential enzyme that is involved in the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. Among the products of SGMS1, we found new transcripts, circular RNAs (circRNAs), that contain sequences of the gene's 5' untranslated region (5'UTR). Some of them include the gene's coding region and fragments of introns. An analysis of the abundance of circRNAs in human tissues showed that the largest transcripts were predominantly found in different parts of the brain. circRNAs of rat and mouse sphingomyelin synthase 1 orthologous genes were detected and are highly similar to the human SGMS1 gene transcripts. A quantitative analysis of the abundance of such transcripts also revealed their elevated amount in the brain. A computational analysis of sequences of human circRNAs showed their high potential of binding microRNAs (miRNAs), including the miRNAs that form complexes with Ago proteins and the mRNA of SGMS1. We assume that the circRNAs identified here participate in the regulation of the function of the SGMS1 gene in the brain.

All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity.

Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.

Modeling sphingomyelin synthase 1 driven reaction at the Golgi apparatus can explain data by inclusion of a positive feedback mechanism.

Here we present a minimal mathematical model for the sphingomyelin synthase 1 (SMS1) driven conversion of ceramide to sphingomyelin based on chemical reaction kinetics. We demonstrate via mathematical analysis that this model is not able to qualitatively reproduce experimental measurements on lipid compositions after altering SMS1 activity. We prove that a positive feedback mechanism from the products to the reactants of the reaction is one possible model extension to explain these specific experimental data. The proposed mechanism in fact exists in vivo via protein kinase D and the ceramide transfer protein CERT. The model is further evaluated by additional observations from the literature.

ASMase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading.

BACKGROUND & AIMS: The pathogenesis of alcohol-induced liver disease (ALD) is poorly understood. Here, we examined the role of acid sphingomyelinase (ASMase) in alcohol induced hepatic endoplasmic reticulum (ER) stress, a key mechanism of ALD. METHODS: We examined ER stress, lipogenesis, hyperhomocysteinemia, mitochondrial cholesterol (mChol) trafficking and susceptibility to LPS and concanavalin-A in ASMase(-)(/-) mice fed alcohol. RESULTS: Alcohol feeding increased SREBP-1c, DGAT-2, and FAS mRNA in ASMase(+/+) but not in ASMase(-/-) mice. Compared to ASMase(+/+) mice, ASMase(-/-) mice exhibited decreased expression of ER stress markers induced by alcohol, but the level of tunicamycin-mediated upregulation of ER stress markers and steatosis was similar in both types of mice. The increase in homocysteine levels induced by alcohol feeding was comparable in both ASMase(+/+) and ASMase(-/-) mice. Exogenous ASMase, but not neutral SMase, induced ER stress by perturbing ER Ca(2+) homeostasis. Moreover, alcohol-induced mChol loading and StARD1 overexpression were blunted in ASMase(-/-) mice. Tunicamycin upregulated StARD1 expression and this outcome was abrogated by tauroursodeoxycholic acid. Alcohol-induced liver injury and sensitization to LPS and concanavalin-A were prevented in ASMase(-/-) mice. These effects were reproduced in alcohol-fed TNFR1/R2(-/-) mice. Moreover, ASMase does not impair hepatic regeneration following partial hepatectomy. Of relevance, liver samples from patients with alcoholic hepatitis exhibited increased expression of ASMase, StARD1, and ER stress markers. CONCLUSIONS: Our data indicate that ASMase is critical for alcohol-induced ER stress, and provide a rationale for further clinical investigation in ALD.