Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank TM coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alkSMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30 -36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn 2؉ . Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank TM . In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ectoenzyme related to the NPP family with specific features essential for its SMase activity. Sphingomyelin (SM)1 is a component of all mammalian cell membranes particularly the plasma membrane and the lysosomal membrane. SM is also a dietary component and is mainly present in milk, eggs, meat, and marine products (1, 2). Hydrolysis of SM generates ceramide, sphingosine, and sphingosine 1-phosphate that have regulatory effects on numerous cellular functions such as proliferation, differentiation, and apoptosis (3, 4). At least five types of sphingomyelinase (SMase) have been identified, of which acid and neutral SMases have been cloned (5-9). An enzyme that catalyzes hydrolysis of SM with optimal alkaline pH was first identified in the intestinal content of human and intestinal mucosa of rat and pig by Nilsson (10) and was named alkaline SMase (alkSMase) thereafter (11). Previous studies indicated that alkSMase may be responsible for digestion of dietary SM and for hydrolysis of endogenous SM derived from bile and from the brush borders of sloughed mucosal cells.SM metabolism in the intestine may have implications in colon cancer development. Dietary supplement with SM and ceramide analogues...

Sphingomyelin (SM) metabolism in the gut may have an impact on colon cancer development. In this study, we purified alkaline sphingomyelinase (alk-SMase) from human intestinal content, and studied its location in the mucosa, expression in colon cancer, and function on colon cancer cells. The enzyme was purified by a series of chromatographies. The molecular mass of the enzyme is 60 kDa, optimal pH is 8.5, and isoelectric point is 6.6. Under optimal conditions, 1 mg of the enzyme hydrolyzed 11 mM SM per hour. The properties of the enzyme are similar to those of rat intestinal alk-SMase but not to those of bacterial neutral SMase. Immunogold electronmicroscopy identified the enzyme on the microvillar membrane in endosome-like structures and in the Golgi complexes of human enterocytes. The expression and the activity of the enzyme were decreased in parallel in human colon cancer tissues compared with the adjacent normal tissue. The enzyme inhibited DNA biosynthesis and cell proliferation dose dependently and caused a reduction of SM in HT29 cells. Intestinal alk-SMase is localized in the enterocytes, downregulated in human colon cancer, and may have antiproliferative effects on colon cancer cells. -Duan, R

Sphingomyelin (SM) metabolism in the gut has been implicated in colonic tumorigenesis. Intestinal alkaline sphingomyelinase (alk-SMase) hydrolyses SM in the intestinal content and at the brush border. The enzyme activity is decreased in the tissues of human colorectal tumours. This study examines whether site or chain-mutation of alk-SMase occurs in colon cancer HT-29 cells and Caco-2 cells. Total RNA was isolated and the cDNA of alk-SMase was amplified by RT-PCR. The size of the cDNA from HT-29 cells was smaller than that of the wild-type cDNA. DNA sequencing identified a deletion of exon 4 in alk-SMase cDNA in HT-29 cells. No mutation in genomic alk-SMase DNA from exon 3 to 5 was identified. The exon 4 deletion was caused by a shift of RNA splice site in chromosome 17q25. In Caco-2 cells, no mutation of alk-SMase cDNA was identified. Transient expression in COS-7 cells showed that the enzyme from the cDNA in HT-29 cells had little alk-SMase activity whereas that in Caco-2 cells was as active as the wild-type alk-SMase. The deleted region included residue His353, which is predicted to form a substrate-binding site of alk-SMase. H353A substitution resulted in a protein with no alk-SMase activity. In monolayer cultured Caco-2 cells and HT-29 cells the alk-SMase activities were low. However, to culture the cells under polarizing conditions increased alk-SMase activity and reduced SM level in Caco-2 cells. The alk-SMase activity varied in parallel with alkaline phosphatase activity. In conclusion, we identified an inactive deletion in alk-SMase in HT-29 cells, and a differentiation-related expression of the enzyme in Caco-2 cells. The results provide a molecular mechanism related to previous findings of reduced alk-SMase activity in human colon cancers.

Exosome‐derived miRNAs are regarded as biomarkers for the diagnosis and prognosis of many human cancers. However, its function in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, differentially expressed miRNAs from urinal exosomes were identified using next‐generation sequencing (NGS) and verified using urine samples of ccRCC patients and healthy donors. Then, the exosomes were analysed in early‐stage ccRCC patients, healthy individuals and patients suffering from other urinary system cancers. Thereafter, the target gene of the miRNA was detected. Its biological function was investigated in vitro and in vivo. The results showed that miR‐30c‐5p could be amplified in a stable manner. Its expression pattern was significantly different only between ccRCC patients and healthy control individuals, but not compared with that of other urinary system cancers, which indicated its specificity for ccRCC. Additionally, the overexpression of miR‐30c‐5p inhibited ccRCC progression in vitro and in vivo. Heat‐shock protein 5 (HSPA5) was found to be a direct target gene of miR‐30c‐5p. The depletion of HSPA5 caused by miR‐30c‐5p inhibition reversed the promoting effect of ccRCC growth. In conclusion, urinary exosomal miR‐30c‐5p acts as a potential diagnostic biomarker of early‐stage ccRCC and may be able to modulate the expression of HSPA5, which is correlated with the progression of ccRCC.

and dietary products such as milk, eggs, meat, and fi sh. Our daily intake of SM is about 300 mg ( 1 ). SM in the diet has been shown to inhibit the colonic tumorigenesis in mice treated with chemical carcinogens ( 2 ), to promote the development of the intestinal mucosa in new born rats ( 3 ), and to inhibit cholesterol absorption in the gut ( 4,5 ). Because most of these effects can be reproduced or linked to ceramide, a hydrolytic product of SM ( 6, 7 ), it is important to study the enzymes that hydrolyze SM and generate ceramide in the gut.More than 40 years ago, Nilsson ( 8 ) identifi ed a type of enzyme in the intestinal mucosa that hydrolyses SM and generates ceramide at alkaline pH. This enzyme was thereafter named alkaline sphingomyelinase (alk-SMase) ( 9 ). Due to the fi ndings of important biological effects of sphingolipids in the last two decades ( 10-12 ), we performed several studies on alk-SMase. We have purifi ed the protein ( 13,14 ), cloned the gene ( 15, 16 ), and found potential implications of the enzyme in SM digestion ( 17 ), cell proliferation ( 18 ), colonic infl ammation ( 19 ), and cholesterol absorption ( 7 ). We also found decreased alk-SMase activity in colonic diseases such as colon cancer and colitis (20)(21)(22), and identifi ed inactive abnormal splicing forms of the enzyme in human colon and liver cancer cells ( 23,24 ).Besides alk-SMase, other SMases such as acid and neutral SMases were also identifi ed in the intestinal tract ( 9 ). A cloning study showed that alk-SMase shares no structural similarities with either acid or neutral SMase but belongs to the ectonucleotide pyrophosphatase/phosphodiesterase (NPP) family ( 15 ). Being a novel member of this family, alk-SMase is also called NPP7. Manuscript received 16 November 2010 and in revised form 20 December 2010. Published, JLR Papers in Press, December 21, 2010 DOI 10.1194 Abbreviations: alk-SMase, alkaline sphingomyelinase; APN, aminopeptidase N; BSSL, bile salt stimulated lipase; Cre-LoxP, Cre-recombinase-Locus of X over P1; ES, embryonic stem; KO, knockout; NPP, ectonucleotide pyrophosphatase/phosphodiesterase; TG, triglyceride; WT, wild-type.

Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophosphatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide in the intestinal tract. The enzyme may protect the intestinal mucosa from inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase can hydrolyse and inactivate PAF. [ 3 H]Octadecyl-labelled PAF was incubated with purified rat intestinal alk-SMase or recombinant human alk-SMase expressed in COS-7 cells. The hydrolytic products were assayed with TLC and MS. We found that alkSMase cleaved the phosphocholine head group from PAF and generated 1-O-alkyl-2-acetyl-sn-glycerol. Differing from the activity against SM, the activity against PAF was optimal at pH 7.5, inhibited by EDTA and stimulated by 0.1-0.25 mM Zn 2+ . The activity was abolished by site mutation of the predicted metalbinding sites that are conserved in all NPP members. Similar to the activity against SM, the activity against PAF was dependent on bile salt, particularly taurocholate and taurochenodeoxycholate. The V max for PAF hydrolysis was 374 µmol · h −1 · (mg of protein) −1 . The hydrolysis of PAF and SM could be inhibited by the presence of SM and PAF respectively, the inhibition of PAF hydrolysis by SM being stronger. The PAF-induced MAPK (mitogen-activated protein kinase) activation and IL-8 (interleukin 8) release in HT-29 cells, and chemotaxis in leucocytes were abolished by alk-SMase treatment. In conclusion, alk-SMase hydrolyses and inactivates PAF by a phospholipase C activity. The finding reveals a novel function, by which alk-SMase may counteract the development of intestinal inflammation and colon cancer.

These findings suggest that PFN1 plays a critical role in gastric carcinoma progression, and these effects are likely mediated through the integrin β1/FAK pathway.

Mounting evidence indicates that microRNAs (miRNAs) are involved in multiple processes of osteogenic differentiation. MicroRNA-101 (miR-101), identified as a tumor suppressor, has been implicated in the pathogenesis of several types of cancer. However, the expression of miR-101 and its roles in the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) remain unclear. We found that the miR-101 expression level was significantly increased during the osteogenic differentiation of hBMSCs. MiR-101 depletion suppressed osteogenic differentiation, whereas the overexpression of miR-101 was sufficient to promote this process. We further demonstrated that enhancer of zeste homolog 2 (EZH2) was a target gene of miR-101. EZH2 overexpression and depletion reversed the promoting or suppressing effect of osteogenic differentiation of hBMSCs, respectively, caused by miR-101. In addition, we showed that miR-101 overexpression promoted the expression of Wnt genes, resulting in the activation of the Wnt/β-catenin signaling pathway by targeting EZH2, while the activity of β-catenin and the Wnt/β-catenin signaling pathway was inhibited by ICG-001, a β-Catenin inhibitor, which reversed the promoting effect of miR-101. Finally, miR-101 also promotes in vivo bone formation by hBMSCs. Collectively, these data suggest that miR-101 is induced by osteogenic stimuli and promotes osteogenic differentiation at least partly by targeting the EZH2/Wnt/β-Catenin signaling pathway.