Identification of one exon deletion of intestinal alkaline sphingomyelinase in colon cancer HT-29 cells and a differentiation-related expression of the wild-type enzyme in Caco-2 cells

Wu, Jun; Cheng, Yajun; Nilsson, Åke; Duan, Rui‐Dong

doi:10.1093/carcin/bgh140

Cited by 50 publications

(62 citation statements)

References 31 publications

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“…The data suggest that bile acids constitute important regulatory factors influencing enterocyte proliferation, differentiation, migration, and loss. Alk-SMase is exclusively expressed in differentiated enterocytes [27] and the dissociation of the differentiated cells into the lumen is expected to have fundamental influence on the Alk-SMase levels in the gut. This may explain partly the reduced Alk-SMase and N-CDase activities in the small intestinal lumen caused by bile diversion.…”

Section: Resultsmentioning

confidence: 99%

Effects of bile diversion in rats on intestinal sphingomyelinases and ceramidase

Duan

Verkade

Cheng

et al. 2007

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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1 Summary:Alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase (N-CDase) in the intestinal microvillar membrane are responsible for dietary sphingomyelin digestion. The activities of the enzymes require the presence of bile salt, and the enzymes can be released into the gut lumen in active forms by bile salts and trypsin. It is unclear to what extent that the intestinal presence of bile salts is critical for the intraluminal activity of these enzymes. We compared the activities of Alk-SMase, N-CDase, and other types of SMases in control and permanently bile diverted rats. In the intestinal tract of control rats, the activity of Alk-SMase was profoundly higher than those of acid and neutral SMases. Bile diversion reduced Alk-SMase activity by 85% in the small intestinal content, and by 68% in the faeces, but did not significantly change the activity in the intestinal mucosa. Western blot showed a marked reduction of the enzyme in the intestinal lumen but not mucosa. N-CDase activities both in the intestinal mucosa and content were reduced by bile diversion. Bile diversion also decreased aminopeptidase N activity in the content and increased that in the mucosa, but had no effects on that of alkaline phosphatase. In conclusion, the presence of bile salts is important for maintaining high intraluminal levels of Alk-SMase and N-CDase, two key enzymes for hydrolysis of sphingomyelin in the gut. We speculate that the sphingomyelin hydrolysis in cholestatic conditions is impaired not only by reduced hydrolytic activity but also by deficient dissociation of the enzymes from the membrane.

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Section: Resultsmentioning

confidence: 99%

Effects of bile diversion in rats on intestinal sphingomyelinases and ceramidase

Duan

Verkade

Cheng

et al. 2007

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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“…In the human gut, alkaline SMase (alk-SMase) is the predominant isoform expressed (Nilsson and Duan, 1999). It is a transmembrane ectoenzyme that localizes both to mucosal surfaces and intracellular vesicles in situ and in HT-29 cells (Duan et al, 2003), although the latter possess a deleterious mutation in the alk-SMase gene (Wu et al, 2004). Immunoblot analysis of T84 extracts using antibodies raised against alk-SMase (Cheng et al, 2002) detects a protein band with a molecular mass of~60-65 kDa (Fig.…”

Section: Expression and Distribution Of Smase In Intestinal Epitheliamentioning

confidence: 99%

Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

Saslowsky

Lencer

2007

Cell Microbiol

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SummaryCholera toxin (CT) enters host cells by binding to ganglioside GM1 in the apical plasma membrane (PM). GM1 carries CT retrograde from the PM to the endoplasmic reticulum (ER), where a portion of the toxin, the A1-chain, retro-translocates to the cytosol, causing disease. Trafficking in this pathway appears to depend on the association of CT-GM1 complexes with sphingomyelin (SM)-and cholesterol-rich membrane microdomains termed lipid rafts. Here, we find that in polarized intestinal epithelia, the conversion of apical membrane SM to ceramide by bacterial sphingomyelinase attenuates CT toxicity, consistent with the lipid raft hypothesis. The effect is reversible, specific to toxin entry via the apical membrane, and recapitulated by the addition of exogenous long-chain ceramides. Conversion of apical membrane SM to ceramide inhibits the efficiency of toxin endocytosis, but retrograde trafficking from the apical PM to the Golgi and ER is not affected. This result suggests that the cause for toxin resistance occurs at steps required for retro-translocation of the CT A1-chain to the cytosol.

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“…(international units)/ml penicillin and 100 µg/ml streptomycin on 6-well plates as described in [22]. Before stimulation, the cells were adapted for serum-free medium for 2 h, followed by adding PAF and alkSMase-treated PAF to a final concentration of 100 pmol/ml.…”

Section: Mapk Assaymentioning

confidence: 99%

“…Recent studies have shown decreased activity of the enzyme in the tissues of human long-standing ulcerative colitis and colonic adenoma and carcinomas, suggesting a potential role of the enzyme in both colonic inflammation and carcinogenesis [19][20][21]. An alternative splicing of alk-SMase mRNA, which leads to inactivation of the enzyme, has been found in colon cancer HT-29 cells [22].…”

Section: Introductionmentioning

confidence: 99%

Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity

Nilsson

Jönsson

et al. 2006

Biochemical Journal

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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.

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Identification of one exon deletion of intestinal alkaline sphingomyelinase in colon cancer HT-29 cells and a differentiation-related expression of the wild-type enzyme in Caco-2 cells

Cited by 50 publications

References 31 publications

Effects of bile diversion in rats on intestinal sphingomyelinases and ceramidase

Effects of bile diversion in rats on intestinal sphingomyelinases and ceramidase

Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity

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