Biomedical subjects

Rui-Dong Duan

Publications and source records attributed to Rui-Dong Duan.

At least 19 recordsLinked to original sources

PubMed2007

Human meconium contains significant amounts of alkaline sphingomyelinase, neutral ceramidase, and sphingolipid metabolites.

Intestinal alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase may catalyze the hydrolysis of endogenous sphingomyelin (SM) and milk SM in human-milk fed infants. The enzymes generate sphingolipid metabolites that may influence gut maturation. Alk-SMase also inactivates platelet-activating factor (PAF) that is involved in the pathogenesis of necrotizing enterocolitis (NEC). We examined whether the two enzymes are expressed in both preterm and term infants and analyzed Alk-SMase, neutral ceramidase, SM, and sphingolipid metabolites in meconium. Meconium was collected from 46 preterm (gestational ages 23-36 wk) and 38 term infants (gestational ages 37-42 wk) and analyzed for Alk-SMase using C-choline-labeled SM and for neutral ceramidase using C-octanoyl-sphingosine as substrates. Molecular species of SM, ceramide, and sphingosine were analyzed by high-performance liquid chromatography mass spectroscopy. Meconium contained significant levels of Alk-SMase and ceramidase at all gestational ages. It also contained 16-24 carbon molecular species of SM, palmitoyl- and stearoyl-sphingosine, and sphingosine. There were positive correlations between levels of SM and ceramide and between ceramide and sphingosine levels. In conclusion, Alk-SMase and ceramidase are expressed in the gut of both preterm and term newborn infants and may generate bioactive sphingolipid messengers.

Amidohydrolases↗

PubMed2006

Alkaline sphingomyelinase: an old enzyme with novel implications.

Alkaline sphingomyelinase (alk-SMase) is present in the intestinal tract and additionally human bile. It hydrolyses sphingomyelin in both intestinal lumen and the mucosal membrane in a specific bile salt dependent manner. The enzyme was discovered 36 years ago but got real attention only in the last decade, when sphingomyelin metabolism was realized to be a source of multiple lipid messengers, and when dietary sphingomyelin was found to inhibit colonic tumorigenesis in animals. The enzyme shares no structural similarity with other SMases and belongs to the nucleotide pyrophosphatase/phosphodiesterase family. The enzyme is of specific properties, such as bile salt dependency, trypsin resistance, high stability, and tissue specific expression. In the colon, the enzyme may play antiproliferative and antiinflammatory roles through generating ceramide, reducing the formation of lysophosphatidic acid, and inactivating platelet-activating factor. The enzyme is down regulated in human long-standing ulcerative colitis and colonic adenocarcinoma, and mutation of the enzyme has been found in colon cancer cells. In the small intestine, alk-SMase is the key enzyme for sphingomyelin digestion. The hydrolysis of sphingomyelin may affect the cholesterol uptake and have impact on sphingomyelin levels in plasma lipoproteins. The review summarizes the new information of alk-SMase from biochemical, cell and molecular biological studies in the last decade and evaluates its potential implications in development of colon cancer, inflammatory bowel diseases, and atherosclerosis.

Animals↗

PubMed2006

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

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. [3H]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 Zn2+. The activity was abolished by site mutation of the predicted metal-binding 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 mumol x h(-1) x (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.

Animals↗

PubMed2005

Ursodeoxycholic acid differentially affects three types of sphingomyelinase in human colon cancer Caco 2 cells.

We investigated the effects of UDCA on sphingomyelinase (SMase) in Caco 2 cells cultured in monolayer and polarized conditions. Alkaline SMase activity was high in polarized cells whereas, acid and neutral SMase activities were high in monolayer cells. In polarized cells, UDCA increased alkaline SMase expression and caspase 3 activity but had no effect on acid and neutral SMases. In monolayer cells, UDCA reduced both acid and neutral SMase activities, inhibited cell proliferation, but had little effect on alkaline SMase and caspase 3 activities. In conclusion, UDCA differentially affects SMase activity, cell proliferation, and apoptosis in colonic cells depending on the cell conditions.

Apoptosis↗

PubMed2005

Absorption and lipoprotein transport of sphingomyelin.

Dietary sphingomyelin (SM) is hydrolyzed by intestinal alkaline sphingomyelinase and neutral ceramidase to sphingosine, which is absorbed and converted to palmitic acid and acylated into chylomicron triglycerides (TGs). SM digestion is slow and is affected by luminal factors such as bile salt, cholesterol, and other lipids. In the gut, SM and its metabolites may influence TG hydrolysis, cholesterol absorption, lipoprotein formation, and mucosal growth. SM accounts for approximately 20% of the phospholipids in human plasma lipoproteins, of which two-thirds are in LDL and VLDL. It is secreted in chylomicrons and VLDL and transferred into HDL via the ABCA1 transporter. Plasma SM increases after periods of large lipid loads, during suckling, and in type II hypercholesterolemia, cholesterol-fed animals, and apolipoprotein E-deficient mice. SM is thus an important amphiphilic component when plasma lipoprotein pools expand in response to large lipid loads or metabolic abnormalities. It inhibits lipoprotein lipase and LCAT as well as the interaction of lipoproteins with receptors and counteracts LDL oxidation. The turnover of plasma SM is greater than can be accounted for by the turnover of LDL and HDL particles. Some SM must be degraded via receptor-mediated catabolism of chylomicron and VLDL remnants and by scavenger receptor class B type I receptor-mediated transfer into cells.

Aging↗

PubMed2005

Acid sphingomyelinase is induced by butyrate but does not initiate the anticancer effect of butyrate in HT29 and HepG2 cells.

Butyric acid and sphingomyelin (SM) affect colonic tumorigenesis. We examined the potential link between butyrate stimulation and SM metabolism in colonic and hepatic cancer cell lines. After incubating HT29 and HepG2 cells with butyrate and other short-chain fatty acids, we found that butyrate increased acid but not neutral or alkaline sphingomyelinase (SMase) activity by 10- to 20-fold. The effects occurred after 16 h of incubation and were associated with reduced SM and phosphatidylcholine contents and increased ceramide levels. Northern blotting showed increased acid SMase mRNA levels in these cells after butyrate stimulation. Propionate was less potent, and acetate had no effect. No similar changes of acid phosphatase could be identified. At concentrations that increased acid SMase expression, butyrate inhibited cell proliferation, activated caspase 3, and induced apoptosis. However, the antiproliferative and apoptotic effects of butyrate preceded the changes of acid SMase and were not affected by knocking down acid SMase expression by small, interfering RNA. In addition, butyrate-induced acid SMase expression was not affected by blocking the caspase pathway. In conclusion, butyrate regulates SM metabolism by stimulating acid SMase expression in colon and liver cancer cells, but the increased acid SMase is not a critical mechanism for initiating the anticancer effects of butyrate.

Antineoplastic Agents↗

PubMed2005

Cloning of alkaline sphingomyelinase from rat intestinal mucosa and adjusting of the hypothetical protein XP_221184 in GenBank.

Intestinal alkaline sphingomyelinase (alk-SMase) digests sphingomyelin and the process may influence colonic tumorigenesis and cholesterol absorption. We recently identified the gene of human alk-SMase and cloned the cDNA. Cross-species screening of homology in GenBank found a hypothetical rat protein, XP_221184, with 491 amino acid residues, which shares 73% identity with human alk-SMase. Based on the cDNA sequence of this protein, we cloned a cDNA from rat intestinal mucosa by RT-PCR. The cloned cDNA encodes a protein with 439 amino acid residues and higher (85%) identity with human alk-SMase. The cloned cDNA differed from the XP_221184 cDNA in splice sites linking exons 2 and 3, and exons 3 and 4, respectively. In the sequence of the cloned protein, the predicted activity motif, sphingomyelin binding sites, and potential glycosylation sites in human alk-SMase are all conserved. To confirm the cloned protein is the real form of alk-SMase, native alk-SMase was purified from rat intestine and subjected to proteolytic digestion followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and electrospray ionization (ESI) tandem mass spectrometry. Seven tryptic peptides were found to match the cloned protein sequence. Transient expression of the cloned cDNA linked with a myc tag in COS-7 cells demonstrated high SMase activity, with an optimal pH at 9.0 and a specific dependence on taurocholate and taurochenodeoxycholate. The expressed protein reacted with both anti-myc and anti-human alk-SMase antibodies. Northern blotting of rat tissues revealed high levels of mRNA in jejunum but not in other tissues. In conclusion, we cloned rat alk-SMase cDNA from rat intestine, adjusted the putative rat alk-SMase protein in GenBank, and confirmed the specific expression of the gene in the small intestine.

Amino Acid Sequence↗

PubMed2005

Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis.

Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme activity. Immunogold labelling showed that the wild-type enzyme was mainly located in the plasma membrane, whereas the C-terminal domain-truncated enzyme was released into the medium. Deglycosylation blocked the release of the enzyme that accumulated in endosome-like structures. The enzyme activity was also decreased by mutations of the residues forming the putative metal-binding sites and the active core. Substitution of the active core sequence with that of NPP or mutation of T75 in the core abolished the enzyme activity against sphingomyelin but failed to render the enzyme NPP active. Our results indicate that alk-SMase activity is severely affected by defective N-glycosylation and structural alterations of the putative metal-binding sites and the predicted active core.

Animals↗

PubMed2004

Inhibition of cancer cell proliferation in vitro by fruit and berry extracts and correlations with antioxidant levels.

The effects of 10 different extracts of fruits and berries on cell proliferation of colon cancer cells HT29 and breast cancer cells MCF-7 were investigated. The fruits and berries used were rosehips, blueberries, black currant, black chokeberries, apple, sea buckthorn, plum, lingonberries, cherries, and raspberries. The extracts decreased the proliferation of both colon cancer cells HT29 and breast cancer cells MCF-7, and the effect was concentration dependent. The inhibition effect for the highest concentration of the extracts varied 2-3-fold among the species, and it was in the ranges of 46-74% (average = 62%) for the HT29 cells and 24-68% (average = 52%) for the MCF-7 cells. There were great differences in the content of the analyzed antioxidants in the extracts. The level of the vitamin C content varied almost 100-fold, and the content of total carotenoids varied almost 150-fold among the species. Also in the composition and content of flavonols, hydroxycinnamic acids, anthocyanins, and phenolics were found great differences among the 10 species. The inhibition of cancer cell proliferation seen in these experiments correlated with levels of some carotenoids and with vitamin C levels, present at levels that can be found in human tissues. The same inhibition of cell proliferation could not be found by ascorbate standard alone. This correlation might indicate a synergistic effect of vitamin C and other substances. In MCF-7 cells, the anthocyanins may contribute to the inhibition of proliferation.

Antioxidants↗

PubMed2004

Rat intestinal ceramidase: purification, properties, and physiological relevance.

Neutral ceramidase activity has previously been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine but has never been fully characterized. We have purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9% NaCl and 3 mM sodium taurodeoxycholate. Using a combination of acetone precipitation and ion-exchange, hydrophobic-interaction, and gel chromatographies, we obtained a homogenous enzyme protein with a molecular mass of approximately 116 kDa. The enzyme acts on both [14)]octanoyl- and [14C]palmitoyl-sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate but is inhibited by 2 mM or more of other bile salts. It is a glycosylated protein stable to trypsin and chymotrypsin exposure, is not influenced by Ca2+, Mg2+, or Mn2+, and is inhibited by Zn2+ and Cu2+. Mass fragmentographic analysis identified 12 fragments covering 17.5% of the sequence for neutral/alkaline ceramidase 2 purified (Mitsutake S, Tani M, Okino N, Mori K, Ichinose S, Omori A, Iida H, Nakamura T, and Ito M. J Biol Chem 276: 26249-262459, 2001) from rat kidney and located in apical membrane of renal tubular cells. Intestinal and kidney ceramidases also have similar molecular mass and ion dependence. Intestinal ceramidase thus is a neutral ceramidase 2 released by bile salts and resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.

Amidohydrolases↗

PubMed2004

Pancreatic trypsin cleaves intestinal alkaline sphingomyelinase from mucosa and enhances the sphingomyelinase activity.

Sphingomyelin (SM) hydrolysis in the gut has implications in colonic tumorigenesis and cholesterol absorption. It is triggered by intestinal alkaline sphingomyelinase (Alk-SMase) that is present in the intestinal mucosa and content. The mechanism by which the enzyme is released into the lumen is not clear. We studied whether trypsin can dissociate Alk-SMase from the mucosa and affect its activity. During luminal perfusion of rat intestine, addition of trypsin to the buffer increased Alk-SMase activity in the perfusate output by about threefold. Treating COS-7 cells transfected with Alk-SMase cDNA with trypsin increased the SMase activity in the medium and reduced that in the cell lysate dose dependently. The appearance of Alk-SMase in the perfusate and culture medium was confirmed by Western blot analysis. The effect of trypsin was blocked by trypsin inhibitor, and neither chymotrypsin nor elastase had a similar effect. We also expressed the full length and COOH-terminal truncated Alk-SMase in COS-7 cells and found that the activity of the full-length enzyme is mainly in the cells, whereas that of the truncated form is mainly in the medium. Both forms were active, but only the activity of the full-length Alk-SMase was enhanced by trypsin. By linking a poly-His tag to the constructed cDNA, we found that the first tryptic site Arg440 upstream of the signal anchor was attacked by trypsin. In conclusion, trypsin cleaves the Alk-SMase at the COOH terminal, releases it from mucosa, and meanwhile enhances its activity. The findings indicate a physiological role of trypsin in SM digestion.

Alkalies↗

PubMed2004

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.

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.

Alkaline Phosphatase↗

PubMed2004

Psyllium and fat in diets differentially affect the activities and expressions of colonic sphingomyelinases and caspase in mice.

Dietary fibre and fat affect colonic tumourigenesis and inflammation. Sphingomyelin metabolism may have implications for the pathogenesis of colonic tumours and ulcerative colitis. The present study examined the effects of psyllium and fat on the enzymes responsible for sphingomyelin metabolism and apoptosis in the colon. Mice were fed control, psyllium-containing (100 g/kg), high-fat (313 g/kg, 53 % energy as fat) or high-fat plus psyllium diets for 4 weeks. The activities of acid, neutral and alkaline sphingomyelinase (SMase), neutral ceramidase, and caspase 3, 8 and 9 in colonic mucosa were determined. The expressions of alkaline SMase and caspase 3 were examined. The psyllium-containing diet was found to increase significantly the activities of alkaline SMase and caspase 3 and decreased those of acid SMase and neutral ceramidase. The high-fat diet had opposite effects on these enzymes and attenuated the effects of psyllium. Western blotting showed that psyllium increased and high-fat decreased the levels of alkaline SMase and caspase 3 in colonic mucosa. The change in caspase 3 activity was positively correlated with that of alkaline SMase and negatively with acid SMase. No similar changes of acid and alkaline phosphatase activities in the colon or acid and neutral SMase activity in the liver were identified. In conclusion, colonic sphingomyelin metabolism and apoptosis were affected by psyllium and fat in an opposite manner. The results may have implications for colorectal tumourigenesis and inflammation.

Acid Phosphatase↗

PubMed2003

Purified intestinal alkaline sphingomyelinase inhibits proliferation without inducing apoptosis in HT-29 colon carcinoma cells.

PURPOSE: Sphingomyelin (SM) hydrolysis by sphingomyelinase (SMase) has become an important signalling pathway, with the product ceramide implicated in regulation of cell growth, differentiation and apoptosis. Alkaline SMase is specifically located in the intestinal tract. Marked reductions of the enzyme activity have been found in sporadic colorectal carcinomas and in both adenomas and flat mucosa of patients with familial adenomatous polyposis, indicating an anti-proliferative role in colonic cell growth. METHODS: We examined the effects of a purified alkaline SMase from rat intestine and a bacterial neutral SMase on cell growth parameters in HT-29 colonic carcinoma cells. RESULTS: Alkaline SMase was found to inhibit proliferation of HT-29 cells in both dose-dependent and time-dependent manners. The threshold concentration of the enzyme was approximately 2.5 microU/ml, and the maximum effect was obtained at approximately 20 microU/ml, which inhibited the cell growth by 50%. The inhibition occurred rapidly, and maximum effect was reached after 12 h of incubation. Dose-dependent inhibition of DNA synthesis was also demonstrated. The effect of alkaline SMase was preceded and accompanied by increased hydrolysis of SM and production of ceramide. Neutral SMase with equivalent hydrolytic capacity did not inhibit cell growth. Alkaline SMase did not induce apoptosis in HT-29 cells. Alkaline SMase did not inhibit growth of IEC-6 cells. CONCLUSION: Alkaline SMase, at doses that induce SM hydrolysis, inhibits growth of colon cancer cells. The inhibition is attributed to an anti-proliferative effect rather than an apoptotic effect.

Animals↗

PubMed2003

Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family.

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 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 alk-SMase. 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 Zn2+. 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. In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ecto-enzyme related to the NPP family with specific features essential for its SMase activity.

Adenosine Triphosphate↗

PubMed2003

Purification, localization, and expression of human intestinal alkaline sphingomyelinase.

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, down-regulated in human colon cancer, and may have antiproliferative effects on colon cancer cells.

Apoptosis↗

PubMed2003

Reduction in alkaline sphingomyelinase in colorectal tumorigenesis is not related to the APC gene mutation.

BACKGROUND AND AIMS: The sphingomyelin pathway is an important intracellular mechanism in regulating cell growth. The first step in this pathway is catalysed by sphingomyelinases. Alkaline sphingomyelinase is specifically located in the intestinal tract. Markedly reduced alkaline sphingomyelinase activities have been found in sporadic colorectal tumours and in familial adenomatous polyposis adenomas. Since the adenomatous polyposis coli (APC) gene is mutated in about 80% of sporadic colorectal tumors, and familial adenomatous polyposis is the consequence of a germline mutation of the same gene, we examined whether low alkaline sphingomyelinase activity is linked to APC gene mutations. PATIENTS AND METHODS: Both germline and sporadic adenomatous polyposis coli gene mutations were studied. Alkaline, neutral, and acid sphingomyelinase activities were measured in the intestinal mucosa and content of multiple intestinal neoplasia mice, a murine model of familial adenomatous polyposis and compared to control mice. Alkaline sphingomyelinase activity was also measured in 11 human rectal tumors with APC gene mutation and compared with 9 control tumors without mutation. RESULTS: Alkaline, neutral, and acid sphingomyelinase activities were present in the small intestine and colon in both mice types with no differences in hydrolytic capacity or distribution pattern. In sporadic rectal tumors similar alkaline sphingomyelinase activities were identified in tumors with somatic APC gene mutations as in samples without mutations. In the tumors without detectable APC mutations beta-catenin was analyzed, but no mutation was detected. CONCLUSION: Alkaline sphingomyelinase is not directly linked to adenomatous polyposis coli gene mutations.

Adenomatous Polyposis Coli↗

PubMed2003

Development of intestinal alkaline sphingomyelinase in rat fetus and newborn rat.

Sphingomyelin metabolism is a novel signal transduction pathway related to cell differentiation, proliferation, and apoptosis. Alkaline sphingomyelinase (alk-SMase) is specifically present in the intestinal tract of many species. The enzyme is important in digestion of dietary sphingomyelin. Milk is the only exogenous source of sphingomyelin for an infant, and digestion of milk sphingomyelin may be important for development of intestinal mucosa. It is unknown whether alk-SMase is present before birth and whether it changes after birth and during the suckling period. We studied activities, expression, and distribution of alk-SMase in rat fetus and newborn. The changes of acid and neutral SMase as well as alkaline phosphatase were analyzed for comparison. Little activity of alk-SMase was identified up to gestation day 20, but increased 10 times during the following 2 d. After birth, the activity continued to increase during the following 4 wk. Western blot using IgY antibody against rat alk-SMase failed to identify the enzyme at gestation day 20 but clearly showed the protein at day 22. The distribution pattern of the enzyme along the intestinal tract in fetus was largely the same as in adult animals, but became more pronounced after birth. Short-term weaning had no effect on alk-SMase activity. The activities of acid and neutral SMase were high at gestation day 20 and decreased significantly before birth. The changes of alk-SMase also differed from those of alkaline phosphatase, another brush border enzyme. Thus, we conclude that alk-SMase is rapidly expressed during the last days of gestation and that the newborn rat acquires the ability to digest milk sphingomyelin early in life.

Alkaline Phosphatase↗