Our aim was to use quantitative and qualitative analyses to gain further insight into the role of ceramide in cystic fibrosis (CF). Sphingolipid ceramide is a known inflammatory mediator, and its ...accumulation in inflamed lung has been reported in different types of emphysema, chronic obstructive pulmonary disease and CF. CF is caused by a mutation of the chloride channel and associated with hyperinflammation of the respiratory airways and high susceptibility to ongoing infections. We have previously demonstrated that de novo ceramide synthesis is enhanced in lung inflammation and sustains
Pseudomonas aeruginosa
pulmonary infection in a CF murine model. We used liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) imaging coupled with mass spectrometry, confocal laser scan microscopy and histology analyses to reveal otherwise undecipherable information. We demonstrated that (i) upregulated ceramide synthesis in the alveoli is strictly related to alveolar infection and inflammation, (ii) alveolar ceramide (C16) can be specifically targeted by nanocarrier delivery of the ceramide synthesis inhibitor myriocin (Myr) and (iii) Myr is able to downmodulate pro-inflammatory lyso-PC, favouring an increase in anti-inflammatory PCs. We concluded that Myr modulates alveolar lipids milieu, reducing hyperinflammation and favouring anti-microbial effective response in CF mouse model.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
β1,3-Galactosyltransferase β3Gal-T5 is highly expressed in the colons of humans and certain primates due to a retroviral long
terminal repeat (LTR) acting as a strong promoter. Because this ...promoter is inactive in other human tissues or mice, we attempted
to understand how adoption of a retrotransposon allowed the gene to acquire tissue-specific expression. We identified three
novel 5â²-UTRs of β3Gal-T5 mRNA, types A, B, and C, and found widespread expression of the type A transcript at much lower
levels than the LTR transcript, the expression of which is restricted to organs of the gastrointestinal tract. Expression
of the type C 5â²-UTR transcript was mostly restricted to the ileum, where it was expressed at high levels. We cloned the 5â²-flanking
regions of both types A and B 5â²-UTRs, found deletion constructs functionally active as promoters, and identified CCAAT-binding
factor (CBF) and hepatocyte nuclear factor 1 (HNF-1) as the principal nuclear factors controlling the promoters of types A
and B 5â²-UTR transcripts, respectively. The CCAAT-binding factor binding site and the entire downstream sequence driving the
expression of type A transcripts in humans are structurally and functionally conserved in mice, where they constitute a uniqueβ3Gal-T5
promoter that appears to be the ancestral promoter of the gene. The HNF-1 binding motif of the second human promoter is identical
to the HNF-1/Cdx binding motif of the LTR promoter but is in the antisense orientation, resulting in much lower binding affinity
and promoter strength. These data may explain the successful insertion of the transposon during evolution.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Beta1,3-galactosyltransferase beta3Gal-T5 is highly expressed in the colons of humans and certain primates due to a retroviral long terminal repeat (LTR) acting as a strong promoter. Because this ...promoter is inactive in other human tissues or mice, we attempted to understand how adoption of a retrotransposon allowed the gene to acquire tissue-specific expression. We identified three novel 5'-UTRs of beta3Gal-T5 mRNA, types A, B, and C, and found widespread expression of the type A transcript at much lower levels than the LTR transcript, the expression of which is restricted to organs of the gastrointestinal tract. Expression of the type C 5'-UTR transcript was mostly restricted to the ileum, where it was expressed at high levels. We cloned the 5'-flanking regions of both types A and B 5'-UTRs, found deletion constructs functionally active as promoters, and identified CCAAT-binding factor (CBF) and hepatocyte nuclear factor 1 (HNF-1) as the principal nuclear factors controlling the promoters of types A and B 5'-UTR transcripts, respectively. The CCAAT-binding factor binding site and the entire downstream sequence driving the expression of type A transcripts in humans are structurally and functionally conserved in mice, where they constitute a uniquebeta3Gal-T5 promoter that appears to be the ancestral promoter of the gene. The HNF-1 binding motif of the second human promoter is identical to the HNF-1/Cdx binding motif of the LTR promoter but is in the antisense orientation, resulting in much lower binding affinity and promoter strength. These data may explain the successful insertion of the transposon during evolution.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The native promoter of β1,3 galactosyltransferase β3Gal-T5 contributes to the expression of the enzyme and its oligosaccharide products, such as Lewis antigens, in many tissues. It is mainly ...sensitive to nuclear factor NF-Y and located nearby two CpG islands. To elucidate the regulation of the native promoter, we analyzed NF-Y protein and β3Gal-T5 mRNA, and found that NF-Y is scarcely modulated among various cell lines and biopsies from normal or cancerous colon. Conversely, β3Gal-T5 expression levels vary in the cell lines and are strongly down-regulated in colon cancer. We also performed quantitative methylation analysis of β3Gal-T5 CpG islands and found an inverse correlation between mRNA expression and DNA methylation. In particular, the methylation levels of both islands are always increased in cancer, with respect to the corresponding normal counterpart, in matched normal and tumor samples of colon and breast origin. Moreover, treatment with chromatin remodeling agents 5-aza-2′deoxycytidine and trichostatin A does not restore transcription in completely negative cells, but only increases expression in basally positive cells. However, methylation analysis after 5-aza-2′deoxycytidine treatment revealed partial demethylation of both islands in all treated cells. Finally, chromatin immunoprecipitation assays on β3Gal-T5 promoter showed that histone H3K4 trymethylation, H3K79 dimethylation, and H3K9-14 acetylation are high in cells expressing the transcript, and very low in those negative, while H4K20 trimethylation and H3K27 dimethylation are the opposite. We conclude that complex epigenetic modulation underlies the regulation of β3Gal-T5 native promoter.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The native promoter of beta 1,3 galactosyltransferase beta 3Gal-T5 contributes to the expression of the enzyme and its oligosaccharide products, such as Lewis antigens, in many tissues. It is mainly ...sensitive to nuclear factor NF-Y and located nearby two CpG islands. To elucidate the regulation of the native promoter, we analyzed NF-Y protein and beta 3Gal-T5 mRNA, and found that NF-Y is scarcely modulated among various cell lines and biopsies from normal or cancerous colon. Conversely, beta 3Gal-T5 expression levels vary in the cell lines and are strongly down-regulated in colon cancer. We also performed quantitative methylation analysis of beta 3Gal-T5 CpG islands and found an inverse correlation between mRNA expression and DNA methylation. In particular, the methylation levels of both islands are always increased in cancer, with respect to the corresponding normal counterpart, in matched normal and tumor samples of colon and breast origin. Moreover, treatment with chromatin remodeling agents 5-aza-2\'deoxycytidine and trichostatin A does not restore transcription in completely negative cells, but only increases expression in basally positive cells. However, methylation analysis after 5-aza-2\'deoxycytidine treatment revealed partial demethylation of both islands in all treated cells. Finally, chromatin immunoprecipitation assays on beta 3Gal-T5 promoter showed that histone H3K4 trymethylation, H3K79 dimethylation, and H3K9-14 acetylation are high in cells expressing the transcript, and very low in those negative, while H4K20 trimethylation and H3K27 dimethylation are the opposite. We conclude that complex epigenetic modulation underlies the regulation of beta 3Gal-T5 native promoter.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We investigated the role of β3Gal‐T5, a member of the β1,3galactosyltransferase (β1,3Gal‐T) family, in cancer‐associated glycosylation, focusing on the expression of sialyl‐Lewis a (sLea, the epitope ...of CA19.9 antigen), poly N‐acetyllactosamines, and sialyl‐Lewis x (sLex) antigen. A clone permanently expressing an antisense fragment of β3Gal‐T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both β1,3Gal‐T activity and sLea expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O‐glycans. Endo‐β‐galactosidase mostly releases NeuAcα2‐3Galβ1‐3Fucα1‐4GlcNAcβ1‐3Gal and NeuAcα2‐3Galβ1‐3GlcNAcβ1‐3Gal from such O‐glycans of BxPC3 membranes, but GlcNAcβ1‐3Gal and type 2 chain oligosaccharides, including NeuAcα2‐3Galβ1‐4Fucα1‐3GlcNAcβ1‐3Gal, from those of the antisense clone. Furthermore, BxPC3 cells secrete sLea in the culture media but not sLex, while antisense clone secretes mostly sLex, and accumulation of both antigens is prevented by benzyl‐α‐GalNAc. These data indicate that β3Gal‐T5 suppression turns synthesis of type 1 chain O‐glycans to poly N‐acetyllactosamine elongation and termination by sLex. In other cell lines and clones, β3Gal‐T5 transcript, β1,3Gal‐T activity, and sLea antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that β3Gal‐T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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