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Transcriptomic analysis unveils correlations between regulative apoptotic caspases and genes of cholesterol homeostasis in human brain.

Picco R, Tomasella A, Fogolari F, Brancolini C - PLoS ONE (2014)

Bottom Line: In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis.We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver.For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Biological Sciences, Università degli Studi di Udine, Udine, Italy.

ABSTRACT
Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Silencing of caspase-2 expression in cultured glioblastoma cells allows the up-regulation of a limited number of genes, among which some are related to cholesterol homeostasis. Lysosomal Acid Lipase A (LIPA) was up-regulated in two different cell lines in response to caspase-2 down-regulation and cells silenced for caspase-2 exhibit reduced cholesterol staining in the lipid droplets. We expanded this observation by large-scale analysis of mRNA expression. All caspases were analyzed in terms of co-expression in comparison with 166 genes involved in cholesterol homeostasis. In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis. These correlations resulted in altered GBM (Glioblastoma Multiforme), in particular for CASP1. We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver. For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging.

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Cholesterol distribution in cells silenced for caspase-2 expression.A. U87MG cells were fixed and processed for immunofluorescence. Epifluorescence microscopy followed by deconvolution analysis was used to visualize different subcellular compartments and cholesterol distribution. With arrows indicate LDs and green arrows LDs encircled by lysosomes. Scale bar 40 µM. B. U87MG cells were transfected with siRNA against caspase-2 or control siRNA as indicated. 48 h later cells fixed and stained with filipin to visualize the intracellular distribution of cholesterol. Arrows point to LDs. Scale bar 30 µM. C. Quantitative analysis of LDs presence in U87MG and IMR90-E1A cells transfected with siRNA against caspase-2 or control siRNA. Data are presented as mean ± SD; n = 3.
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pone-0110610-g002: Cholesterol distribution in cells silenced for caspase-2 expression.A. U87MG cells were fixed and processed for immunofluorescence. Epifluorescence microscopy followed by deconvolution analysis was used to visualize different subcellular compartments and cholesterol distribution. With arrows indicate LDs and green arrows LDs encircled by lysosomes. Scale bar 40 µM. B. U87MG cells were transfected with siRNA against caspase-2 or control siRNA as indicated. 48 h later cells fixed and stained with filipin to visualize the intracellular distribution of cholesterol. Arrows point to LDs. Scale bar 30 µM. C. Quantitative analysis of LDs presence in U87MG and IMR90-E1A cells transfected with siRNA against caspase-2 or control siRNA. Data are presented as mean ± SD; n = 3.

Mentions: Having evidences of the existence of a connection linking caspase-2 to some genes of the cholesterol pathway, we compared the subcellular distribution of cholesterol after labeling of IMR90-E1A and U87MG cells with filipin. Fluorescence staining was detectable in the plasma membrane (PM) and in intracellular membrane structures (Fig 2A). As expected co-localization studies using GM130, transferrin receptor and LBPA, as markers respectively of, Golgi apparatus, late endosomal compartment and endosomal/recycling compartment evidenced that cholesterol can be detected in all these organelles. In addition, intense filipin fluorescence staining was present in regular spherical structures that do not co-localize with the used markers and that can be identified as LDs (lipid droplets).


Transcriptomic analysis unveils correlations between regulative apoptotic caspases and genes of cholesterol homeostasis in human brain.

Picco R, Tomasella A, Fogolari F, Brancolini C - PLoS ONE (2014)

Cholesterol distribution in cells silenced for caspase-2 expression.A. U87MG cells were fixed and processed for immunofluorescence. Epifluorescence microscopy followed by deconvolution analysis was used to visualize different subcellular compartments and cholesterol distribution. With arrows indicate LDs and green arrows LDs encircled by lysosomes. Scale bar 40 µM. B. U87MG cells were transfected with siRNA against caspase-2 or control siRNA as indicated. 48 h later cells fixed and stained with filipin to visualize the intracellular distribution of cholesterol. Arrows point to LDs. Scale bar 30 µM. C. Quantitative analysis of LDs presence in U87MG and IMR90-E1A cells transfected with siRNA against caspase-2 or control siRNA. Data are presented as mean ± SD; n = 3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199739&req=5

pone-0110610-g002: Cholesterol distribution in cells silenced for caspase-2 expression.A. U87MG cells were fixed and processed for immunofluorescence. Epifluorescence microscopy followed by deconvolution analysis was used to visualize different subcellular compartments and cholesterol distribution. With arrows indicate LDs and green arrows LDs encircled by lysosomes. Scale bar 40 µM. B. U87MG cells were transfected with siRNA against caspase-2 or control siRNA as indicated. 48 h later cells fixed and stained with filipin to visualize the intracellular distribution of cholesterol. Arrows point to LDs. Scale bar 30 µM. C. Quantitative analysis of LDs presence in U87MG and IMR90-E1A cells transfected with siRNA against caspase-2 or control siRNA. Data are presented as mean ± SD; n = 3.
Mentions: Having evidences of the existence of a connection linking caspase-2 to some genes of the cholesterol pathway, we compared the subcellular distribution of cholesterol after labeling of IMR90-E1A and U87MG cells with filipin. Fluorescence staining was detectable in the plasma membrane (PM) and in intracellular membrane structures (Fig 2A). As expected co-localization studies using GM130, transferrin receptor and LBPA, as markers respectively of, Golgi apparatus, late endosomal compartment and endosomal/recycling compartment evidenced that cholesterol can be detected in all these organelles. In addition, intense filipin fluorescence staining was present in regular spherical structures that do not co-localize with the used markers and that can be identified as LDs (lipid droplets).

Bottom Line: In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis.We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver.For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Biological Sciences, Università degli Studi di Udine, Udine, Italy.

ABSTRACT
Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Silencing of caspase-2 expression in cultured glioblastoma cells allows the up-regulation of a limited number of genes, among which some are related to cholesterol homeostasis. Lysosomal Acid Lipase A (LIPA) was up-regulated in two different cell lines in response to caspase-2 down-regulation and cells silenced for caspase-2 exhibit reduced cholesterol staining in the lipid droplets. We expanded this observation by large-scale analysis of mRNA expression. All caspases were analyzed in terms of co-expression in comparison with 166 genes involved in cholesterol homeostasis. In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis. These correlations resulted in altered GBM (Glioblastoma Multiforme), in particular for CASP1. We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver. For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging.

Show MeSH
Related in: MedlinePlus