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Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells.

Xu Y, Tertilt C, Krause A, Quadri LE, Crystal RG, Worgall S - Respir. Res. (2009)

Bottom Line: Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC.Following exposure to P. aeruginosa, expression of 3beta-hydroxysterol-Delta7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Weill Cornell Medical College, New York, USA. yax2002@med.cornell.edu

ABSTRACT

Background: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC) are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown.

Methods: This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT) mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection.

Results: CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP), a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3beta-hydroxysterol-Delta7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.

Conclusion: These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.

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Cav1 and SREBP expression in DC from WT and CF mice. A. RNA was extracted from DC from WT and CF mice and Cav1 gene expression was measured by Real-time RT-PCR. Relative expression levels in the samples were calculated using the ΔΔCt method, with GAPDH as internal normalization control. The y-axis represents Cav1 cDNA transcription level in terms of relative quantity value (RQ). B. Western analysis of Cav1 and SREBP in DC from WT and CF mice and corresponding GAPDH expression. C. Quantification of Cav1 and SREBP expression by image intensity analysis normalized to GAPDH. D. Luciferase assay of SRE transcriptional activity in CF and WT DC. DC were infected with AdZ-SRE-luc for 48 h and harvested for luci-ferase assay and β-galactosidase assay. Data is shown luciferase activity (RLU) normalized to β-galactosidase. Shown is the mean ± SEM of three of independent samples. *denotes p < 0.05, ** denotes p < 0.01.
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Figure 4: Cav1 and SREBP expression in DC from WT and CF mice. A. RNA was extracted from DC from WT and CF mice and Cav1 gene expression was measured by Real-time RT-PCR. Relative expression levels in the samples were calculated using the ΔΔCt method, with GAPDH as internal normalization control. The y-axis represents Cav1 cDNA transcription level in terms of relative quantity value (RQ). B. Western analysis of Cav1 and SREBP in DC from WT and CF mice and corresponding GAPDH expression. C. Quantification of Cav1 and SREBP expression by image intensity analysis normalized to GAPDH. D. Luciferase assay of SRE transcriptional activity in CF and WT DC. DC were infected with AdZ-SRE-luc for 48 h and harvested for luci-ferase assay and β-galactosidase assay. Data is shown luciferase activity (RLU) normalized to β-galactosidase. Shown is the mean ± SEM of three of independent samples. *denotes p < 0.05, ** denotes p < 0.01.

Mentions: Seven genes were down-regulated in DC from CF mice with more than 1.5-fold change [see Additional file 2]. The expression level of the membrane lipid raft protein Cav1 in DC from the CF mice was 4.1-fold decreased compared to the WT mice. This finding was confirmed with real-time RT-PCR which showed a 50-fold reduction of the Cav1 mRNA level in the CF DC compared to WT DC (p < 0.01, Figure 4A). Cav1 protein was almost undetectable in CF DC (Figure 4B) and quantification of Cav1 protein expression level indicated a 6.2-fold lower expression in CF DC compared to WT DC (p < 0.01, Figure 4C). Cav1 is known to be negatively regulated by sterol regulatory element binding protein (SREBP) [30-32], therefore we further compared the expression and activity levels of SREBP in DC from CF and WT mice. SREBP functions as a transcription factor that binds and regulates the sterol regulatory element (SRE) containing promoter. The activation of SREBP requires the proteolytic cleavage to release the active form into nucleus and regulate the target genes [42]. The cleavage of SREBP protein was increased in the CF DC (Figure 4B) and quantification of the active form of SREBP demonstrated a 4.3-fold higher expression in DC of CF mice compared to WT mice (p < 0.05, Figure 4C). The transcriptional activity of SRE was increased in CF DC infected with AdZ-SRE-luc, an Ad vector expressing an SRE-promoter linked to a luciferase reporter, compared to WT controls infected with AdZ-SRE-luc (p < 0.01, Figure 4D), suggesting that SREBP activity was increased in the CF DC.


Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells.

Xu Y, Tertilt C, Krause A, Quadri LE, Crystal RG, Worgall S - Respir. Res. (2009)

Cav1 and SREBP expression in DC from WT and CF mice. A. RNA was extracted from DC from WT and CF mice and Cav1 gene expression was measured by Real-time RT-PCR. Relative expression levels in the samples were calculated using the ΔΔCt method, with GAPDH as internal normalization control. The y-axis represents Cav1 cDNA transcription level in terms of relative quantity value (RQ). B. Western analysis of Cav1 and SREBP in DC from WT and CF mice and corresponding GAPDH expression. C. Quantification of Cav1 and SREBP expression by image intensity analysis normalized to GAPDH. D. Luciferase assay of SRE transcriptional activity in CF and WT DC. DC were infected with AdZ-SRE-luc for 48 h and harvested for luci-ferase assay and β-galactosidase assay. Data is shown luciferase activity (RLU) normalized to β-galactosidase. Shown is the mean ± SEM of three of independent samples. *denotes p < 0.05, ** denotes p < 0.01.
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Figure 4: Cav1 and SREBP expression in DC from WT and CF mice. A. RNA was extracted from DC from WT and CF mice and Cav1 gene expression was measured by Real-time RT-PCR. Relative expression levels in the samples were calculated using the ΔΔCt method, with GAPDH as internal normalization control. The y-axis represents Cav1 cDNA transcription level in terms of relative quantity value (RQ). B. Western analysis of Cav1 and SREBP in DC from WT and CF mice and corresponding GAPDH expression. C. Quantification of Cav1 and SREBP expression by image intensity analysis normalized to GAPDH. D. Luciferase assay of SRE transcriptional activity in CF and WT DC. DC were infected with AdZ-SRE-luc for 48 h and harvested for luci-ferase assay and β-galactosidase assay. Data is shown luciferase activity (RLU) normalized to β-galactosidase. Shown is the mean ± SEM of three of independent samples. *denotes p < 0.05, ** denotes p < 0.01.
Mentions: Seven genes were down-regulated in DC from CF mice with more than 1.5-fold change [see Additional file 2]. The expression level of the membrane lipid raft protein Cav1 in DC from the CF mice was 4.1-fold decreased compared to the WT mice. This finding was confirmed with real-time RT-PCR which showed a 50-fold reduction of the Cav1 mRNA level in the CF DC compared to WT DC (p < 0.01, Figure 4A). Cav1 protein was almost undetectable in CF DC (Figure 4B) and quantification of Cav1 protein expression level indicated a 6.2-fold lower expression in CF DC compared to WT DC (p < 0.01, Figure 4C). Cav1 is known to be negatively regulated by sterol regulatory element binding protein (SREBP) [30-32], therefore we further compared the expression and activity levels of SREBP in DC from CF and WT mice. SREBP functions as a transcription factor that binds and regulates the sterol regulatory element (SRE) containing promoter. The activation of SREBP requires the proteolytic cleavage to release the active form into nucleus and regulate the target genes [42]. The cleavage of SREBP protein was increased in the CF DC (Figure 4B) and quantification of the active form of SREBP demonstrated a 4.3-fold higher expression in DC of CF mice compared to WT mice (p < 0.05, Figure 4C). The transcriptional activity of SRE was increased in CF DC infected with AdZ-SRE-luc, an Ad vector expressing an SRE-promoter linked to a luciferase reporter, compared to WT controls infected with AdZ-SRE-luc (p < 0.01, Figure 4D), suggesting that SREBP activity was increased in the CF DC.

Bottom Line: Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC.Following exposure to P. aeruginosa, expression of 3beta-hydroxysterol-Delta7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Weill Cornell Medical College, New York, USA. yax2002@med.cornell.edu

ABSTRACT

Background: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC) are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown.

Methods: This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT) mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection.

Results: CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP), a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3beta-hydroxysterol-Delta7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.

Conclusion: These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.

Show MeSH
Related in: MedlinePlus