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Primary 1,25-dihydroxyvitamin D3 response of the interleukin 8 gene cluster in human monocyte- and macrophage-like cells.

Ryynänen J, Carlberg C - PLoS ONE (2013)

Bottom Line: Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site.In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR.Our observation provides further evidence for the immune-related functions of vitamin D.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

ABSTRACT
Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)2D3 target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.

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Primary 1,25(OH)2D3 target genes of the CXCL gene cluster in undifferentiated THP-1 cells.With samples obtained from THP-1 cells qPCR was performed to determine the basal expression, relative to the housekeeping gene RPLP0, of the nine genes of the CXCL gene cluster and each four flanking genes (A) and the change of expression of CXCL8 (B), CXCL6 (C) and CXCL1 (D) in response to incubation with 10 nM 1,25(OH)2D3 over a time period of 8 h. Columns (A) and data points (B-D) represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of the mRNA induction by the stimuli (* p < 0.05; ** p < 0.01; *** p < 0.001).
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pone-0078170-g002: Primary 1,25(OH)2D3 target genes of the CXCL gene cluster in undifferentiated THP-1 cells.With samples obtained from THP-1 cells qPCR was performed to determine the basal expression, relative to the housekeeping gene RPLP0, of the nine genes of the CXCL gene cluster and each four flanking genes (A) and the change of expression of CXCL8 (B), CXCL6 (C) and CXCL1 (D) in response to incubation with 10 nM 1,25(OH)2D3 over a time period of 8 h. Columns (A) and data points (B-D) represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of the mRNA induction by the stimuli (* p < 0.05; ** p < 0.01; *** p < 0.001).

Mentions: In order to get an overview on the relative basal expression of the members of the CXCL cluster and their upstream and downstream flanking genes, we performed qPCR in non-stimulated, undifferentiated THP-1 cells (Figure 2A). Within the CXCL cluster we could detect the expression of only CXCL8, CXCL6 and CXCL1: CXCL8 is 28- and 18-times higher expressed than CXCL6 and CXCL1, respectively. In addition, from the upstream flanking genes of the CXCL cluster the albumin (ALB) gene and from the downstream flanking the genes methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2-like (MTHFD2L) and amphiregulin (AREG) are expressed in undifferentiated THP-1 cells. Next we stimulated the cells with 1,25(OH)2D3 and performed qPCR for the six expressed genes, in order to evaluate their possible primary response to the VDR ligand. Interestingly, the detailed time courses indicated that CXCL8 (Figure 2B), CXCL6 (Figure 2C) and CXCL1 (Figure 2D) are already significantly up-regulated 1 h after onset of stimulation with 1,25(OH)2D3 and reach after 8 h an induction of 9.1-fold for CXCL8, 3.7-fold for both CXCL6 and CXCL1, respectively. In contrast, the flanking genes ALB, MTHFD2L and AREG display no significant response to 1,25(OH)2D3 (data not shown).


Primary 1,25-dihydroxyvitamin D3 response of the interleukin 8 gene cluster in human monocyte- and macrophage-like cells.

Ryynänen J, Carlberg C - PLoS ONE (2013)

Primary 1,25(OH)2D3 target genes of the CXCL gene cluster in undifferentiated THP-1 cells.With samples obtained from THP-1 cells qPCR was performed to determine the basal expression, relative to the housekeeping gene RPLP0, of the nine genes of the CXCL gene cluster and each four flanking genes (A) and the change of expression of CXCL8 (B), CXCL6 (C) and CXCL1 (D) in response to incubation with 10 nM 1,25(OH)2D3 over a time period of 8 h. Columns (A) and data points (B-D) represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of the mRNA induction by the stimuli (* p < 0.05; ** p < 0.01; *** p < 0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824026&req=5

pone-0078170-g002: Primary 1,25(OH)2D3 target genes of the CXCL gene cluster in undifferentiated THP-1 cells.With samples obtained from THP-1 cells qPCR was performed to determine the basal expression, relative to the housekeeping gene RPLP0, of the nine genes of the CXCL gene cluster and each four flanking genes (A) and the change of expression of CXCL8 (B), CXCL6 (C) and CXCL1 (D) in response to incubation with 10 nM 1,25(OH)2D3 over a time period of 8 h. Columns (A) and data points (B-D) represent the means of at least three independent experiments and the bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of the mRNA induction by the stimuli (* p < 0.05; ** p < 0.01; *** p < 0.001).
Mentions: In order to get an overview on the relative basal expression of the members of the CXCL cluster and their upstream and downstream flanking genes, we performed qPCR in non-stimulated, undifferentiated THP-1 cells (Figure 2A). Within the CXCL cluster we could detect the expression of only CXCL8, CXCL6 and CXCL1: CXCL8 is 28- and 18-times higher expressed than CXCL6 and CXCL1, respectively. In addition, from the upstream flanking genes of the CXCL cluster the albumin (ALB) gene and from the downstream flanking the genes methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2-like (MTHFD2L) and amphiregulin (AREG) are expressed in undifferentiated THP-1 cells. Next we stimulated the cells with 1,25(OH)2D3 and performed qPCR for the six expressed genes, in order to evaluate their possible primary response to the VDR ligand. Interestingly, the detailed time courses indicated that CXCL8 (Figure 2B), CXCL6 (Figure 2C) and CXCL1 (Figure 2D) are already significantly up-regulated 1 h after onset of stimulation with 1,25(OH)2D3 and reach after 8 h an induction of 9.1-fold for CXCL8, 3.7-fold for both CXCL6 and CXCL1, respectively. In contrast, the flanking genes ALB, MTHFD2L and AREG display no significant response to 1,25(OH)2D3 (data not shown).

Bottom Line: Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site.In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR.Our observation provides further evidence for the immune-related functions of vitamin D.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

ABSTRACT
Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)2D3 target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.

Show MeSH
Related in: MedlinePlus