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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 PMA-differentiated THP-1 cells.With samples obtained from PMA-differentiated 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 1,25(OH)2D3 (* p < 0.05; ** p < 0.01; *** p < 0.001).
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pone-0078170-g004: Primary 1,25(OH)2D3 target genes of the CXCL gene cluster in PMA-differentiated THP-1 cells.With samples obtained from PMA-differentiated 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 1,25(OH)2D3 (* p < 0.05; ** p < 0.01; *** p < 0.001).

Mentions: The phorbol ester PMA is known to differentiate in suspension growing THP-1 cells into adherent M2-type macrophage-like cells [52]. In such PMA-differentiated THP-1 cells we used qPCR to compare the basal expression of the genes of the CXCL cluster and their flanking genes (Figure 4A). In addition to the genes CXCL8, CXCL6, CXCL1, ALB, MTHFD2L and AREG, which are already expressed in undifferentiated THP-1 cells (Figure 2), we found the expression of CXCL7 and CXCL3. Also in differentiated THP-1 cells CXCL8 displays the highest expression within the investigated genomic region and showed 443-fold higher mRNA levels than CXCL6, 114-fold higher than CXCL1, 48-fold more than CXCL7 and a 67-fold excess compared to CXCL3. Moreover, compared to undifferentiated cells, in PMA-differentiated THP-1 cells CXCL8 is 33-times higher expressed. Detailed 8 h time course experiments in PMA-differentiated THP-1 cells showed that CXCL8 (Figure 4B), CXCL6 (Figure 4C) and CXCL1 (Figure 4D) are primary 1,25(OH)2D3 target genes also in this cellular model. However, in these macrophage-like cells all three CXCL genes are less inducible than in undifferentiated THP-1 (monocyte-like) cells: even after 8 h stimulation with 1,25(OH)2D3 the induction of CXCL8 is only 1.9-fold, that of CXCL6 is 3.3-fold and and that of CXCL1 is 3.0-fold. Furthermore, for all three genes a significant induction by 1,25(OH)2D3 was detected only after 2.5 to 3.5 h stimulation, i.e. clearly delayed compared to undifferentiated THP-1 cells. For comparison, in the same time course experiments the genes CXCL7 and CXCL3 showed no significant response to 1,25(OH)2D3 (Figure S2). Moreover, also in PMA-differentiated THP-1 cells the flanking genes ALB, MTHFD2L and AREG do not shown any early response to treatment with 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 PMA-differentiated THP-1 cells.With samples obtained from PMA-differentiated 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 1,25(OH)2D3 (* 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-g004: Primary 1,25(OH)2D3 target genes of the CXCL gene cluster in PMA-differentiated THP-1 cells.With samples obtained from PMA-differentiated 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 1,25(OH)2D3 (* p < 0.05; ** p < 0.01; *** p < 0.001).
Mentions: The phorbol ester PMA is known to differentiate in suspension growing THP-1 cells into adherent M2-type macrophage-like cells [52]. In such PMA-differentiated THP-1 cells we used qPCR to compare the basal expression of the genes of the CXCL cluster and their flanking genes (Figure 4A). In addition to the genes CXCL8, CXCL6, CXCL1, ALB, MTHFD2L and AREG, which are already expressed in undifferentiated THP-1 cells (Figure 2), we found the expression of CXCL7 and CXCL3. Also in differentiated THP-1 cells CXCL8 displays the highest expression within the investigated genomic region and showed 443-fold higher mRNA levels than CXCL6, 114-fold higher than CXCL1, 48-fold more than CXCL7 and a 67-fold excess compared to CXCL3. Moreover, compared to undifferentiated cells, in PMA-differentiated THP-1 cells CXCL8 is 33-times higher expressed. Detailed 8 h time course experiments in PMA-differentiated THP-1 cells showed that CXCL8 (Figure 4B), CXCL6 (Figure 4C) and CXCL1 (Figure 4D) are primary 1,25(OH)2D3 target genes also in this cellular model. However, in these macrophage-like cells all three CXCL genes are less inducible than in undifferentiated THP-1 (monocyte-like) cells: even after 8 h stimulation with 1,25(OH)2D3 the induction of CXCL8 is only 1.9-fold, that of CXCL6 is 3.3-fold and and that of CXCL1 is 3.0-fold. Furthermore, for all three genes a significant induction by 1,25(OH)2D3 was detected only after 2.5 to 3.5 h stimulation, i.e. clearly delayed compared to undifferentiated THP-1 cells. For comparison, in the same time course experiments the genes CXCL7 and CXCL3 showed no significant response to 1,25(OH)2D3 (Figure S2). Moreover, also in PMA-differentiated THP-1 cells the flanking genes ALB, MTHFD2L and AREG do not shown any early response to treatment with 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