Because RA and RARs are implicated in regulating intestinal expression of BCMO1, it was suggested that RARs and ISX independently control BCMO1 gene activity (18). However, BCMO1 expression is not altered in selleck catalog ISX-deficient mice subjected to vitamin A restriction (18), suggesting instead that the effect of RA on BCMO1 expression is dependent on ISX. Therefore, we reasoned that ISX rather than BCMO1 expression is directly controlled by RA and RARs. To test this hypothesis, we treated human colonic CaCo-2 cells with RA or the synthetic RAR agonist, TTNPB. After 4 h, we isolated total RNA and determined ISX mRNA expression by qRT-PCR analysis. In both RA- and RAR agonist-treated CaCo-2 cells, ISX mRNA increased 4�C6 fold over levels to those in untreated or vehicle-treated control cells (Fig.

1A). To provide further evidence for RAR regulation of ISX, we treated CaCo-2 cells with LE540, a well-known RAR pan-antagonist. This treatment prevented RA-dependent induction of ISX mRNA expression (Supplemental Fig. S1). To examine whether induction of ISX was a direct effect or whether it involved the synthesis of other protein factors, we treated CaCo-2 cells with RA in the presence of the protein synthesis inhibitor cycloheximide (20 ��g/ml). RA still induced ISX expression ~4-fold in the presence of cycloheximide, indicating direct transcriptional regulation (Fig. 1B). We also performed immunohistochemical staining to determine whether RA also increased ISX expression at the protein level in CaCo-2 cells. In this case, CaCo-2 cells were treated with RA for 12 h and then cells were fixed and stained with an ISX-specific antiserum.

By confocal imaging, we detected strong staining for ISX in the nucleus of RA-treated CaCo-2 cells. In contrast, vehicle-treated control CaCo-2 cells showed only weak ISX staining (Fig. 1C). This analysis provides evidence that ISX is a RA-inducible target gene, an activity that likely reflects direct positive control of RARs. Figure 1. RA induces ISX expression through RARs in CaCo-2 cells. CaCo-2 cells were seeded in DMEM and 10% FBS. After allowing the cells to adhere for 24 h, cells were treated with 1 ��M RA or 1 ��M 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic … RARs directly bind to the ISX promoter RARs function by binding to specific retinoic acid response elements (RAREs) within regulatory regions of target genes.

We used NubiScanV 2.0 Carfilzomib and AliBaba 2.1 software to identify putative RAREs in the human ISX gene. This program predicted a DR-5 RARE located 180 bp upstream of the ATG start codon (Fig. 2A). To investigate whether RARs associate with this element in CaCo-2 cells, we performed ChIP assays using antiserum specific for human RARs and two primer pairs designed for detection of the putative RARE in precipitated DNA fractions (Fig. 2A).