Abstract
Cytokines have been implicated in the pathogenesis of inflammatory cholestasis. This is due to transcriptional down-regulation of hepatic transporters including the Na(+)/bile acid cotransporter, ntcp, and the multispecific organic anion exporter, mrp2. We have recently shown that ntcp suppression by lipopolysaccharide in vivo is caused by down-regulation of transactivators including the previously uncharacterized Footprint B-binding protein. Both the ntcp FpB element and the mrp2 promoter contain potential retinoid-response elements. We hypothesized that retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers would activate these two genes and that cytokines that reduce bile flow might do so by suppressing nuclear levels of these transactivators. Retinoid transactivation and interleukin-1beta down-regulation of the ntcp and mrp2 promoters were mapped to RXRalpha:RARalpha-response elements. Gel mobility shift assays demonstrated specific binding of RXRalpha:RARalpha heterodimers to the ntcp and mrp2 retinoid-response elements. The RXRalpha:RARalpha complex was down-regulated by IL-1beta in HepG2 cells. An unexpected finding was that an adjacent CAAT-enhancer-binding protein element was required for maximal transactivation of the ntcp promoter by RXRalpha:RARalpha. Taken together, these studies demonstrate regulation of two hepatobiliary transporter genes by RXRalpha:RARalpha and describe a mechanism which likely contributes to their down-regulation during inflammation.
Highlights
A broader appreciation of the role of lipopolysaccharide (LPS)1 and associated cytokines in the pathogenesis of a num
Reduced function of the ntcp transporter leads to decreased bile acid uptake by the hepatocyte, whereas reduced function of the mrp2 transporter leads to impaired excretion of bilirubin, glutathione, and numerous organic anions, as well as to reduction of the bile salt-independent component of bile flow [7, 9]
Administration of LPS to rats reproduces the cholestasis, which is a common clinical feature of sepsis, as well as many immunological, viral, and toxic liver diseases [7]. This is primarily due to transcriptional suppression of hepatocyte transporters, including the bile acid uptake protein, ntcp, and the multispecific organic anion pump, mrp2 [11, 12]
Summary
Sequence consensus; 5Ј-CGCTTGATGACTCAG3Ј consensus; 5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј ntcp ntϪ11/ϩ8; 5Ј-TGCTGGTTAATCTTTTATTT-3Ј ntcp nt Ϫ81/Ϫ62; 5Ј-CAGGAAACTTGAGCAAGGTA-3Ј consensus; 5Ј-TGCAGATTGCGCAATCTGCA-3Ј ntcp nt Ϫ56/Ϫ37; 5Ј-TCCGGGGCATAAGGTTATGG-3Ј consensus; 5Ј-GCTTCAAGGTCACCAGGTCAGAGAG-3Ј ntcp nt Ϫ56/Ϫ37; 5Ј-TCCGGttaATAAGtggATGG-3Ј mrp nt Ϫ422/ Ϫ398; 5Ј-GGGTATTTAACATCTCTGTGAACTC-3Ј consensus; 5Ј-GCTTCAGGTCACCAGGAGGTCAGAGAG-3Ј including RXR, and their respective target genes are suppressed by endotoxin and associated cytokines at the transcriptional level [26, 27]. We identify FpB BP as a RXR␣:RAR␣ heterodimer and characterize RXR␣:RAR␣-response elements within the rat ntcp and mrp gene promoters. These response elements mediate induction by retinoids and suppression by IL-1 of gene expression. A proposed molecular mechanism of ntcp and mrp acute phase transcriptional suppression via IL-1-induced reduction in the concentration of nuclear RXR␣:RAR␣ heterodimers is described. These mechanisms likely contribute to the reduction in transcription of these two genes during the acute phase response
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