Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

Abstract

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNFbo, C/EBPcx, HNF3ct, HNF3P, or HNF3y but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-junlc-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.Serum albumin is synthesized exclusively by hepatocytes in all mammals, and its expression is regulated mainly at the transcriptional level. Albumin expression begins with initiation of liver organogenesis and increases gradually with development (53,57,59). Whereas the level of albumin transcription is very high in normal adult liver, down regulation of albumin transcription has been observed in a variety of physiological and pathophysiological conditions, e.g., during the acute-phase reaction and liver regeneration (45,53,55,57). The rate of albumin transcription decreases markedly after hepatocytes are isolated from liver tissue and is low even in well-differentiated hepatoma cell lines compared with that in liver (10, 11). It has also been shown that transcription of albumin is subject to regulation by a number of hormones such as glucocorticoids (47,53,62) and insulin (42, 53). The molecular mechanisms by which these different signals may regulate albumin transcription are still largely unknown.Previous work has shown that the albumin promoter is able to direct high-level cell-type-specific transcription of albumin in some hepatoma cell lines (32, 48). However, the activity of the promoter is undetectable when it is introduced into transgenic mice, where a liver-specific transcriptional enhancer located at -10 kb is required for albumin transcription (6, 50). In contrast, the albumin enhancer is not functional in fetal liver, in regenerating liver,...

Hepatic specification of the gut endoderm in vitro: cell signaling and transcriptional control.

“…7B, lane 4), and the binding activity was confined to the embryo liver and not the head (data not shown). Hu and Isom (1994) reported that AP-1 could bind the albumin enhancer between the eG and eH sites, and we found that an AP-1 site could compete for eYbinding activity (Fig. 7B, lane 2).…”

“…6C). Muta tion of any of these sites inactivates the enhancer (Zaret et al 1990;Liu et al 1991;Hu and Isom 1994), and thus they define the enhancer's core. In vivo footprinting of adult liver chromatin showed that eG and eFi, as well as eF and eX, sites for two less char acterized binding proteins, are occupied within the con text of one of the aforementioned nucleoprotein particles (McPherson et al 1993).…”

Identification of novel molecular candidates for fatty liver in the hyperlipidemic mouse model, HcB19

“…Abdullah et al (28) showed binding of fatty acids to AP-1 (c-jun), which resulted in suppression of the expression of CPS1. Altered transcription can also occur in other genes that have a similar (putative) AP-1 enhancer site, such as ALB (29) and PCCA (30), and may be a generalized mechanism relevant to the development of fatty liver. Of note, CPS-1, ALB, and PCCA were all lower in HcB19 mice ( Table 2).…”

Role for growth factors and extracellular matrix in controlling differentiation of prospectively isolated hepatic stem cells

“…Because such growth inhibition is irrelevant to the transcriptional activity of C/EBPα (Harris et al, 2001), another mechanism should exist to control transcription of genes important for liver development. Actually, in the transcriptional control regions of ALB and αAT, liver-enriched transcription factors such as HNF1 (Baumhueter et al, 1990), HNF3 and HNF4 (Costa et al, 1989), and widely distributed proteins such as activator protein 1 (AP1) (Hu et al, 1994) bind to regulate these genes along with C/EBP proteins. The currently proposed cascade of sequential transcriptional control of hepatic stem cell differentiation, however, is still unreliable.…”