AFP gene expression after acute diethylnitrosamine intoxication is not Afr2 regulated.

The level of alpha-fetoprotein (AFP) gene expression during liver regeneration in mice is regulated by the Afr2 gene. C3H/HeJ mice express 10-fold higher levels of AFP than C57BL/6J mice. We show that AFP gene expression is not Afr2 regulated after intoxication with the carcinogen diethylnitrosamine (DEN). Peak levels of AFP gene expression of the 2 strains were identical, although reached at different times following treatment. Analysis of the expression of AFP derived transgenes not subject to Afr2 regulation and genetic analyses showed that the difference in timing of peak AFP gene expression after DEN intoxication was independent of Afr2 regulation.

Afr2 regulation occurs cell-autonomously in vitro but is not conferred on episomal DNA in transient assays.

Oncofetal antigens such as alpha-fetoprotein (AFP) are expressed in regenerating liver. The level of AFP gene expression during liver regeneration is regulated by the unlinked, autosomal gene, Alpha-fetoprotein regulator 2 (Afr2). C3H/HeJ (Afr2A/A) mice express 10-fold higher levels of AFP than C57BL/6J (Afr2B/B) mice. Here we show that primary hepatocytes isolated from C3H/HeJ and C57BL/6J mice exhibit differential expression of the endogenous AFP gene, which was attributed to the Afr2 gene locus and indicative of a cell-autonomous mechanism. We show that the Afr2-Response Element (ARE), between 1010 and 838 base pairs upstream of the AFP transcriptional start site, did not modulate reporter gene expression in transfection assays of Hep G2, Hep 3B, Hepa 1.6, and HeLa cell lines. Reporter gene expression in transiently transfected primary hepatocytes was also ARE-independent. Finally, gene expression from reporter constructs delivered by hydrodynamics-based transfection to the livers of C3H/HeJ and C57BL/6J mice after CCl4-induced liver regeneration was ARE-independent. In conclusion, ARE-dependent transcription was not found in transient assays performed in three different systems, two of which retained regulation of the endogenous AFP gene, suggesting that the ARE may not function as a simple transcription factor recognition site.

Inhibiting proteasomal degradation of microsomal triglyceride transfer protein prevents CCl4-induced steatosis.

Carbon tetrachloride (CCl(4)) interferes with triglyceride secretion and causes steatosis, fibrosis, and necrosis. In mice, CCl(4) decreased plasma triglyceride-rich lipoproteins, increased cellular lipids, and reduced microsomal triglyceride transfer protein (MTP) without diminishing mRNA levels. Similarly, CCl(4) decreased apoB-lipoprotein production and MTP activity but had no effect on mRNA levels in primary enterocytes and colon carcinoma and hepatoma cells. CCl(4) did not affect MTP synthesis but induced post-translational degradation involving ubiquitinylation and proteasomes in McA-RH7777 cells. By contrast, MTP inhibitor increased cellular lipids without affecting MTP protein. MTP was covalently modified when cells were incubated with (14)CCl(4). This modification was prevented by the inhibition of P450 oxygenases, indicating that CCl(3)(.) generated by these enzymes targets MTP for degradation. To determine whether inhibition of proteolysis could prevent CCl(4) toxicity, mice were fed with CCl(4) with or without lactacystin. Lactacystin increased ubiquitinylated MTP and prevented lipid accumulation in tissues. Thus, CCl(4) induces post-translational degradation without affecting lipid transfer activity, whereas MTP antagonist inhibits lipid transfer activity without causing its destruction. These studies identify MTP as a major target of CCl(4) and its degradation as a novel mechanism involved in the onset of steatosis, suggesting that inhibition of proteolysis may prevent some forms of steatosis.

Family members p53 and p73 act together in chromatin modification and direct repression of alpha-fetoprotein transcription.

Aberrant expression of the alpha-fetoprotein (AFP) gene is a diagnostic tumor marker of hepatocellular carcinoma. We find that AFP gene expression is repressed by the TP53 family member p73 during normal hepatic development and when p73alpha or p73beta is introduced into cultured hepatoma cells that express AFP. Transient co-transfection of p53 family members showed that p53 and transactivating (TA)-p73, but not TA-p63, repress endogenous AFP transcription additively or independently. p53-independent functions of p73 are further supported by delayed, p73-associated compensation of AFP repression during development of the p53-null mouse. Chromatin immunoprecipitation assays of normal and p53-null mouse liver tissue showed that TA-p73 binds at a previously identified p53 repressor site (-860/-830) within the distal promoter of AFP at a level equivalent to p53 in wild type liver, with increased binding of TA-p73 to chromatin in the absence of p53. Sequential chromatin immunoprecipitation analyses revealed that TA-p73 and p53 bind simultaneously to their shared regulatory site in wild type liver. Like the founding family member p53, TA-p73 represses AFP expression by chromatin structure alteration, targeting reduction of acetylated histone H3 lysine 9 and increased dimethylated histone H3 lysine 9 levels. However, chromatin-bound TA-p73 is associated with elevated di- and tri-methylated histone H3 lysine 4 levels in p53-null liver and hepatoma cells, concomitant with a reduced ability to repress transcription compared with p53.