Conditional hepatocarcinogenesis in mice expressing SV 40 early sequences.

We closely mimicked the in vivo setting in which sporadic hepatocarcinoma occurs by establishing a transgenic mouse model carrying regulatable SV40 early sequences under the control of the regulatory sequences of the human antithrombin III gene that confer hepatic expression. In this system, floxed dormant oncogenic sequences became functional after excision due to adenoviral expression of Cre recombinase or the stable transgenic expression in liver of a tamoxifen-inducible Cre. Hepatic oncogene expression was switched on by both methods, leading to the development of hepatocellular carcinoma. This model could be useful for investigating the key steps of the preneoplastic process, to identify suitable targets for the testing of new therapies.

A tamoxifen-inducible chimeric Cre recombinase specifically effective in the fetal and adult mouse liver.

The spatiotemporal control of somatic mutagenesis in mice is considered a promising step to determine the function of a given gene product in a defined population of cells at any given time during animal life and also to generate better mouse models of human diseases. To introduce defined mutations in a temporally controlled manner in the liver, we established transgenic mice expressing a tamoxifen-inducible Cre recombinase under the control of the transthyretin promoter (TTR-Cre ind). The recombinase activity was examined on 2 different floxed alleles by crossing TTR-Cre ind mice with either the reporter strain ROSA 26 or with homozygous mice carrying floxed catalytic alpha2 subunit of the adenosine monophosphate (AMP)-activated protein kinase gene. By placing 2 mutated hormone-binding domains of murine estrogen receptor (Mer) at both termini of the Cre, we show that the fusion protein is active only on administration of the synthetic estrogen antagonist 4-hydroxytamoxifen (4-OHT) without any background in the absence of the inducing agent. The recombination is specific of the fetal and adult liver, and we show that the efficiency of recombination reached 80% to 100% after treatment with 4-OHT. In conclusion, TTR-Cre ind transgenic mice represent a valuable tool for temporally controlling the desired gene modifications in vivo in the fetal and adult liver. This would certainly help to understand the physiologic functions of genes in the liver, to create various mouse models mimicking human diseases, and to contribute to liver cancer-specific suicide gene therapy studies.

In vivo functional characterization of the aldolase B gene enhancer.

A 400-bp intronic enhancer fragment in conjunction with the proximal promoter of the aldolase B gene provided correct tissue-specific expression in transgenic mice together with hormonal regulation in the liver. We investigated in vivo and in cultured cells the contribution of the intronic regulatory sequences and their interaction with the promoter elements in controlling aldolase B gene expression. Transgene activity was completely abolished by disruption of the two hepatocyte nuclear factor 1 (HNF1) binding sites in the enhancer, whereas mutation of one HNF1 site had no effect in the liver but strongly decreased activity in the kidney. Our data show that the HNF1 binding site(s) in the enhancer were key regulators of aldolase B transgene expression both in the liver and kidney. Deletion of the CCAAT/enhancer-binding protein site in the promoter completely abolished the enhancer function in HepG2 cells. These results suggest that expression of the aldolase B gene in the liver requires cooperative interactions between CCAAT/enhancer-binding protein and HNF1. Deletion of the HNF4 binding site in the enhancer suppressed expression in both liver and kidney in half of the transgenic lines, suggesting that this element might play a role in chromatin opening at the insertion site. We firmly establish that the endogenous aldolase B gene's first response to glucagon or cyclic AMP exposure was a transient increase in the expression in the liver, followed by a secondary decline in the transcription, as previously reported. This response was reproduced by all transgenes studied, indicating that neither HNF1 nor HNF4 binding sites in the enhancer were involved in this biphasic cyclic AMP response.

Severe iron deficiency anemia in transgenic mice expressing liver hepcidin.

We recently reported the hemochromatosis-like phenotype observed in our Usf2 knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the Usf2 knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the Usf2 knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hepcidin action.