Iron overload promotes Cyclin D1 expression and alters cell cycle in mouse hepatocytes
Introduction
Liver iron overload, such as observed in genetic hemochromatosis, but also during some chronic liver diseases not primarily related to iron overload diseases, including those of alcoholic or viral origin, may favour the development of hepatic lesions, including fibrosis, cirrhosis and hepatocellular carcinoma [1], [2], [3]. These data suggests that excessive iron content is, in humans, a potential risk factor contributing to the development of hepatocellular carcinoma.
The mechanisms involved in the occurrence of such lesions during iron overload are not completely understood. On one hand, cellular oxidative stress induced by iron excess could cause alterations on membrane lipids, proteins and DNA [4] favouring functional organelle impairment and genetic mutations, such as the mutation in p53 gene [5], a key gene of cell cycle control.
On the other hand, during iron overload, a misregulation of hepatic genes could participate to the development of lesions. Some of them, including hepcidin[6], [7], take place in the iron metabolism control. Some others, such as TGF-beta and alpha-1-collagen-1[8] participate to the extracellular matrix formation or to the control of oxidative stress, like GSTA4[9], [10].
A number of arguments suggest that cell cycle control is disturbed during iron overload. Modification of hepatocyte ploidy as well as an increase in liver volume have been frequently reported in vivo during iron overload [11], [12], [13]. However, during iron overload, deregulation of genes involved in cell cycle progression is not well characterized.
Therefore, the aim of our study was to identify hepatic genes regulated by iron overload and involved in cell cycle, which therefore could play a role in hepatocarcinogenesis.
For this purpose, we used mouse iron overload models, in which, as expected, we observed both hepatocyte polyploidisation and hepatomegaly. A macroarray strategy led us to the identification of the iron-induced Cyclin D1 gene overexpression. This result was confirmed at the protein level. The Cyclin D1 gene encodes a cyclin associated to the irreversible involvement of hepatocyte from quiescence to cell cycle progression. Cyclin D1 overexpression was associated with a rise of in vitro hepatocyte DNA synthesis, reflecting abnormal cell cycle control. These data suggests that hepatocyte Cyclin D1 overexpression during iron overload could participate to hepatocarcinogenesis.
Section snippets
Animals
Five or 7-week-old BALB/cJ and C57BL/6 male mice (CERJ, Le Genest St Isle, France) were maintained under standard conditions of temperature and light according to French law and regulations. They received standard AO3 diet (UAR, France). BALB/cJ mice were iron-overloaded for 8 months by addition of 3% carbonyl–iron in the diet. Controls did not have iron supplementation. Iron–dextran overload consisted in a single subcutaneous injection of iron–dextran (1 g iron/kg body weight) on 7-week-old
Assessment of liver iron overload
Iron overload exposure was well tolerated. Liver iron concentration was strongly increased, more than 10 fold, in all iron-overloaded animals compared to controls (Fig. 1A). In the iron–dextran model, we noticed a small decrease in liver iron concentration with iron exposure duration. As expected [12], [19], in carbonyl–iron and 2 months iron–dextran models, iron overload was mainly localised in hepatocytes (Fig. 1B), contrasting with a massive accumulation of iron in Kupffer cells after one
Discussion
We identify, for the first time, hepatic overexpression of Cyclin D1 both at mRNA and protein levels during liver iron overload, and correlate this induction with abnormalities in hepatocyte cell cycle progression, hepatocyte ploidy and liver mass (see Table 3).
To date, the relationship between iron and cell cycle has been mainly studied during iron depletion. Iron chelators induce a cell cycle blockage mainly during G1-, S- or G2-phases [22], [23]. This may be related to the deficiency of iron
Acknowledgements
We thank Medhi Alizadeh of Etablissement Français du Sang de Bretagne for helpful technical advice. This work was supported by the French Ministry of Research and Technology (MBT), the Région Bretagne BC and PRIR 139, the Association Fer et Foie, and the QLRT-2001-00444 EC contract.
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