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To the Editor:

An interesting report by Streetz et al. published in HEPATOLOGY described the production of hepatocyte conditional gp130 knockout mice subjected to chronic liver injury by repeated carbon tetrachloride (CCl4) treatment.1 Chronic injury induces liver regeneration by recruitment of cells from a precursor, either from a stem cell or a progenitor cell known as the oval cell. Numerous mouse studies such as that described by Streetz et al.1 utilize albumin-alpha-fetoprotein (AFP) promoter and/or enhancer sequences to achieve hepatocyte specific gene deletions by expressing Cre recombinase.2 The albumin-AFP promoter and/or enhancers are used, as AFP is an early marker of hepatocyte commitment. First detected at 8.5 days gestation in hepatoblasts, it is highly expressed in perinatal liver and diminishes to low or undetectable levels in the adult mouse.3 Albumin production commences around 9.5 days gestation and is maintained throughout development.4

We have previously shown that the choline-deficient, ethionine-supplemented diet (CDE) induces liver progenitor oval cells in rodents and that these cells express albumin and AFP.5, 6 Oval cells induced by CCl4 will express albumin and AFP.7 We therefore raise the possibility that the interpretation of many hepatocyte-specific gene targeting experiments may be compromised by this situation. A quiescent pool of oval cells exists in the liver and will respond to promitogenic signals initiated by chronic liver damage. In the study by Streetz et al.,1 repeated CCl4 treatment may activate oval cell proliferation.7 Therefore, oval cells derived from the endogenous pool will also be deficient for gp130. The protective role of gp130 stimulation as observed by Streetz et al.1 might well be explained by gp130 stimulation of oval cells, which results in cellular proliferation (see below). This phenomenon has not been considered in the publication. We wish to alert your readership that conditional gene targeting utilizing Cre recombinase driven by the albumin-AFP promoter/enhancer sequences will inactivate the gene of interest in hepatocytes and oval cells.

We recently produced novel data implicating the combination of interleukin (IL)-6 and its soluble IL-6R (sIL-6R) (hyper IL-6)8 in oval cell activation (Fig. 1). IL-6 signalling via the sIL-6R requires gp130 to exert its effect. Therefore, in the publication by Streetz et al.,1 it is likely that the knockout of gp130 in progenitor oval cells may compromise their activation during chronic liver injury. Although few oval cells are present in normal liver, their presence can confound experiments that do not involve chronic liver injury. In a mouse model where simian virus 40 (SV40) large T antigen is overexpressed under the control of the albumin promoter, hepatocellular carcinoma occurs9. It is accepted that SV40 large T antigen expressed in hepatocytes is responsible for the hepatocellular carcinoma. Since overexpression of SV40 large T antigen induces oval cell proliferation and carcinoma, which are positive for the oval cell marker A6,10 it's possible that the tumorigenic process is initiated in an oval cell.

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Figure 1. The combination of interleukin (IL)-6 and sIL-6R (hyper IL-6) enhances oval cell activation. (A) Proliferation of a hyper IL-6 (50ng/mL) stimulated immortalized oval cell line as determined by the MTT assay. (B) Male C57BL/6 mice were subjected to the choline-deficient, ethionine-supplemented diet for 2 weeks and injected intraperitoneally every 2 days with Hanks Balanced Salt Solution or Hyper IL-6 (4 μg). Livers were excised and sections stained for the oval cell marker A6. Each data point represents results from 1 mouse.

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In conclusion, experiments utilizing the albumin and AFP promoter/enhancers to activate or inactivate genes need to consider that oval cells as well as hepatocytes may be affected.

References

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  • 1
    Streetz KL, Tacke F, Leifeld L, Wustefeld T, Graw A, Klein C, et al. Interleukin 6/gp130-dependent pathways are protective during chronic liver diseases. HEPATOLOGY 2003; 38: 218229.
  • 2
    Kellendonk C, Opherk C, Anlag K, Schutz G, Tronche F. Hepatocyte-specific expression of Cre recombinase. Genesis 2000; 26: 151153.
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    Shiojiri N. Enzymo- and immunocytochemical analyses of the differentiation of liver cells in the prenatal mouse. J Embryol Exp Morphol 1981; 62: 139152.
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    Cascio S, Zaret KS. Hepatocyte differentiation initiates during endodermal-mesenchymal interactions prior to liver formation. Development 1991; 113: 217225.
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    Tian YW, Smith PG, Yeoh GC. The oval-shaped cell as a candidate for a liver stem cell in embryonic, neonatal and precancerous liver: identification based on morphology and immunohistochemical staining for albumin and pyruvate kinase isoenzyme expression. Histochem Cell Biol 1997; 107: 243250.
  • 6
    Smith PG, Tee LB, Yeoh GC. Appearance of oval cells in the liver of rats after long-term exposure to ethanol. HEPATOLOGY 1996; 23: 145154.
  • 7
    Engelhardt NV, Baranov VN, Lazareva MN, Goussev AI. Ultrastructural localization of alpha-fetoprotein (AFP) in regenerating mouse liver poisoned with CCl4. 1. Reexpression of AFP in differentiated hepatocytes. Histochemistry 1984; 80: 401407.
  • 8
    Fischer M, Goldschmitt J, Peschel C, Kallen KJ, Brakenhoff JPJ, Wollmer A, Grötzinger J, and Rose-John S. A bioactive designer cytokine for human hematopoietic progenitor cell expansion. Nat Biotechnol 1997; 15: 142145.
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    Hino O, Kitagawa T, Nomura K, Ohtake K, Cui L, Furuta Y, Aizawa S. Hepatocarcinogenesis in transgenic mice carrying albumin-promoted SV40 T antigen gene. Jpn J Cancer Res 1991; 82: 12261233.
  • 10
    Bennoun M, Rissel M, Engelhardt N, Guillouzo A, Briand P, Weber-Benarous A. Oval cell proliferation in early stages of hepatocarcinogenesis in simian virus 40 large T transgenic mice. Am J Pathol 1993; 143: 13261336.

Vance B. Matthews*, Stefan Rose-John†, George C. T. Yeoh*, * The University of Western Australia Centre for Medical Research Western Australian Institute for Medical Research Perth, Australia Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences Crawley, Australia, † Biochemistry Institute Christian Albrechts University Kiel, Germany.