The galectin-3 gene promoter binding proteins in the liver of rats 48-h post-treatment with CCl4
Introduction
Galectin-3 (Gal-3) is a member of the family of β-galactoside specific soluble lectins, galectins (Albrandt et al., 1987, Barondes et al., 1994, Hirabayashi et al., 2002, Liu et al., 2002, Yang et al., 1996). The functions of Gal-3 have been assigned to a variety of regulatory events in cells; in addition to functions in cell adhesion, immune responses, and RNA processing, and as a receptor of the advanced glycation endproduct, RAGE, the anti-apoptotic activity of Gal-3 is thought to be important in maintaining cell viability (Liu et al., 2002, Yang et al., 1996). During development, Gal-3 is detectable in most types of cells including the hepatocytes in human and mouse embryos, (Fowlis et al., 1995, van den Brule et al., 1997), present in rat neonate livers by 9 days after birth, but fell to a trace level in the adult liver (Cerra et al., 1985). Our previous Western and Northern blot analyses confirmed trace amounts of both polypeptide and mRNA for Gal-3 in normal livers of adult rats (Yamazaki et al., 2001). These properties in Gal-3 biosynthesis during development suggest that Gal-3 is involved in the normal processes of the development and differentiation of various types of cells including hepatocytes. However, mice in which there is a homozygously disrupted Gal-3 gene are apparently normal in their embryonic and post-natal development (Colnot et al., 1998, Hsu et al., 2000). Roles of the hepatic Gal-3 remain to be examined.
The liver actively catabolizes xenobiotic compounds by microsomal drug-metabolizing enzymes each comprising a member of the cytochrome P450 family as a terminal oxygenase, since these are representative pathways for liver-specific metabolism. CCl4 catabolism is initiated by cytochrome P450 isoforms, CYP2E1 and CYP1A2, yielding toxic substances that deleteriously injure hepatocytes (Plaa, 2000, Raucy et al., 1993, Wong et al., 1998). The appropriate dose of CCl4 causes pericentral hepatocyte death approximately 24 h after its administration. The pericentral lesion thus formed can be restored to a normal architecture by reparation and regeneration of the cellular components during an approximately 2-week period post-treatment (Yamazaki et al., 2001). The reproducible nature of the chemically induced liver injury allows the study, at a tissue level, of the proteins involved in the regulation of hepatocyte death and regeneration. On the other hand, acetaminophen and some other therapeutics can similarly injure the liver in humans (Clark et al., 1973, Meadows, 2001, Zhang et al., 2002), implying that drug-induced liver injury is a serious problem in patients under particular conditions. Therefore, identification of proteins that protect and ameliorate the injury is useful for understanding the mechanisms of serious liver injury and developing suitable treatments, as well as the understanding of mechanisms for viability control in hepatocytes.
In our rat model, histological analysis demonstrated that pericentral hepatocyte death occurs around 24 h after CCl4 administration, and cellular components begin to proliferate, probably from periportal regions, at a period later than 72 h (Yamazaki et al., 2001). Moreover, albumin mRNA turnover changed properties along with this time course (Morigasaki et al., 2000). Albumin mRNA level, which decreased to 20% of the normal level at a 10-h period by its destabilization and transcription suppression of its gene, had begun to increase at 48 h conversely by albumin mRNA stabilization and increased albumin gene transcription, implying that the impaired functions of hepatocytes began to be restored around 48 h (Morigasaki et al., 2000).
Considering and taking advantage of the chronological sequence of the reversible liver injury induced by CCl4, we attempted to survey proteins that change their properties at periods for reparation of the liver injury. Western blot analysis using an anti-phosphotyrosine antibody clearly showed increased production of a 30-kDa protein in the liver 48- to 72-h post-treatment (Yamazaki et al., 2001), periods for repair of the impaired functions (Morigasaki et al., 2000). We identified this polypeptide as Gal-3. The Gal-3 induced was predominantly present in the cytosolic fraction of the liver and in the cytoplasm of periportal hepatocytes (Yamazaki et al., 2001).
Gal-3 is present at a trace level in the normal liver. Its increased production accompanied a greater than 60-fold increase in the amounts of Gal-3 mRNA, and could be ascribed to increased Gal-3 gene transcription in response to pathological conditions (Yamazaki et al., 2001). One putative role of Gal-3 induced in the liver, therefore, was assigned to the reparation and survival of the injured hepatocytes. The lag time to the appearance of large amounts of Gal-3 was a characteristic involved in Gal-3 induction by CCl4 administration; that is, CCl4 did not appear to directly trigger Gal-3 induction. This property further suggested that a change in the availability of factors required to increase Gal-3 gene transcription was a critical responses in hepatocytes to pathological conditions.
There have been several published studies on Gal-3 gene transcription (Rosenberg et al., 1993, Kadrofske et al., 1998, Stock et al., 2003). However, the relationship between transcription regulation directing the Gal-3 gene and Gal-3 induction in the liver is largely unknown. In the present study, we tried to identify the rat Gal-3 gene promoter and characterize its binding proteins present especially in the injured liver and compared them with those in Ac2F rat hepatoma cells in culture and in livers of normal rats. The present paper describes several structural features of the rat Gal-3 gene promoter and its binding proteins including a novel protein, rat Purβ.
Section snippets
Materials
Male Wistar rats at 7 weeks of age were fasted overnight, and received a single dose of 50% (W / W) CCl4 in olive oil at 2 ml/kg of body weight by intragastric administration. The rats had free access to a normal laboratory chow and water until use, and were killed at appropriate periods. Ac2F rat hepatoma cells from the Health Science Research Resources Bank (Osaka, Japan) were cultured in minimum essential medium containing 10% fetal bovine serum. Rat Gal-3 cDNA previously cloned (Yamazaki et
Transcription start site of the rat Gal-3 gene
A 19-kb genomic DNA fragment cloned with a Gal-3 cDNA probe was mapped by XhoI. Fragments yielded at four internal XhoI sites were 1.4-, 1.7-, 4.1-, 7.9- and 3.8-kb in lengths and from 5′ to 3′ in orientation (Fig. 1A). Southern blot analysis using [32P]-primer 1 as a probe revealed and nucleotide sequencing confirmed that the 1.4-kb fragment arising from a 5′-end region by digestion with XhoI (Fig. 1A and B) encodes nucleotides 1 to 36 of the Gal-3 cDNA cloned by us (Yamazaki et al., 2001) in
Discussion
CCl4 specifically injures the liver, since CYP2E1 and 1A2, which are required for the initial activation of CCl4, solely function in hepatocytes (Plaa, 2000, Raucy et al., 1993, Wong et al., 1998). Reparation of injured cells, and proliferation and differentiation of repaired cells to cellular components of the liver may precede restoration from pericentral hepatocyte necrosis to a normal lobular architecture during a 2-week period post-treatment of rats with a single dose of CCl4. Taking
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Present address: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.