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Abstract

Regucalcin, which was discovered in 1978 as a calcium-regulatory protein, plays a multifunctional role as a suppressor in signal transduction in various types of cells and tissues. The regucalcin gene (rgn) is localized on the X chromosome. Regucalcin was found to suppress nuclear deoxyribonucleic acid and ribonucleic acid synthesis in liver cells. Overexpression of endogenous regucalcin revealed suppressive effects on proliferation in the modeled rat hepatoma cells due to regulating the gene expression of various proteins that are involved in cell cycle and carcinogenesis. Regucalcin mRNA expression was uniquely downregulated in the development of carcinogenesis in the liver of rats in vivo. The expressions of regucalcin mRNA was found to depress in human hepatoma tissues. The suppressed regucalcin gene expression was found to be associated with progression of hepatocarcinogenesis by proteosome analysis. The regucalcin gene may be a target in the therapy of hepatocarcinoma.

Key words: regucalcin, cell signaling, nuclear regulation, cell proliferation, hepatocarcinogenesis

Introduction

Regucalcin was discovered in 1978 as a Ca²⁺-binding protein that contains no EF-hand motif of Ca²⁺-binding domain [1-4]. The regucalcin gene is localized on the X chromosome [5,6]. The organization of regucalcin gene consists of seven exons and six introns [7]. Regucalcin and its gene (rgn) are identified in over 15 species consisting of regucalcin family in vertebrate and invertebrate species [4,8,9]. Various transcription factors have been shown to regulate transcription activity of the regucalcin gene expression that is mediated through Ca²⁺ and other signal systems [9].

Regucalcin plays a multifunctional role in cell regulation of various types of cells and tissues; regulation of intracellular Ca²⁺ homeostasis and suppressions of various signal transductions, protein synthesis, cell proliferation and apoptosis [10-13]. Regucalcin, which is present in the cytoplasm, is translocated into the nucleus of various types of cells, and it plays a pivotal role in the regulation of nuclear function including ssuspression of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, inhibition of various protein kinases and phosphatases activities, and regulation of the gene expression of various proteins [14]. Regucalcin is proposed to play a pivotal role in maintaining cell homeostasis for stimulation of various factors including hormones and cytokines [10,11].

Moreover, regucalcin is found to be involved in various pathophysiologic states including liver disease, kidney disorder, heart failure, brain disease, osteoporosis, and lipid metabolism [15-19]. Interestingly, the expressions of regucalcin mRNA was found to depress in human hepatoma tissues [12]. Such suppression was associated with progression of hepatocarcinogenesis by proteosome analysis [20,21]. The regucalcin gene may be a target in the therapy of hepatocarcinoma. This review will discuss the recent findings regarding to involvement of regucalcin in hepatocarcinogenesis.

Regucalcin regulates liver nuclear function

Regucalcin is greatly in the cytoplasm of liver cells, and the protein is translocated into the nucleus through protein kinase C-dependent signaling mechanism [14]. Nuclear translocation of regucalcin was not regulated through adenosine 5’-triphosphate and guanosine 5’-triphosphate that are required for nuclear import of proteins [22], and it was not changed with the lectin wheat germ agglutinin that suppresses transport of nuclear protein [23]. Nuclear translocation of regucalcin was not related to nuclear localization signal that is responsible for selection for intranuclear active transport. Regucalcin may be passively transported to the nucleus through nuclear pore in cells, since the molecular weight of regucalcin is about 33 kDa [4]. Regucalcin has also been shown to localize in the nuclei of the cloned normal rat kidney proximal tubular epithelial NRK52E cells with immunocytochemical analysis [24]. This nuclear localization of regucalcin was found to enhance through hormonal Ca2+-signaling dependent process that is involved protein kinase C [24].

Regucalcin has been shown to bind protein and DNA in the nucleus [25]. Regucalcin regulated various enzyme activities in the nucleus. Endonuclease is responsible for DNA fragmentation occurring during programmed cell death (apoptosis) and certain forms of chemically induced cell killing [26]. Regucalcin revealed suppressive effects on Ca²⁺-activated DNA fragmentation due to inhibiting endonuclease activity in isolated rat liver nuclei [27]. Smal GTPase Ran (ras-related nuclear protein) is required for protein export from the nucleus and protein import into the nucleus [28]. Regucalcin inhibited GTPase activity in rat liver nucleus [22]. Process of signal transduction from the cytoplasm to nucleus in liver cells is mediated through various protein kinases and protein phosphatases. Regucalcin suppressed the activities of tyrosine kinase, protein kinase C and Ca²⁺/calmodulin-dependent protein kinase, which are enhanced in the cytoplasm and nucleus obtained from regenerating rat liver with proliferating cells in vivo [29]. The activity of nuclear Ca²⁺-dependent protein kinases was increased in the presence of anti-regucalcin monoclonal antibody in the enzyme reaction mixture, and such increases are completely depressed with addition of regucalcin [29]. Nuclear endogenous regucalcin played a suppressive role in the regulation of protein tyrosine phosphatases using anti-regucalcin monoclonal antibody in the reaction mixture [30]. Regucalcin was demonstrated to play a pivotal role in the regulation of the activity of various enzymes in the nucleus.

Regucalcin was found to reveal suppressive effects on DNA and RNA synthesis activity in the nuclei of normal rat liver and regenerating rat liver in vivo [31-34]. Regucalcin may suppress the enhancement of nuclear DNA and RNA synthesis in proliferating liver cells in vivo. The presence of anti-regucalcin monoclonal antibody in the reaction mixture causes an increase in nuclear DNA synthesis activity [32]. This increase was completely depressed in the presence of regucalcin [32]. Endogenous regucalcin was found to reveal a suppressive effect on DNA synthesis in the nuclei of rat liver. The effect of regucalcin in decreasing nuclear RNA synthesis activity in normal rat liver was not seen in the presence of α-amanitin, an inhibitor of RNA polymerase II and III [33,34], suggesting that this suppressive effect is partly resulted from the inhibitory action on RNA polymerase II and III. Thus, regucalcin was demonstrated to reveal direct inhibitory effects on nuclar DNA and RNA polymerase a