Differential expression of CD44 isoforms during liver regeneration in rats
Introduction
CD44 is a multifunctional cell surface glycoprotein [1] involved in lymphocyte homing and activation, inflammation, tumor growth and metastasis [2]. CD44 standard form (CD44s) is expressed in lymphocytes, most mesenchymal and epithelial cells, and it is detected at high levels in some tumor cells [3]. CD44 acts as a receptor for hyaluronic acid (HA) and other extracellular matrix (ECM) components. An important feature of CD44 is the intracellular domain where the phosphorylation of two serine residues seems to be crucial for the activation of different signal transduction pathways [4], [5]. The transmembrane interaction between CD44 and the cytoskeleton is essential in the onset of oncogenesis and tumor progression [6]. Its heterogeneity is due to alternative splicing and post-translational modifications that affect the extracellular domain [7], [8], [9]. The analysis of the CD44 genomic structure in the rat, mouse and human has revealed that the alternative splicing involves the differential use of at least ten exons [9], [10]. The differential incorporation of these exons that gives rise to CD44 variants plays an important role in the process of tumor metastasis. It has been demonstrated that the stable transfection of a specific spliced form of CD44, containing the isoform pMeta, into a non-metastatic cell line confers metastatic properties [11]. This isoform differs from the standard form in that it includes an extra domain of 162 amino acids (224–385). Functional experiments conducted by repeated injection in vivo of a monoclonal antibody directed against this specific epitope of the CD44 isoform or by a soluble human CD44-immunoblogulin fusion protein prevent metastasis in target organs by blocking lymphogenic invasion [12], [13]. The presence of exon v6 in different isoforms of CD44 has been found not only in activated T-cells [14], but also in different tumors, which seems to confer metastatic characteristics [15], [16], [17]. The presence of isoforms containing v6 has been associated in humans with tumor development and the metastatic potential of colorectal cancer, squamocellular carcinoma and breast cancer [18], [19], [20]. In a similar analysis of hepatocellular carcinoma, the presence of variant proteins containing v3 and v6 has been demonstrated [21].
One of the most useful experimental tools to study the proliferation in vivo is represented by liver regeneration. In the rat, following 70% hepatectomy, the remaining hepatocytes begin to proliferate rapidly, entering the S-phase after 12–18 h [22]. DNA synthesis in non-parenchymal cells (NPC) begins 24 h later. The cells continue to proliferate until the original cellular mass is reconstituted. In rats, the hepatic mass is restored in about 10–15 days [23], [24]. Many factors and signal transduction pathways have been shown to play a role in regulating the hepatocyte proliferation that follows partial hepatectomy (PH) [25], [26], [27], [28]. Growth factors, hormones, transcription factors and biochemical changes in parenchymal cells have all been implicated as regulators of regeneration, but until now, the mechanisms controlling this proliferative process have been poorly understood [23], [29], [30], [31]. Molecules involved in cell adhesion and the ECM, which influence differentiation, growth, cell–cell interactions and cellular polarity, play an important role in the regenerating liver [32], [33]. Studies on the expression of fibronectin and α5-β1-integrin have demonstrated a 6–8-fold increase at 12–24 h after PH, suggesting that fibronectin mediates adhesion between hepatocytes and the ECM during the regenerating process [34], [35]. Other authors have studied components of the ECM, e.g. laminin, the expression of which is modulated during hepatic regeneration; however, their precise function is poorly understood [33], [34], [35], [36].
In this study, we investigated the qualitative modifications of CD44 in regenerating liver, which represents a powerful model system for studying cellular proliferation in vivo. We analyzed the modulation of CD44 gene expression and post-transcriptional modifications of CD44 in a proliferating system. We examined the expression of different isoforms containing exon v6 in the regenerating liver. Furthermore, we studied CD44 expression in the hepatoma H-35 cell line, the properties of which closely resemble those of proliferating hepatocytes during liver regeneration [37].
Section snippets
Animals
The experiments were performed on 3-month-old male Sprague–Dawley rats. The animals received humane care according to National Institute of Health (NIH) guidelines. The surgical procedures were performed between 08:00 and 24:00 h under ether anesthesia. The animals were partially hepatectomized, according to Higgins and Anderson's procedure, removing approximately 70% of the liver mass [38]. As a control, sham operations (SO) were performed (transverse abdominal incision followed by digital
CD44 expression during liver regeneration
To study the role of CD44 in liver regeneration, we first analyzed its expression in rat liver at different times following PH. For this purpose, Poly(A)+ mRNA was analyzed by Northern blotting and hybridized with a cDNA probe specific for CD44. Our results revealed the presence of two transcripts, the levels of which were not modified during the first 72 h following PH (Fig. 1A). The expression of CD44 protein in normal and regenerating liver was also analyzed by Western blotting using a mouse
Discussion
The rat regenerating liver provides an excellent model to study a proliferative process in vivo. In the present study, we described a possible role played by CD44 and its isoform, CD44v6, in liver regeneration and in the regulation of proliferation. Many authors have shown that CD44v6 is expressed in a variety of tumors [46]. The ability to metastasize in some tumors is linked to the expression of v6 in different CD44 isoforms [46]. Recently, the presence of a spliced form of CD44 containing v6
Acknowledgements
The authors would like to thank the other members of their laboratory, D. Bartoli, D. Piobbico, M. Castelli, S. Brancorsini and A. Giacomucci for fruitful discussions and the preparation of this paper. The authors are grateful to P. Sassone-Corsi, N.S. Foulkes, E. Lalli, G.M. Fimia and A. Columbano for critical reading and suggestion on the manuscript. Thanks are extended to S. Pagnotta and M. Coli for the excellent technical assistance and to Dr J.W. Koontz for the gift of the H-35 hepatoma
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