Extracellular matrix (ECM) is of great importance for the survival, differentiation, and normal function of cells within a tissue (Kim et al., 1997
). This is particularly true for hepatocytes, the parenchymal cells of the liver. ECM is of key importance for determining differentiation and proliferation of hepatocytes in culture and in vivo
(Block et al., 1996
; Kim et al., 1997
; Michalopoulos, 2007
; Rudolph et al., 1999
). ECM remodeling is an essential part of liver regeneration after partial hepatectomy (Kim et al., 1997
). Signals from the ECM are transmitted to the interior of the cell via integrins (Hehlgans et al., 2007
). Recently, there has been much progress in determining mechanisms by which integrins deliver their signals inside the cell. A major mediator of integrin signaling is integrin-linked kinase (ILK) (McDonald et al., 2008
). ILK is a Ser/Thr kinase that is emerging as a key regulator of cell-ECM adhesions. Activation of ILK, either by integrin clustering or by growth factors, affects multiple cell signaling pathways that regulate different processes, such as survival, differentiation, proliferation, migration, and angiogenesis (Hehlgans et al., 2007
; McDonald et al., 2008
). Previous studies in our laboratory have shown that hepatocytes in primary culture lose their characteristic gene expression patterns (Block et al., 1996
). They can be stimulated to proliferate under the influence of hepatocyte growth factor (HGF) and/or epidermal growth factor (EGF). Addition of artificial ECM to hepatocytes in culture (e.g., Matrigel, Type I collagen gels) restores full differentiation and inhibits hepatocyte proliferation (Block et al., 1996
). Because it is practically impossible to eliminate ECM from an intact organ, elimination of the proteins responsible for transmission of the ECM signals to hepatocytes became a feasible alternative when ILK loxP/loxP mice became available. Integrin signaling involves multiple components and interactions with other receptors, etc. There are two proteins, however, primarily involved with transmission of the integrin signal, focal adhesion kinase and ILK (Hehlgans et al., 2007
; van Nimwegen and van de Water, 2007
). Thus, liver-targeted elimination of ILK disrupts in part the integrin signal.
Recently, we have been successful in eliminating the ILK gene specifically from hepatocytes (Gkretsi et al., 2008
). Liver histology in the ILK/liver−/− mice is indistinguishable at birth from the wild type (WT) except for a decrease in the number of bile ductules. At 2–3 weeks after birth, hepatocyte plates in the ILK/liver−/− mice are irregular with clusters of multiple cells surrounded by irregular sinusoids. At 6 weeks and thereafter, there are multiple hepatocyte mitoses and apoptosis in the ILK/liver−/− mice. By the end of 30 weeks, the livers of ILK/liver−/− mice are almost 30% larger than the WT mice (Gkretsi et al., 2008
). These 30-week-old ILK/liver−/− mice were subjected to 70% partial hepatectomy. Whereas the WT livers returned to exactly the same liver weight as prehepatectomy, the livers of ILK/liver−/− mice gained additional weight (59% increase). The increase in resting liver weight and the apparent “overgrowth” of the regenerating liver in the ILK/liver−/− mice shows that in absence of matrix signaling (as a result of removal of ILK), termination of liver regeneration does not function properly and liver grows to a much larger size (Apte et al., 2009
). Thus, this study highlights essential role of ECM-mediated signaling via ILK in regulation of both liver regeneration and its termination.
Studies from several investigators have shown, however, that the hepatic enlargement induced by chemical xenobiotic mitogens (such as phenobarbital [PB], dilantin, diazepam, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCBOPOP), peroxisome proliferators, etc.) (Columbano and Shinozuka, 1996
) proceeds through very different signaling mechanisms in comparison to liver regeneration. Growth factors associated with liver regeneration are minimally involved, and many of the cell cycle–associated genes induced at the early stages of liver regeneration do not play a part in the hepatic enlargement induced by chemical mitogens. Thus, we wanted to explore whether the enhanced proliferative response and defective termination of proliferation seen in ILK/liver−/− mice in liver regeneration also occurs in hepatic enlargement induced by chemical mitogens. Given the dissimilarities of the two growth responses (Columbano and Shinozuka, 1996
), we wanted to explore whether the enhanced proliferative response and defective termination of proliferation seen in ILK/liver−/− mice in liver regeneration also occurs in hepatic enlargement induced by chemical mitogens. The present study investigated the role of ILK in hepatocyte proliferation induced by PB, which is known to induce hyperplasia and hypertrophy of hepatocytes, resulting in hepatomegaly in mice and humans. Based on the previous studies in our laboratory on regeneration of liver in ILK/liver−/− mice, we hypothesized that ILK/liver−/− mice would also respond with increased and prolonged proliferative response to PB resulting in massive hepatomegaly. Our data indeed demonstrate that ECM proteins communicate with the signaling machinery of dividing cells in part via ILK to regulate hepatocyte proliferation and termination of the proliferative response not only in liver regeneration but also in response to hepatomegaly induced by xenobiotic chemical mitogens.