Despite the dramatic effects of specific chemical mitogens such as TCPOBOP on the liver, the signaling pathways responsible for limiting the chemically induced hypertrophic and hyperplastic responses remain largely unknown. Studies in our laboratory have shown the role of ECM in inhibiting hepatocyte proliferation. (16
) 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 ILKloxP/loxP
mice became available. FAK (focal adhesion kinase), Mig2 and ILK are three proteins, primarily involved with transmission of the integrin signals. Our results demonstrate that the final size of the liver due to the TCPOBOP-induced hypertrophic and hyperplastic response is to a significant level dependent on ILK. Livers deficient in ILK show prolonged and sustained proliferative response to TCPOBOP. They also show higher liver/body weight ratios as compared to the WT counterpart given the same treatment.
TCBOPOP is the strongest chemical mitogen (5
) for the liver. The final liver sizes in normal mice following stimulation by TCBOPOP are the largest know in terms of liver to body weight ratio. The observed further enhancements in liver size and weight after TCBOPOP in the ILK/Liver −/− mice is to our knowledge the largest recorded for mice of that age. Numerous studies have demonstrated that the size of the liver, though highly susceptible to hormonal and nutritional responses, is overall adjusted to appropriate levels for the size of the body of the animal. We have used the term “hepatostat” to characterize this phenomenon.(27
) Our recent studies have implicated extracellular and pericellular matrix as involved in this process. Interference with ECM/integrin signaling by elimination of hepatocyte ILK has led to a higher
“hepatostat” in three different models of growth, such as liver regeneration after partial hepatectomy (18
), phenobarbital (19
) and now TCBOPOP. On the other hand, over-expression of the pericellular protein glypican 3 (GPC3) in hepatocytes led to a lower hepatostat (Am. J. Pathology, in press), consistent with the growth suppressing effects of GPC3.(28
) Our current studies underscore the important role of ECM as overall regulator of the hepatostat by mechanisms which need to further studied.
The hepatomegaly induced by TCPOBOP is known to be CAR dependent. (1
) We found considerable differences in the activation of CAR in the WT and ILK/liver−/− mice. While the WT mice showed an early strong activation of CAR the ILK/liver−/− mice showed a lower but a prolonged activation. It is very likely that the prolonged activation of CAR in the ILK/liver−/− mice is to compensate for the lower activation of CAR at early time points. Why removal of ILK from the hepatocytes leads to lower activation of CAR is worthy of further investigation.
We next investigated the mechanisms behind this prolonged proliferative response in the ILK/liver−/− mice. Promitogenic proteins like cyclin d1, HGF and Yap show sustained induction in the ILK/liver−/− mice. The protein c-myc has been implicated in various aspects of liver proliferation such as that observed in liver regeneration, growth and tumorigenesis. (29
) Recent study have shown(1
) c-myc as a key component of the TCPOBOP-induced hepatocyte proliferation. In our study also we saw increased and sustained induction of c-myc in the ILK/liver−/− mice as compared to the WT mice. It is possible that the increased and sustained proliferation seen in the ILK/liver−/− is in part c-myc dependent. Mitoinhibitory molecule like TGFβ1 was also lower (day 2 and 5) in the ILK/liver−/− mice as compared to WT mice. Taken together the ILK/liver−/− mice have a sustained and prolonged induction of Promitogenic signaling. It is important to understand that given the multiplicity of changes accompanying removal of ILK, it is not easy to assign the defect in termination of TCPOBOP-induced hepatocyte proliferation to any specific single signaling system. The cybernetic interconnections between the different signaling systems are quite complex. It is reasonable to postulate, however, that the “hyperproliferation” seen in these mice is a consequence of the adaptations resulting from one central event, the disruption or alteration of the signaling of ECM to hepatocytes because of the removal of ILK.
Our studies show that the overall CAR function and expression are different in the ILK/liver−/− mice. While it is possible that there is a direct interaction between ILK and CAR, the changes in hepatocyte differentiation and function after elimination of ILK are so complex that it is highly likely that the effects on CAR are indirect. For a perspective, please note Figure 6 of reference 16
In summary, these results demonstrate a central role of ECM signaling via ILK in terminating TCPOBOP-induced hepatocyte proliferation. Overall, these studies provide critical information on the mechanisms by which matrix defines and controls hepatocyte proliferation the liver. This work, however, has implications, not just for liver, but also for all tissue biology. Matrix defines the extracellular environment and regulates cellular function and growth in all tissues including liver, which has been one of the best tissue paradigms to investigate the complex interactions between matrix and different aspects of growth and differentiation.