George K. Michalopoulos, MD, PhD

Our laboratory is investigating the mechanisms leading to initiation and termination of liver regeneration and their relevance to hepatic neoplasia. We have found that a key process associated with initiation is activation of extracellular matrix (ECM) remodeling by urokinase, leading to local and systemic release of activated HGF. Both HGF and EGF receptors are activated by tyrosine phosphorylation at 30 minutes after initiation of the regenerative process. HGF and the ligands of EGFR are unique in their capacity to stimulate hepatocyte DNA synthesis in primary cultures and in unoperated animals. Other regulating signaling systems include TNF, IL6, norepinephrine, bile acids, leptin, serotonin, etc. These signaling systems differ from HGF and EGFR ligands in that they do not stimulate hepatocyte DNA synthesis in vivo or in culture. Their role is essential, however, in coordinating the intracellular signals that mediate entry of hepatocytes into DNA synthesis. ECM signaling is also important in termination of liver regeneration and in adjustment of the final weight of the regenerated liver. Mice with hepatocyte-specific elimination of ILK (integrin linked kinase) have defective integrin signaling and they do not properly terminate the regenerative process. The final liver weight in the hepatocyte-specific ILK KO mice is about 60% higher than the weight at the time of hepatectomy. These mice also respond with excessive liver enlargement in response to chemical xenobiotics such as phenobarbital and TCPOBOP. Regulation of nuclear receptors and Yap protein (target of the Hippo kinase mammalian homologs MST1 and MST2) as well as alterations in hepatic integrins and extracellular matrix proteins appear to be involved. The GPI linked protein known as Glypican 3 (GPC3), produced excessively by hepatocellular carcinomas, is also involved in termination of proliferation of hepatocytes and non-parenchymal cells. We have found that GPC3, in conjunction with the tetraspanin CD81, exerts anti-proliferative signals towards the end of regeneration. Mice over-expressing GPC3 have severe defects in liver regeneration.

The signaling systems operating in liver regeneration may also be involved in hepatic neoplasia, especially hepatocellular carcinoma (HCC). In a recent study of genomics of HCC we discovered that the most frequent small deletions occur in genes controlling important signaling pathways associated with liver regeneration. The gene LSP1 was associated with the most genomic alterations (51 of 98 cases of HCC examined) and it controls the RAF-MEK-ERK pathway. PTPRD, the second most frequently gene (28/98 cases) controls activation of STAT3, an important signaling molecular for hepatocyte proliferation. MAML2 and RSU1, also majorly affected, control Notch and ILK signaling. All these are hepatocyte growth suppressor genes and their deletions accelerate growth of neoplastic hepatocytes.

Further studies from our lab using chimeric livers in rats and organoid cultures have demonstrated that in severely suppressed proliferation of biliary cells, periportal hepatocytes have the capacity to trans-differentiate to biliary epithelial cells and rescue the biliary compartment. We also verified that the process of transdifferentiation in which biliary cells and hepatocytes acquire transcription factors of each other also occurs in human liver failure and chronic biliary disease. Overall, our laboratory is exploring pathways of organ regeneration using liver as a quintessential paradigm. The studies have opened new understandings in liver regeneration and control of the "hepatostat" program regulating liver size. They have also connected signaling pathways altered in neoplasia to pathways related to liver regeneration and established the capacity of mature epithelial cells (biliary and hepatocytic) to function as stem cells for each other.