Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Abstract

Glypicans are heparan sulfate proteoglycans that are bound to the cell surface by glycosylphosphatidylinositol. While six members of the glypican family are known in mammals, our study focused on glypican 3 (GPC3). Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and postnatal liver and other organ overgrowth. GPC3 is overexpressed in human hepatocellular carcinoma; however, its role in normal liver regeneration and hepatocyte proliferation is unknown. Here we investigated the role of GPC3 in hepatocyte proliferation. GPC3 mRNA and protein levels begin to increase 2 days after hepatectomy with peak expression levels by day 5. In hepatocyte cultures, GPC3 reaches a plateau when hepatocyte proliferation decreases. In vitro studies using Morpholino oligonucleotides showed that blocking GPC3 expression promoted hepatocyte growth. Yeast two-hybrid assays revealed that GPC3 interacts with CD81, a member of the tetraspanin family that is reported to be involved in hepatitis C virus infection and cell proliferation. We found that CD81 levels also increased 2 days after partial hepatectomy and toward the end of regeneration. Immunofluorescence showed that CD81 and GPC3 colocalize by 2 and 6 days after hepatectomy. Co-immunoprecipitation validated the interaction of GPC3 and CD81. Our results indicate that GPC3 may be a negative regulator of liver regeneration and hepatocyte proliferation, and that this regulation may involve CD81.