Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol-anchored, cell-surface heparan sulfate proteoglycans. The GPC3 gene is located on the X chromosome, and is highly expressed during embryogenesis and organogenesis. Loss-of-function mutations of GPC3 in humans result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and post-natal liver and other organ overgrowth. GPC3 is one of the most over-expressed proteins in human hepatocellular carcinoma and is used as a novel diagnostic marker. However, its role in normal liver regeneration is still not well characterized. In this study, we investigated the role and effects of GPC3 in hepatocyte proliferation and liver regeneration, using 2/3 partial hepatectomy (PHx) model in rats and hepatocyte-targeted GPC3 transgenic mice. We found in rats that GPC3 mRNA and protein increase in a time frame which coincides with the termination of proliferative activities of either hepatocytes (day 2 after PHx and day 8-12 in culture) or non-parenchymal cells (day 5-6 after PHx). Blocking GPC3 expression using morpholino oligonucleotides promoted rat hepatocyte growth in vitro. We further generated GPC3 transgenic mice with hepatocyte-targeted over-expression of GPC3. These transgenic mice develop normally compared with their non-transgenic littermates, but have a suppressed rate of hepatocyte proliferation and liver regeneration after 2/3 PHx. Therefore we hypothesize that GPC3 is a negative regulator of hepatocyte proliferation and liver regeneration. The yeast two-hybrid assay revealed that GPC3 interacts with several interesting proteins including CD81, a cell membrane tetraspanin. CD81 levels changed in the same manner as GPC3 after rat PHx, and their interaction was confirmed by co-immunoprecipitation and co-immunofluorescence. The co-localization of GPC3 and CD81 after PHx indicates an important regulator interaction between the two proteins. Moreover, gene array analysis revealed a series of changes in the expression profiles in GPC3 transgenic mice. After PHx, a panel of cell cycle related genes and some oncogenes are either up- or down-regulated, which was confirmed by western blotting. Our results indicate that GPC3 plays a negative regulatory role in hepatocyte proliferation and liver regeneration in rats and hepatocyte-targeted transgenic mice, in which several potential proteins and multiple pathways are involved and affected.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07302010-140531 |
| Date | 13 August 2010 |
| Creators | Liu, Bowen |
| Contributors | Wendy M. Mars, Chuanyue Wu, George K. Michalopoulos, Jian-hua Luo, Satdarshan P. Singh Monga, James L. Funderburgh |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07302010-140531/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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