TGF-ß signal pathway activation regulates restoration of quiescence in the regenerating liver and of the liver mass:body weight ratio after partial hepatectomy

Judith Romero-Gallo, Anna Chytil, William Russell, Shiva Gautam, Mark Magnuson, Harold Moses, William M. Grady 

Departments of Medicine, Cancer Biology, Pediatrics, and Biostatistics; Vanderbilt University Medical School, Nashville, TN

Background: The transforming growth factor ß (TGF-ß) signaling pathway, which is mediated through the TGF-ß receptor complex consisting of the type I and type II TGF-ß receptors (TGFBR1 and TGFBR2), is essential for normal growth and development as well as for stress responses in the liver.  Stimulation of this pathway can regulate cell growth and death through 1) affecting cell cycle progression, 2) mediating differentiation, and 3) inducing apoptosis in epithelial cells.  TGF-ß signaling has been shown to play a role in 1) the regulation of liver cell proliferation, 2) the regulation of liver epithelial cell apoptosis, and 3) the formation of hepatocellular carcinomas (HCC); however, the in vivo effects of TGF-ß on liver regeneration, liver mass regulation, and transformation are largely unknown.  We have generated a novel in vivo system for studying TGF-ß mediated effects on cell proliferation in the liver and now present results demonstrating the effect of loss of Tgfbr2 on liver epithelial cell proliferation in the setting of compensatory hyperplasia. 

Methods: Because of the complex nature and multiple functions of TGF-ß and the TGF-ß signaling pathway, we have developed a novel hepatocyte-specific Tgfbr2 knock-out mouse, the Tgfbr2^hepko mouse, in order to study the physiologically relevant effects and mechanisms of TGF-ß signaling in vivo. Tgfbr2^hepko mice were generated by crossing Tgfbr2^flx/flx mice with Alb-Cre mice.  Alb-cre Tgfbr2^flx/flx and Tgfbr2^flx/flx mice were then subjected to 70% partial hepatectomy and harvested at serial timepoints after hepatectomy, ranging from 24 hours to 1 month.  The harvested livers were then assessed for hepatocyte proliferation by Brdu staining and for liver mass: body weight ratio.   Protein lysates were prepared from the livers and subject to western blot and IP:western blot analysis to assess the cell cycle associated proteins. In addition, the mRNA expression of specific cell cycle genes was assessed by quantitative RT-PCR. 

Results:  Proliferation is greater at 24hrs and 1 week after partial hepatectomy in the Tgfbr2^hepko mice as compared to the Tgfbr2^flx/flx mice consistent with a model that TGF-ß signaling suppresses mitogenesis in the regenerating liver. At one month, the livers in both the Alb-cre Tgfbr2^flx/flx and Tgfbr2^flx/flx mice were in the quiescent state suggesting that other mechanisms can eventually compensate for lack of TGF-ß signaling in the hepatocytes to eventually stop hepatocyte proliferation.  Importantly, we also found that liver mass: body weight ratio was increased in the Tgfbr2^hepko mice at 1 week and at 4 weeks after partial hepatectomy implicating TGF-ß as a physiologically important mechanism for regulating the restoration of appropriate liver mass in the setting of compensatory hyperplasia. In order to determine the mechanism through which TGFBR2 regulates proliferation after partial hepatectomy, we have assessed the expression of cell cycle associated proteins and pocket proteins in lysates taken at the serial time points after partial hepatectomy.  We have observed decreased expression of p130, increased phosphorylation of p130, and increased expression of cyclin E in the livers from the Tgfbr2^hepko mice compared to the Tgfbr2^flx/flx mice. 

Conclusions: Through the use of the Alb-cre Tgfbr2^flx/flx mouse model, we have demonstrated that TGF-ß signaling in hepatocytes suppresses mitoggenesis after partial hepatectomy.  Tgfbr2 appears to play a role in regulating the restoration of the quiescent state in the regenerating liver and also involved in regulating the liver mass:body weight ratio.  These effects are associated with changes in p130 expression and phosphorylation and cyclin E expression suggesting that these proteins are the relevant cell cycle proteins that are being regulated by TGF-ß to inhibit liver regeneration. 

Acknowledgements: This research was supported by funding from the NIDDK (RO1 DK60669) and the Digestive Disease Research Center at Vanderbilt University Medical Center.