Karen Ho, Nhue Do, Hasan Otu, Martin J. Dib, Ernest Terwiliger, Seth J. Karp
Brigham and Women’s Hospital; Boston, MA
PURPOSE OF STUDY
Tob1 is an antiproliferative molecule and null mutants exhibit high rates of spontaneous liver neoplasm. We hypothesized that Tob1 may function as a constitutive suppressor of hepatocyte proliferation and that loss of Tob1 leads to enhanced liver regeneration after partial hepatectomy (phx).
Phx were performed in wildtype (wt) and Tob1 knockout (Tob1-/-) mice. Tob1 gene and protein levels were determined by real time RT-PCR and Western blotting. Restoration of liver parenchyma was measured as a function of liver:body weight. Ki67 immunostaining was a marker of hepatocyte proliferation. Expression of cell cycle proteins cyclin D1 and cyclin A and TGF? signaling pathway members Smad 1/5/8 was detected by Western blotting. A microarray analysis was used to determine differential regulation of biological pathways between wt and Tob1-/- livers. In vivo expression of Tob1 was manipulated with a novel, recombinant AAV8-based, hepatocyte-specific vector we developed.
SUMMARY OF RESULTS:
Tob1 gene and protein levels are downregulated 6 and 36 hours, respectively, after phx . At baseline, wt and Tob1-/- livers are grossly equivalent. Tob1-/- livers demonstrate enhanced DNA replication and more rapid restoration of liver mass after phx. In vivo overexpression of Tob1 in wt mice increased Ki67 staining; restoration of Tob1 in Tob1-/- mice reduced Ki67 staining in a dose-dependent manner. Tob1 has been shown to interact with Smad proteins, but we observed that nuclear and cytoplasmic localization of Smads 1/5/8 in wt and Tob1-/- mice after phx are equivalent, suggesting that Tob1 signaling is downstream of Smad signaling.
By varying the expression of Tob1, the rate of liver regeneration after phx can be controlled. These results support a new paradigm in liver regeneration, i.e. that downregulation of a constitutively expressed anti-proliferative molecule is required for normal liver regeneration. This further establishes the importance of modulating inhibitory pathways to control the rate of liver regeneration. Implications of these findings include therapeutic targets to enhance liver regeneration and control liver cancer.