In this study, we demonstrate that homocysteine inhibits cell proliferation via impairment of normal cell cycle regulation in hepatocytes. Thus, our results provide important insights into the molecular mechanism underlying hyperhomocysteinemia-mediated impairment of liver regeneration.
It has been documented that homocysteine induces growth arrest or apoptosis though ER stress in endothelial cells 
. Up-regulation of CHOP probably plays a potential role in linking homocysteine-mediated ER stress to alterations in endothelial growth and proliferation 
. Likewise, elevated expression of CHOP has been considered as a critical molecule for hepatic apoptosis in a murine model of ethanol feeding 
. However, there is still a missing link between the induction of CHOP and cellular dysfunction. In this study, we demonstrate that homocysteine (a) up-regulates TRB3 expression in a CHOP-dependent manner, (b) attenuates the phosphorylation of Akt, (c) enhances the expression of p53 and p21Cip1
, and (d) inhibites cell proliferation. Thus, our results provide the evidence that inducible expression of TRB3 by homocysteine-induced ER stress is responsible for cell growth arrest.
TRB3 is a mammalian homologue of the Drosophila Tribbles, a protein identified as a regulator of cell cycles in Drosophila 
. It has been shown that Tribbles mediates the degradation of string/CDC25 by the ubiquitin-dependent pathway in Drosophil
. However, there is no evidence that TRB3 is involved in protein degradation in mammals 
. TRB3 has been shown to interact directly with Akt and suppress the phosphorylation of this kinase in livers 
. A recent study has confirmed that TRB3 is a novel target gene of CHOP involved in the coordination of apoptosis during ER stress 
. Our recent study indicates that expression of TRB3 is elevated in livers of mice with hyperhomocysteinemia 
. In the current study, homocysteine induces TRB3 expression through the ER stress pathway in cultured hepatocytes. Knockdown of TRB3 expression by shRNA partially blunts cell growth arrest in HepG2 cells. Thus, TRB3 is a critical component in homocysteine-mediated cell growth arrest in hepatocytes. He et al. 
have reported that both the protein and the mRNA levels of TRB3 are elevated in hepatic tissues in rats fed with ethanol. The mechanisms underlying ethanol effects on TRB3 expression remain unclear. Since ethanol also induces hepatic dysfunction through the ER stress pathway, it is likely that upregulation of TRB3 is also mediated by CHOP.
It has been well established that the PI3K/Akt signaling pathway is important to promote cell survival and proliferation 
. Suhara et al. 
have reported that homocysteine significantly inhibits Akt activity in endothelial cells. Activation of Akt by infection of adenoviral construct expressing the constitutively active form of Akt reversed endothelial apoptosis induced by homocysteine. Although the mechanism underlying homocysteine-mediated inactivation of Akt remains unknown, these results suggests that the Akt-signaling is a novel target for homocysteine-induced endothelial cytotoxicity in vitro. In the current study, we demonstrate that homocysteine reduces phosphorylation of Akt (Ser473) through a TRB3-dependent pathway in hepatocytes, indicating that cell proliferation inhibited by homocysteine results from inactivation of Akt. The fact that LiCl significantly suppresses cell growth arrest and dephosphorylation of Akt induced by homocysteine supports this view.
We observe that the protein levels of p53 are increased after homocysteine treatment in hepatocytes and HepG2 cells. In contrast, homocysteine has no effect on cell proliferation in Hep3B, which does not express p53 gene. Furthermore, inhibition of p53 activity by its inhibitor pifithrin-α or knockdown of p53 expression significantly suppresses homocysteine-induced cell growth arrest. The fact that Akt can phosphorylate MDM2 and enhance MDM2-mediated degradation of p53 by ubiquitination 
may provide a ready explanation of our observation that homocysteine up-regulates protein levels, but not mRNA levels, of p53. p21Cip1
, which is a target gene of p53, plays a role in the G1 transition by inhibiting cyclin E/Cdk2 
. Homocysteine markedly induces the expression of p21Cip1
in HepG2, but not in Hep3B, indicating that p21Cip1
is the downstream molecule of p53 in homocysteine-induced cell growth arrest. Consistent with our in vitro results, a significant increase in expression of p53 and p21Cip1
is observed in livers of mice with hyperhomocysteinemia 
. Thus, p53/p21Cip1
pathway is essential in homocysteine-induced cell growth arrest. Like p21Cip1
is also associated with a cell-cycle arrest through its inhibitory effect on cyclin E/Cdk2 complexes 
. Although homocysteine up-regulates the expression of p27kip1
, it does not affect cellular proliferation in Hep3B cells. These results rule out a role of p27kip1
for this process.
In the current study, marked induction of ER stress and cell arrest requires supra-physiological concentrations of homocysteine (0.5–1.0 mM) in vitro
. Previous studies also demonstrate that the concentrations of homocysteine to produce significant effects in vitro
and in vivo
are different 
. The reasonable explanation is that the conditions of in vitro
studies are different from physiologic conditions 
. Homocysteine has a short half-life time in vitro
, whereas the level of homocysteine is constant for a long time in vivo
. Furthermore, it has been shown that a twofold to sixfold transient increase (approximately 4–12 µmol/g) in intracellular homocysteine, requires an extracellular homocysteine concentration of 1 to 5 mM in HepG2 cells 
. Thus, Zhang et al. have suggested that it is not the extracellular but the intracellular level of homocysteine that causes ER stress 
In conclusion, our study demonstrates that homocysteine inhibits hepatocyte proliferation during hepatic regeneration. Homocysteine up-regulates TRB3 expression through the ER stress pathway, which in turn inhibits the phosphorylation of Akt, resulting in an increase in the protein levels of p53. p53 induces the expression of p21 Cip1, thus inhibiting hepatocyte proliferation. These results may enhance our understanding of the direct link between hyperhomocysteinemia and hepatic dysfunction.