Amino acids regulate hepatocyte proliferation through modulation of cyclin D1 expression.

The mechanisms by which amino acids regulate the cell cycle are not well characterized. In this study, we examined the control of hepatocyte proliferation by amino acids and protein intake. In short-term culture, hepatocytes demonstrated normal entry into S phase and cell cycle protein expression in the absence of essential amino acids. However, deprivation of a set of nonessential amino acids (NEAA) potently inhibited cell cycle progression and selectively down-regulated the expression of proliferation-control proteins. Notably, NEAA withdrawal after the mitogen restriction point still inhibited entry into S phase, suggesting that these amino acids regulate a distinct checkpoint. Cyclin D1, an important mediator of hepatocyte proliferation, was markedly inhibited at the transcriptional level by NEAA deprivation, and transfection with cyclin D1 (but not cyclin E) overcame the cell cycle arrest. As previously shown, protein-deprived mice demonstrated impaired hepatocyte proliferation in vivo after 70% partial hepatectomy. The expression of cyclin D1 and downstream cell cycle proteins after partial hepatectomy was inhibited in these mice. Transfection with cyclin D1 in vivo triggered hepatocyte DNA synthesis and the expression of S phase proteins in the absence of dietary protein. Cyclin D1 also induced global protein synthesis in NEAA-deprived hepatocytes and promoted liver growth in vivo in the setting of protein deprivation. These results indicate that cyclin D1 is a key target of amino acid signaling in hepatocytes.

Evidence that cyclin D1 mediates both growth and proliferation downstream of TOR in hepatocytes.

Signaling through the target of rapamycin is required for increased protein synthesis, cell growth, and proliferation in response to growth factors. However, the downstream mediators of these responses, and the elements linking growth and proliferation, have not been fully elucidated. Rapamycin inhibits hepatocyte proliferation in culture and liver regeneration in vivo. In cultured rat hepatocytes, rapamycin prevented the up-regulation of cyclin D1 as well as proteins acting downstream in the cell cycle. Transfection with cyclin D1 or E2F2, but not cyclin E or activated Akt, overcame the rapamycin-mediated cell cycle arrest. Rapamycin also inhibited the induction of global protein synthesis after growth factor stimulation, and cyclin D1 overcame this inhibition. Rapamycin inhibited hepatocyte proliferation and cyclin D1 expression in the mouse liver after 70% partial hepatectomy. In rapamycin-treated mice, transfection with cyclin D1 induced hepatocyte proliferation, increased hepatocyte cell size, and promoted growth of the liver. These results suggest that cyclin D1 is a key mediator of increased protein synthesis, cell growth, and proliferation downstream of target of rapamycin in mitogen-stimulated hepatocytes.

Differential regulation of cyclins D1 and D3 in hepatocyte proliferation.

Substantial evidence suggests that cyclin D1 plays a pivotal role in the control of the hepatocyte cell cycle in response to mitogenic stimuli, whereas the closely related protein cyclin D3 has not been extensively evaluated. In the current study, we examined the regulation of cyclins D1 and D3 during hepatocyte proliferation in vivo after 70% partial hepatectomy (PH) and in culture. In contrast to cyclin D1, which was nearly undetectable in quiescent liver and substantially up-regulated after PH, cyclin D3 was constitutively expressed and induced only modestly. In the regenerating liver, the concentration of cyclin D3 was only about 10% of that of cyclin D1. Cyclin D1 formed complexes primarily with cyclin-dependent kinase 4 (cdk4), which were markedly activated in the regenerating liver and readily sequestered the cell cycle inhibitory proteins, p21 and p27. Cyclin D3 bound to both cdk4 and cdk6. Cyclin D3/cdk6 activity was readily detectable in quiescent liver and changed little after PH, and this complex appeared to play a minor role in sequestering p21 and p27. In cultured hepatocytes, epidermal growth factor or insulin had little effect, but the combination of these agents substantially induced cyclin D1 and cell cycle progression. Inhibition of Mek1 or phosphoinositide 3-kinase markedly inhibited cyclin D1 expression and replication. In contrast, cyclin D3 was expressed in the absence of mitogens and was only modestly affected by these manipulations. In addition, growth-inhibitory extracellular matrix conditions inhibited cyclin D1 but not cyclin D3 expression. In conclusion, these results support the concept that cyclin D1 is critically regulated by extracellular stimuli that control proliferation, whereas cyclin D3 is regulated through different pathways and plays a distinct role in the liver.