10 Therefore, we undertook a comprehensive approach to determine potential roles of eNOS

in hepatocyte proliferation by conducting a systematic analysis of hepatocyte proliferative response, based on the sequential induction of key cyclins that drive cell-cycle Y-27632 cell line progression at the G1, G1/S, and G2/M phases, as well as BrdU-incorporation kinetics over 24-96 hours. Corresponding to early events, hepatocyte cell-cycle progression and proliferation in response to PH were significantly attenuated in eNOS−/− livers. Most notably, cyclin E and cyclin A protein induction in response to PH were dependent on eNOS expression. Peak BrdU incorporation, reflecting active DNA synthesis, was observed at 45 hours post-PH, which was significantly attenuated in eNOS−/− mice. It should be noted that analysis of BrdU incorporation over extended post-PH periods confirms the lack of compensatory or delayed DNA synthetic responses in eNOS−/− livers and highlights the functional significance of eNOS in hepatocyte proliferation in response to PH. We did not

observe any compensatory increase in iNOS protein or mRNA expression in eNOS−/− livers subjected to PH. Our studies confirm C646 chemical structure previous observations that the iNOS isoform does not compensate for eNOS deficiency in regenerating livers.25 Despite distinct impairments in hepatocyte proliferation, liver growth at 8 days post-PH was comparable between WT and eNOS−/−

livers. During liver regeneration, numerous cytokines, growth factors, and transcription factors are activated and function synergistically, which, in turn, initiate and promote hepatocyte proliferation, angiogenesis, and organ growth.1, 2, 26 Our findings validate the current consensus that multiple redundant mechanisms are in place to ensure liver regeneration in response to hepatic insufficiency, and that no single factor is independently sufficient to induce and complete liver regeneration. Several factors may account for attenuated hepatocyte proliferation in eNOS−/− livers. First, efficient transcriptional activation of cyclins, such as cyclins D1, E, and A, are dependent on c-Jun/AP-1 activity. Second, the ERK-signaling Astemizole cascade is involved in the regulation of G1 phase progression in liver regeneration.27 The present study reveals a novel role of eNOS-mediated signaling on ERK activation in regenerating livers, as evidenced by impaired MEK/ERK signaling in eNOS−/− mice early (5 minutes) after liver resection. Third, early activation of MMPs, such as MMP-9, is essential for the dissolution of ECM and proteolytic activation latent growth factors, such as HGF.28-30 Interestingly, our findings suggest that eNOS signaling influences the early induction of MMP-9 in regenerating livers, as evidenced by the delay and dysregulation of MMP-9 protein expression in the remnant livers of eNOS−/−.