3A ). Twenty-four hours after PH, the levels of p-EGFR, p-ERK1/2, and p-AKT appeared to be further elevated in mig-6 knockout mice, indicating that the enhanced JNK inhibitor hepatocyte proliferation at these early time points might be due to amplified EGFR signaling (Fig. 3A,B). Notably, at the 36-hour time point, the levels of p-ERK1/2 declined, whereas EGFR and AKT remained activated in mig-6 knockout livers, suggesting that hepatocyte proliferation might be driven by EGFR-AKT signaling. In addition, we found that total EGFR protein levels were increased in mig-6 knockout mice, suggesting that loss of mig-6 enhances EGFR protein

stability (Fig. 3A,B). Interestingly, EGFR up-regulation seems to occur through a posttranslational mechanism, because EGFR messenger RNA levels were unchanged in mig-6 knockout and wild-type animals (Fig. 3C). In addition, we found increased levels of p-Rb in the regenerating liver of mig-6 knockout mice (Fig.

3A), which might stimulate the expression of genes required for S-phase entry. Furthermore, elevated activity of the activator protein-1 transcription factor c-Jun, which is known to be a key regulator of liver regeneration,21 was detected in mig-6 knockout livers. Interestingly, the EGFR ligand HB-EGF but not TGFα is up-regulated at the transcriptional level at 0, 24, and 36 hours after PH (Fig. 3C), suggesting that HB-EGF may activate the EGFR. Because mig-6 is known to be a negative regulator of all EGF receptors, we examined the expression levels of ErbB2, ErbB3, GSK-3 inhibitor and ErbB4 in regenerating mig-6 knockout and wild-type livers. In line with published data,22 we could not detect ErbB2 nor ErbB4 expression, whereas ErbB3 was weakly expressed (data not shown) suggesting that mig-6 is a specific negative regulator of EGFR signaling in hepatocytes. Notably, 48 hours after PH the activation of the EGFR pathway is comparable between knockout and wild-type control mice

(Fig. 3A-C), suggesting Linifanib (ABT-869) that the EGFR is eventually inactivated by a mig-6–independent mechanism and that mig-6 is dispensable for EGFR regulation at later time points during liver regeneration. To study the effect of mig-6 on EGFR function in human liver cancer cell lines, we stimulated HepG2 and Hs 817.T cells with EGF for the indicated time points (Fig. 4A ). EGF stimulation led to a strong and continuous induction of mig-6 expression (Fig. 4A). Interestingly, mig-6 induction correlates with a rapid decrease in EGFR phosphorylation and expression, as well as a reduction in p-ERK1/2 levels. Importantly, mig-6 is able to bind to the activated form of the EGFR, thereby most likely regulating EGFR activity (Fig. 4B). To better understand the role of mig-6 in human liver cancer cell lines, we down-regulated mig-6 by specific siRNAs in HepG2 cells and examined EGFR signaling. Suppression of mig-6 led to elevated EGFR activity upon EGF stimulation (Fig. 5A ).