IKK2 hepatocyte specific deletion alters cell cycle progression during liver regeneration

Aims: The liver is the only organ able to restore its mass and function after liver injury. NF-kB is involved in many cellular responses. In non-stimulated cells, NF-kB is sequestred in the cytoplasm by the inhibitory protein IkB. Upon stimulation, IkB is phosphorylated by the IKK complex, allowing NF-kB activation and translocation in the nucleus. The IKK complex consists of two catalytic subunits IKK1 and IKK2, and the regulatory subunit NEMO.

Aim: We studied the impact of hepatocyte-specific IKK2 deletion on liver regeneration as NF-kB is involved in early events of hepatocyte proliferation.

Methods: IKK2 constitutive knock-out mice die embryonically. Thus, we generated C57/BL6 hepatocyte-specific IKK2 knock-out (IKK2Δhepa) mouse using the Cre/LoxP system. 70% partial hepatectomy (PH) was performed on IKK2f/f mice (wild-type) and IKK2Δhepa mice (knock-out), and liver regeneration was studied.

Results: All mice survived after PH. In order to understand the role of IKK2 deletion during liver regeneration, we first analysed the priming phase. TNF mediated priming of hepatocytes and its ability to induce extracellular matrix remodelling via MMP9, are key events during liver regeneration. TNF and MMP-9 were expressed earlier in IKK2Δhepa mice compared to IKK2f/f mice, suggesting an earlier priming in the knock-out animals. Accordingly, BrdU analysis showed earlier proliferation in IKK2Δhepa mice compared to IKK2f/f mice. Real Time PCR and Western Blot analysis of cyclin D, E and A further confirmed this finding, as all the cyclins were expressed earlier in IKK2Δhepa mice compared to IKK2f/f mice. Unexpectedly, no difference in cyclin B expression was observed. The cyclin B-dependent kinase protein CDC2 is a G/2M activator and western blot analysis of this protein showed a reduced CDC2 activity in IKK2Δhepa mice assuming a mitosis miregulation. In addition, phospho histone H3 immuno-histochemistry showed a prolonged mitotic phase in IKK2Δhepa mice compared to IKK2f/f mice.

Conclusions: Our data demonstrate that hepatocyte-specific IKK2 deletion alters cell cycle progression by triggering an earlier priming phase and proliferative response, while the mitosis phase is misregulated.