Expression of Cyclin E1 and Cdk2 in Hepatic Stellate Cells is critical for initiation and progression of liver fibrosis in mice

Background and aims Liver fibrogenesis is a wound healing process characterized by the accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). Initiation of liver fibrosis involves cell cycle re-entry and thus activation and proliferation of normally quiescent HSCs. It is believed that the cyclin-dependent kinase 2 (Cdk2) together with its regulatory subunit Cyclin E controls cell cycle re-entry. We have recently shown that constitutive ablation of Cyclin E1 (CcnE1) in mice inhibited liver fibrogenesis. However, the effector cells of CcnE1 during fibrogenesis are not known so far and it has also not been clarified yet, whether the pro-fibrotic effect of CcnE1 depends on the kinase activity of Cdk2. Thus the aim of the present study was to evaluate the contribution of CcnE1 and Cdk2 specifically in HSCs for liver fibrogenesis. Methods We generated HSC-specific knockout mice for CcnE1 (CcnE1 ΔHSC ) and Cdk2 (Cdk2 ΔHSC ) by crossing floxed ( i.e. CcnE1 f/f /Cdk2 f/f ) mice with transgenic mice expressing cre-recombinase under the control of the L-rat promoter. Liver fibrosis was induced by treating mice with a combination of Diethylnitrosamin (DEN) and CCl 4 or with CCl 4 only according to established protocols. For pharmacological inhibition of Cdk2 kinase activity the pan-Cdk inhibitor CR8 was applied in vitro on HSC cell lines (LX-2, human; GRX, murine) and in primary murine HSCs. Results Genetic ablation of Cdk2 in HSCs significantly reduced fibrogenesis in the liver after CCl 4 treatment when compared to cre-negative littermates. Accordingly, Cdk2 ΔHSC mice showed a significantly reduced HSC activation in the liver. Similarly, CcnE1 ΔHSC mice revealed significantly reduced liver fibrosis after DEN/CCl 4 treatment. In good agreement with these findings, treatment with CR8 inhibited kinase activity, proliferation, survival and pro-fibrotic activation of HSCs ( i.e. primary cells and cell lines) in vitro . Conclusion The pro-fibrotic properties of HSCs depend on functional Cdk2 and CcnE1. This suggests that cell cycle re-activation of naïve HSCs in vivo requires functional CcnE1/Cdk2 kinase activity. We conclude that pharmacological inhibition of Cdk2 kinase activity in HSC could be an alternative approach to treat liver fibrosis. Publication History Article published online: 08 September 2020 © Georg Thieme Verlag KG Stuttgart · New York