Genetic or pharmacological inhibition of cyclin-dependent kinase 2 in Hepatic Stellate Cells acts anti-fibrotic

Question:

Liver fibrosis is a wound healing process in response to chronic liver injury, characterized by regenerative proliferation of hepatocytes and the accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of the normally quiescent HSCs controlled by cyclins and associated cyclin-dependent kinases (Cdks). Cdk2 mediates the entry and progression through S-phase (i.e. DNA synthesis) in complex with E-and A-type cyclins. We have recently demonstrated that Cyclin E1 is essential for liver fibrogenesis in mice. However, so far it was not known, if this function was dependent on Cdk2. Thus, in this study we tested the requirement for Cdk2 and related kinases in HSCs.

Methods:

We generated conditional, HSC-specific Cdk2 knockout mice using transgenic cre-expression under control of the L-rat promoter. These mice were challenged with CCl4 for 6 weeks and subsequently investigated for liver fibrosis. For pharmacological in vitro analyses we used the pan-Cdk inhibitor CR8 with highest specificity against Cdk2 and Cdk1. This drug was tested for anti-fibrotic and cytotoxic properties on human (LX-2) and murine (GRX) HSC cell lines as well as on primary murine HSCs and hepatocytes.

Results:

Genetic ablation of Cdk2 specifically in HSCs significantly reduced collagen accumulation in the liver after CCl4 treatment. This suggests that cell cycle re-activation of naïve HSCs in vivo requires functional Cdk2 presumably in complex with Cyclin E1. In order to translate these findings into a pre-clinical therapeutic model, we challenged human LX-2 and murine GRX cells with the pan-Cdk inhibitor CR8. CR8 treatment substantially reduced Cdk kinase activity in both cell lines. In addition CR8 inhibited proliferation, survival and pro-fibrotic activation in both LX-2 and GRX cells, and also triggered DNA damage and cell cycle arrest. Importantly, we identified effective CR8 dosages mediating anti-fibrotic effects in primary murine HSCs without affecting cell cycle activity and survival in primary hepatocytes.

Conclusion:

The pro-fibrotic properties of HSCs depend on functional Cdk2 in vivo. In addition, pharmacological pan-Cdk inhibition reduced the fibrogenic functions of HSC lines and primary HSCs without affecting the regenerative capacity of hepatocytes in vitro. We thus conclude that HSC-specific inhibition of Cdk2 could be a novel therapeutic approach to treat liver fibrosis.