Background and Aims: Liver regeneration is a biological process essential for the restoration of liver mass, homeostasis and function after an injury. During liver regeneration, proteolytic activity is timely regulated in infiltrating and liver resident cells depending on the cellular demands and participates in apoptosis, proliferation and the activation and repression of growth factors, amongst others. Despite our growing understanding of the roles played by lysosomal proteases in liver disease, our knowledge of their role in liver regeneration remains still very limited. Thus, the aim of this study was to analyse cathepsin D (CtsD) cell-specific role in hepatocytes during liver regeneration. Methods: We generated and validated a novel knock-out mouse strain by breeding Albumin-Cre (hepatocytes) mice with CtsD floxed mice. Partial hepatectomy (PhX) was performed for 72 hours in CtsDF/F and CtsDDHep mice. Liver damage was determined by serum ALT. CtsD deletion was assessed by IF and RTPCR. CtsD activity was determined by an enzymatic assay. Mitotic bodies and BrdU positive cells were analysed and quantified in liver tissue sections using Fiji software. Gene expression for apoptotic and cell cycle markers was determined by RT-qPCR in total liver. Results: CtsD cell-specific deletion in hepatocytes was validated by CtsD WB in hepatocytes and IF in liver tissue section from CtsDDHep mice. Of note, CtsD expression remained unaffected in liver non-parenchymal cells in CtsDDHep mice. Next, PhX was performed for 72h and CtsD deletion in CtsDDHep livers was confirmed by gene expression and IF. Despite CtsD deletion in hepatocytes did not affect liver damaged (ALT), the hepatic regeneration rate or the number of mitotic bodies, the liver BrdU incorporation was significantly increased in CtsDDHep mice after PhX indicating an increase in cell proliferation. In agreement, gene expression analysis of PhX livers revealed a significant increase of genes involved in proliferation and cell cycle, such as FoxMB1 and Cyclin B1, without changes in apoptotic genes (BCL2, NOXA, BAK1 and FADD) in PhX-CtsDDHep mice versus PhX-CtsDF/F ones. Conclusion: CtsD contributes to the control of hepatocyte proliferation and cell cycle during liver regeneration after partial hepatectomy.

Resumen del trabajo presentado en el XXXIII Congrés de la Societat Catalana de Digestologia, celebrado en Lleida, del 25 al 27 de enero de 2024