Inhibition of Phosphoinositide 3‐kinase delta (PIK3CD) Suppresses Hepatocyte Proliferation by More than 50% in the Regenerating Liver after Partial Hepatectomy

Abstract

Background and Objective The extracellular matrix (ECM) is important for survival, differentiation, and normal functioning of cells within the liver; integrins are key signaling receptors in this process. Previous data from our lab has shown that with hepatocyte specific knockout of integrin linked kinase (hILK KO), there is hepatocyte proliferation, increased matrix deposition, and unorganized biliary cell/ductal proliferation; this led us to investigate the signaling pathways downstream of hILK KO that might be responsible for the observed phenotype. Through this, we uncovered a possible central role of Phosphoinositide 3‐kinase (PI3K) delta (PIK3CD), a protein thought to be immune specific and absent in hepatocytes. The objective of this study was to determine the role PIK3CD has in the liver. From literature searches and our initial data collection, our hypothesis was that the inhibition of PIK3CD would suppress hepatocyte proliferation. Methods Data array analyses were performed on livers from 7‐ and 14‐week old chronic hILK KO and WT mice. Partial Hepatectomies (PHx) were performed on C57BL/6J mice injected IP with vehicle control or the PIK3CD inhibitor, Cal‐101, at 10mg/kg, two days prior to PHx, and every day following until harvest. Days 0, 1, 2, 4 and 7 post‐surgery were examined through immunohistological and western blot analyses. RNA was generated and pooled (n=6) for each condition and timepoint and sent out for analyses. Additionally, in vitro cell culture was performed using primary hepatocytes isolated from hILK WT mice. Hepatocytes were seeded at 300K cells per well, with media changed every other day and treated with or without 5uM CAL‐101 over the course of 6 days. BrdU was added 2 hours prior to harvest of cells to assess proliferation. Results Data array analyses on ILK KO and WT animals by IPA identified PIK3CD as common to several pathways in the ILK KO mice. WT mice that were treated with the PIK3CD inhibitor, Cal‐101, exhibited a significant decrease in hepatocyte proliferation on days 1, 2, 4, and 7 after PHx, compared to vehicle control, through quantification of nuclear Ki67 staining. By day 7, proliferation subsided in both the control and Cal‐101 mice and liver to body weight ratios were similar. Additionally, western blot analyses revealed differences in p‐AKT and p‐ERK on days 0,1, and 2 after PHx, as well as changes in p‐MET at days 0 and 1. In vitro culture data also supported a role for PIK3CD in hepatocyte proliferation as a significant decrease was observed in primary hepatocytes treated with Cal‐101 compared to control over a 6‐day culture period. Conclusion This data shows a previously unknown essential role for PIK3CD in controlling hepatocyte proliferation in the regenerating liver.