p-Ezrin (Thr567) regulates Hippo pathway and Yap in liver

It has been shown that up regulation activity of CD81(TAPA-1, the portal of entry of Hepatitis C virus) by agonistic antibody results in phosphorylation of Ezrin. We have previously shown that in liver, Ezrin phosphorylation occurs via Syk kinase, causing suppression of hippo intensity, therefore increases sequential Yap activity. The opposite occurs when Glypican-3 (GPC3) or E2 protein of HCV bind to CD81. Mice over-expressing GPC3 in hepatocytes have decreased p-Ezrin(Thr567) and Yap, increased Hippo activity and suppressed liver regeneration. The role of Ezrin in these processes has been speculated, but not proven. We now provide dynamic picture of Ezrin regulates Hippo pathway and Yap. Forced expression of plasmids expressing mutant Ezrin (T567D) (which mimics p-Ezrin(Thr567)) suppressed Hippo activity and activated Yap signaling. And this mutant Ezrin drive more cell proliferation to cell division through up regulated Yap activity in vitro and in vivo. CD81 loses expression, while p-Ezrin(Thr567) increases in JM1 and JM2 hepatocellular carcinoma (HCC) cells. Administration with compound NSC668394, a characterized p-Ezrin(Thr567) antagonist, caused significant decrease in HCC cell proliferation. We additionally present evidence that pEzrin(T567) is also controlled by EGFR and MET. Conclusions: Ezrin phosphorylation, mediated by CD81 associated Syk kinase, is directly involved in regulation of Hippo pathway, Yap levels and growth rates of normal and neoplastic hepatocytes. The finding has mechanistic and potentially therapeutic applications in understanding and regulating growth of hepatocytes and HCC and HCV pathogenesis. We used microarrays to detail the global programme of gene expression in GFP positive hepatocytes of FVB mice adminstration with EzrinT567D plasmids Six- to eight-week-old wild-type FVB/N mice (Charles River, Wilmington, MA) were used. For hydrodynamic tail vein injection, PT3EF5α EzrinT567D (10μg) and pCMV/SB (0.8μg) were diluted into 2ml 0.9% NaCl solution, sterile filtered with 0.2μm filters, and then injected into mouse lateral tail vein within 5–7 seconds. Isolation of primary hepatocytes from liver was performed according to the two-step collagenase digestion 1 week after plasmids injection. All fluorescence-activated cell sorting (FACS) analyses were performed with a FACS Aria II-SORP (BD Biosciences, San Jose, CA) equipped with a 130-μm nozzle and running DiVa software version 8.0.2 (BD Biosciences). FACS plots were generated with FlowJo software version 9.9.6 (TreeStar, Ashland, OR). Microarray analysis was performed on RNAs isolated from the GFP - (i.e. EzrinT567D negative) and GFP + (i.e. EzrinT567D positive) group from isolated hepatocytes for study, using the GeneChip® Mouse Genome 430 2.0 Array Set 430 (Affymetrix, Santa Clara, CA).