ABSTRACT
The major life-saving treatment for severe acute liver injury (ALI) leading to acute liver failure (ALF) is a liver
transplant. However, there is an enduring shortage of healthy donor livers. Moreover, even when a suitable
liver can be obtained, transplant recipients face myriad life-threatening risks like graft rejection, infection, renal
failure, and vascular problems, leading to death in 10-20% within a year post-transplant. Clearly, better
treatments are needed. However, poor understanding of the mechanisms of regeneration specifically in ALI is
a major obstacle to achieving that goal. While prior work in tissue regeneration has focused on the roles of
proteins like growth factors and cytokines, our laboratory is interested in the underexplored effects of lipid
second messengers. In particular, our work has revealed that the glycerophospholipid phosphatidic acid (PA)
is a critical regeneration signal in the mouse model of acetaminophen (APAP) overdose, which is the most
common cause of ALF in the US. Our data show that PA mediates regeneration signaling by inhibiting
glycogen synthase kinase 3β (GSK3β), which normally suppresses hepatocyte proliferation and therefore
tissue repair. Now, our objective in this R01 project is to improve understanding of the regeneration process by
determining exactly how PA regulates GSK3β. We hypothesize that PA accumulates specifically in lipid
membranes – especially in the endoplasmic reticulum – after ALI and coordinately recruits GSK3β and liver
kinase b 1 (LKB1) so that LKB1 phosphorylates GSK3β Ser9 and thereby inhibits GSK3β activity, which
removes the brakes on regeneration. We will test our hypothesis in two specific aims. Specific Aim 1: Detail
the temporal, zonal, and subcellular localization and physical interactions of PA and GSK3β. Our prior
work shows that PA deactivates GSK3β during regeneration after acetaminophen (APAP)-induced ALI. In
addition, our preliminary data indicate that GSK3β can directly bind to PA. Here, we will use mass
spectrometry imaging (MSI), triple quadrupole mass spectrometry, immunohistochemistry, and lipid-binding
tests to characterize the location and binding of PA and GSK3β in hepatocytes in vitro and after APAP
hepatotoxicity in mice. Specific Aim 2: Define the role of LKB1 in GSK3β phosphorylation and liver
regeneration in the mouse model of APAP-induced ALI. Our data show that LKB1 co-precipitates with
GSK3β. Furthermore, other groups have demonstrated that LKB1 binds to PA. Thus, it is highly plausible that
binding to PA brings LKB1 and GSK3β together. We will use similar methods from Specific Aim 1 to describe
the distribution of LKB1 during regeneration. We will then use genetic approaches to disrupt LKB1 signaling in
mice and explore the effect on both GSK3β Ser9 phosphorylation and liver regeneration in APAP-induced ALI.
Finally, we will assess the relative contributions of PA signaling and conventional Wnt signaling for regulation
of β-catenin. Overall, the critical mechanistic insights produced in this project will be valuable to develop new
therapeutic approaches for ALF, as well as other high-priority diseases involving GSK3β.