Friday, July 26, 2024
7/26/2024
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.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
