Donghun Shin, Ph.D. - US grants

DESCRIPTION (provided by applicant): Chronic liver diseases are the 12th leading cause of mortality and among the most common causes of morbidity in the U.S. with 5.5 million people suffering from the diseases. The liver has an enormous capacity to regenerate itself, but this capacity is greatly reduced in the diseased liver, making liver transplantation the only effective treatment for end-stage chronic liver diseases. The shortage of donor livers however makes this therapy extremely limited, thus necessitating alternative therapies. Augmenting innate liver regeneration can be an alternative therapy because it may mitigate the diseases and improve the quality of life. During innate liver regeneration, regenerated hepatocytes can be derived from preexisting hepatocytes or biliary epithelial cells (BECs). BEC-driven liver regeneration occurs when hepatocyte-driven liver regeneration is compromised, which is the case in patients with chronic liver diseases. It appears that BEC-driven liver regeneration was initiated but failed to b complete in the patients. Understanding of the entire process of BEC-driven liver regeneration should provide significant insights into how to complete this process in liver patients as therapeutics. Thus, we developed an innovative zebrafish liver regeneration model in which regenerated hepatocytes are exclusively derived from BECs. Using this model, we found that pharmacological inhibition of Bmp signaling impaired BEC-driven liver regeneration. Based on our preliminary studies, we hypothesize that Bmp signaling plays multiple roles in BEC-driven liver regeneration. We will test this hypothesis by pursuing the following three specific aims. Aim 1: We will delineate temporal characteristics of BEC-driven liver regeneration in our zebrafish model by testing our working hypothesis: BECs first proliferate, dedifferentiate into hepatoblast-like cells, the equivalent of oval cells in rodent liver injury models, and then redifferentiate ino hepatocytes that actively proliferate to recover liver mass. Aim 2: We will determine the roles of Bmp signaling in BEC-driven liver regeneration, by blocking or enhancing Bmp signaling at distinct time-windows during hepatocyte ablation and liver regeneration and by examining BEC-driven liver regeneration in smad5 mutants. Aim 3: We will determine the role of Id2a, a member of the inhibitor of differentiation family of transcription factors, which is known to be the direc target of Bmp signaling in several tissues, in BEC-driven liver regeneration by testing the working hypothesis that Id2a mediates the effect of Bmp signaling on BEC-driven liver regeneration. The accomplishment of the proposed work will significantly advance the field of liver regeneration by revealing the mechanisms by which Bmp signaling regulates BEC-driven liver regeneration. Furthermore, they will provide novel insights into how to augment and complete BEC-driven liver regeneration in patients with chronic liver diseases as therapeutics.

This project will map disease pathways for biliary atresia (BA), which has failed all therapy and requires liver transplantation (LTx), using genetic susceptibility as a common basis. BA causes liver failure at birth and accounts for up to half of all pediatric LTx, worldwide, but has an uncertain etiology. BA leads to scarred and atretic bile ducts, which fail to drain the liver. Some children also have anomalous left-right patterning of extrahepatic organs. Preliminary genome-wide association study (GWAS) of single nucleotide polymorphisms (SNPs) in BA cases, and other evidence from human BA and experimental models lead us to propose the following specific aims: Aim 1. We will confirm whether BA associates with SNPs in manosidase-1-?-2 (MAN1A2), and in genes which signal via hedgehog, epidermal and transforming growth factors and genes for ciliogenesis. These pathways are abnormal in liver from BA cases, zebrafish with knockdown of man1a2, and Man1a2 -/- (knockout) mice. We will identify novel potentially causal variants with targeted sequencing of significant SNP loci. Aim 2. We will confirm whether knockdown of man1a2 and other candidates induces biliary dysgenesis and cardiac and hepatic heterotaxy in zebrafish, impaired ciliogenesis in the ex vivo mouse airway epithelia model of ciliogenesis, and dysregulation of the abovementioned developmental pathways in the liver transcriptome. Aim 3. We will construct putative pathways for BA with those significant SNP loci and dysregulated genes which show interactions in an integrated systems analysis of data from Aims 1 and 2. These loci will also implicate aberrant inflammatory and stress responses. We will corroborate pathways with combined perturbations of a developmental gene with an inflammatory and a stress response gene in zebrafish and ex vivo ciliogenesis models. We expect that aberrant developmental, inflammatory and stress response signaling contributes to hepatic and extrahepatic BA. We will genotype the largest homogeneous cohort of 800 Caucasian BA cases with LTx from three of the world's largest pediatric LTx centers: the Children's Hospital of Pittsburgh, King's College Hospital, London, UK, Birmingham Children's Hospital, UK. We will perform whole genome transcriptome (RNA) sequencing of liver samples from 80 of these cases. We will use experimental and bioinformatics resources of the Universities of Pittsburgh and California, San Diego, and the Center for Applied Genomics, Philadelphia. At the end of our project, we will have expanded our knowledge about pathogenesis of BA and related birth defects and identified candidate strategies to prevent or overcome delayed biliary morphogenesis. The international collaboration of leaders in hepatology (Kelly, Dhawan, Squires), transplant surgery (Sharif, Sindhi), genomics (Weeks, Hakonarson, Higgs), pathology (Ranganathan) and systems biology/bioinformatics (Subramanian, Higgs) is well suited to this task.

Abstract Chronic liver diseases are the 12th leading cause of mortality and among the most common causes of morbidity in the U.S. with 5.5 million people suffering from the diseases. Currently, liver transplantation is the only effective treatment for end-stage liver diseases; however, the shortage of donor livers makes this therapy extremely limited, thus necessitating alternative therapies. Promoting innate liver regeneration in chronic liver diseases is an attractive alternative. Upon liver injury, hepatocytes proliferate to yield more hepatocytes to restore lost liver mass and maintain liver function. However, when hepatocyte proliferation is compromised, a phenomenon observed in advanced liver diseases, liver progenitor cells (LPCs) are activated and these LPCs expand and eventually differentiate into hepatocytes. Thus, it is crucial to understand the molecular mechanisms of LPC- driven liver regeneration, which will provide significant insights into promoting this process as a pro-regenerative therapy for advanced liver diseases. Particularly, given the prevalence of LPCs in chronically diseased livers, promoting LPC differentiation into functional hepatocytes will be a promising pro-regenerative therapy. We have established a zebrafish liver injury model in which hepatocyte-specific overexpression of oncogenes induces oncogene-induced hepatocyte damage, such as senescence and apoptosis, followed by inflammation, LPC activation, fibrosis and eventually LPC-mediated liver repair. Using this chronic liver injury model as a screening tool for identifying small molecules that can promote LPC differentiation into hepatocytes, we discovered that treatment with EGFR inhibitors promoted LPC differentiation into hepatocytes, thereby enhancing liver repair/recovery. In addition to the zebrafish model, we have established a mouse liver injury model for LPC- driven liver regeneration. This mouse model allows us to determine if EGFR inhibition can promote LPC differentiation into hepatocytes in mammals as in fish. Here, we propose to determine the effect of EGFR inhibition on LPC differentiation and the role of EGFR signaling in LPC-driven liver regeneration by pursuing three specific aims. Aim 1: Using two zebrafish models of hepatocyte-specific oncogene overexpression, we will elucidate the process of LPC-driven liver regeneration in oncogene-induced liver damage settings. Aim 2: Using the zebrafish and mouse liver injury models for LPC-driven liver regeneration, we will determine the effects of EGFR inhibition on LPC differentiation into hepatocytes and subsequent liver recovery. Aim 3: We will determine the molecular mechanisms controlling LPC differentiation by investigating the role of EGFR and Sox9 in this differentiation process. Successful accomplishment of the proposed work will not only significantly advance the mechanistic understanding of liver regeneration in the diseased liver, but also lay the groundwork for use of EGFR inhibitors as a promising pro-regenerative agent to augment LPC-driven liver regeneration in patients with advanced liver diseases.

Chronic liver diseases are among the leading causes of mortality and morbidity in the U.S., with 5.5 million people suffering from these diseases. Currently, liver transplantation is the only definitive treatment for end-stage liver diseases; however, the shortage of donor livers makes this therapy extremely limited. Augmenting innate liver regeneration in advanced liver diseases is an attractive therapeutic alternative. To develop such a therapy, it is crucial to understand the molecular mechanisms of liver regeneration, particularly in the diseased liver. Upon liver injury, hepatocytes proliferate to yield more hepatocytes to restore lost liver mass and maintain liver function. However, when hepatocyte proliferation is compromised, a phenomenon observed in advanced liver diseases, or when massive hepatocyte necrosis occurs, liver progenitor cells (LPCs) are activated and these LPCs expand and are able to differentiate into hepatocytes. A correlation between disease severity and LPC numbers in patients with chronic liver diseases suggests the occurrence of LPC activation in the diseased livers but its poor differentiation into hepatocytes. In addition, LPCs secrete pro-inflammatory, pro-fibrogenic cytokines that can perpetuate inflammation and contribute to subsequent fibrosis. Thus, augmenting innate LPC-driven liver regeneration is expected to have beneficial effects in liver patients by generating more functional hepatocytes and by concomitantly reducing inflammation and fibrosis. Despite this significance, the molecular basis of LPC- driven liver regeneration remains poorly understood. Our long-term goal is to completely delineate the molecular mechanisms underlying LPC-driven liver regeneration. In pursuit of this goal, during the previous grant cycle, we elucidated the crucial role of bone morphogenetic protein (BMP) signaling in LPC-driven liver regeneration; in this renewal grant application, we propose to determine how the nuclear receptor farnesoid X receptor (FXR) regulates LPC-driven liver regeneration. We have established both zebrafish and mouse liver injury models for LPC-driven liver regeneration. Using the zebrafish model, we performed chemical screening and discovered that treatment with a synthetic FXR agonist, GW4064, impaired LPC-driven regeneration. Given the beneficial effects of FXR agonists on hepatic steatosis, fibrosis, and hepatocyte-driven liver regeneration and the multiple clinical trials of the agonists, their negative effect on LPC-driven liver regeneration is unexpected and surprising, justifying an extensive mechanistic investigation. Based on our preliminary findings, we hypothesize that FXR activation impairs LPC-driven liver regeneration by repressing the PI3K-AKT-mTOR pathway. We will test this hypothesis by elucidating the effects of FXR activation and suppression on LPC-driven liver regeneration (Aim 1) and by determining the role of the PTEN-PI3K-AKT-mTOR axis in the regeneration process (Aim 2). Successful accomplishment of the proposed work will not only significantly advance the mechanistic understanding of liver regeneration in diseased livers, but also support a more cautionary administration of FXR agonists for treating patients with advanced liver diseases.