UCSF

Chronic liver disease is characterized by persistent hepatocellular injury, chronic inflammation, extracellular matrix remodeling, increased tissue rigidity, and activation of cellular pathways that contribute to fibrosis, cirrhosis, hepatocellular carcinoma, and ultimately liver failure. Although the molecular mechanisms regulating liver injury have been investigated extensively, less is known about how hepatocytes sense and respond to the changing mechanical environment that accompanies the progression of fibrosis. Increasing evidence suggests that increased extracellular matrix stiffness functions not only as a consequence of fibrosis but also as an active regulator of cellular behavior and function through mechanotransduction. This dissertation advances our understanding of hepatocyte mechanobiology through three aims focused on identifying mechanosensitive pathway components that mediate hepatocyte dysfunction in a stiffened matrix and characterizing the biomechanical environment in which these pathways operate in human liver.Aim 1 investigates hepatocyte-specific Rho-associated coiled-coil containing protein kinase (ROCK) signaling in a murine model of porphyria-associated liver disease. Hepatocyte-specific deletion of both ROCK isoforms, ROCK1 and ROCK2, followed by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-mediated liver injury, demonstrated that ROCK signaling is essential for hepatocyte survival during chronic hepatic injury. Mechanistic analyses revealed a significant increase in cyclin-dependent kinase inhibitor 1A (p21) expression accompanied by caspase-3 activation, suggesting cell-cycle arrest, impaired proliferative responses, and enhanced apoptosis in ROCK-deficient hepatocytes. This work establishes ROCK as a critical regulator of hepatocyte adaptation and survival during injury. Aim 2 examines protein kinase C-related kinase, also known as Protein Kinase N (PKN), another downstream effector of the RhoA GTPase involved in cytoskeletal regulation, proliferation, and migration. Building on observations from the ROCK studies, small-molecule inhibition using Y-27632 improved the expression of Hepatocyte Nuclear Factor 4 Alpha (HNF4α), an important master transcriptional regulator of hepatocyte identity and function, in hepatocytes seeded on a stiff matrix. Studies have shown that Y-27632 targets both ROCK and PKN with similar potency, motivating our investigation of the role of PKN in hepatocyte mechanotransduction. Using additional small-molecule inhibitors of PKN and siRNA-mediated knockdown, we found that PKN2 inhibition improved the expression of HNF4α and several of its target genes while decreasing the expression of Yes-associated protein (YAP) target genes involved in hepatocyte dedifferentiation. Aim 3 characterizes the mechanical properties of normal and fibrotic human liver tissue using atomic force microscopy (AFM). Liver specimens obtained from a patient with hepatitis C virus infection and from a healthy control liver were analyzed. Direct measurements demonstrated substantial mechanical heterogeneity within the fibrotic liver, with increased stiffness in fibrotic septa, compared with the lower stiffness values observed in normal liver. These findings establish the biomechanical landscape encountered by hepatocytes under normal conditions and during chronic liver disease and provide a physiologically relevant context for mechanosensitive signaling pathways. They also establish a protocol for future studies comparing healthy and fibrotic human liver tissue across different disease etiologies and patient demographics. Together, these studies support a model in which the impact of chronic liver injury on hepatocytes is governed by interactions between extracellular mechanical forces and intracellular signaling networks. Progressive fibrosis alters the biomechanical properties of the liver, while Rho-dependent pathways, including ROCK and PKN, enable hepatocytes to sense, interpret, and respond to these changing conditions. By integrating mechanistic studies of hepatocyte signaling with direct measurements of human liver biomechanics, this dissertation advances our understanding of the role of hepatocyte mechanosignaling in a stiffened environment, identifies pathways that may contribute to chronic liver disease progression, and identifies PKN2 as a potential target for preserving hepatocyte identity and function in chronic liver disease.