HIPK2 role in the pathogenesis of liver fibrosis

Fibrosis represents a pathological outcome common to many chronic inflammatory diseases and is characterized by excessive deposition and remodeling of the extracellular matrix (ECM) following dysregulation of physiological wound-healing responses. While transient ECM deposition is required to restore tissue integrity after acute injury, persistent damage leads to sustained matrix accumulation, ECM remodeling, tissue dysfunction, and ultimately organ failure. In liver, fibrosis is a defining feature of chronic liver diseases, which represent a leading cause of morbidity and mortality worldwide. Chronic hepatic injury sustains inflammation and drives a self-perpetuating fibrogenic response involving both activation of hepatic stellate cells (HSCs) into myofibroblasts and qualitative remodeling of the hepatic ECM, which contributes to disease progression by modulating cell behavior, tissue stiffness, and inflammatory signaling. During fibrosis, this remodeling involves both fibrillar collagens, such as collagen I and III, and non-fibrillar collagens, including collagen IV and VI, which regulate basement membrane integrity, matrix architecture, and cell-matrix interactions. Thus, fibrosis progression involves qualitative changes in collagen type and localization that profoundly alter the hepatic microenvironment. Non-alcoholic steatohepatitis (NASH) is a major cause of chronic liver disease and is closely associated with the global rise in metabolic disorders, including obesity, insulin resistance, and type 2 diabetes. Altered hepatocellular lipid handling and lipid droplet (LD) accumulation represent a relevant cellular feature of NASH. Excessive influx of free fatty acids disrupts hepatic lipid metabolism and promotes the accumulation of toxic lipid species, leading to lipotoxicity, organelle dysfunction, and cellular stress. Lipotoxic injury promotes the release of damage-associated molecular patterns, activating innate immune responses and sustaining chronic inflammation that drives fibrogenesis. Despite the clinical relevance of liver fibrosis, the lack of effective treatments makes the identification of molecular factors regulating fibrosis a crucial need. Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase that functions as a transcriptional co-regulator and signal transducer through the phosphorylation of downstream substrates. In addition to its role in stress responses and in controlling cellular processes such as proliferation, apoptosis, and development, HIPK2 has been shown to play divergent roles in fibrotic diseases depending on the organ and pathological context. In kidney and heart, HIPK2 promotes fibrogenesis by enhancing profibrotic signaling pathways leading to increased ECM deposition and tissue remodeling. 9 In contrast, in the lung, HIPK2 overexpression induces fibroblast apoptosis and suppresses pulmonary fibrosis through inhibition of the Wnt/β-catenin signaling pathway. More recently, HIPK2 has been implicated in the regulation of fibrotic microenvironments in early pancreatic neoplasia, where its inactivation reshapes stromal composition and attenuates disease progression. These observations indicate that HIPK2 regulates tissue remodeling in a context-dependent manner. Despite its established roles in other fibrotic and pathological contexts, the contribution of HIPK2 to liver fibrosis remains largely unexplored. Given its role in integrating stress signals and modulating profibrotic pathways and stromal remodeling, HIPK2 represents a potential regulator of liver fibrosis. In this study, we aim to investigate the role of HIPK2 in liver fibrogenesis using complementary in vivo and in vitro approaches. Specifically, we assess the contribution of HIPK2 to the in vivo development of liver fibrosis and determine whether HIPK2 acts as a general regulator of fibrogenesis or mediates etiology-specific mechanisms depending on the underlying cause of liver damage. In parallel, we dissect the cell-autonomous role of HIPK2 in HSC activation and identify HIPK2-dependent mechanisms involved in fibrogenesis. We generated a hepatocyte-specific Hipk2 knockout mouse model (Hipk2-KO) and induced liver fibrosis using two complementary approaches: a choline-deficient amino acid-defined diet supplemented with cholesterol to model chronic metabolic stress, recapitulating features of human NASH, and chronic carbon tetrachloride (CCl₄) administration to induce toxin-mediated hepatocellular injury, driven by oxidative stress and direct hepatocyte damage. These models enabled a direct comparison of HIPK2 function in metabolic versus toxin-driven fibrogenesis. Hipk2-KO mice did not show significant differences in fibrosis induced by chronic hepatotoxic injury. Following CCl₄ treatment, collagen deposition, αSMA expression, and HSC activation were comparable between Hipk2-KO and -WT mice, indicating that HIPK2 is dispensable for fibrosis driven by oxidative hepatocyte injury. In contrast, in the CDAA model of chronic metabolic stress, Hipk2-KO mice showed a significant attenuation of liver fibrosis at advanced stages of disease progression. Despite the reduced collagen deposition, the canonical HSC activation marker αSMA, assessed by immunostaining and Acta2 expression, was comparable between genotypes, indicating that Hipk2 deletion does not impair HSC activation or myofibroblast differentiation. At early stages of CDAA feeding, Hipk2-KO livers exhibited reduced mRNA expression of pro-inflammatory cytokines such as Il6 and Tnfα, consistent with an attenuated 10 inflammatory response to CDAA-induced hepatocellular stress. Transcriptomic analyses revealed alterations in ECM components, with significant downregulation of non-fibrillar collagens, including collagen IV and collagen VI, suggesting that Hipk2 influences the qualitative composition of the fibrotic ECM. In parallel, transcriptomic profiling revealed coordinated changes in hepatic lipid metabolic gene expression in Hipk2-KO livers, consistent with enhanced fatty acid oxidation and reduced de novo lipogenesis, indicating a shift toward more protective fatty acid handling. Hipk2-KO organoids cleared excess fatty acids more efficiently, resulting in lower LD accumulation. Notably, a similar reduction in LD accumulation was observed in Hipk2-WT organoids upon pharmacological inhibition of HIPK2. Collectively, these findings identify HIPK2 as a context-dependent regulator of liver fibrogenesis that modulates hepatic adaptation to chronic metabolic stress. At early stages, Hipk2-KO livers display coordinated transcriptional shifts in metabolic, inflammatory, and non-fibrillar ECM processes that limit fibrogenic progression, with functional consequences becoming evident only after prolonged CDAA feeding. This pattern suggests that Hipk2 modulates the hepatic microenvironment influencing the liver response to metabolic injury-driven fibrotic progression. This study highlights HIPK2 as a modulator of NASH-induced liver fibrosis and supports its potential as a therapeutic target in metabolic liver disease-associated fibrogenesis.